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3rd COST ACTION CM1307 CONFERENCE ● SOCEPA ● SEFIG

Antiparasitic Chemotherapy for

Human and Veterinary Use

3rd COST Action CM1307 Conference / SOCEPA / SEFIG Joint Meeting

& WG2 and WG3 Meeting

October 24‐26, 2016 Madrid, Spain

3rd COST Action CM1307 Conference ● SOCEPA ● SEFIG, Madrid, 2016

Chemotherapy towards diseases caused by endoparasites: Antiparasitic Chemotherapy for Human and Veterinary use| 1

COST Action CM1307

Chemotherapy towards diseases caused by endoparasites: Antiparasitic Chemotherapy for

Human and Veterinary Use

3rd COST Action CM1307 Conference / SOCEPA / SEFIG Joint Meeting

& WG2 and WG3 Meeting

October 24-26, 2016 Madrid, Spain



The Joint COST Action CM1307 3rd Conference / Sociedad Española de Parasitología (SOCEPA) / Sociedad Española de Farmacia Industrial y Galénica (SEFIG)/ WG2 and WG3 Meeting has been organized by the UCM Research Group ICPVet, Faculty of Veterinary Medicine, Universidad Complutense de Madrid, Madrid, Spain. Conference and WG2 and WG3 will be held in the Hotel Tryp Chamartín, Calle Mauricio Ravel 10, 28046 Madrid (http://www.melia.com/en/hotels/spain/madrid/tryp-madrid-chamartin-hotel/index.html).

This meeting is focused on different topics covering the scope of the COST Action CM1307. Invited conferences and talks have been organized in several sessions:

Molecules and targets I

Molecules and targets II

Molecules and targets III

Molecules and targets IV

Natural products

Drug delivery I

Drug delivery II

Drug delivery III

Screening models

Chemotherapy of parasitic diseases in human and veterinary medicine

The meeting also includes posters and a round table discussion at the end of the 3rd Conference COST CM1307/SEFIG/SOCEPA meeting.

The meeting will be followed by the meetings of working groups WG2 (Medicinal Chemistry) and WG3 (Natural products) and the meeting of Management Committee of the COST Action.

http://www.melia.com/en/hotels/spain/madrid/tryp-madrid-chamartin-hotel/index.html

http://www.melia.com/en/hotels/spain/madrid/tryp-madrid-chamartin-hotel/index.html



Scientific Committee Philippe M. Loiseau (University of Paris-SUD-CNRS, France)

R. Luise Krauth-Siegel (University of Heidelberg, Germany)

Harry P. de Koning (University of Glasgow, United Kingdom)

M. Paola Costi (University of Modena and Reggio Emilia, Italy)

Tomaž Šolmajer (University of Ljubljana, Slovenia)

Vassilios Roussis (National and Kapodistrian University of Athens, Greece)

Thomas J. Schmidt (University of Münster, Germany)

Francisco J. Otero-Espinar (University of Santiago de Compostela, Spain)

Fred Opperdoes (De Duve Institute, Belgium)

Ana Tomás (University of Porto, Portugal)

Juan M. Irache (University of Navarra, Spain)

Organizing Committee María Jesús Corral (University Complutense of Madrid, Spain) Ana Isabel Olías-Molero (University Complutense of Madrid, Spain) M. Dolores Jiménez-Antón (University Complutense of Madrid, Spain)

Alberto Gutiérrez-Dionisio (University Complutense of Madrid, Spain)

José María Alunda (University Complutense of Madrid, Spain) Philippe M. Loiseau (University of Paris-SUD-CNRS, France)

Sponsors The meeting has been sponsored by the Universidad Complutense of Madrid,

SOCEPA and the Colegio de Veterinarios de Madrid (Colvema).



Scientific Programme October 23rd 18:30 Get-together meeting: cocktail –“vino español” October 24th OPENING OF THE CONFERENCE 9:00 Otero-Espinar F.J., President SEFIG Valladares B., President SOCEPA Loiseau P.M., Chair COST Action CM1307 OPENING LECTURE 9:20 Descoteaux A., INRS- Institut Armand-Frappier, Laval, Canada (COST invited speaker)

Leishmania targets the host cell membrane fusion machinery to modulate immune responses

MOLECULES AND TARGETS I Chairpersons A. Descoteaux, A. Tomas 10:00 Loiseau P.M., University of Paris-Sud, France Design of antileishmanial agents targeting host cell vesicular trafficking 10:20 Pomel S., University of Paris-Sud, France

GDP-mannose pyrophosphorylase: a therapeutic target for the selection of new specific antileishmanial agents

10:40 Mangalagiu I.I., Alexandru Ioan Cuza University of Iasi, Romania New azaheterocycles derivatives of potential interest in leishmaniasis 11:00 Stevanović S., University of Belgrade, Serbia Design of novel NADH dehydrogenase inhibitors as antileishmanial agents 11:20-11:50 Coffee break and poster viewing



MOLECULES AND TARGETS II Chairpersons E. Davioud-Charvet, R. Leurs 11:50 Kivrak A., Yuzuncu Yil University, Turkey

Synthesis of novel thienocarbazole derivatives via intramolecular cyclization reactions

12:10 Botta M., University of Siena, Italy

New promising targets vs neglected Tropical Diseases (Leishmaniasis and Trypanosomiasis)

12:30 García-Sosa A.T., University of Tartu, Estonia Designing inhibitors with pan-activity and poly-pharmacology for neglected diseases and parasites

12:50-14:00 Lunch and poster viewing MOLECULES AND TARGETS III Chairpersons H. de Koning, F. Opperdoes 14:00 Krauth-Siegel R.L., Biochemie-Zentrum der Universität Heidelberg, Germany

Small redoxins as target molecules in antitrypanosomal drug development 14:20 Leurs R., VU University Amsterdam, The Netherlands

Phosphodiesterase inhibitors as a potential treatment for Neglected Parasitic Diseases

14:40 Dardonville C., Instituto de Química Médica IQM-CSIC, Spain

Targeting trypanosome alternative oxidase (TAO) inhibitors to mitochondria: original targeting for a unique target

15:00 de Koning H.P., University of Glasgow, United Kingdom

Trypanocydal action of bisphosphonium salts through a mitochondrial target in bloodstream form of Trypanosoma brucei

15:20-16:00 Coffee break and poster viewing



MOLECULES AND TARGETS IV Chairpersons R.L. Krauth-Siegel, P. Loiseau 16:00 Mukherjee B., University of Geneva, Switzerland Structure-function relationships of Toxoplasma gondii aspartyl protease 3 16:20 Doligalska M., University of Warsaw, Poland On the way to find a cure for infection with Babesia microti 16:40 de Koning H.P., University of Glasgow, United Kingdom

Potential antischistosomal activity of PDE inhibitors using in vitro Schistosoma mansoni worm killing

17:00 Leontovyč A., The Czech Academy of Sciences, Czech Republic

Secreted serine protease SmSP2 of the blood fluke Schistosoma mansoni: biochemical characterization, localization and host protein processing

17:20 Cordeiro-da-Silva A., I3S, IBMC, University of Porto, Portugal

Trypanosomatid ribose 5-phosphate isomerase structures and fragment screening revels novel lead compound series

17:40 Free time* *18:00: Walking free tour: a visit to monumental Madrid



October 25th NATURAL PRODUCTS Chairpersons T.J. Schmidt, V. Roussis 09:00 Schmidt T.J., University of Münster, Germany

European box tree (Buxus sempervirens L.) contains alkaloids with potent and selective anti-protozoal activity

09:20 Biedermann D., The Czech Academy of Sciences, Czech Republic The effect of flavonolignans on the Mesocestoides vogae (Cestoda) tetrathyridia 09:40 Gemma S., University of Siena, Italy

From the natural compound dihydroplakortin to synthetic bicyclic and bridged endoperoxides active against chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum parasites

10:00 Ebiloma G.U., University of Glasgow, United Kingdom

Antiprotozoal compounds from Nigerian medicinal plants: identification and mode-of-action studies

DRUG DELIVERY I Chairpersons P. Couvreur, J.J. Torrado 10:20 Couvreur P., University of Paris-Sud, France (COST Invited speaker)

Nanotechnologies for the treatment of severe diseases

11:00-11:20 Coffee break and poster viewing

DRUG DELIVERY II Chairpersons P. Couvreur, J.J. Torrado 11:20 Otero-Espinar F.J., University of Santiago de Compostela, Spain (SEFIG

sponsored speaker) Development and in vivo efficacy of biocompatible drug-loaded microspheres against Cryptosporidium parvum

11:40 Serrano D.R., University Complutense of Madrid, Spain Is the oral delivery of Amphotericin B possible to treat parasitic diseases? 12:00 Lopes F., University of Lisbon, Portugal

Nanoencapsulation of tetraoxane-based double drugs with antileishmanial activity



DRUG DELIVERY III Chairpersons F.J. Otero-Espinar, L. Maes 12:20 Golenser J., The Hebrew University of Jerusalem, Israel

Inhibition of Schistosoma mansoni development in mice by slow release of artemisone

12:40 Ucisik M.H., Istanbul Medipol University, Turkey

Emulsomes: A tool for delivery of anti-leishmanial BNIP derivatives to macrophages

13:00 Torrado J.J., University Complutense of Madrid, Spain (SEFIG sponsored

speaker) Cyclodextrins in antiparasitic drug formulations 13:30-14:30. Lunch and poster viewing SCREENING MODELS Chairpersons C.R. Caffrey, T. Solmajer 14:30 Caffrey C.R., University of California San Diego, USA An automated screening technology for the schistosome helminth parasite 14:50 Hendrickx S., University of Antwerp, Belgium

In vitro “time-to-kill” assay to assess the cidal activity dynamics of current reference drugs against Leishmania donovani and L.infantum

15:10 Caljon G., University of Antwerp, Belgium Is it important to include the insect vector to evaluate the potential of a drug? EMERGING ISSUES Chairpersons M.P. Costi, J.Mª Alunda 15:30 Vivancos V., University Miguel Hernández Elche, Spain (SEFIG sponsored

speaker) Drug absorption modifications in giardiasis 15:50 Costi M.P., University of Modena and Reggio Emilia, Italy

Fragment-based drug discovery fosters the identification of new leads against Trypanosoma brucei PTR1

16:10-16:30. Coffee break and poster viewing



CHEMOTHERAPY OF PARASITIC DISEASES IN HUMAN AND VETERINARY MEDICINE Chairpersons K. Pfister, J.M. Harrington 16:30 Pfister K., LMU Munich, Germany (COST invited speaker) Anti-parasitic treatment in veterinary medicine: a big challenge..! 17:10 Harrington J.M., Merial Inc., USA

Past, present and future of endoparasiticides at Merial 17:50-19:00. Round table discussion: Resistance, therapeutic failure and beyond Moderator F. Gamarro, Instituto de Parasitología y Biomedicina López-Neyra, CSIC (IPBLN-CSIC), Granada, Spain Pfister K., LMU Munich, Germany Maes L., University of Antwerp, Belgium Hervás P., Veterindustria, Spain Alunda J.M., University Complutense of Madrid, Spain Loiseau P.M., University of Paris-Sud, France Otero Espinar F.J., SEFIG, University of Santiago de Compostela, Spain López Medrano F., Hospital 12 de Octubre Madrid, Spain Harrington J.M., Merial Inc, USA

Selzer P., Boehringer Ingelheim Animal Health, Germany 19:00 Concluding remarks and closure of the annual plenary COST CM1307

Conference and Joint meeting with SEFIG and SOCEPA 20:30 Gala dinner Restaurante “La Chalana” Paseo de la Castellana, 179

Madrid 28015 October 26th 9:00-12:00 Meetings of the COST Working groups WG2 (Medicinal Chemistry) and

WG3 (Natural products). Separate meetings and common discussion. 12:00-12:45 Quick lunch for WG participants and MC members 12:45-14:30 COST Management Committee meeting (only for MC members and

MC substitutes) 14:30 Farewell



Poster Outline (24th & 25th October 2016)

P.1. Palmieri N., University of Tartu, Estonia Identification of protein kinase inhibitors in Cystoisospora suis by genomic-based virtual screening P.2. Vasilache V., Alexandru Ioan Cuza University of Iasi, Romania New bis-pyridazine derivatives of potential interest in leishmaniasis P.3. Sarlauskas J., Vilnius University, Lithuania Preliminary in vitro studies of antiprotozoal activity of some heterocyclic N-oxides and N,N’-dioxides P.4. Horn M., The Czech Academy of Sciences, Czech Republic Structural basis for vinyl sulfone inhibition of the SmCB1 drug target from the human blood fluke P.5. Gomes-Alves A.G., I3S, IBMC, University of Porto, University of Minho, Portugal Anti-Leishmania activity of a series of Quinolin-4(1H)-imines P.6. Gil C., Centro de Investigaciones Biológicas, CSIC, Spain Development of new quinone derivatives against Leishmania P.7. Natto M.J., University of Glasgow, United Kingdom Molecular characterisation and cloning of Novel Equilibrative Nucleoside Transporter family members in Trichomonas vaginalis P.8. Peric M., University of Zagreb, Croatia Anti-Toxoplasma activity of novel macrolide hybrid derivatives P.9. Loiseau P.M., University of Paris-Sud, France Anti-malarial combination therapy: synergistic effect between an antisense strategy and different antimalarial drugs in resistant strains of Plamodium falciparum P.10. Gil C., Centro de Investigaciones Biológicas, CSIC, Spain Drug repurposing of human kinase inhibitors as new hits against Leishmania P.11. Baltas M., CNRS, University Paul Sabatier, France Microwave-assisted and conventional 1,3-dipolar cycloaddition reactions to the synthesis of some benzimidazole/(benzo)indolizine hybrids: a comparative study P.12. André-Barrès C., CNRS, University Paul Sabatier, France DFT studies of autoxidation of 2-alkylidene-1,3-cyclohaxadione leading to bicyclic-hemiketal endoperoxides P.13. Calogeropoulou T., National Hellenic Research Foundation, Greece Dinitroaniline-Ether Phospholipid Hybrids P.14. Castro G., University of Porto, Portugal In vitro anti-parasitic activity of marine cyanobacterial extracts against Leishmania, Giardia and Trichomonas



P.15. Dea-Ayuela M.A., University CEU Cardenal Herrera, Spain Efficacy of oral and parenteral Amphotericin B systems against experimental Trypanosoma cruzi infection P.16. Serrano D.R., University Complutense of Madrid, Spain Oral Nanomedicines for Chagas Disease P.17. Fernández R., University Complutense of Madrid, Spain Effect of the aggregation state of Amphotericin B on red blood cells P.18. Alunda J.M., University Complutense of Madrid, Spain Antiparasitic chemotherapy in veterinary medicine: challenges, hurdles and opportunities P.19. Espuelas S., University of Navarra, Spain Early preclinical studies of new selenocyanate and diselenide compounds as leishmanicidal agents P.20. Espuelas S., University of Navarra, Spain Topical treatment of CL with paromomycin and anti-TNF-α antibodies: efficacy study in L.major infected BALB/c mice P.21. Bautista L., University Complutense of Madrid, Spain Novel oral formulations of Active Hexose Correlated Compound (AHCC) and their antiparasitic activity in an in vivo model P.22. Roussis V., National and Kapodistrian University of Athens, Greece In vitro activity evaluation of marine metabolites against bloodstream forms of Trypanosoma brucei P.23. Gamarro F., Instituto de Parasitología y Biomedicina López-Neyra, CSIC

(IPBLN-CSIC), Granada, Spain Influence of glutathione and antimony in the ATPase activity of Leishmania LABCG2 transporter P.24. Costi M.P., University of Modena and Reggio Emilia, Italy The NMTRyPI - New Medicines for Trypanosomatidic Infections – drug discovery platform P.25. Gandhi H., University College Cork, Ireland Modelling, synthesis and evaluation of novel quinine analogues-new drugs for Chagas disease P.26. Boije af Gennäs G., University of Helsinki, Finland Membrane-bound pyrophosphatases – A novel approach to target pathogenic protozoan parasite P.27. Costi M.P., University of Modena and Reggio Emilia, Italy Synergy activities on Neglected Tropical Diseases drug discovery within FP7 EU context



P.27. Davioud-Charvet E., University of Strasbourg, France Repurposing and old anti-arthritis golden drug, auranofin, and its anticancer GoPi-sugar surrogate for the treatment of human parasitic diseases: from Leishmania to helminth infections



OPENING LECTURE



Leishmania targets the host cell membrane fusion machinery to modulate immune responses

Albert Descoteaux

INRS- Institut Armand-Frappier, Laval, QC, Canada

Successful vacuolar pathogens have developed sophisticated strategies to hijack the

endomembrane system of host cells and evade antimicrobial responses. The protozoan parasite Leishmania, the causative agent of leishmaniases in humans, is particularly adept at transforming the macrophage into a hospitable host cell. As they establish within phagocytes, Leishmania promastigotes release molecules that sabotage host cell microbicidal and immune functions. Lipophosphoglycan (LPG) and GP63 are two virulence factors involved in this process. Hence, we previously established that insertion of LPG from L. donovani promastigotes into host cell lipid microdomains causes remodeling of the parasitophorous vacuole, delays its maturation into a highly microbicidal phagolysosome, and prevents recruitment of the v-ATPase. We also discovered that the surface metalloprotease GP63 enables Leishmania promastigotes to target the macrophage membrane fusion machinery, to create an intracellular compartment favorable to the establishment of infection and to manipulate host immune responses. To achieve this, LPG and GP63 must traffic from the parasitophorous vacuole to the cytoplasm. We obtained evidence that the mechanism associated to this trafficking process involves the host cell endoplasmic reticulum-Golgi intermediate compartment. Collectively, these studies provide insights into the mechanisms of Leishmania pathogenesis.

Supported by the Canadian Institutes of Health Research



MOLECULES AND TARGETS I



Design of antileishmanial agents targeting host cell vesicular trafficking

Sébastien Pomel1, Sandrine Cojean1, Vanessa Liévin Le Moal1, Yu Wu1,

Valérie Nicolas1, Julien Barbier2, Jean-Christophe Cintrat2, Joel Vacus3, Daniel Gillet2, Philippe M. Loiseau1

1 Antiparasitic Chemotherapy, UMR 8076 CNRS BioCIS, Université Paris-Sud,

Chatenay-Malabry, France ([email protected]) 2 SIMOPRO, CEA Saclay, France

3 Drugabilis, Chatenay-Malabry, France

Leishmaniases are a complex of tropical and sub-tropical diseases provoked by Leishmania protozoan parasites transmitted by the sandfly vector and presenting different clinical expressions. Visceral leishmaniases provoked by L. donovani in India and in Africa and L. infantum in the Mediterranean basin are lethal when untreated; muco-cutaneous leishmaniasis in South-America caused by L. braziliensis is sub-lethal but seriously invalidating whereas cutaneous leishmaniasis caused by L. major or L. tropica in Middle-East areas is self-curing leaving disgraceful facial scars.

The anti-leishmanial chemotherapy is expensive, aspecific therefore toxic, and drug resistance is usual or at risk, mainly to antimonials, the most classical drugs, and recently to miltefosine and amphotericin B.

New therapeutic approach are necessary. As parasites such as Leishmania successfully strive in host cells because they divert the intracellular trafficking machinery to maintain their parasitophorous vacuole in which they proliferate, inhibitors of key elements of this diverted machinery should handicap the parasite survival.

From an identification by HTS of compounds blocking the retrograde transport of toxins and active against Leishmania amazonensis, this study aims to identify a drug-candidate for the treatment of leishmaniasis having the following characteristics:

- An original mechanism of action that interferes with vesicle trafficking in host-cell impairing the development of the vacuole in which the parasite proliferates

- No direct and intrinsic antiparasitic activity on the parasite itself in order to reduce the risk of drug resistance emergence

- A suitable drugability for oral or intravenous administration

A chemical series exhibits interesting activities both in vitro and in vivo on the Leishmania infantum/Balb/C mice model according to a flow-chart that has been established to select the most promising compounds. A confocal imaging approach showed that some compounds exert a blockage of the fusion between Leishmania infantum parasitophorous vacuoles and lysosomes during Leishmania infection. Two compounds were active by oral route at 20 mg/kg/day x 5, but significantly less active than miltefosine.

This work was supported by the French National Research Agency (ANR) LabEx LERMIT R3 to P.L. and NRBC Programme n°I11.5 to Yu Wu



GDP-mannose Pyrophosphorylase: a therapeutic target for the selection of new specific antileishmanial agents

W. Mao 1, P.Daligaux 1, N.Lazar 2, C.Cavé 1, H.van Tilbeurgh 2, P.M.Loiseau 1

and S.Pomel 1

1 Université Paris-Sud, Faculté de Phamarcie, UMR CNRS 8076 BioCIS, Châtenay-Malabry

2 Université Paris-Sud, UMR CNRS 9198, Institut de Biologie Intégrative de la Cellule E-mail: [email protected]

Leishmaniases are neglected tropical diseases caused by the protozoan parasite

belonging to the genus Leishmania, and transmitted by an insect vector, the sandfly. Currently, about 350 million people are threatened in 88 countries by leishmaniases, with around 2 million new cases each year. The few existing treatments are very limited because of their cost, toxicity, besides increasing problems of drug resistance. In this context, the development of new antileishmanial drugs specifically directed against a therapeutic target becomes crucial. The GDP-Mannose Pyrophosphorylase (GDP-MP) has been validated as a promising therapeutic target since it was previously shown to be essential for parasite survival in macrophages both in vitro and in vivo. In this work, recombinant GDP-MPs from human and three Leishmania species responsible for either the visceral (L. donovani) or the cutaneous (L. mexicana) form of leishmaniasis were produced and purified in order to determine and compare their enzymatic properties. Previous comparative molecular modelling and docking analyses of human and leishmanial GDP-MPs allowed to design and synthesize 100 compounds derivated from the substrate GDP-mannose. The activity of these compounds was evaluated on the purified enzymes as well as on axenic and intramacrophage amastigotes of L. donovani and L. mexicana. Two compounds presented promising activities on both the purified enzymes and the parasites. Further crystallization studies will allow to determine new pharmacomodulations in order to improve the affinity and specificity of the compounds for the leishmanial GDP-MPs.



New azaheterocycles derivatives of potential interest in leishmaniasis

Ionel I. Mangalagiu

“Alexandru Ioan Cuza” University of Iasi, Faculty of Chemistry, Bd. Carol 11, 700506 Iasi,

Romania. E-mail: [email protected]

Nitrogen derivatives are “privileged structures” in drug design, optoelectronics, etc.,

the azaheterocycle scaffold being a core skeleton for multiple purposes.

Imidazoquinolines (IMQ), especially imiquimod, resiquimod and gardiquimod, represent a class of drugs used in cutaneous leishmaniasis, several mechanisms being described for their antileishmania activity: directly activate macrophages , TLR7 and/or TLR8 agonists, etc. On the other hand, pentamide is a second-line drug largely used in leishmaniasis.

The emphasis of this work consist in design, synthesis and characterization of new azaheterocycles derivatives of potential interest in leishmaniasis.

N

N

NCOOEt

O

Z

Z= -OR, -NH2, -NH-NH2

Z=Y

Y= H, Cl,

Br, OCH3

, C6H5

N

N

COOEtO

Z

I II

O

NN

NN

O

O

ZZ

CY= - OR, -NH2 - NH-NH2Z=

O

Y

III

Acknowledgements. Authors are thankful to COST Action CM1307 and to CNCS Bucharest, Romania, project PN-II-DE-PCE-2011-3-0038, no. 268/05.10.2011, for financial support.



Design of novel NADH dehydrogenase inhibitors as antileishmanial agents

Strahinja Stevanović1, Andrej Perdih2, Sanja Glišić1, Tomaž Šolmajer2

1Center of Multidisciplinary Research, Institute of Nuclear Sciences “Vinča”, University of Belgrade, Mike Petrovića Alasa 12-14, 11001 Belgrade, Serbia; 2National Institute of

Chemistry, Hajdrihova 19 1001 Ljubljana, Slovenia. E-mail: [email protected]

Alternative NADH dehydrogenase (NDH2) is essential enzyme of the Leishmania

Infantum respiratory systhem. These enzymes catalyze transference of electrons from NADH to ubiquinone molecule, without coupled proton pumping.

Previous studies of NADH-II: rubiquinone oxydoreductase (NDH2) crystal structure reported that there are two close ubiqinone UQ binding sites, which are responsible for mechanism of oxydoreduction in presence of cofactors NADH and FAD. We developed novel NADH dehydrogenase (Leishmania Infantum) 3D model structure based on homologus models1 using advanced remote homology detection methods (Phyre2 server2). In order to select commertially available compounds for potential inhibitor activity against LiNDH2, we trained pharmacophore models based on homologus NDH2 activity profile of inhibitors of the quinone class, from another study (Plasmodium Falciparum) 3. Approximately 550,000 commercially available compounds were screened using LigandScout4 and compounds with desired pharmacophoric features had been selected. Afterwards, docking screening in GOLD Suite5, using genetic algorithm, tested selected compounds fittings into selected UQ binding site in LiNDH2 homology model. Resulting compounds list is consensus from docking scores, pharmacophore fit scores and visual examination of structure-based and ligand-based designs. We selected 24 hits with variety of scaffolds for further testing.

References:

1. Feng Y, Li W, Li J, Wang J, Ge J, Xu D et al. Structural insight into the type-II mitochondrial NADH dehydrogenases. Nature 2012 491(7424):478-482.

2. The Phyre2 web portal for protein modeling, prediction and analysisKelley LA et al. Nature Protocols 2015 10: 845-858

3. Biagini G, Fisher N, Shone A, Mubaraki M, Srivastava A, Hill A et al. Generation of quinolone antimalarials targeting the Plasmodium falciparum mitochondrial respiratory chain for the treatment and prophylaxis of malaria. Proceedings of the National Academy of Sciences 2012 109(21):8298-8303.

4. Wolber GLanger T. LigandScout: 3-D Pharmacophores Derived from Protein-Bound Ligands and Their Use as Virtual Screening Filters. Journal of Chemical Information and Modeling 2005 45(1):160-169.

5. Jones G, Willett P, Glen R, Leach A, Taylor R. Development and validation of a genetic algorithm for flexible docking. Journal of Molecular Biology 1997 267(3):727-748.



MOLECULES AND TARGETS II



Synthesis of Novel Thienocarbazole derivatives via intramolecular cyclization reactions

Arif Kivrak

Department of Chemistry, Yuzuncu Yil University, 65080 Van, TURKEY

E-mail: [email protected]

Carbazoles and derivatives have emerged as central candidates for pharmaceutical applications since they show remarkable analgesic, anti-inflammatory, anti-bacterial antiparasitic, antitussive, hypoglycemic, antitumor and/or anticancer activities. Many synthetic methods have been used for the synthesis of new heterocyclic compounds. They have also been isolated from different kinds of plants and used to treat different diseases for many years. On the other hand, there are a few studies for thienocarbazoles which may have critical biological properties. Thieno[c]carbazoles are bioisosters of pyridocarbazoles which have a broad range of biological activities including anticancer, antibacterial and antifungal properties. In this study, novel methodologies for the synthesis of potentially biologically active thieno[c]carbazoles was developed by using intramolecular cyclization reactions.



New Promising Targets vs Neglected Tropical Diseases (Leishmaniasis and Trypanosomiasis)

Giusy Tassone1, Laura Friggeri1, Mattia Mori1,2, Claudio Zamperini1 Fernanda

A.H. Batista3, Julio Cesar Borges3, Kevin Read4, Manu De Rycker5 and Maurizio Botta6

1 University of Siena, Department of Biotechnology, Chemistry and Pharmacy, Siena.

Italy; 2Instituto Italiano di Tecnologia, Center for Life Nano Science@Sapienza, Roma. Italy; 3Universidade de São Paulo (USP), Instituto de Química de São Carlos, Grupo de Biologia Molecular e Bioquímica, São Carlos. Brazil; 4University of Dundee, School of Life Sciences,

Biological Chemistry and Drug Discovery. United Kingdom; 5University of Dundee, Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery. United Kingdom;

6 Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, Philadelphia. USA


Leishmaniasis and trypanosomiasis are two of the most important neglected tropical diseases. These diseases affect largely the poorest side of population leaving in developing countries and more than 20 million people are infected worldwide. [1] Currently available therapy against these diseases is not satisfactory, as it relies on non-specific, toxic and not effective drugs. [2] Therefore it is necessary to develop a new specific drug therapy against these diseases. In a medicinal chemistry-oriented strategy for identifying new scaffolds potentially active against Leishmania donovani and Trypanosoma cruzi, we selected 80 compounds with different chemical scaffolds, belonging to our in house library. These compounds were tested against Leishmania spp, and the obtained data demonstrated that eight molecules showed interesting antileishmanial activity. Four potential Hit to Lead: KLDS-20, -67, -2, -61 showing interesting activity with pEC50 (log(EC50[M])) values approximately of 5 (IC50=10 μM), were identified. These compounds were subjected to the experimental determination of ADME proprieties. These studies have shown a good permeability profile and many compounds have shown a high metabolic stability. Currently, some studies to increase the activity of compounds with similar structure to KLD20 are running, in order to increase its solubility, improve the ADME characteristics but without altering the pharmacological activity. Preliminary results of our 80 compounds vs amastigote stage of Trypanosoma cruzi show that some compounds emerge as potential antitrypanosomal compounds. The most promising compounds are the ones with a Trypanosoma cruzi maximum effect > 90% (100% means that the compound is as good at clearing parasites as Nifurtimox). In particular, the compound KLD47 shows a Trypanosoma cruzi maximum effect of 94%, a pEC50 of 5.2 and a cytotoxic effect of 22%.

In addition, we selected Heat shock protein 90 (Hsp90) of Leishmania braziliensis as a potential specific target for the development of a specific drug therapy against leishmaniasis. [3] Heat shock protein is an ubiquitous protein functioning as molecular chaperone that stabilizes client proteins in a folded and functional state. Hsp90 plays a significant role in the life cycle control of the protozoan parasite Leishmania braziliensis and is essential for survival and proliferation of the intracellular mammalian stage, the amastigote. At first we set up a robust structure-based approach boosted by similarity

mailto:[email protected]



search and docking-based virtual screening studies for identifying small molecule inhibitors of N-terminal domain of Leishmania braziliensis Hsp90. These compounds were investigated in vitro for anti-protozoa activity and some of them emerged as potential ligands of Hsp90. In particular, for one of them a Kd of about 17 μM has been determined. To date, the crystallographic structure of Leishmania braziliensis is not available. For this reason we have initiated the process of determining the three-dimensional structure of the protein in order to obtain insight into its structure and protein-inhibitors complexes. The protein was overexpressed in Escherichia coli strain BL21 and the purification trial was carried out using a Nickel affinity column at levels and purities sufficient for performing the crystallization trials that currently are ongoing.

References [1] Li, Q. et al, Parasitology research. 2009, 105 (6), 1539-48 [2] McGwire, B. S.et al, QJM : monthly journal of the Association of Physicians. 2014,

107 (1), 7-14 [3] Silva KP et al, Biochim Biophys Acta. 2013;1834(1):351-6



Designing inhibitors with pan-activity and poly-pharmacology for neglected diseases and parasites

Alfonso T. García-Sosa 1

1Institute of Chemistry, University of Tartu, Ravila 14a, Tartu 54011, Estonia


A variety of neglected diseases present as comorbidity. In addition, compounds that could treat several diseases would be highly desirable from an affordability point of view, as well as for compliance to treatment regimen, and delaying drug-resistance development. There are reports of compounds that can inhibit several diseases at the same time, such as leishmaniasis, Chagas disease, and sleeping sickness.[1]

Arginase is a critical component for several Leishmania organisms.[2] Virtual screening using a database of natural products with EIIP/AQVN and 3D QSAR filters, as well as docking to Leishmania and human arginase structures in addition to anti-targets, has been performed resulting in a list of compounds with desirable properties. A known inhibitor, flavonoid, natural compound was recovered among this list.

Cystoisospora suis is an intestinal parasite present in pigs and responsible for large losses. Virtual screening was conducted on the target CDPK1 (Toxoplasma gondii homolog), with a list of compounds selected for further testing.

1. Khare, S.; Nagle, A. S.; Biggart, A.; et al. “Proteasome inhibition for treatment of

leishmaniasis, Chagas disease, and sleeping sickness”, Nature, 2016, doi:10.1038/nature19339

2. Glisic S., Sencanski M., Perovic V., Stevanovic S., García-Sosa A. T., "Arginase flavonoid anti-leishmanial in silico inhibitors flagged against anti-targets", Molecules, 2016, Vol. 21, Iss. 5, 589. doi:10.3390/molecules21050589

Acknowledgments Dr. Sanja Glisic, Dr. Milan Sencanski, and Dr. Nicola Palmieri for collab. Estonian Ministry of Science and Education, Grant Number: IUT34-14 EU COST Action CM1307 Targeted chemotherapy towards diseases caused by endoparasites EU COST Action CA15135 Multi-target paradigm for innovative ligand identification in the drug discovery process (MuTaLig)



MOLECULES AND TARGETS III



Small Redoxins as Target Molecules in Antitrypanosomal Drug Development

Samantha Ebersoll1, Blessing Musunda1, Marcelo A. Comini2 and

R. Luise Krauth-Siegel1

1Biochemie-Zentrum der Universität Heidelberg (BZH), Im Neuenheimer Feld 328, D69120

Heidelberg, Germany, E-mail: [email protected]; 2Group Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, Mataojo 2020, CP

11400, Montevideo, Uruguay.

In all organisms, redoxins are involved in the redox regulation of a huge number of cellular processes. Typical representatives are thioredoxins and glutaredoxins (Grxs), proteins with a molecular mass of 10-15 kDa and a redox active CXXC motif. Work of the last few years revealed the small oxidoreductases as targets of different antitumor drugs.

For maintaining the intracellular thiol redox homeostasis, trypanosomatids employ trypanothione [T(SH)2] which is kept reduced by trypanothione reductase (TR). T(SH)2 is the direct electron donor for a variety of vital pathways, most of the reactions being mediated by the parasite specific tryparedoxin (Tpx). The T(SH)2/Tpx couple is the electron donor for ribonucleotide reductase, methionine sulfoxide reductase and the enzymes that catalyze the detoxification of peroxides. Tpx is the central cytosolic redoxin. The oxidoreductase is essential and its inactivation is an attractive approach for the development of novel antitrypanosomal drugs.

Here I will also discuss the role of two Grxs of African trypanosomes which are located in the cytosol and mitochondrial intermembrane space. Both proteins are kept reduced by T(SH)2. They catalyze the thiol/disulfide exchange between glutathione disulfide and T(SH)2 as well as the deglutathionylation of different model components. The cytosolic Grx1 accounts for the major part of the total deglutathionylation capacity of the parasite. Deletion of both grx1 and grx2 alleles, respectively, did not result in any proliferation defect of bloodstream parasites, even not under various stress conditions. Apparently, these redoxins are dispensable and thus unlikely to represent putative drug targets. Intriguingly, when rising the culture temperature from 37 °C to 39 °C, proliferation of the Grx1- and Grx2-deficient bloodstream parasites is even significantly less affected compared to that of wildtype cells indicating that the proteins play a regulatory role in the thermotolerance of the parasites (Musunda et al. 2015, Mol. Biochem. Parasitol. 204, 93-105).




Phosphodiesterase Inhibitors as a Potential Treatment for Neglected Parasitic Diseases

Rob Leurs

Division of Medicinal Chemistry, Faculty of Science, Amsterdam Institute for Molecules,

Medicines and Systems (AIMMS), VU University Amsterdam, the Netherlands. E-mail: [email protected], [email protected]

Cyclic nucleotide phosphodiesterases (PDEs) have emerged as attractive molecular targets for a novel treatment for a variety of Neglected Parasitic diseases, including African trypanosomiasis, Chagas disease, and malaria. For example, both genetic knock-down and chemical inhibition of PDE activity resulted in halted proliferation and eventually elimination of Trypanosoma brucei (Tbr), the causative agent of African sleeping sickness. The vast knowledge and generated expertise within the field of human PDEs provides a shortcut to high-affinity inhibitors of parasitic PDEs. We have brought together a public-private consortium with PDE experts, medicinal chemists and parasitologists to effectively target parasitic PDEs.

The PDE4NPD project is an EU-funded platform to develop new chemical entities targeting parasitic PDEs. The PDE4NPD project is supported by the European Union 7th Framework Program (FP7/2007-2013) under grant agreement n° 602666 and involves ten consortium members and research labs in seven countries (www.PDE4NPD.eu). In this presentation we will show our progress in developing approaches to combat parasitic diseases by both a phenotypic and target-based approaches.



http://www.pde4npd.eu/



Targeting trypanosome alternative oxidase (TAO) inhibitors to mitochondria: original targeting for a unique target

Christophe Dardonville1, Francisco José Fueyo González1, Godwin U. Ebiloma2,

Carolina Izquierdo García1, Victor Bruggeman1, José María Sánchez Villamañán1, Anne Donachie2, Emmanuel Balogun3, Kiyoshi Kita3,

Harry P. de Koning2

1 Instituto de Química Médica, IQM–CSIC, Juan de la Cierva 3, E–28006 Madrid, Spain.

2 Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.

3 Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Japan.

During their life-cycle, trypanosomes adapt their energy metabolism to the availability

of nutrients in their environment. Hence, procyclic forms of T. brucei have a fully functional respiratory chain and synthesize ATP by oxidative phosphorylation in the mitochondrion. In contrast, respiration of bloodstream forms (BSF) of T. brucei (i.e. the human-infective form) relies exclusively on glycolysis for energy production. The trypanosome alternative oxidase (TAO) is the sole terminal oxidase enzyme to re-oxidize NADH accumulated during glycolysis. It is a cyanide-resistant and cytochrome-independent ubiquinol oxidase which is sensitive to the specific inhibitors salicylhydroxamic acid (SHAM) and ascofuranone. This enzyme which is essential to the viability of BSF trypanosomes and has no counterpart in the mammalian host is a potential target for chemotherapy.

To boost the activity of a TAO inhibitor, 4-(decyloxy)-2-hydroxybenzoic acid (1), which was inactive against T. brucei in a whole cell assay, we investigated a chemical strategy consisting of the conjugation of the inhibitor with lipophilic cations (LC) that can cross lipid bilayers by non-carrier mediated transport, and thus accumulate specifically into mitochondria, driven by the plasma and mitochondrial transmembrane potentials (negative inside). This design afforded several LC–TAO inhibitor conjugates active in the submicromolar to low nanomolar range against wild type and resistant strains of African trypanosomes (T. b. brucei, T. congolense). Selectivity over human cells was >500 and reached >23,000 for one compound. Studies of the effects on purified TAO, parasite respiration, mitochondrial membrane potential (Ψm), and cell cycle showed that the compounds effectively target TAO in vitro. Hence, the LC-carrier strategy successfully delivers specific antiparasitic drugs to their mitochondrial target. This chemical approach could be applied to other mitochondrial targets validated for antiparasitic drug discovery, including against intracellular parasites.

Acknowledgements. This work was funded by the Spanish Ministerio de Economia y Competitividad (SAF2015-66690-R). G. U. Ebiloma was supported by a TET-fund studentship from the government of Nigeria and by a Mac Robertson Travel Scholarship from the College of Medical, Veterinary and Life Sciences of the University of Glasgow.



Trypanocidal action of bisphosphonium salts through a mitochondrial target in bloodstream form Trypanosoma brucei

Abdulsalam A. M. Alkhaldi,1 Jan Martinek, 2 Brian Panicucci,2 Christophe

Dardonville,3 Alena Zíková 2 and Harry P de Koning 1

1 Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom; 2 Institute of Parasitology, Biology

Centre & Faculty of Science, University of South Bohemia, České Budějovice, Czech Republic; 3 Instituto de Química Médica, IQM–CSIC, Madrid, Spain.

Lipophilic bisphosphonium salts are among the most promising antiprotozoal leads

currently under investigation. As part of their preclinical evaluation we here report on their mode of action against African trypanosomes, the etiological agents of sleeping sickness. The bisphosphonium compounds CD38 and AHI-9 exhibited rapid inhibition of T. brucei growth, apparently the result of cell cycle arrest that blocked the replication of mitochondrial DNA, contained in the kinetoplast, thereby preventing the initiation of S-phase. Incubation with either compound led to a rapid reduction in mitochondrial membrane potential, and ATP levels decreased by approximately 50% within 1 h. Between 4 and 8 h, cellular calcium levels increased, consistent with release from the depolarized mitochondria. Within the mitochondria, the Succinate Dehydrogenase complex (SDH) was investigated as a target for bisphosphonium salts, but while its subunit 1 (SDH1) was present at low levels in the bloodstream form trypanosomes, the assembled complex was hardly detectable. RNAi knockdown of the SDH1 subunit produced no growth phenotype, either in bloodstream or in the procyclic (insect) forms and we conclude that in trypanosomes SDH is not the target for bisphosphonium salts. Instead, the compounds inhibited ATP production in intact mitochondria, as well as the purified F1 ATPase, to a level that was similar to 1 mM azide. Co-incubation with azide and bisphosphonium compounds did not inhibit ATPase activity more than either product alone. The results show that, in Trypanosoma brucei, bisphosphonium compounds do not principally act on succinate dehydrogenase but on the mitochondrial FoF1 ATPase. This study also provides the first comprehensive investigation of the Succinate Dehydrogenase Complex in bloodstream and procyclic Trypanosoma brucei.



MOLECULES AND TARGETS IV



Structure-function relationships of Toxoplasma gondii aspartyl protease 3

Budhaditya Mukherjee1, Sunil Kumar Dogga1, Marq JB1, Paco Pino1, Francesca

Tessaro2, Ruben Hartkoorn3, Gianpaolo Chiriano2, Leonardo Scapozza2 and Dominique Soldati-Favre1

1Department of Microbiology and Molecular Medicine. Faculty of Medicine. University of Geneva, CMU, 1 rue Michel-Servet, 1211 Geneva 4 Switzerland.

2Pharmaceutical Biochemistry, School of Pharmaceutical Sciences, University of Lausanne, University of Geneva, Switzerland. Quai Ernest-Ansermet 30, 1211 Genève 4 – Switzerland.

3Institut Pasteur de Lille, Lille France Email: [email protected]

Toxoplasma gondii is a member of the phylum Apicomplexa and an important

pathogen for humans and animals. Active entry and egress from infected host cells are key steps in the lytic cycle of this obligate intracellular parasite. Adhesins, perforins and proteases are discharged by the regulated secretory organelles called micronemes and critically contribute to parasite motility invasion and egress. The T. gondii aspartyl protease 3 (TgASP3) resides in the endosomal-like compartment (ELC) and acts as a maturase that processes at least one microneme protein, TgMIC6, which traffics through the ELC as part of its targeting to the organelles. Conditional deletion of TgASP3 gene severely impairs invasion and egress without impacting on parasite intracellular growth or gliding motility.

A peptidomimetic inhibitor based on a hydroxy-ethyl-amine scaffold (compound 49c); developed against the Plasmodium aspartyl protease Plasmepsin II exhibits an IC50 of ~ 0.6 µM against T. gondii. Importantly 49c recapitulates TgASP3 depletion phenotypes and inhibits TgASP3 activity in vitro. 49c also blocks malaria parasite egress from infected erythrocytes at subnaomolar concentrations and presumably targets the orthologues of TgASP3, Plasmepsin IX and X but not the hemoglobin degrading enzyme Plasmepsin II.

The catalytic dyad of the aspartyl proteases is composed of two aspartic acid residues, acting as proton donor and acceptor when cleaving the peptide bond. The flap and flap-like structures of several Plasmepsins have been reported to be highly flexible in both free and ligand-bound form and to critically modulate the access of the binding cavity to various inhibitors. Compared modeled structures of various Plasmepsins and TgASP3 in presence of 49c predicted that key conserved phenylanine residues in the flap could account for the sensitivity of ASP3 to 49c. Expression of ASP3 mutated in these residues in parasites lacking ASP3 confirmed this prediction with an increased IC50 value to ~ 2 µM.



On the way to find a cure for infection with B. microti

Joanna SZYMCZAK, Katarzyna Donskow-Łysoniewska, Maria DOLIGALSKA

Department of Parasitology, Institute of Zoology, Faculty of Biology, University of Warsaw Address: Ilji Miecznikowa 1, 02-096 Warszawa, Poland

Email: [email protected]

Babesiosis is an emerging, tick-borne disease caused by intraerythrocytic parasite Babesia microti. In immunocompetent individuals B. microti infection is typically asymptomatic or appears as a mild flu-like disease that quickly resolves. Immunocompromised patients, particularly those suffered from B-cell lymphoid malignancies and treated with rituximab, experience severe, persistent and relapsing babesiosis. In these individuals B. microti infection may persist despite multiple courses of treatment with standard antiprotozoal drugs. The increasing incidence of human babesiosis caused by B. microti coupled with a growing number of immunosuppressed people who live or travel in areas where babesiosis is endemic, emphasize the need for new therapeutics for this protozoan infection with more effective mechanisms of action.

The results of our study revealed that in experimental B. microti infection, B cells interact in a complex duet with CD4+ T cells. The cooperation between B cells and T CD4+ cells affects the activation, differentiation and effector functions of both cell populations over the course of babesiosis. The Inhibited interaction between B cells and CD4+ T cells resulted in impaired immune response and consequently, may lead to increased susceptibility to babesia infection.

It is known that rituximab induces a profound and long-term depletion of CD20+ B cells, including the majority of the B-cell lineages, before plasma cell differentiation. These data suggest that the relapse of acute, symptomatic babesiosis in patients with B-cell lymphoid malignancies treated with rituximab may be a result of abnormal interactions between B cells and CD4+ T cells.

Accumulating evidence demonstrates that B cells and CD4+ T cells play a critical role in determining host propensity to cause acute versus persistent disease. Understanding the multiple roles of B cells and CD4+ T cells in regulating cellular and humoral immune responses to parasite may be helpful for the development of an effective vaccine or novel therapeutics against B. microti that can potentially modulate CD4+ T-cell-B-cell interactions for better infection outcome. Further, identifying the possible mechanism involving B cells that is responsible for babesiosis relapse may provide important clues for contributing to optimal treatment for immunocompromised patients infected with B. microti.

Project was co-financed by ESF under the Operational Programme Human Capital, POKL.04.03.00-00-060/12 and Principal Research/2015 of Faculty of Biology, University of Warsaw.




Potential antischistosomal activity of PDE inhibitors using in vitro Schistosoma mansoni worm killing

Sanaa S. Botros1; Samia William1, Abdel-Nasser Sabra1; Geert J. Sterk2; Irene G. Salado3; Koen Augustyns3; Victor Sebastian4; Nuria E. Campillo4; Carmen

Gil4; Louis Maes3; Jane C. Munday5; Rob Leurs1; Harry P. De Koning5

1Theodor Bilharz Research Institute1, Warrak El-Hadar, Imbaba, P.O. Box 30, Giza, Egypt 2Vrije Universiteit Amsterdam, The Netherlands (VUA).

3Universiteit van Antwerpen, Belgium (UA). 4Centro de InvestigacionesBiológicas (CSIC), Madrid, Spain.

5University of Glasgow, UK. Email: [email protected]

We report the testing of 135 non-toxic phosphodiesterase inhibitors developed at

VUA, UA and CSIC, with molecular weights between 234 and 606, for their potential antischistosomal activity. The compounds were assessed for killing of adult and early mature Schistosoma mansoni in vitro; female ovipositing capacity and worm coupling.

Findings of 2-3 repeat experiments revealed potential antischistosomal activities against adult mature schistosomes, expressed as worm killing/and or sluggish worm movement for 19 compounds. However, the effect was recorded using high concentrations of 100 µM and 50 µM, resulting in worm killing of 17%-100% and 8%-64%, respectively. For 74% of the compounds we observed at least some worm killing, with survivors showing sluggish movement. For a further 5% of compounds we observed sluggish worm movement but no worm killing.

In 18 out of 19 promising compounds, only male worms were affected and 100% of those were killed. Meanwhile insult to early mature worms was more pronounced: The percentage worm killing recorded at 25 µM of test compound was 19%-44%, with the insult directed against male worms only.

A subset of the compounds was tested for worm uncoupling with absence of ova, and this was recorded for 74% of the compounds at concentrations of 100 µM and 50 µM; 11% and 5% of the compounds showed the same profile at concentrations of 25 µM and 10 µM, respectively.

The most promising compound was the NPD-000223 (VUA): this compound showed 100%, 64%, 25% and 7% worm killing at concentrations of 100 µM, 50 µM, 25 µM and 10 µM.

Expression and cloning analysis of PDEs in S. mansoni adult and early mature worms revealed higher expression of Sm4A, Sm4C and Sm11 in adult and early mature male worms than in female worms. Sm9C is highly expressed in juvenile male.

This work is part of the PDE4NPD consortium supported by Framework Program 7of the European Commission No: 602666




Secreted serine protease SmSP2 of the blood fluke Schistosoma mansoni: biochemical characterization,

localization and host protein processing

Adrian Leontovyč1, Lenka Ulrychová1, Anthony J. O’Donoghue2, Lucie Marešová1, Jiří Vondrášek1, Conor R. Caffrey2, Michael Mareš1, Martin Horn1,

Jan Dvořák1,3

1Institute of Organic Chemistry and Biochemistry, the Czech Academy of Sciences, Prague, Czech Republic

2Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA 92093, USA

3Institute of Molecular Genetics, the Czech Academy of Sciences, Prague, Czech Republic

Schistosomiasis caused by parasitic blood flukes of the genus Schistosoma is the

second most important parasitic infection after malaria with more than 240 million people infected. There is an urgent need to identify novel anti-schistosomal targets for therapeutic interventions. Our work is focused on S. mansoni serine protease 2 (SmSP2). It was localized in the tegument and esophageal glands, ovaries, testes and vitelaria of adult schistosomes by immunofluorescence microscopy and in situ RNA hybridization. Enzyme activity measurements and immunoblotting identified SmSP2 in the excretory/secretory products. Recombinant SmSP2 was produced in the Pichia pastoris expression system and its cleavage specificity was investigated using combinatorial substrate libraries and 3D model analysis. SmSP2 was found to activate plasmin, the key component of the fibrinolytic system, and releases vasoregulatory kinins from kininogen. Our results suggest that SmSP2 plays a role in host-parasite interactions and represents a potential target for inhibitory drugs.

This work was supported by the grant LD15101 and the project InterBioMed LO1302 from the Ministry of Education, Youth and Sports of the Czech Republic, the institutional project RVO 61388963, the Academy of Sciences of the Czech Republic, Center for Discovery and Innovation in Parasitic Diseases.

http://en.wikipedia.org/wiki/Schistosoma



Trypanosomatid Ribose 5-phosphate isomerase structures and fragment screening reveals novel lead compound series

Nathalie Trouche5, Céline Ronin5, Joana Tavares1,2, Monica Silva1,2, Nuno Santarem1,2,

Joana Faria1,2, Emily A. Dickie4, Louise L. Major4, Fabrice Ciesielski5, Dominique Roecklin5, Emmanuel Klein5, Christina Muller5, Terry K, Smith4, Anabela Cordeiro-da-

Silva1,2,3* & Paola Ciapetti5

1Parasite Disease group, IBMC - Instituto de Biologia Celular e Molecular, Porto, Portugal; 2Instituto de Investigação e Inovação em Saúde, Porto, Portugal; 3Faculdade de

Farmácia da Universidade do Porto, Porto, Portugal 4Biomedical Sciences Research Complex, University of St Andrews, North Haugh, St Andrews,

Fife, KY16 9ST, UK 5NovAliX, BioParc, 850 Boulevard Sébastien Brant, BP30170 F-67405 Illkirch, France


Ribose 5-phosphate isomerase is an enzyme involved in the non-oxidative branch of

the pentose phosphate pathway, that catalyses the inter-conversion of D-ribose 5-phosphate and D-ribulose 5-phosphate. This enzyme in the trypanosomatids: Leishmania infantum and Trypanosome brucei has been genetically validated as essential and therefore a candidate drug target.

Fragment libraries were screened using the thermal shift technology, which originate promising hits. Subsequently the most interesting fragments were tested against the RIP enzyme assays as well as their trypanocidal effect on Leishmania infantum and on Trypanosoma brucei parasites.

X-ray crystal structures of both the L. infantum and T. brucei ribose 5-phosphate isomerase were obtained complexed with potential inhibitors. Structural comparisons with human and parasitic ribose 5-phosphate isomerase complexes reveal interesting differences in the binding modes of these inhibitors.

Overall, these results are of general interest since they open the way to novel structure-based drug design for these and other Neglected tropical diseases.

The research leading to these results has received funding from: the European Community’s Seventh Framework Programme under grant agreement No.602773 (Project KINDRED)



NATURAL PRODUCTS



The effect of flavonolignans on the Mesocestoides vogae (Cestoda) tetrathyridia

Gabriela Hrčková1, David Biedermann2, Terézia Mačák-Kubašková1

1Parazitologický ústav SAV, Slovak Academy of Sciences, Hlinkova 3, 040 01 Košice,

Slovakia; 2Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, Praha 4-Krč, 142 20

Milk thistle (Silybum marianum L. Gaertn (Asteraceae)) is medicinally used at least

from 14th century.[1] Today it is a source of silymarin – standardized extract of its fruits – which is used as nutraceutical and for treatment of the liver problem. Silymarin contains several flavonolignans, among them silybin (SB) and silychristin (SCH) are the most abundant. 2,3-dehydrosilybin (DHSB) can be prepared from silybin, which tend to posses better activity but it is also more toxic. Recently antiparasitic activity of those compounds were reported.[2]

SB and SCH were isolated from silymarin using Sephadex LH-20 collumn chromatography and DHSB was prepared by oxidation with iodine in boiling acetic acid.

Larval stage, tetrathyridium, of cestode Mesocestoides vogae possess the ability to proliferate asexually in various hosts including cold-blooded animals and mammals (mice, dogs, cats). Since the first passage in laboratory mice, M. vogae larvae have been distributed to several laboratories in the world. Due to many biological and molecular similarities with other larval cestode infections it was recommended by WHO (1996) as a suitable model for the slower developing metacestode infections. We developed the system for long-term in vitro cultivation of the larvae [3].

We examined activity of SB, SCH and DHSB at concentrations of 5 and 50 μM on larvae incubated up to 7 days in RPMI media under hypoxic conditions. Only DHSB had the significant larvicidal effect seen already after 72 h of incubation. All compounds modulated metabolic activity in mitochondria. After 24 h, only DHSB at concentration of 50 μM reduced metabolic activity what correlated with time-dependent inhibition of GST activity and concentration of enzyme in larvae.

In conclusion our preliminary data showed that selected flavonolignans have different mechanisms of activity on molecular targets in M. vogae model eukaryotic parasite and 2,3-dehydrosilybin exerted profound larvicidal effect in vitro.

The study was partially supported by MAD project between AV ČR and SAV no. 16-13 and by MŠMT project LD 15081.

1. Biedermann D, Vavrikova E, Cvak L, Kren V. Chemistry of silybin. Natural Product Reports 2014;31:1138-1157

2. Rabia, I.; Nagy, F.; Ali, E.; Mohamed, A.; El-Assal, F.; El-Amir, A. Effect of treatment with antifibrotic drugs in combination with PZQ in immunized Schistosoma mansoni infected murine model. International Journal of Infectious Diseases 14, S16-S17.

3. Vendelova E, Hrckova G, Lutz M.B, Brehm K, J. NONO Komguep J.N. In vitro culture of Mesocestoides corti metacestodes and isolation of immunomodulatory excretory–secretory products. Parasite Immunology, 2016; 38: 403–413



From the natural compound dihydroplakortin to synthetic bicyclic and bridged endoperoxides active against chloroquine-

sensitive and chloroquine-resistant P. falciparum parasites

Sandra Gemma, Luisa Di Cerbo, Alessandra Vallone, Gloria Alfano, Simone Brogi, Giuseppe Campiani, Stefania Butini, Sarah D’Alessandro, Silvia Parapini,

Nicoletta Basilico, Donatella Taramelli

Department of Biotechnology, Chemistry and Pharmacy, University of Siena, via Aldo Moro 2, 53100, Siena, Italy.


Cyclic peroxides such as 1,2-dioxolanes, 1,2,4-trioxanes and 1,2- dioxanes are a class of organic compounds with interesting pharmacological properties and widely represented in nature. Artemisinin is an endoperoxide-based natural product, which is highly effective against clinically relevant P. falciparum strains responsible for human malaria. Currently, the so-called artemisinin-based combination therapies are employed as first line treatment in most malaria endemic countries, adhering to WHO recommendations. However, lower susceptibility to artemisinins is being reported from highly malaria endemic regions. So, novel peroxides characterized by different structural features could delay the potential selection of P. falciparum resistant strains. Moreover, the cost associated with the extraction of this drug or with synthetic precursors from natural sources prompted researchers to develop synthetic peroxides as low-cost alternatives to artemisinins. Here we describe the development of novel series of endoperoxides as synthetic analogues of the natural product dihydroplakortin. Suitable and straightforward synthetic procedures for the preparation of bicyclic, bridged and spirocyclic endoperoxides have been developed. The peroxides presented here are more potent antiplasmodials than dihydroplakortin itself and they showed antimalarial activity in vivo.




Anti-protozoal compounds from Nigerian medicinal plants: identification and mode-of-action studies

Godwin U. Ebiloma1, Evangelos Katsoulis1, John Igoli2, Alexander I. Gray2, and

Harry P. de Koning1

1Institute of Infection, immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, UK. 2Strathclyde Institute of Pharmacy and Biomedical

Sciences, University of Strathclyde, Glasgow. Email: [email protected]

African trypanosomiasis is a disease caused by infection of humans and animals with

parasites called trypanosomes, usually through the bite of infected tsetse flies. Unfortunately, the current drugs are ineffective due to drug resistance and efforts towards new drug development are inadequate. Using in vitro models of Trypanosoma brucei, we used a multiple approach towards the identification of new lead compounds and evaluate their potency, based on extracts from traditional medicinal plants from West Africa. The result shows that neither the crude extracts or the active compounds isolated from them are toxic to Human Embryonic Kidney (HEK) cells, whereas promising activity was found against Trypanosoma brucei, a drug sensitive wild type strain Trypanosoma brucei (s427-WT) and a multi-drug resistant strain, B48, which lacks both the TbAT1/P2 transporter and the high affinity pentamidine transporter (HAPT). The isolated compounds displayed the same activity against both trypanosomes strains tested (EC50 ~ 0.3 µg/ml). Fluorescence microscopic assessment of DNA configuration revealed cell cycle defects after 8 hours of incubation with the natural compounds: DNA synthesis could not be initiated, leading to a dramatic reduction of cells in the S phase. DNA fragmentation became evident after 10 hours of incubation with compound HDK-20, visualised by flow cytometry and Terminal deoxynucleotidyl transferase dUTP Nick-End Labelling (TUNEL) assay, which reveal up to 80% of cells with DNA fragmentation after 12 hours. Compounds HDK-20 and HDK-40 also induced a fast and profound depolarisation of the parasites’ mitochondrial membrane potential after 1 hour of incubation and this continued until a near complete depolarization was achieved after 12 hours. Intracellular ATP levels of the T. brucei were also measured and were found to be depleted. Metabolomic assessments of T. brucei cells did not reveal the targeting of any specific metabolic pathway. Since the isolated compounds have almost no toxicity against human cells but are very active in vitro against multidrug-resistant trypanosomes, these compounds could serve as lead compounds towards the identification of more efficient anti-trypanosome drugs.



DRUG DELIVERY I



Nanotechnologies for the treatment of severe diseases

Patrick COUVREUR

University of Paris-Sud, Institut Galien, UMR CNRS 8612, 5 rue J-B Clément F-92296 Chatenay-Malabry (France)

Even if new molecules are discovered to treat severe diseases, the clinical use and

efficacy of conventional chemotherapeutics is hampered by the following limitations: (i) drug resistance at the tissue level due to physiological barriers (non cellular based mechanisms), (ii) drug resistance at the cellular level (cellular mechanisms), and (iii) non specific distribution, biotransformation and rapid clearance of the drugs in the body. It is therefore of importance to develop nanodevices able to overcome these limitations.

This will be illustrated by various nanomedicine platforms developed in the laboratory: the design of biodegradable nanoparticles loaded with doxorubicin for the treatment of the resistant hepatocarcinoma (a nanomedicine currently in phase III clinical trials) (1), the construction of nanoparticles made of metal oxide frameworks (NanoMOFs) (10) and the “squalenoylation” (2), a technology that takes advantage of squalene's dynamically folded conformation to link this natural and biocompatible lipid to anticancer (3), antimicrobial (4) or neuroprotective compounds (5) in order to achieve the spontaneous formation of nanoassemblies (100–300 nm) in water, without the aid of surfactants. The design of “multidrug” nanoparticles combining in the same nanodevice chemotherapy and imaging (ie., “nanotheranostics”) or various drugs with complementary biological targets will be also discussed (6). Finally, it will be shown that the construction of nanodevices sensitive to endogenous (ie. pH, ionic strenght, enzymes etc.) or exogenous (ie., magnetic or electric field, light, ultrasounds etc.) stimuli may allow the spatio-temporal controlled delivery of drugs and overcome resistance to current treatments (7). The possibility to use other terpenes (natural or synthetic) than squalene to design nanoparticles for the treatment of resistant intracellular infections (8) or cancer will be discussed, too (9).

References 1. L. Barraud et al., J. Hepatology, 42, 736-743 (2005) 2. P. Couvreur et al., Nano Letters, 6, 2544-2548 (2006) 3. A. Maksimenko et al., Proceedings of the National Academy of Science, 111 (2) E217- E226 (2014) 4. N. Semiramoth et al., ACS Nano, 6, 3820-3831 (2012) 5. A. Gaudin et al., Nature Nanotechnology, 9, 1054-1063 (2014) 6. A. Maksimenko et al., ACS Nano, 8, 2018-2032 (2014) 7. S. Mura et al., Nature Materials, 12, 991-1003 (2013) 8. N. Abed et al., Scientific Reports (Nature), doi: 10.1038/srep13500 (2015) 9. S. Harisson et al., Angewandte Chemie Int. Edition, 52, 1678-1682 (2013) 10. Horcajada P et al., Nature Materials. 9, 172-178 (2010)



DRUG DELIVERY II



Development and in vivo efficacy of biocompatible drug-loaded microspheres against C. parvum

E. Blanco García1,, J. Blanco Méndez1 , F. J. Otero Espinar1, H. Gómez Couso2,

E. Ares Mazás2, A. Luzardo Álvarez1

1Departamento de Farmacología, Farmacia y Tecnología Farmacéutica. Universidad de Santiago de Compostela. Spain; 2 Departamento de Microbiología y Parasitología. Universidad

de Santiago de Compostela. Spain. E-mail: [email protected]

Human cryptosporidiosis is one of the most commonly diagnosed protozoan-

associated intestinal diseases worldwide. It is recognised as one of the main causes of diarrhoeal in immunocompromised hosts (children, AIDS patients) as an opportunistic pathogen [1]. Up to now, there is no any completely efficient treatment. Based on previous work [2], an alternative therapy against Cryptosporidium parvum using bioadhesive Paromomycin and Diloxanide Furoate (DF)-loaded microspheres have been developed. Microspheres (MS) were prepared using chitosan (CHI) and poly(vinyl alcohol) (PVA) and two types of cyclodextrins (β-CD and DM-β-CD) for the potential use of treating cryptosporidiosis. Microparticle formulations were characterized in terms of size, surface charge, drug release and morphology. In vivo bioadhesion properties of CHI/PVA microspheres were also evaluated. In addition, the in vivo efficacy of CHI/PVA microspheres against C. parvum was tested in neonatal mouse model of cryptosporidiosis.

Microspheres prepared by spray-drying showed spherical shape, diameters between 6.67 ± 0.11 and 18.78 ± 0.07 µm and positively surface charged. The bioadhesion studies demonstrated that MS remained attached at +16h (post-infection) to the intestinal cells as detected by fluorescence. The study of efficacy of treatment determined in mice receiving orally administered microspheres with and without drug showed significantly lower parasite loads compared with the control mice.

Our results suggest that microspheres appear to be a safe and simple system to be used in an anticryptosporidial treatment. This work demonstrated the high potential of using bioadhesive chitosan/PVA microspheres for the possible application in the antiparasitic drug delivery by oral route in the treatment or prevention of C. parvum infections.

[1] Bouzid, M. et al., 2013. Clin Microbiol Rev. 26, 115–34. [2] Luzardo-Álvarez, A. et. 2012. Eur. J. Pharm. Sci. 47, 215-227.




Is the oral delivery of Amphotericin B possible to treat parasitic diseases?

D. R. Serrano1, A. Lalatsa2, MA. Dea-Ayuela3, P. Bilbao-Ramos1, NL. Garrett4,

J. Moger4, J. Guarro5, J. Capilla5, MP. Ballesteros1, AG. Schätzlein6,║, F. Bolas1, JJ. Torrado1, IF. Uchegbu6,║

1 School of Pharmacy, Complutense University of Madrid, Plaza Ramon y Cajal s/n, Madrid,

28040, Spain. 2 School of Pharmacy and Biomedical Sciences, University of Portsmouth, PO1 2DT, UK. 3 School of Health Sciences, Universidad Cardenal Herrera-CEU, Moncada, Valencia, 46113, Spain. 4 School of Physics, University of Exeter, Stocker Road, Exeter, EX4 4QL, UK. 5 Facultat de Medicina, IISPV, Universitat Rovira i Virgili, Reus, 43201, Spain. 6 UCL School of

Pharmacy, University of London, 29-39, Brunswick Square, London, WC1N 1AX, UK. ║ Nanomerics Ltd., 14 Approach Road, St. Albans, Hertfordshire, AL1 1SR, UK.

INTRODUCTION. Amphotericin B (AmB) is one of the drugs of choice for visceral

leishmaniasis (VL) which is fatal if left untreated. However, AmB provokes severe adverse effects such as nephrotoxicity and infusion-related side effects that limit its use in clinical practice. Marketed formulations are only parenterally available due to its poor aqueous solubility and permeability which obliges to prolonged hospitalization. The development of an AmB oral nanomedicine would expand the treatment access.

METHODS. AmB was encapsulated into quaternary ammonium palmitoyl glycol chitosan (GCPQ) nanoparticles [1]. Biodistribution in plasma and in major target organs such as spleen, liver, lung and bone marrow following single or multiple oral nanoparticle administration was evaluated in mice and dogs. Anti-leishmanial activity in infected mice was assessed in order to correlate with the increase in oral bioavailability. Multimodal multiphoton microscopy was used to image AmB in major target organs.

RESULTS. AmB levels reached in liver (Fig. 1), spleen and lungs were higher with respect to kidney concentration obtaining a favourable risk- benefit ratio (Fig. 2). The oral bioavalability of AmB was 24.7% in mice. AmB-GCPQ nanoparticles also enabled oral absorption in dogs. The oral administration resulted in 98.9±1.2% and 92.1±7.5% suppression of parasites in liver and spleen respectively.



CONCLUSIONS. An oral therapy for 10 consecutive days at 5 mg kg-1 of AmB-GCPQ exhibited a similar potency to parenterally administered AmBisome® against Leishmania parasites in liver and spleen. The high activity linked with a low toxicity results in an effective and safe oral AmB therapy which it can expand the access for the treatment of VL especially in developing countries.

REFERENCES. Serrano DR et a. Mol Pharm 2015, 12:420-431.



Nanoencapsulation of tetraoxane-based double drugs with antileishmanial activity

João P. Quintas, Manuela Carvalheiro, António J. Almeida and Francisca Lopes Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa,

Lisbon, Portugal, Email: [email protected]

Leishmania genera stand out by their complex redox metabolism, depending on the

flavoenzyme trypanothione reductase (TR) as a defence against oxidative stress, by neutralizing hydrogen peroxide produced by macrophages during infection [1]. Thus, the development of potent inhibitors of TR could lead to new drugs to treat the various forms of leishmaniasis.

In this context, we propose novel endoperoxide-based hybrid compounds capable of selectively release TR inhibitors inside the amastigotes. This approach is applicable to any infectious agents that acquire high levels of iron at critical steps of their life cycle, such as Leishmania, which is dependent on an iron pool for amastigote differentiation and virulence [2]. It provides an unprecedented class of antiparasitic compounds able of disrupting the redox balance through two different and potentially synergistic mechanisms, leading to high levels of ROS and ultimately to parasite death. We selected 1,2,4,5-tetraoxanes, which are reductively activated by iron(II)-heme to form carbon-centered radicals, ROS and carbonyl species [3].

Solid lipid nanoparticles (SLN) are efficient colloidal drug carriers mainly due to their stability profile, ease of scalability and cost efficacy. Due to their particulate nature and inherent structure SLN exhibit good potential in the treatment of parasitic infections [4]. Furthermore, SLN are rapidly cleared by MPS (mononuclear phagocyte system) leading to passive targeting to liver and spleen, which would be quite useful for the incorporation and selective delivery of novel TR inhibitors. Here, we report the synthesis of novel hybrid tetraoxane-based agents, their loading in SLN and the in vitro activity of the double-acting compounds and loaded SLN particles against leishmania amastigotes in THP-1 cells. The implications in for future development into useful antileishmanial agents will be discussed.

1. Tovar J, Cunningham ML, Smith AC, Croft SL, Fairlamb AH (1998). Down-regulation of

Leishmania donovani trypanothione reductase by heterologous expression of a trans-dominant mutant homologue: effect on parasite intracellular survival. PNAS, 95: 5311-5316.

2. Flannery AR, Renberg RL, Andrews NW (2013). Pathways of iron acquisition and utilization in leishmania. Curr Opin Microbiol. 16: 716-721.

3. Oliveira R, Guedes RC, Meireles P, Albuquerque IS, Goncalves LM, Pires E, Bronze MR, Gut J, Rosenthal PJ, Prudencio M, Moreira R, O'Neill PM, Lopes F (2014). Tetraoxane-pyrimidine nitrile hybrids as dual stage antimalarials. J. Med. Chem. 57: 4916-4923.

4. Lopes RM, Pereira J, Esteves A, Gaspar MM, Carvalheiro M, Eleutério CV, Gonçalves LMD, Jiménez-Ruiz A, Almeida AJ, Cruz MEM (2016). Lipid-based nanoformulations of trifluralin analogs in the management of Leishmania infantum infections. Nanomedicine (Lond), 11: 153-170.



DRUG DELIVERY III



Inhibition of Schistosoma mansoni development in mice by slow release of artemisone

D. Gold1, M. Alian2, A. Domb2, Y. Karawani3, M. Jbarien3, J. Chollet4, R. K.

Haynes5, V. Buchholz6, A. Greiner6, J. Golenser3 1Department of Clinical Microbiology and Immunology, Sackler Faculty of Medicine, Tel Aviv University; 2School of Pharmacy, and 3The Kuvin Center for the Study of Infectious and Tropical Diseases, The Department of Microbiology and Molecular

Genetics; Faculty of Medicine, Hebrew University of Jerusalem, Israel; 4Swiss Tropical Institute, Basel, Switzerland; 5Centre of Excellence for Pharmaceutical Sciences, North-West University, South Africa; 6Macromolecular Chemistry II, University of

Bayreuth, Germany Schistosomes are parasitic helminths, most important in terms of socio-economic and

public health in tropical and subtropical areas. Schistosomiasis causes skin allergies, intestinal, liver and urinary pathologies. Chronic disease may also lead to cancer. In addition, there are often systemic symptoms, such as retarded growth, slowing of cognitive development and the effect of continuous low-level blood loss. Current treatment is based on the anti-helminthic drug praziquantel (PZQ). PZQ affects only the adult stages of schistosomes. Also, unfortunately, following its widespread use, there are reports of PZQ resistance.

It is our purpose to test a drug, which could serve as a potential alternative or complement to PZQ, and also as a means of treating infections at an earlier, pre-granuloma schistosome stage. Derivatives of artemisinin, effective anti-malarials, have been indicated as potential alternatives, because both plasmodia and schistosomes are blood-dwelling and blood-feeding parasites. The mechanism of action of artemisinins is ascribed inter alia to oxidative effects of the peroxide on intracellular reductants such as reduced flavins that lead to formation of cytotoxic reactive oxygen species. In this work we used the artemisinin derivative artemisone, which has improved pharmacokinetics and anti-plasmodial activity, and reduced toxicity compared to other artemisinin derivatives that are in current use.

We infected adult mice by subcutaneous injection of S. mansoni cercariae and treated them at various times post infection by the following methods: a. artemisone suspension administered by gavage; b. subcutaneous injection of a gel containing a known concentration of artemisone; c. subcutaneous insertion of the drug incorporated in a solid polymer; d. intraperitoneal injection of the drug solubilized in DMSO. Drug insertion in gel and solid polymer was performed to enable slow release of the artemisone that was verified in a bioassay system of sensitive malaria parasites. Treatment was performed once, twice or thrice, usually starting about three weeks post infection with schistosomes. The results were estimated by counting the adult worms, males and females, surgically extracted from the mice. In most cases we found strong anti-schistosome effects, mainly following repetitive treatments – either by injection of the drug absorbed in the polymers or by gavage of its suspension. The results indicate that artemisone has a potential anti-schistosome activity. Its main importance in this context, however, is its effectiveness in treating hosts harboring juvenile schistosomes, before egg-deposition and induction of deleterious immune responses.

We thank Cipla for the kind donation of artemisone.



Emulsomes: A Tool for Delivery of anti-leishmanial BNIP Derivatives to Macrophages

Zeynep Islek1, Mustafa Güzel2, Fikrettin Sahin1, Mehmet H. Ucisik3,*

1 Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, Istanbul, Turkey; 2 Department of Medical Pharmacology, International School of Medicine,

Istanbul Medipol University, Istanbul, Turkey; 3 Department of Biomedical Engineering, School of Engineering and Natural Sciences, Istanbul Medipol University, Istanbul, Turkey

Corresponding Author; Phone: +90 216 681 5154; E-mail: [email protected]

Similar to other parasitic diseases, chemotherapy is the most efficient strategy for

leishmaniasis. However, the high toxicity of many antiparasitic compounds restricts their utility, and the emergence of drug resistant strains often impairs the lifespan of a given drug.

Among alternative drug candidates, bisnapthalimidopropyl (BNIP) derivatives have been recently shown to have anti-leishmanial activities, which even surpass the standard and most common Amphotericin B therapy [1]. However BNIP derivatives have some drawbacks including low aqueous solubility and toxicity. Addressing these limitations, this study applies two diverse technologies including medical chemistry approach together with the structure-based drug design, and nanotechnological drug delivery approach. The former approach will focus on design of new BNIP derivatives that have higher efficacy and bioavailability, whereas the latter will be used to deliver the drug specifically to the parasite, thereby decreasing the side effects of the chemotherapy, in particular on macrophages.

The delivery of BNIP derivatives into the macrophages will be achieved by encapsulating the active molecule in a lipid- based nanocarrier system, so-called emulsomes [2]. Emulsome is preferred mainly because of its four major features. Firstly, owing a solid lipid core like the solid lipid nanoparticles, emulsome may offer high loading capacities for hydrophobic substances such as BNIP [2,3]. Secondly, composed of only lipids and in the absence of any surfactants, emulsome is highly biocompatible [3]. Thirdly, the solid character of the nanocarrier provides a prolonged drug release profile, which can be controlled, or tuned, by the selection of the lipid composition as well as by surface modifications [4]. Lastly, but most importantly, the natural feature of lipids allows emulsome to accumulate in the organs of the reticuloendothelial system (RES) instead of the kidney, which will not only largely reduce toxicity, but will also improve the anti-leishmaniasis efficacy of the loaded drug, as parazites are also located in the organs of RES.

The development of new active BNIP derivatives and the emulsome-BNIP nanoformulations facilitating the targeted delivery to the macrophages is expected to substantially contribute to the improvements in treating parasitic disease Leishmaniasis in European region as well as worldwide.




References [1] Tavares J., Quaissi A., Lin P.K.T., Loureiro I., Kaur S., Roy N., Cordeiro-da-Silva A., ChemMedChem (2010), 5, 140-147

[2] Ucisik M.H., Küpcü S., Debreczeny M., Schuster B., Sleytr U.B., Small (2013), 9, 2895- 2904.

[3] Ucisik M.H., Küpcü S., Schuster B., Sleytr U.B., J. Nanobiotechnology (2013), 11, 37.

[4] Ucisik M.H., Küpcü S., Breitwieser A., Gelbmann N., Schuster B., Sleytr U.B., Colloids Surf. B. (2015),132-139.

Acknowledgement This study is supported by Tübitak EU-COST project no. 115Z846 and integrated to the COST action CM1307 entitled “Targeted chemotherapy towards diseases caused



Cyclodextrins in antiparasitic drug formulations

Juan José Torrado

Universidad Complutense de Madrid, School of Pharmacy, Plaza Ramón y Cajal, 28040, Madrid (Spain), Email: [email protected]

Cyclodextrins are cyclic ring oligosaccharides of six, seven or eight glucose molecules

forming α, β and γ-cyclodextrins, respectively. These cyclic oligosaccharides are characterized by a pore structure an inner lipophilic diameter of 0.5-0.8 nm. The hydrophilic hydroxyl groups are located on the outside structure of these cyclic oligosaccharides while a lipophilic core region is formed. Lipophilic low soluble drug compounds can be inserted in this lipophilic core part of the cyclodextrins. Furthermore, on the drug-cyclodextrin mixtures the lipophilic drug molecules are stably distributed in extremely small particles. This type of distribution increases their surface area enhancing the relative solubility. These drug-cyclodextrin mixtures can form different complex interactions with different properties. Two of the most important characteristics of these drug-cyclodextrin complexes are the solubility improvement and the increase of stability.

For many lipophilic drugs its low water solubility characteristics compromises its oral absorption. Inclusion of this type of drugs into cyclodextrins enhances its oral bioavailability. Moreover, the inclusion of the drug molecules inside the oligosaccharide ring can avoid or delay chemical degradation reactions. So, chemical stability of the drug molecules can be improved by complexation with cyclodextrins.

Examples of how different types of cyclodextrins can be useful in antiparasitic drug formulation are obtained from the scientific literature. The pharmacokinetic and pharmacodynamic characteristics of drug-cyclodextrin complexes for the treatment of different parasitic diseases are described. Examples include the following diseases:

- Leishmaniosis - Trypanosomiasis - Malaria - Trypanosomiasis - Cryptosporidiosis - Toxoplasmosis - Trichinellosis



SCREENING MODELS



An automated screening technology for the schistosome helminth parasite

Conor R. Caffrey1, Steven Chen2, Brian M. Suzuki1, Rahul Singh3, Michelle R.

Arkin2

1Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA;

2Small Molecule Discovery Center, Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA;

3Department of Computer Science, San Francisco State University, San Francisco, CA, USA

To accelerate drug discovery for schistosomiasis, we have developed an automated high-throughput and high-content drug screening platform to quantify the chemically induced responses of Schistosoma mansoni. I will describe the challenges overcome to standardize the preparation and handling of the parasite. These include interfacing it with an automated instrumentation environment - image acquisition, object segmentation and tracking, and feature extraction. We tested the platform with a number of anthelmintic drugs to measure a range of static and dynamic phenotypes as a function of time and concentration. We developed a user interface to visualize and interrogate the data, which are maintained in a customized database. We combined the high-dimensional data into a single metric output suitable for primary first pass library screening. The platform increases throughput, improves rigor and will support the identification of targets and mechanisms of action.

Supported by NIH grants R01AI089896 and R21AI107390.



In vitro ‘time-to-kill’ assay to assess the cidal activity dynamics of current reference drugs against Leishmania donovani and

L. infantum.

Louis Maes, Jolien Beyers, Annelies Mondelaers, Magali Van den Kerkhof, Eline Eberhardt, Guy Caljon, Sarah Hendrickx

University of Antwerp, Universiteitsplein 1, 2610 Wilrijk (Antwerp),


INTRODUCTION: Despite a continued search for novel antileishmanial drugs, treatment options remain restricted to a few standard drugs, e.g. antimonials, miltefosine, amphotericin B and paromomycin. Although already used now for several decades, their action mechanism still remains partly hypothetical and their dynamics of cidal action and time-to-kill are still fairly poorly documented.

METHODS: An in vitro time-to-kill assay on intracellular amastigotes of the laboratory reference strains Leishmania donovani (MHOM/ET/67/L82) and L. infantum (MHOM/MA(BE)/67/ITMAP263) evaluated the cidal action dynamics of the listed reference drugs at three different concentrations: at IC50, 2x IC50 and the near cytotoxic dose level (CC90: determined on MRC-5 cells). This assay focused at identifying the minimal exposure time needed to completely eliminate viable intracellular amastigotes, using the standard microscopic Giemsa-assay and the promastigote back-transformation assay.

RESULTS: While 100% reduction was microscopically apparent for most drugs, the promastigote back-transformation assay clearly demonstrated a concentration- and time-dependent cidal mechanism. The time-to-kill at 2x IC50 was >240h for pentavalent antimony (77 µg eq./ml), 96h for trivalent antimony (44 µg eq./ml), 168-192h for miltefosine (10 µM), 192h for paromomycin (100 µM) and 192-216h for amphotericin B (2 µM). No major differences were noted between both Leishmania species.

CONCLUSIONS: Evaluation of the concentration- and time-dependent cidal activity using the promastigote back-transformation assay revealed striking differences in efficacy of the antileishmania reference drugs. This assay allows in-depth pharmacodynamic evaluation of novel drug leads in comparison to the existing antileishmanial drug repertoire.



Is it important to include the insect vector to evaluate the potential of a drug?

Louis Maes, Eline Eberhardt, Mabille Dorien, Lieselotte Van Bockstal, Annelies

Mondelaers, Magali Van den Kerkhof, Sarah Hendrickx, Guy Caljon

University of Antwerp, Universiteitsplein 1, 2610 Wilrijk (Antwerp), Email: [email protected]

African trypanosomes and Leishmania belong to the same family of protozoan parasites (Trypanosomatidae) and share the feature of being transmitted by the bites of blood feeding insects, tsetse and sand flies respectively. The role these bugs in the parasite transmission cycles clearly extends beyond a role as flying syringes.

These insect vectors create a very peculiar host-parasite interphase which involves the deposition of parasites in the specific microenvironment of the host dermis together with a range of pharmacologically and immunologically active components from the anterior part of the insect alimentary tract and/or from the salivary glands. Recent findings for tsetse fly transmitted trypanosomes and a review of the sand fly contribution to Leishmania transmission indicate some distinctive features of infections initiated through the natural route. Parasites benefit from an altered vector behavior resulting in multiple inoculations and from co-inoculated components and recruited host innate immune cells for an enhanced host colonization process. Parasites were also found to interact with somatic cells such as adipocytes and are able to establish a dermal parasite population proximal to the site of infection initiation.

Despite the compelling evidence of the importance of the vector component, host-parasite interactions such as parasite virulence (infectivity and fitness), disease-associated pathology, and treatment efficacy are nearly exclusively studied in laboratory rodent models that exclude the insect vector. It can be assumed that the specific cellular interactions with immune and somatic cells and the parasite tissue tropism resulting from a bite-mediated transmission could impact treatment efficacy and relapse rate depending on the specific drug characteristics. Collectively, these observations advocate for including the insect vector in animal models to evaluate drug efficacy.



EMERGING ISSUES



Drug absorption modifications in Giardiasis

Verónica Vivancos1; Isabel Gonzalez-Alvarez1, Marta Gonzalez-Alvarez1, María A. Dea2, María del Val Bermejo1

1Departamento de Ingeniería. Área Farmacia y Tecnología Farmacéutica. Universidad Miguel Hernández de Elche. 2. Departamento de parasitología. Universidad Cardenal Herrera CEU,

Valencia [email protected];[email protected];[email protected];[email protected]

Giardia intestinalis is a flagellated protozoon able to colonize the small intestine of

many vertebrates, including humans. It is the causative agent of giardiasis, one of the most common reason of parasitic gastrointestinal disease. In children is especially important as diarrhea and malabsorption are responsible for anemia, stunting and cognitive delay. Several mechanisms could be involved infection, most notably impaired intercellular junctions and apoptosis of host cells. After that, it is observed enterocytes damage, loss of brush border of intestines, shortening of microvilli and impaired epithelial barrier function.

The effect of Giardia on the absorption of nutrients and vitamins has been widely studied, although there are few studies conducted on how this parasite affects drug absorption. Craft et al., found that the absorption of some antibiotics decreased in patients with giardiasis, compromising the effectiveness and safety of drug therapy. This is dangerous for paediatric patients because small changes in the absorbed dose can lead to toxicity or undertherapeutic drug concentrations. A reliable experimental model for determining the effect that this parasite has on the absorption of drugs is necessary, especially for drugs with narrow therapeutic window.

Drug absorption experiments were performed using in vitro and in situ models of the intestinal barrier in the presence and absence of G.intestinalis. The parasite was added to Caco-2 cells. The transport study was performed in apical to basal direction. Closed loop in situ perfusion method using the duodenum section (Doluisios´technique) was also performed. The in vitro Permeability coefficients were obtained by linear regression of the accumulated amounts versus time in the acceptor chamber after checking sink conditions maintenance. The apparent first order absorption rate coefficients (ka) from in situ experiments were obtained by non-linear fitting of a monoexponential equation to the luminal concentrations versus time.

The results suggest that Giardia intestinalis modifies the permeability of the drugs tested, which is consistent with the in vivo findings observed in other drugs or nutrients. The growth of Giardia intestinalis involves the formation of a monolayer that adheres to the intestinal membrane and prevents the correct performance of biological transport mechanisms. The permeability is reduced in drugs absorbed by passive diffusion due to steric hindrance exerted by the parasite. Conversely, the absorption is increased in drugs transported via the paracellular pathway due to the damage of tight junctions caused by G. intestinalis. Furthermore, active carriers are blocked in the presence of G. intestinalis causing a decrease in permeability if the drug is carried by influx, or an increase in permeability if it is carried by efflux.

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References: Rópolo, A. S. & Touz, M. C. A lesson in survival, by Giardia lamblia. Scientific World Journal. 10, 2019-31 (2010). Banik, S., Renner Viveros, P., Seeber, F., Klotz, C., Ignatius, R. & Aebischer, T. Giardia duodenalis arginine deiminase modulates the phenotype and cytokine secretion of human dendritic cells by depletion of arginine and formation of ammonia. Infect. Immun. 81, 2309-17 (2013).

Acknowledgements: EUROPEAN COMISSION: DCI ALA/19.09.01/10/21526/245-297/ALFA 111(2010)29: Red-Biofarma



Fragment-Based Drug Discovery fosters the identification of new leads against Trypanosoma brucei PTR1

Maria Paola Costi1, Cecilia Pozzi2, Joanna Panecka5, Sheraz Gul3, Anabela Cordeiro da Silva4, Luca Costantino1, Pasquale Linciano1, Rebecca Wadee,

Stefano Mangani2.

1Università degli Studi di Modena e Reggio Emilia, Via Campi 103, 41125 Modena, Italy. 2Università di Siena, Via Aldo Moro 2 – 53100 Siena, Italy, 3Fraunhofer Institute for

Molecular Biology and Applied Ecology ScreeningPort (Fraunhofer-IME SP), Schnackenburgallee 114, D-22525 Hamburg, Germany.4IBMC - Instituto de Biologia Molecular

e Celular, Universidade do Porto, Oporto, Portogual.5Heidelberg Institute for Theoretical Studies (HITS), Schloss-Wolfsbrunnenweg 35, 69118 Heidelberg, Germany.

According to the World Health Organization (WHO), parasitic diseases, such as

African (sleeping sickness) and American (Chagas disease) trypanosomiasis and Leishmaniasis, affect over three billion people in the world. There is a clear necessity to discover new targets and new drugs to treat these diseases. Trypanosomatids lack the ability to synthesize folates de novo and are totally dependent on the salvage of extracellular folates for growth. However, antifolates cannot be used in the therapy of trypanosomatidic infections because dihydrofolate reductase (DHFR) inhibition is compensated by pteridine reductase-1 (PTR1). PTR1 is involved in the reduction of biopterin but can also reduce folates, thereby safeguarding the cell survival. Therefore, PTR1 is a promising target for the design and the development of new antiparasitic drugs. [1] To address these issues, we are employing a fragment-based drug design (FBDD) approach to drug discovery. Crystallographic screening revealed the binding modes of several pteridine-like fragments with inhibition constants (Ki) values ranging between 10-3-10-4 M. Subsequent structure-based design, guided by previously published structural data, resulted in two series of compounds with high affinity and good ligand efficiency. Structure–activity relationships were established, and more potent compounds were designed and synthesized using fragment growth and fragment linking strategies. All the synthesized compounds were evaluated for their ability to inhibit parasitic PTR1 as well as other parasitic folate-dependent enzymes (TS and DHFR) in enzymatic assays. The early toxicity profiles were also determined. All assays were performed using HTS technologies. Seven new crystallographic structures of ternary complexes of developed compounds with TbPTR1 and NADPH were also obtained. The determined poses support the design by reproducing the poses and by retaining the key interactions of the initial binding fragments. Additional binding regions surrounding the PTR1 active site were also identified. The compounds were tested against different parasites as single agents and in combination with methotrexate, revealing good antiparasitic activity, selectivity and synergy against T. brucei. The progress of the compounds through the pipeline reduced the liabilities of the drug candidates, allowing us to identify the best candidates for further in vivo studies, such as the determination of the pharmacokinetic and antiparasitic activity in mouse models. The resultssuggest that the FBDD approach provides a route to high-quality lead candidates.



This work was carried out by the NMTrypI (New medicine for Trypanosomatidic Infections, www.nmtrypi.eu) consortium with funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement No. 603240.

Reference [1] Cavazzuti, A., et al., Discovery of potent pteridine reductase inhibitors to guide antiparasite drug development. Proc Natl Acad Sci U S A, 2008. 105(5): p. 1448-53.

http://www.nmtrypi.eu/



CHEMOTHERAPY OF PARASITIC DISEASES IN HUMAN AND

VETERINARY MEDICINE



Anti-parasitic treatment in veterinary medicine: a big challenge..!

Kurt Pfister

Prof. Dr. med. vet., Dip EVPC

Parasite Consulting GmbH, CH-3006 Berne – Switzerland &

Comparative Tropical Medicine and Parasitology, LMU Munich - Germany www.parasiten-bekaempfung.ch

Parasites are widely spread in domestic and wild animals all over the world, and quite

many are of considerable relevance in veterinary medicine.

It is primordial to notice that the major impact of veterinary parasites is much more their pathogenic potential and therewith associated clinical and pathologic consequences for the animals including the economic losses for the owners, rather than their presence in a host or their overall prevalence. For many parasites, this is even the case with regard to their zoonotic potential. It is thus obvious that such aspects have several consequences for the decision whether or not to initiate an antiparasitic treatment. In many respect, endemic outbreaks of certain parasite infections in livestock but also in other animals have often caused high lethal rates and therewith associated tremendous economic losses.

The discovery and introduction of highly efficacious drugs in the 1960-1970’s (BZM, Pyrimidines, Imidazothiazoles) and subsequently of the AVM (1980’s) has given rise to the most welcomed idea of the so-called mass treatment strategy. To some extent this was initially even thought to have the potential to eliminate (or even eradicate) some parasites of greater pathologic/clinical importance. However, it became relatively qjuickly evident that one of the most severe (negative) consequence of this „routine type of treatment over years“ was a selection for specific drug-resistant parasite populations. The development of resistance became rather quickly relevant for some pathogenic nematodes in small ruminants (sheep and goats) and equids, but is meanwhile also widely detectable in various cattle and pig parasites as well as in other animal species.

Another important issue regarding mass treatment is the so-called „over-treatment“of animals at a too young age (or entire flocks: e.g. against D. viviparus or O. ostertagi in cattle, etc.) which has led to an insufficient immune reaction and consequently in many cases to subsequent lethal disease outbreaks (due to a lacking immune reaction).

A quite important handicap in veterinary parasitology – for several parasite species – is the lack of authority-registered drugs and, according to the animal species (in particular for livestock), drug-related withdrawal periods for their products (milk, meat etc.) have to be respected. Consequently, such hurdles have to be taken into consideration, too.

http://www.parasiten-bekaempfung.ch/



What can we conclude from all the presented aspects and what kind of consequences should we consider? In order to better deal with this we have to consider:

- the way of treatment - the status of resistance - the biology (reachability of the parasite in a high metabolic activity stage) - the specific diagnosis and appropriate parasite identification - the seasonal occurrence (epidemiology) - in some cases rather a metaphylactic instead of a prophylactic treatment - the economic (and maybe the zoonotic) importance of the parasite

infection to be treated - the immune competence of the given parasite species

An alternative treatment approach to the above focuses either predominantly on animals with a high environmental contamination rate (e. g. egg production) or, on animals which are highly affected, i. e. which carry a high, clinico - pathologically relevant parasite burden. This approach – typically evidence-based - is called „selective treatment“, is epidemiologically appropriate and includes often also an adaptation of management aspects for the given flock/herd/farm/pet family. There are various types of selective treatment, but most importantly, this procedure allows to maintain at the same time a refugium of drug-sensitive parasites on a given entity/surface.

However, there are also a lot of parasites (e. g. many coccidia species, various gastrointestinal cestodes, trematodes and nematodes) which are of a high prevalence but which do not need any treatment because of their low-level pathologic potential.



Past, Present and Future of Endoparasiticides at Merial

John M. Harrington

Merial, Inc., Duluth, GA, USA., [email protected]

While Merial has existed in an independent form since 1997, its origins can be traced back decades further and include fundamental discoveries in anthelmintic chemotherapy and introduction of products that revolutionized production and companion animal health. Paramount to these products are the macrocyclic lactones exemplified by ivermectin. The discovery of ivermectin and subsequent development of related compounds such as eprinomectin hold important lessons for the anthelmintic discovery and development process today. While these compounds laid the foundations for Merial’s current leadership in animal health parasiticides, Merial’s worldwide presence also facilitates our leadership in solutions to animal diseases caused by protozoan parasites. A historical footprint, industrial collaborations and regional hubs in African countries allow us to provide the only complete program for prevention and treatment of animal trypanosomiasis. Looking forward, Merial’s pharmaceutical discovery group is poised to deliver new solutions to issues such as resistance by finding molecules with new modes of action. To do so, we utilize diverse technologies including phenotypic and target based screening and engage in collaborations with academic research groups, crop-protection and human health partners. With these efforts we will uphold the high standards of parasiticide discovery and development that are at the basis of Merial’s origins.



POSTERS



P.1. Identification of protein kinase inhibitors in Cystoisospora suis by genomic-based virtual screening

Alfonso Garcia-Sosa1, Nicola Palmieri2

1 Institute of Chemistry, University of Tartu, Ülikooli 18, 50090 Tartu, Estonia –

email: [email protected] ; 2 Institute of Parasitology, Department of Pathobiology, University of Veterinary Medicine, Veterinärplatz 1, A-1210, Vienna, Austria –

email: [email protected]

Cystoisospora suis is a protozoan parasite (phylum Apicomplexa) that causes enteritis and diarrhea in suckling piglets and is responsible for significant economic losses in swine production across Europe and worldwide. While this disease is currently treated with toltrazuril, drug costs and drug resistance are of increasing concern. Protein kinases are attractive drug targets in Apicomplexa, as they have pleiotropic roles in many essential cellular processes. However, the screening for kinase inhibitors among thousands of molecules can be a daunting task, thus virtual screening methods can be a powerful way to scan large libraries of molecules. Here we sequenced and annotated the 84Mb genome of C. suis and identified a set of 318 protein kinases through functional annotation analyses. Comparative searches with the Protein Data Bank identified the presence of an ortholog 3D protein structure for one of these kinases (CDPK1) in the closely related species Toxoplasma gondii. By using this structure as a template we constructed a homology 3D model for the C. suis CDPK1 and used it for scanning a library of commercially available drug compounds by molecular docking techniques, in order to find potential inhibitors. We identified 60 natural compounds with low Glide docking scores including two interesting candidates with low molecular weight: 2-(4-hydroxyphenyl)ethyl and a gallotannine. These molecules will be tested on an established in vitro model of C. suis through invasion-inhibition assays. Our study showed that is possible to pinpoint molecules with bioactive function in a non-model organism using an in-silico approach based on genomics and proteomics data.





P.2. New bis-pyridazine derivatives of potential interest in leishmaniasis

Violeta Vasilache,2,3 Dorina Mantu,1 Vasilichia Antoci,1 Ionel I. Mangalagiu1

1 “Alexandru Ioan Cuza” University of Iasi, Faculty of Chemistry, Bd. Carol 11, 700506 Iasi, Romania; 2“Alexandru Ioan Cuza” University of Iasi, CERNESIM Research Center, Bd. Carol 11, 700506 Iasi, Romania; 3“Stefan cel Mare” University Suceava, Faculty of Food Engineering, Str.

Universitatii 13, Suceava, Romania.

Cutaneous Leishmaniasis is the most common form of leishmaniasis.

Current treatments for cutaneous leishmaniasis are performed with success using classical drugs such us pentamide and imidazoquinolines. However, their use is limited by their toxicity, side effects, relatively high cost, discomfort and the emergence of drug resistance. This is why new approaches are urgently needed.

Nitrogen derivatives are “privileged structures” in drug design, optoelectronics, etc., the azaheterocycle scaffold being a core skeleton for multiple purposes.

Pentamide is a second-line drug largely used in treatment against cutaneous leishmaniasis caused by Leishmania major infection.

The emphasis of this work consist in design, synthesis and characterization of new bis-pyridazine derivatives of potential interest in leishmaniasis, analogues of pentamide.

O O

HNNH2

NHH2N

Pentamide O

NN

NN

O

O

ZZ

CY= - OR, -NH2 - NH-NH2Z=

OY

The in vitro anti-leishmaniasis tests are under going.

Acknowledgements. Authors are thankful to COST Action: CM1307 and to CNCS Bucharest, Romania, project PN-II-DE-PCE-2011-3-0038, no. 268/05.10.2011, for financial support.



P.3.Preliminary in vitro studies of antiprotozoal activity of some heterocyclic N-oxides and N,N’-dioxides

Jonas SARLAUSKAS1,Philippe GRELLIER2, Diego BENITEZ3, Marcelo A.

COMINI3, Louis MAES4, Jurgen JOOSSENS5, Sandrine COJEAN6 , Philippe LOISEAU6, and Narimantas CENAS1

1Department of Xenobiotics Biochemistry of Institute of Biochemistry, Vilnius University, Sauletekio av. 7, Vilnius, LT-10257, Lithuania; 2Laboratoire USM 0504 Biologie Fonctionnelle des Protozoaires, Département RDDM, Museum National d'Histoire Naturelle, FRE 3206 CNRS, 61 rue Buffon, Paris Cedex 05, France; 3Redox Biology of Trypanosomes Laboratory, Institut Pasteur de Montevideo, Mataojo 2020, Montevideo, Uruguay; 4Laboratory of Microbiology, Parasitology and Hygiene (LMPH), Departments of Pharmaceutical and Biomedical Sciences,University of Antwerp (UA), Universiteitsplein 1, B-2610 Antwerp, Belgium; 5Medicinal Chemistry (UAMC), Department of Pharmaceutical Sciences, University of Antwerp (UA), Universiteitsplein 1, B-2610 Antwerp, Belgium; 6Groupe Chimiotherapie Antiparasitaire UMR 8076 CNRS Faculte de Pharmacie Universite de Paris-Sud 11, France


In this continuation of our collaborative research [1-3] we have studied in vitro antiprotozoal activity of some compounds from the three selected chemical classes of potentially interesting redox active agents: substituted benzofuroxans, pyridine N- oxides and quinoxaline N, N’-dioxides. The library of all studied compounds were synthesized in Vilnius University by one of the authors (J.S.) The aim of the present research was to investigate the main structure-activity relationships and to detect the most perspective pharmacophoric functional groups for the further structure optimization.

Generally, it was found that a number of functional groups located at specific positions of aromatic ring in evaluated heterocyclic molecules can serve as promising pharmacophores: nitro, halogens (Cl, Br), CF3 and in some particular cases, CH3 and alkoxy/ alkylendioxy groups. A potential impact of enzymatic bio-reduction products (1,2-benzoquinondioxime (o-BODOX) and 2,3-diaminophenazine (PHADA) [5]) and their derivatives as an active metabolites of benzofuroxans on antiparasitic activity has been proposed in the present work for the first time.

In this preliminary study, the most effective compounds were found: against Plasmodium falciparum str. FcB1 (chloroquine resistant strain) - JSQ-33 (pyridine N-oxide derivative, IC50 =5.4 µM), against Trypanosoma brucei brucei str. 427 – JOSHA-20 (benzofuroxan derivative, IC50=0.3 µM), against Trypanosoma cruzi Tulahuen CL2 strain – LXBD-096 (benzofuroxan nitroderivative, IC50=4.5 µM) and against Leishmania donovani - JSLT-09 (pyridine N-oxide derivative, IC50=1.6 µM).

1. Grellier, P. et al, Arch Biochem Biophys. 2010; 394(1):32-39. 2. Sarlauskas, J. et

al: http://www.vin.bg.ac.rs/180/cost_cm1307/CM1307_Belgrade_oct_2015_book_of_abstracts.pdf, p.57

3. Benitez D. et al. PLOS Neglected Tropical Diseases. 2016, 10: e0004617. 4. Sarlauskas J. et al, Int. J. Mol. Sci., 2014, 15: 23307-23331.

http://www.vin.bg.ac.rs/180/cost_cm1307/CM1307_Belgrade_oct_2015_book_of_abstracts.pdf

http://www.vin.bg.ac.rs/180/cost_cm1307/CM1307_Belgrade_oct_2015_book_of_abstracts.pdf



P.4. Structural basis for vinyl sulfone inhibition of the SmCB1 drug target from the human blood fluke

Adela Jilkova1, Martin Horn1, Petra Rubesova1, Pavla Fajtova1, Pavlina Rezacova1, Jiri Brynda1, James H. McKerrow2, Conor R. Caffrey2 and

Michael Mares1

1Institute of Organic Chemistry and Biochemistry, The Czech Academy of Sciences, Flemingovo

nam. 2,16610 Prague, Czech Republic; 2Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California,

San Diego, San Diego, CA 92093, USA

Schistosomiasis caused by parasitic blood flukes of the genus Schistosoma afflicts over 240 million people worldwide. Schistosoma mansoni cathepsin B1 (SmCB1) is a gut-associated peptidase that digests host blood proteins as a source of nutrients. In our recent work we demonstrated that SmCB1 is a drug target for vinyl sulfone peptidomimetic inhibitors. Now we performed a detailed analysis with a unique set of 30 vinyl sulfone derivatives with diverse substituents. The inhibitors were screened in vitro against recombinant SmCB1 and ex vivo against cultivated S. mansoni. Two most effective inhibitors in terms of IC50 values and parasite suppression were complexed with SmCB1, and high resolution crystal structures were determined. Analysis of 3D structures and inhibition profiling identify key binding interactions and provide insight into SmCB1 inhibition specificity. Our work provides a footing for the rational design of anti-schistosomal chemotherapeutics.

This work was supported by the grant LD15101 and the project InterBioMed LO1302 from the Ministry of Education, Youth and Sports of the Czech Republic, the institutional project RVO 61388963, the Academy of Sciences of the Czech Republic, and the Skaggs School of Pharmacy and Pharmaceutical Sciences.



P.5. Anti-Leishmania activity of a series of Quinolin-4(1H)-imines

Ana G. Gomes-Alves1,2,3, Margarida Duarte1,2, Tânia Cruz1,2, Rui Moreira4, Ana

S. Ressurreição4,*, Ana M. Tomás1,5,*

1 i3S - Instituto de Investigação e Inovação em Saúde, Porto, Portugal. 2IBMC - Instituto de Biologia Molecular e Celular, Porto, Portugal. 3University do Minho, Braga, Portugal.

4iMed.ULisboa - Research Institute for Medicines, Lisboa, Portugal. 5 ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Porto, Portugal.

E-mail: *[email protected]; [email protected]

Leishmaniasis comprise a set of neglected tropical diseases, caused by trypanosomatid protozoan parasites of the genus Leishmania. Depending on the host immune response and on the Leishmania species, leishmaniasis can range from a nonlethal cutaneous form (e.g. by L. major and L. amazonensis) to a fatal visceral condition (e.g. by L. infantum). Current treatments for leishmaniasis rely on inadequate chemotherapeutics with poor efficacy and high host toxicity forcing people to stop therapy. This leads to disease relapse and emergence of resistant strains. Besides this, the high costs associated to most of the available therapeutic options are far from suitable for developing countries.

This absence of satisfactory treatments prompts the discovery of new compounds with leishmanicidal activity. Using a high content-based platform previously implemented in the lab, we screened about 40 different compounds against intracellular L. infantum (in bone marrow-derived macrophages). From this screening, a set of quinolin-4(1H)-imines, which were previously shown to display antiplasmodial activity [1], emerged as the most interesting family of compounds, the most active ones presenting half maximal inhibitory concentrations (IC50) around 1µM and selectivity indexes of 7-11 (host cell cytotoxicity evaluated also in bone marrow-derived macrophages). Quinolin-4(1H)-imines active against L. infantum were also tested against intracellular L. major and L. amazonensis but we observed a lower activity against the cutaneous forms of the disease (IC50 two to three times higher when compared to L. infantum).

Some quinoline derivatives were previously suggested to have their mechanism of action on the disruption of normal mitochondrial function in Plasmodium falciparum [2].

Quinolin-4(1H)-imines were initially designed to target mitochondrial cytochrome bc1 [1]. The inhibitory effect on the basal oxygen consumption of intact L. infantum amastigotes, suggests that the mode of action of these compounds could also include inhibition of cytochrome bc1 and/or other respiratory chain enzymes.

In short, this study suggests that quinolin-4(1H)-imines might be an interesting chemotype in the search of new anti-Leishmania leads.

Ana G. Alves and Ana S. Ressurreição are financed by the Portuguese Foundation for Science and Technology with PhD (SFRH/BD/93766/2013) and FCT Investigator Starting (IF/01034/2014) Grants, respectively.

[1] a) Ressurreição, et al. Med. Chem. 2013, 56, 7679; b) Rodrigues, et al Med. Chem. 2013, 56, 4811. [2] Teguh, et al. Med. Chem. 2013, 56, 6200.




P.6. Development of new quinone derivatives against Leishmania

Carmen Gil, Maria Ángeles Abengózar, Sara Sandoval, Víctor Sebastián,

Ana Martínez, Nuria E. Campillo, Luis Rivas

Centro de Investigaciones Biológicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain Contact: [email protected]

From a human health perspective, leishmaniasis is the second protozoan disease in

importance, only superseded by malaria. The disease encompasses the wide range of clinical pathologies produced by the infection with different protozoan species of the genus Leishmania. Globally, the disease accounts for 10-12 million people infected worldwide, with an incidence of 1.5 million new cases per year. Despite the considerable advances carried out in the last years, nowadays none human vaccine is currently available, and the efforts to curtail dissemination by vector and reservoir control are far less than satisfactory. This leaves chemotherapy as the sole method to combat efficiently the disease. The chemotherapeutic arsenal is quite limited and its efficacy is increasingly eroded by growing resistance, aside from the severe side effects associated to many of them. Furthermore, the high cost for their implementation is unaffordable for the bulk of the patients, belonging to low-income countries. Thus, development of new drugs is urgently required.1

In this work the search of new drugs for leishmaniasis was based on a phenotypic-based approach using as source of new hits our in-house chemical library. A new class of quinone derivatives has emerged as potential hits for this disease and a medicinal chemistry optimization program is ongoing. Furthermore, initial results on their mechanism of action supported the importance of the bioenergetic collapse of the parasite induced by the quinones, with a rapid drop of intracellular ATP levels in the parasites and decrease of the respiration rate in Leishmania donovani promastigotes. Its role in the lethal mechanism of these quinones and SAR studies with a limited set of compounds will be discussed.

Acknowledgments: This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO, project no. SAF2015-65740), Redes de Investigación Cooperativa Instituto de Salud Carlos III RICET (RD12/0018/0007 & RD16/0027/0010) and FEDER funds.

1 Nagle, A. S.; Khare, S.; Kumar, A. B.; Supek, F.; Buchynskyy, A.; Mathison, C. J.; Chennamaneni, N. K.; Pendem, N.; Buckner, F. S.; Gelb, M. H.; Molteni, V. Recent developments in drug discovery for leishmaniasis and human African trypanosomiasis. Chem Rev 2014, 114, 11305-11347.




P.7. Molecular Characterisation and cloning of Novel Equilibrative Nucleoside Transporter family members in

Trichomonas vaginalis

Manal J. Natto, Neils B. Quashi and Harry de P. Koning

Institute of Infection, Immunity and Inflammation, University of Glasgow, UK E-mail: [email protected]

Trichomoniasis is the most common, non-viral sexually transmitted disease (STD),

caused by the amitochondriate protozoan Trichomonas vaginalis. The disease annually affects over 170 million people in the world. Increased resistance of T. vaginalis to metronidazole, the drug of choice for the treatment of the disease, necessitates the development of newer chemical entities with different chemotypes. To date, no high-throughput library screens have been performed against this parasite, but it is possible to identify essential biochemical pathways in this parasite. The nucleoside/nucleobase salvage system of the parasite is an attractive target, because the parasite cannot synthesise either purines or pyrimidines de novo and has to salvage the nutrients from the host through transporters, whereas their human hosts have both purine salvage and synthesis pathways for purines and pyrimidines. Therefore, depriving the parasites of these essential requirements, through controlled blockage of the salvage pathways, is certain to cause parasite death, but should not affect the human host. Nucleoside salvage in the parasite was therefore systematically investigated using the rapid oil stop technique. The results show the existence in T. vaginalis of at least four transporters: with high and low affinity for purine and pyrimidine nucleosides as well as adenine similar to that of the Equilibrative Nucleoside Transporter (ENT) family observed for other protozoans. In order to match the observed transport activities to genes, all 9 T. vaginalis ENT genes were cloned, sequenced, and expressed in Trypanosoma brucei, selecting a strain from which one of the main nucleoside transporters, TbAT1, was already deleted. Each gene was resynthesized in the codon preference of T. brucei since T. vaginalis DNA is very A/T-rich. This will allow us to characterise each of the transporters in isolation. Two identical sets of transfectants are being constructed: one with synthetic but other original open reading frames, and one with the ORFs coupled C-terminally to Green Fluorescent protein to assess cellular localisation of the gene products.



P.8. Anti-toxoplasma activity of novel macrolide hybrid derivatives

Mihaela Peric1, Budhaditya Mukherjee2, Andrea Fajdetic3,4,

Dominique Soldati-Favre2

1 University of Zagreb School of Medicine, Center for Translational and Clinical Research, Salata 2, 10000 Zagreb, Croatia; 2 University of Geneva, Faculty of Medicine, Department of Microbiology and Molecular Medicine, Rue Michel Servet 1 - 1211 Genève 4, Switzerland;

3Fidelta d.o.o., Prilaz baruna Filipovića 29, 10000 Zagreb, Croatia; 4 Present address: Xellia d.o.o (Xellia Ltd), Slavonska avenija 24/6, 10000 Zagreb, Croatia

Novel therapeutics to treat toxoplasma infections are highly needed since currently

available therapeutic options are limited and often associated with safety and parasite resistance issues. Azithromycin is a semi-synthetic macrolide antibiotic, the first member of the azalide class. It is also known for its antimalarial and anti-toxoplasma activity and its clinical usefulness and safety have been well documented. Recently, novel classes of azalide derivatives were reported as potent antimalarial agents. Two of the most promising derivatives, both comprising azithromycin scaffold with chloroquinoline moiety linked at two different positions, were tested in various Toxoplasma gondii in vitro assays and their activity compared to azithromycin and clarithromycin. Both compounds exhibited improved activity over standard macrolide antibiotics. As two macrolide hybrid compounds have the same building blocks their mode of action could be linked to the specific linking position on the azalide scaffold. In conclusion, macrolide hybrid compounds have the potential to be further investigated as promising anti-toxoplasma agents.



P.9. Anti-malarial combination therapy: synergistic effect between an antisense strategy and different anti-malarial

drugs in resistant strains of Plasmodium falciparum

Soulaf Suyyagh-Albouz1, Fernanda Bruxel2, Nicolas Tsapis3, Elias Fattal3, Philippe Loiseau1, Helder Teixeira4, Sandrine Cojean1,5.

1Université Paris-Sud, UMR 8076 BioCIS CNRS, LabEx LERMIT, Châtenay-Malabry

2Universidade Federal do Pampa, Brésil 3Université Paris-Sud, UMR 8612 institut Galien Paris Sud, CNRS, Châtenay-Malabry

4Universidade Rio Grande do Sul, Porto Alegre, Brésil 5CNR Paludisme, Hôpital Bichat Claude Bernard, Paris

In these days, only the Artemisinin-based Combination Therapy still effective in

fighting malaria. But a threat exists in these associations due to the emergence of resistance to artemisinin derivatives in Southeast Asia. Which could lead to a restriction in used anti-malarial drugs as increasing doses is limited to avoid toxicity.

Antisense strategies represent a promising new therapeutic approach targeting nucleic acids. Antisense oligonucleotides (ODN) may be employed to treat malaria. Their limitations were mainly their low intracellular penetration to their target and their rapid degradation. New generations of vectors have helped to enhance the effects of ODN with reduced toxicity. During our studies, we have developed a cationic nanoemulsion (NE) in order to adsorb ODN directed against the topoisomerase II of Plasmodium falciparum. This NE/ODN allowed the inhibition of parasite growth.

To develop a combination therapy, some anti-malarial drugs, whose resistances are proven, were associated with the NE/ODN. We tested our NE/ODN in combination with chloroquine, atovaquone and dihydroartemisinin on the 3D7 strain sensitive to all anti-malarial drugs, the W2 strain resistant to chloroquine and PAV strain resistant to atovaquone. A synergistic effect, no matter which anti-malarial drug was associated with the NE –ODN, was observed. There was also a limited reinfection in presence of the different combinations even in the resistant strains.

Our perspective is to encapsulate the atovaquone inside the NE/ODN due to its lipophilic properties in order to prevent or reverse the resistance, and reduce the dose used by increasing the bioavailability of atovaquone.



P.10. Drug repurposing of human kinase inhibitors as new hits against Leishmania

Carmen Gil, Maria Ángeles Abengózar, Paula Martínez de Iturrate, Víctor

Sebastián, Ana Martínez, Nuria E. Campillo, Luis Rivas

Centro de Investigaciones Biológicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain Contact: [email protected]

Treatment of leishmaniasis, the second human protozoan disease in importance,

relies almost exclusively on chemotherapy, on its turn reduced to a scarce number of drugs. Their efficacy is threatened by rising resistance, and pipeline for new leads is scarcely populated. Due to the low economical level of the bulk of affected population, investment for development of new drugs received a poor investment. Drug repurposing resulted as a fast and low cost approach to add new drugs into leishmaniasis treatment.2

The inhibition of protein kinases of Leishmania constitutes an appealing approach to tackle infections by this parasite. Globally, protein kinases constitute a substantial target of the drug discovery efforts, accounting for nearly a third of the “druggable genome”. In fact, the inhibition of specific protein kinases by small molecules inhibitors has been pharmacologically validated for the treatment of a wide range of therapeutic indications. This fact, together with the growing number of parasite kinases validated as targets for parasitic disease allowed us to surmise that protein kinases are druggable targets for leishmaniasis.3

Based on our previous experience on human GSK-3 and CK1 inhibitors, we have focused our attention on these two kinases also present in Leishmania (GSK3 and CK1.2) as targets for the development of antileishmania drugs. A number of specific human kinase inhibitors with different chemical structures have been tested in a phenotypic assay at low micromolar concentrations, some of them with an adequate specificity index. Their ability to inhibit Leishmania kinases will be further elucidated by experimental and molecular modelling techniques. Acknowledgments: This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO, project no. SAF2015-65740), Redes de Investigación Cooperativa Instituto de Salud Carlos III RICET (RD12/0018/0007 & RD16/0027/0010) and FEDER funds. P. M. acknowledges the contract from the Fondo de Garantía Juvenil (European Social Fund, Youth Employment Initiative) and FEDER funds.

2 Andrews, K. T.; Fisher, G.; Skinner-Adams, T. S. Drug repurposing and human parasitic protozoan diseases. Int J Parasitol Drugs Drug Resist 2014, 4, 95-111. 3 Merritt, C.; Silva, L. E.; Tanner, A. L.; Stuart, K.; Pollastri, M. P. Kinases as druggable targets in trypanosomatid protozoan parasites. Chem Rev 2014, 114, 11280-11304.

http://ec.europa.eu/esf/

http://ec.europa.eu/social/main.jsp?langId=en&catId=89&newsId=1829&furtherNews=yes

http://ec.europa.eu/social/main.jsp?langId=en&catId=89&newsId=1829&furtherNews=yes



P.11. Microwave-assisted and conventional 1,3-dipolar cycloaddition reactions to the synthesis of some

benzimidazole/(benzo)indolizine hybrids: a comparative study

Roumaissa Belguedj1,2, Christophe Menendez2, Michel Baltas2, Abdelmalek Bouraiou1

1Université des Frères Mentouri, Unité de Recherche de Chimie de l’Environnement et

Moléculaire Structurale, Constantine 25000, Algérie ; 2Université Paul Sabatier, Laboratoire de Synthèse et Physico-Chimie de Molécules d’Intérêt Biologique, UMR-CNRS 5068, Toulouse,

France

Indolizines constitute the core structure of many naturally occurring alkaloids such as, (-)-slaframine,1 (-)-dendroprimine, and coniceine. Many indolizine derivatives have important biological activities, including anti-inflammatory, anti-HIV, hypoglycemic, CNS depressant1

The synthetic utility of microwave irradiation has increased considerably in recent years due to its capacity to reduce chemical reaction times, increase yields, and in some cases to lead to outcomes different from those obtained with conventional means2.

In continuation of our research interest in heteroatomic N-ylide involving 1,3-cycloaddition reactions3 we report here the synthesis, spectroscopic identification and the crystal structures of some new benzimidazole/(benzo)indolizine hybrids and benzimidazole/furane compounds Indolizines were obtained via 1,3-dipolar cycloaddition of alkynes with 1-(2’-benzimidazolyl-methyl)(benzo)pyridinium ylide. Optimal conditions under microwave has been elucidated and compared to the conventional ones indicating that reactions for the microwave-assisted 1,3-cycloaddition reaction of 1-(2’ benzimidazolylmethyl) (benzo) pyridinium ylide are faster and higher-yielding. Similarly furanes were obtained via a three component reaction using 1-(2’-benzimidazolylmethyl)(benzo)pyridinium ylides, benzaldehyde derivatives and active methylene compounds (ie. 2,4-pentanedione and ethyl acetoacetate).

The biological activities of some synthesized indolizines and furanes against Leishmania Parasite4 are underway by the team of Pr. P. Loiseau.

NH

N N

Cl

NH

N

N

R2R1

R1 R2

MW

(1) G. S. Singh, E. E. Mmatli, Eur. J. Med. Chem. 2011, 46, 5237. (2) M. Pineiro, T. M. V. D. Pinho e Melo, Eur. J. Org. Chem. 2009, 5287. (3) R. Belguedj, A. Bouraiou*, S. Bouacida, H. Merazig, A Chibani, Z. Naturforsh. 2015, 885. (4) J.F. Marquis, I. Hardy, M. Olivier, Parasitology. 2005, 131, 197.

We thank (Ministère de l’Enseignement Supérieur et de la Recherche Scientifique) Algerie, for PNE program (Programme National Exceptionnel, R.B.) and CNRS/France and the University Toulouse 3 for financial support.



P.12. DFT studies of autoxidation of 2-alkylidene-1,3-cyclohaxadione leading to bicyclic-hemiketal endoperoxides

B. Tuccio,1 C. André-Barrès2

1Institut de Chimie Radicalaire, UMR AMU-CNRS 7273, Laboratoire Chimie Provence, Equipe SACS, Aix-Marseille Université, Campus de Saint Jérôme, 13397 Marseille cedex 20, France 2Laboratoire de Synthèse et de Physicochimie de Molécules d'Intérêt Biologique, UMR CNRS 5068, Université Paul-Sabatier, 118 route de Narbonne, F-31062 Toulouse cedex 04, France

We are interested in antimalarial agents acting as artemisinin and we focused on the

syntheses of new endoperoxides, related to the natural phytohormones known as G factors (G1, G2, G3) issued from the leaves of Eucalyptus grandis. The synthesis is based on spontaneous oxygen uptake on a dienol system furnishing exclusively required endoperoxides.

This autoxidation was investigated by a combined spin trapping/EPR/mass spectrometry approach allowing the characterizing of the diradical species present in pathway B.

Based on these previous results, a theoretical study was undertaken.

Each step of the mechanism of addition of triplet oxygen on the dienolic precursor was calculated by DFT using the restricted or unrestricted B3LYP with 6-311+G(d,p) as set of bases. The geometry and energy of each minima and transition states were characterized, in singlet and triplet state, allowing the description of the complete reaction pathway of the formation of endoperoxides. The crossing between triplet and singlet potential energy surfaces was found. The singlet diradical character changing was also examined along the intrinsic reaction coordinate (IRC) calculations leading to the endoperoxide.

Mathilde Triquigneaux, Laurence Charles, Christiane André-Barrès and Béatrice Tuccio, Org. Biomol. Chem., 2010, 8, 1361-1367. Jérémy Ruiz, Joëlle Azéma, Corinne Payrastre, Michel Baltas, Béatrice Tuccio, Henri Vial, Christiane André-Barrès. Current Topics in Medicinal Chemistry, 2014, 14, 1668-16683.



P.13. Dinitroaniline-Ether Phospholipid Hybrids

M. Roussaki1, K. C. Prousis1, P. Afroudakis1, A. Cordeiro-da Silva2, N. Santarem2, D. Costa2, S. Gul3, J. Clos4, I. M. de Amorim,5 E.S. Barrias,6 W. de

Souza,5 T.M.U. de Carvalho5, T. Calogeropoulou1*

1National Hellenic Research Foundation, Institute of Biology, Medicinal Chemistry and Biotechnology, Athens, Greece; 2Instituto de Investigação e Inovação em Saúde, Universidade

do Porto, Portugal and Instituto de Biologia Molecular e Celular (IBMC) da Universidade do Porto, Portugal; 3Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Hamburg, Germany; 4Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany; 5Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, CCS, UFRJ, Rio

de Janeiro, Brazi; l6Diretoria de Programas, Instituto Nacional de Metrologia, Qualidade e Tecnologia-Inmetro, Xerém, Rio de Janeiro, Brazil


Protozoa are unicellular eukaryotes and represent one of most important sources of parasitic diseases. Every year, more than one million people die from complications from protozoal infections worldwide. Trypanosomatidae protozoa constitute the causative agents of several human diseases such as Chagas disease (Trypanosoma cruzi), sleeping sickness (Trypanosoma brucei) and leishmaniasis (Leishmania sp). These illnesses have been classified by the WHO as neglected diseases, which affect people living in poverty in developing countries and for which no efficient therapy is available.2,3 Miltefosine (hexadecylphosphocholine) is an alkylphosphocholine with demonstrated activity against various parasite species and cancer cells, as well as some pathogenic bacteria and fungi. Moreover, Miltefosine is currently the only oral drug available for the treatment of visceral (VL) and cutaneous leishmaniasis (CL). However, at the therapeutically effective doses, severe gastrointestinal side effects and serious weight loss were observed while, teratogenicity remains a problem.

During the last decade our group has been investigating ring-substituted alkylphosphocholines and we have indicated that introduction of cycloalkane rings in the lipid portion provides compounds with enhanced activity and reduced toxicity.1-3

In the context of more in depth SAR studies we designed and synthesised hybrid molecules which combine in one molecular scaffold two pharmacophores, the dinitroaniline moiety and the ether phospholipid structure.4 This approach would enable us to address two different mechanisms of action, namely the inhibition of the alpha-tubulin of the parasite, targeted by dinitroaniline herbicides such as trifluraline, and the putative molecular targets of alkylphosphocholines.

The new trifluraline-substituted ether phospholipids encompass analogues active against L. infantum, L. donovani and T. cruzi amastigotes as well as T. b. brucei (blood stream form). Extensive ADME-Tox studies demonstrated that the toxicity of the majority of the compounds is very low and much lower than Miltefosine, especially against THP-1 macrophages.



Acknowledgement

This project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement n° 603240 (NMTrypI - New Medicine for Trypanosomatidic Infections). http://www.nmtrypi.eu/

References

1Calogeropoulou et al. (2012), US8,097,752 2Calogeropoulou. et al. Bioorg. Med. Chem. Lett., 2010, 20, 5484-5487. 3Calogeropoulou et al. J. Med. Chem. 2008, 51, 897-908. 4Godinho J.L et al. Exp Parasitol. 2013, 135, 153-165.




P. 14. In vitro Anti-parasitic activity of Marine Cyanobacterial extracts against Leishmania, Giardia and Trichomonas

Gisela Castro,1,2, Sandra Gomes-Pereira3,4, Rita F. Oliveira3, Agostinho Cruz3,

Vítor Vasconcelos1,2, Rosário Martins1,3

1Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto, Portugal; 2Faculty of Sciences, University of Porto, Portugal; 3 Centre of Health and Environmental Research (CISA), Superior School of Health, Polytechnic of Porto, Portugal;4 ISLA-Instituto Politécnico de Gestão e Tecnologia,

Porto-Portugal

Leishmaniosis is a neglected disease that affects the poorest and most vulnerable populations in developing countries. Currently, high rates of clinical failures have been reported associated with the classical treatments like miltefosine. Giardiasis is the most common parasitic diarrheal disease affecting humans. The first-line nitroimidazoles drugs were found to have relevant side effects and single and multidrug resistance to compounds has been reported. Trichomoniasis is sexual transmitted parasitosis that increases the risk of HIV transmission and leads to adverse outcomes of pregnancy. Treatment options are in this case reduced to nitroimidazole compounds. For all the described parasitosis the search for new medicines seems mandatory.

Marine organisms are recognized as source of secondary metabolites promising for drug discovery. Among those, cyanobacteria have come into focus for the production of bioactive compounds with potential phamacological applications namely as antiparasitic. In Portugal, the Interdisciplinary Centre of Marine and Environmental Research (CIIMAR) hosts a cyanobacteria culture collection (LEGE culture collection) composed manly by strains isolated from the portuguese coast. The aim of this work was to evaluate the antiparasitic potential of LEGE cyanobacterial strains against Leishmania infantum, Giardia duodenalis and Trichomonas vaginalis, in vitro. Cyanobacteria crude extracts obtained by a dichloromethane:methanol (2:1) extraction of freeze dried biomass were tested at a final concentration of 1mg/mL, 0,1mg/mL and 0,01mg/mL.

L. infantum promastigotes were cultured in RPMI medium. Parasites at a concentration of 1,5x106 parasites/mL were exposed to the cyanobacteria extracts for 72h. G. duodenalis was cultured in TYI-S-33 medium and T. vaginalis trophozoites were cultured in Diamond-TYM medium. Parasites at a concentration of 5x104 and 2,8x105 parasites/mL respectively were exposed to the extracts for 24 hours at 37ºC.The MTT assay was applied in order to verify an anti-parasitic effect.

Cyanobacteria extract from Leptolyngbya halophila LEGE 06102 and Synechocystis salina LEGE 06099 isolates showed inhibition of Leishmania promastigotes growth. Concerning Giardia parasite a growth inibithion was registered with the two Cyanobium sp. strains, LEGE07175 and LEGE06113. Trichomonas trophozoites were not affected by the presence of cyanobacterial extracts.

These results show that some cyanobacterial strains present activity against Leishmania and Giardia parasites but not against Trichomonas. Further studies are needed in order to confirm their potential antiparasitic effect.



Acknowledgements: This research was partially supported by FCT – Foundation for Science and Technology under the project UID/Multi/04423/2013 and by the Structured Program of R&D&I INNOVMAR - Innovation and Sustainability in the Management and Exploitation of Marine Resources (reference NORTE-01-0145-FEDER-000035, Research Line NOVELMAR), funded by the Northern Regional Operational Program (NORTE2020) through the European Regional Development Fund (ERDF).



P.15. Efficacy of Oral and Parenteral Amphotericin B Systems against Experimental Trypanosoma cruzi Infection

M. Rolón1, D.R. Serrano2, A. Lalatsa3, E. de Pablo2, JJ. Torrado2, M.P. Ballesteros2, A.M. Healy4, C. Vega1, C. Coronel1, F. Bolás-Fernández5,

M.A. Dea-Ayuela6

1Centro para el Desarrollo de la Investigación Científica (CEDIC),Asunción, Paraguay.

2Departamento de Farmacia y Tecnología Farmacéutica, Facultad de Farmacia, Universidad Complutense de Madrid, Spain. 3School of Pharmacy and Biomedical Sciences, University of

Portsmouth, PO1 2DT, UK. 4School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, Ireland. 5Departamento de Parasitología, Facultad de Farmacia, Universidad

Complutense de Madrid, Spain. 6Departamento de Farmacia, Facultad de Ciencias de la Salud, Universidad CEU Cardenal Herrera, Moncada, Valencia, Spain.

INTRODUCTION. Chagas disease is a chronic parasitic infection caused by

Trypanosoma cruzi. Although chemotherapy is available, existing drugs (benznidazol and nifurtimox) are far from ideal. Activity of amphotericin B (AmB) in T. cruzi infections has not been well documented. In this work, oral and parenteral drug delivery systems of AmB with different aggregation states were tested.

METHODS. Dimeric AmB (AmB-NaDC), poly-aggregated AmB and poly-aggregated AmB encapsulated in albumin microspheres (AmB-AME) were prepared and characterised by TEM, SEM, DSC, TGA, FTIR, PXRD as previsouly described [1]. In vitro activity on extracellular and intracellular T.cruzi forms and cytotoxicity on NCTC929 fibroblasts was evaluated. In vivo efficacy was assessed after intravenous and oral administration of AmB-AME and AmB- sodium deoxycholate micelles (AmB-NaDC) respectively [2].

RESULTS. AmB aggregation state was correlated to the λmax of the absorption spectra. Dimeric AmB spectra was characterized by a broad intense band at 328-340 nm while poly-aggregated AmB and AmB-AME displayed bands of smaller intensity at 360–363, 383–385 and 406–420 nm. Different molecular structures were observed in TEM and SEM micrographs (Fig.1). All three formulations displayed promising IC50 values and selectivity index against T.cruzi both epimastigotes and amastigotes. AmB-AME showed a better efficacy after the intravenous administration of three doses of 5 mg/kg. On the contrary, oral administration of AmB-NaDC was the most effective formulation resulting in a >75% reduction of parasitemia when doses higher than 10 mg/kg were administered (Fig. 2).



CONCLUSIONS. Oral administration of AmB-NaDC (> 10 mg/kg) is a greater and inexpensive therapy to trypanosomiasis compared to parenteral AmB-AME allowing treatment access worldwide.

REFERENCES

[1] Serrano DR et al. Int J Pharm. 2013;447:38-46; [2] Vega C, et al. Parasitol Res. 2005;95:296-8.



P.16. Oral Nanomedicines for Chagas Disease

Eustine M. Hanna1, Dolores R. Serrano2, Aikaterini Lalatsa1

1School of Pharmacy and Biomedical Sciences, St Michaels Building, University of Portsmouth, White Swan, Portsmouth, PO1 2DT, UK. 2Departamento de Farmacia y Tecnología

Farmacéutica, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza Ramon y Cajal, Spain, S/N28040.

INTRODUCTION: Chagas disease (CD) is a parasitic zoonosis endemic in most mainland countries of central and South America affecting nearly 10 million people amongst 100 million people that are at high risk. Treatment is only effective if applied at early stages of the disease. The only two marketed drugs (benznidazole (BNZ) and nifurtimox (NFX)) are poorly soluble (BCS Class II) and are both widely criticised because of their low efficacy and oral bioavailbility and serious toxic side effects leading to discontinuation of 20–30% treatments. However, none of them can still be replaced with more effective novel drugs. Thus, reformulating them in more effective oral formulation with reduced frequency of administration and better oral bioavailability is imperative and Self-Nanoemulsifying Drug Delivery Systems (SNEDDS) made from generally regarded as safe (GRAS) excipients can be an easily scalable, safe and cost-effective formulations with increased patient compliance.

METHODS: Quality by Design (QbD) experiments were carried out to identify the optimal ration of Labrasol, Labrafil M1944 and Capryol 90 than would produce nanometric particle size of SNEDDS upon aqueous dispersion and high drug loading. Solid SNEDDS were prepared by adsorption of drug loaded SNEDDS onto solid silica carriers (Syloid 244 FP, or Syloid XDP 3050) and mixing the resulting powder with microcrystalline cellulose and sodium starch glycolate [SNEDDS : solid carriers ratio of 1:3w/w] and compressed into tablets that were overcoated with Eudragit RS PO or Eudragit RL PO to control the dissolution profile. Dissolution testing using flow through-cell was performed. Powder flow properties and tablet characteristics were also investigated and compared to commercially available tablets [Lampit (Bayer) and Rochagan (Roche)]. Accelerated stability studies over a range of temperatures (25-80oC) were conducted over five weeks.

RESULTS: NFX and BNZ loaded optimal SNEDDS illustrated a particle size below 400 nm and good drug loading (2mg/g and 10mg/g respectively or combined). NFX and BNZ loaded SNEDDS enhanced the dissolution rate and increased the solubility in simulated gastrointestinal media compared to commercially available tablets. For SNEDDS loaded with both NFX and BNZ, however, a reduction in the level of drug released was observed possibly due to electrostatic interactions between both drugs. Solid SNEDDS prepared with Syloid 244 FP illustrated optimal flow properties, while those prepared with Syloid XDP 3050 possessed better compressibility properties. However, only overcoated tablets were able to pass the friability test. Model stability data indicated that NFX loaded SNEDDS will only be stable if refrigerated in an airtight vial for one month, while NFX Solid SNEDDS were stable in similar conditions for six months. Combined solid SNEDDS illustrated an increased stability for NFX.

CONCLUSIONS: NFX and BNZ nanomedicines illustrated an improved dissolution profile compared to commercial formulations. Co-formulating both drugs in a single solid SNEDDS leads to an increase in chemical stability of NFX while maintaining a good dissolution profile resulting in a cost-effective, easily scalable and combined effective treatment for CD worldwide.



P.17. Effect of the aggregation state of Amphotericin B on Red Blood Cells

Raquel Fernández1, M. Paloma Ballesteros1, Dolores R. Serrano1

1Departamento de Farmacia y Tecnología Farmacéutica, Facultad de Farmacia, Universidad Complutense de Madrid. Plaza Ramón y Cajal, s/n, 28040-Madrid, Spain.

Introduction: Amphotericin B (AmB) is a macrolide produced by Streptomyces nodosuswith antifungal and antileishmanial activity. This drug joins to the sterols in the cell-membrane forming a pore that destabilises the cell and causes apoptosis. AmB has a higher affinity for the ergosterol in fungal cells and some parasites, such as Leishmania. However, it can also binds to the cholesterol of the mammalian cells leading to toxicity such as haemolysis. This drug is only commercially available in different intravenous formulations.AmBcan be found in three different aggregation states: monomer, dimer or polyaggregate. The aggregation state can be easily identified by UV. The dimer has a characteristic band at 328nm wavelength,the monomer at 406nm wavelength and a mixture of them for the polyaggregated form.AmB was prepared in its three aggregation states and the haemolytic activity was evaluated in vitro.

Methods: Poly-aggregated AmB(1): AmB (50 mg) was added in 10 ml of an aqueous solution

containing 41 mg of sodium deoxycholate, 10 mg of dibasic sodium phosphate and 0.9 mg of monobasic sodium phosphate. The dispersion was stirred until a homogeneous yellow suspension was obtained. Dimeric AmB: Dimeric AmB was prepared similarly to poly-aggregated AmB. However, before adding the drug into the aqueous solution, the pH was adjusted to 12.0 in order to solubilise the drug and subsequently reduced to 7.4.

Monomeric AmB: it was prepared similar to dimeric AmB but using ϒ-cyclodextrin instead of sodium deoxycholate.

Ex vivo Red Blood Cell (RBC)haemolysis assay (2): Diluted RBCs to 4% concentration were added into a 96 well plate (180 µl/well). Formulations were diluted using deionised water. 20 µl of each sample concentration was loaded into the wells in triplicate. For positive control wells, a 20% solution of Triton X-100 (20 µl) was added. For negative control wells, PBS at pH 7.4 (20 µl) was incorporated. Plates were incubated at 37 ⁰C for 1 h. Subsequently, plates were centrifugedand 50 µl of supernatant from each well were transferred into a clear, flat-bottomed 96-well plate. The absorbance of the supernatants was measured using a plate reader at 570 nm. The percentage of haemolysis was calculated using the following equation: Haemolysis (%) = (ABSsample – ABSPBS)/ABSTriton x 100; where ABSPBS is the average of the absorbance from the negative control samples and the ABSTriton is the average of the absorbance from the positive control samples. The concentration of AmB that produces 50% haemolysis at the tested conditions (HC50) was calculated using Compusyn software.

Results: The HC50 for the monomer, dimer and poly-aggregated AmB was: 9.8, 229, 753 µg/ml respectively. The poly-aggregated form was 76-fold less haemolytic than the monomeric form and 3.3-fold less than the dimeric aggregation state.

Conclusions: Monomeric AmB is the most hemolytic aggregation state while the poly-aggregate is the least. Formulations containing poly-aggregated AmB could have a therapeutical advantage over the other aggregation states, considering that AmB formulations are intravenously administered.



References:

(1) D.R. Serrano et al. InternationalJournal of Pharmaceutics, 2013; 447(1-2)38-46.

(2) B.C. Evans, et al. Journal of visualized experiments, (2013); JoVE:e50166.



P.18. Antiparasitic Chemotherapy in Veterinary Medicine: Challenges, Hurdles and Opportunities

María J. Corral, M. Dolores Jiménez-Antón, Ana Isabel Olías-Molero,

José Mª Alunda

Departamento de Sanidad Animal (ICPVet Group), Faculty of Veterinary Medicine, University Complutense of Madrid, Madrid, Spain

It is considered that integrated control systems should be the choice to reduce the

extension and severity of parasitic diseases affecting animals. Biological complexity of parasites and their life cycles and fragmented knowledge of immune system in many animal species make that vaccines against parasitic diseases are very limited. Non medical preventive measures are sometimes unpractical, unacceptable by environmental reasons or unaffordable. Thus veterinary medicine of pets and livestock strongly relies on the use of antiparasitic drugs. However chemotherapy of most parasitic diseases affecting animals, domestic and wild, has important shortcomings. Some of the currently used drugs of choice were synthesized over 50 years ago and no new chemical entities (NCE) are foreseen. For some parasitic diseases there is no available chemotherapy. Socioeconomic changes have increased the demand of products of animal origin and there is growing social awareness of the presence of drug residues in the environment.

Global market and reduced economic margins in farm activities require efficient control systems including affordable prices of antiparasitic drugs and sustainability in environmental terms. Resistant strains to chemotherapeutic agents have been reported in parasitic protozoa, helminths and arthropods and there is spread resistance to all available drugs in gastrointestinal nematodiases of small ruminants. There is a shortage of new drug launches despite the massive investment made by pharmaceutical companies and the public sector. This scarcity is probably rooted on both managerial (e.g. reduction of pharma companies; strict separation of financial and technical departments), social (e.g. more strict regulations on animal experimentation) and scientific reasons (e.g. limited chemical space explored; trenched strategies in drug discovery (DD) pipeline; inadequate laboratory models).

This scenario favors the fine tuning of DD pipeline and the evaluation of potential antiparasitic drugs. Of paramount importance will be the widening of chemical space to get NCE by using different sources (e.g. natural or natural-derived scaffolds); the reevaluation of selection criteria along the DD pipeline; identification of resistance and therapeutic failure; and use of truly predictive animal models. The path from molecular biology to pharmacology and, finally, chemotherapy is longer and more complex than previously thought; and DD should integrate all these steps. It is expected that veterinary medicine, with a deep understanding of the pathophysiology of parasitic diseases, plays a significant role in the development of refined animal models. These predictive models will increase health, welfare and production.



P.19. Early preclinical studies of new selenocyanate and diselenide compounds as leishmanicidal agents

Verónica Alcolea1,2, Esther Moreno3, Elena González-Peñas1, Juan Manuel Irache4, Juan Antonio Palop1,2, Carmen Sanmartín1,2, Socorro Espuelas3,4

1Department of Organic and Pharmaceutical Chemistry, University of Navarra, Pamplona,

Spain; 2 Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona, Spain; 3 Institute of Tropical Health, University of Navarra; 4 Pharmacy and Pharmaceutical Technology

Department, University of Navarra, Pamplona, Spain. [email protected], [email protected], [email protected], [email protected]

Leishmaniasis is a neglected tropical disease caused by the parasite Leishmania spp.

The infection can cause different clinical syndromes which range from a cutaneous form to the affectation of mucocutaneous tissues or visceral organs. Visceral leishmaniasis (VL) is the most severe form, causing 20,000-40,000 deaths per year. Current drugs for the treatment of VL are not completely effective and they show important drawbacks such as severe toxicity, high cost, long-term treatments or resistances. Therefore, there is an urgent need to develop new effective, safe and cheap leishmanicidal agents.

In a previous publication, we synthesized a series of novel heteroaryl selenocyanates and diselenides and they were screened for their activity and selectivity against L. infantum amastigotes (1). Two compounds (2d and 1h) emerged as the lead compounds of these series, showing excellent values of activity and selectivity. In the present work we have evaluated the appropriate dose and route of administration of these two compounds for the in vivo efficacy studies in L. infantum-infected mice. Since the novel compounds exhibited poor values of intestinal permeability, they are not suitable for oral administration. Therefore, the intravenous (i.v.) route has been explored. A lipid nano-emulsion formulation has been developed to allow the i.v. administration of compound 2d. This new formulation has been found to be safe for the i.v. administration of 2d at the dose of 2 mg/kg for five consecutive days. On the other hand, compound 1h showed higher toxicity in vitro and in vivo and thus it has to be administered at the dose of 1 mg/kg. The studies of efficacy in L. infantum infected-BALB/c mice are now in course.

References

(1) Novel heteroaryl selenocyanates and diselenides as potent antileishmanial agents. Y. Baquedano, V. Alcolea, M.A. Toro, K.J. Gutiérrez, P. Nguewa, M. Font, E. Moreno, S. Espuelas, A. Jiménez-Ruiz, J.A. Palop, D. Plano, C. Sanmartín. Antimicrob. Agents Chemother.60:6 (20)



P.20. Topical treatment of CL with paromomycin and anti-TNF-α antibodies: efficacy study in L. major infected BALB/c mice

Juana Schwartz, Esther Moreno, Alba Calvo, Socorro Espuelas

Institute of Tropical Health and Pharmacy and Pharmaceutical Technology Department, University of Navarra, Instituto de Investigación Sanitaria de Navarra (IdiSNA), 31008

Pamplona, Spain [email protected], [email protected], [email protected], sespuelas@unav.

es

The immune responses induced by Leishmania infection determine the disease pathogenesis, as lack of immune response is related to chronification and severity and an effective immune response leads to infection resolution. Thus, an obvious strategy seems to be to modulate the immune response with the aim to boost antileishmanial mechanisms using pro-inflammatory drugs. However, in localized cutaneous (CL) and mucocutaneous leishmaniasis, disease severity has been linked to an excessive production of pro-inflammatory mediators. In both cases, parasites in the affected tissue are scanty and ulceration and tissue destruction are a consequence of the vigorous local inflammatory response. Thus, in order to minimize tissue destruction that leads to scar formation, treatment of the lesions with anti-inflammatory agents could be also an interesting strategy to explore. Despite immunochemotherapy is arising as a promising strategy, clinical studies have shown that the use of immunomodulators alone does not lead to parasite clearance although it improved the response to treatment when combined with leishmanicidal drugs. Therefore, in this work, we proposed combining the antileishmanial drug paromomycin with poly(I:C) (PIC) (pro-inflammatory) or anti-TNFα (anti-inflammatory) to explore the potential of these formulations to both lead to parasite clearance and wound healing.

In our work, we first evaluated the antileishmanial activity of the drugs. Then, skin permeability of the compounds and their anti-inflammatory activity were assessed in a mouse model of IMQ-induced inflammation. Finally, the different drugs and combinations were tested in a L. major BALB/c model of infection. Immunohistochemistry studies were also carried out in skin lesions to observe if there were changes in the expression of genes that are involved in inflammation and wound healing. In our study, combinations of paromomycin with anti-TNFα or PIC were similar in terms of in vitro parasite clearance in infected macrophages. In the BALB/c mouse model of inflammed skin, paromomycin and anti-TNFα applied separately were able to reduce inflammation locally. In infected mice, combination of PM plus PIC or anti-TNFα significantly reduced the parasite burden in skin, lymph nodes, liver, and spleen similarly to PM alone and PM with methylbenzethonium chloride (the marketed formulation). However, in L. major-infected BALB/c mice, the combination therapy of paromomycin with anti-TNFα had a stronger anti-inflammatory activity that was confirmed by the down-regulation of TNF-α, IL-1β, iNOS, IL-17, and CCL3, whereas paromomycin alone only decreased the expression of iNOS and IL-17. Immunohistochemistry studies confirmed a decrease of the neutrophilic infiltrate during infection. Therefore, topical application of formulations combining leishmanicidal drugs with small biological anti-inflammatory molecules could be useful to reduce inflammation and scarring in CL.








P.21. Novel oral formulations of Active Hexose Correlated Compound (AHCC) and its parasitic activity in an in vivo model

Liliana Bautista1, Hajime Fujji2 and Juan J. Torrado1

1Complutense University of Madrid, Faculty of Pharmacy; 2Amino Up Chemical, Sapporo,Japan

AHCC is extracted from Shiitake mushroom (Lentinus edades). AHCC is a functional food; published studies indicate that it has immunostimulatory properties being beneficial in adjuvant therapy in patients with cancer. A scientific collaboration between the pharmaceutical company and the depatament of Pharmaceutical Technology at the Complutense University of Madrid was establisehd in order to develop more hydrophilic oral formulations as an alternative to the lipidic formulations already marketed. These hydrophobic formulations contain large amounts of lipidic excipient with the aim of protecting the lyophilized powder from the humidity which leads to chemical degradation and compromises the product activity. In Japan, commonly, the form of consumption of AHCC is directly from the scahets without water. However, in Western countries the sachets are usually dispersed in water and taken as oral suspensions or extemporary oral solutions. Therefore, a hydrophilic formulation would be more convenient than a hydrophobic formulation to improve the dispersability of the product in water and the patient compliance.

Aims

1. To develop and characterize novel hydrophilic formulations of sachets containing AHCC and the study the stability of the developed formulations in accordance with ICH guidelines.

2. To study the in vivo bioavailability of novel oral formulations of AHCC.

Methodology

The excipients used in this research are approved for use in nutraceutical formulations in most countries. Stability studies were performed according to the ICH Q1A ( R2 ). The in vitro an in vivo activity of the formulations was evaluated.

Results

1.We have developed and characterized novel AHCC hydrophilic formulations in sachets, during stability studies was the caking tendency This effect occurs under extreme conditions of humidity (96% ± 5%) and after 24 months being easily reversible because the formulations are dispersed and solubilized in an aqueous medium. Therefore, we suggest 24 months as expiration date.The improved formulation in sachets from a stability point of view has to contain a relatively low dose of AHCC (0.6 g) with lots of excipients resulting in a final weight of 3.13 g granulate. As packaging materials, aluminium- aluminium sachets are used and is recommended to avoid storage conditions with excessive moisture.

2.The characteristics invivo of AHCC were evaluated by analyzing some of its components: glucose, G1 and isomaltol after oral administration. No hyperglycemic effect was detected even after oral administration of doses up to 5 g of AHCC-FD to healthy



volunteers under fasting conditions. G1 and isomalt are not detected in plasma samples after oral administration of AHCC in humans and mice. We conclude therefore that these molecules are not useful to indicate the oral bioavailability of the formulation.

3.It was observed that AHCC is useful by itself to reduce the number of parasites in an experimental mouse model of T. spiralis. The anthelmintic efficacy of AHCC is proportional to the dose. AHCC administration reduces the duration of the infection (decreasing faster the number of parasites in the intestinal phase) and inflammation (Th1 response decreases which was quantified by interleukins: TNF, and IL-2 IFNγ) compared to the untreated control group.

4.No significant differences in activity was observed after the administration of the same dose of raw material (AHCC-FD) and the developed formulation in sachets concluding that they are pharmacodynamic bioequivalent.

Acknowledgment

Animo Up Chemical.



P. 22. In vitro activity evaluation of marine metabolites against bloodstream forms of Trypanosoma brucei

Maria Harizani1, Sara Costa2, Nuno Santarem 2, Anabela Cordeiro-da-Silva2,3,

Vassilios Roussis1 and Efstathia Ioannou1

1Department of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, National and Kapodistrian University of Athens, Athens 15771, Greece.

2I3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto 4200-135, Portugal.

3Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Porto 4050-313, Portugal.

Parasites of the family Trypanosomatidae are causing serious human diseases,

including leishmaniasis, African sleeping sickness and Chagas disease. The currently used therapeutics are not devoid of problems, such as toxicity, side effects and development of drug resistance, so there is an urgent need for the discovery of new, more effective chemotherapeutics.

Nature, the most prolific source of biological and chemical diversity, has provided mankind with remedies to health problems since the ancient years and continues to be the most promising reservoir of bioactive chemicals for the development of modern drugs. In addition to terrestrial organisms that still remain a promising source of new bioactive metabolites, the marine environment, covering approximately 70% of the Earth’s surface and hosting a largely unexplored biodiversity, offers an enormous resource for the discovery of novel bioactive compounds.

As part of our ongoing studies towards the isolation and structure elucidation of new bioactive metabolites from marine macro- and microorganisms from the East Mediterranean Sea, the antiparasitic activity against Trypanosoma brucei and the cytotoxicity against THP-1 macrophages of a panel of structurally diverse natural products, including halogenated sesquiterpenes, diterpenes, acetogenins and polyethers, were evaluated.

Among the 27 tested metabolites a polyether of actinobacterial origin showed promising activity against T. brucei with IC50 = 240 nM and a selectivity index (SI) of 58. Further testing of structurally related metabolites and pharmacokinetic studies of the bioactive polyether are currently in progress.



P.23. Influence of glutathione and antimony in the ATPase activity of Leishmania LABCG2 transporter

Ana Perea1, Yasuhisa Kimura2*, José Ignacio Manzano1*, Santiago Castanys1#,

Kazumitsu Ueda2,3#, Francisco Gamarro1# * Both authors contributed equally to this work. # Equal senior authors.

1 Instituto de Parasitología y Biomedicina “López-Neyra” (IPBLN-CSIC), Parque Tecnológico de

Ciencias de la Salud, Granada (Spain). Email: [email protected] 2 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto

(Japan) 3 Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Kyoto (Japan)

The Leishmania LABCG2 transporter is involved in protecting these protozoan

parasites against the toxic effects of antimony, probably by efflux as conjugated thiol complexes. However, the effects of glutathione (GSH) and antimony (SbIII) on the ATPase activity of LABCG2 protein have not been described. We have expressed and purified a functional LABCG2 protein using the baculovirus-Sf9 insect cell system. By reconstitution of purified Leishmania LABCG2 in liposomes, we have analyzed the ATPase activity. LABCG2 reconstituted in brain polar extract showed an ATPase activity that was stimulated (3.5-fold) at 30 µM GSH, with Vmax and Km values of 678.6 ± 0.14 nmol Pi/min/mg and 9.02 ± 3.00 µM, respectively. Additionally, we examined the kinetics of ATPase activity of purified LABCG2 after incubation with increasing concentrations of SbIII. As a result, we found an increase in the LABCG2 ATPase activity (3.2-fold), reaching the maximum activity at 30 µM SbIII with Vmax and Km values of 405.3 ± 0.03 nmol Pi/min/mg and 12.84 ± 3.11 µM, respectively. Taken together, these data corroborate the hypothesis that LABCG2 is an active GSH and SbIII transporter. In the future, this achievement will allow the screening of potential substrates and inhibitors of LABCG2, contributing to the understanding of this transporter and its biological role in Leishmania.

This work was supported by the Spanish Grants SAF2015-68042-R (to S.C. and F.G.),

SAF2012-34267 (to F.G.) and Junta de Andalucía, Ref. CTS-7282 (to F.G.).



P. 24. The NMTRyPI - New Medicines for Trypanosomatidic Infections – drug discovery platform

M.P.Costi, J.M.Alunda, J.Clos, A.Cordeiro da-Silva, A.Khalid, S.Gul, S.Mangani,

C.Morales, W.Muller, T.Calogeropoulou, A.Venturelli, R.C.Wade, S.Wrigley.

The NMTrypI Consortium (http://www.nmtrypi.eu/), Email: [email protected]

According to the WHO (1) one billion people are at risk of or are affected by Neglected Tropical Diseases (NTD). Diseases caused by kinetoplastids, HAT, Chagas disease and Leishmaniasis continue to cause major problems in humans (2) and conventional available therapies show several drawbacks. . Furthermore, research on Trypanosomatid diseases is limited and fragmented. Open economy and human relocation by regional wars besides consistently increasing business travel, immigration, worker exchange, and global climate changes alter the geographic distribution of parasites and vectors and further the spread of vector-born diseases. Europe is particularly susceptible to these challenges. To meet the medical need an European project in the drug discovery and development field has been performed. The NMTrypI project takes a multipronged approach to discovering and characterizing new candidate drugs. Some of the achievements are:

1) The development of an alkylphospholipid compound exhibiting improved efficacy and lower toxicity than Miltefosine. A early protein biomarker set to detect the candidate efficacy and giving hints of its potential mechanism of action on a cellular basis has been generated. A SAR around the compound and a backup compound have been developed.

2) Pathogenic protozoa Trypanosoma brucei spp., T.cruzi and Leishmania spp are digenetic (complete their life cycle in two host organisms) and closely related. Thus we have focused on a target that is homologous to in all three of these closely related organisms and has no human homologue: pteridine reductase 1 (PTR1). We validated the concept of the pteridine reductase 1 (PTR1) bypass block discovering several classes of compounds targeted against PTR1, with no activity against the parasite but able to potentiate the effect of a parasitic DHFR inhibitor. These compounds are now under evaluation in vivo.

3) We have implemented a kinetoplastid-based screening platform and efficiently screened over 14000 compounds in phenotypic assays, and 5000 compounds in biochemical assays against 6 targets and three off-targets,as well as an array of early-tox assays. Furthermore, we have performed phenotypic screening against three parasites of the Hypha Mycodiverse library of natural compounds.

4) Synergetic collaborations between four large FP7 EU projects on discovery of drugs for neglected diseases have been established, including sharing an innovative drug discovery pipeline.

5) All data generated are stored and shared using the SEEK platform ((https://fp7h-synergy.h-its.org) and will be made openly available upon publication.



Acknowledgment. This project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement n° 603240 (NMTrypI - New Medicine for Trypanosomatidic Infections). http://www.nmtrypi.eu/

1. http://www.who.int/neglected_diseases/2010report/en/; 2. Ken Stuart, et al. J Clin Invest. 2008.


http://www.who.int/neglected_diseases/2010report/en/



P. 25. Modelling, Synthesis and Evaluation of Novel Quinine Analogues – New Drugs for Chagas Disease

Hirenkumar Gandhi1, Ligia F. Ceole2, Luz H. Villamizar2, Maurilio J. Soares2,

and Timothy P. O’Sullivan1

1Department of Chemistry, Analytical and Biological Chemistry Research Facility, University College Cork, Cork, Ireland

2Laboratory of Cell Biology, Carlos Chagas Institute/Fiocruz, Curitiba, PR, Brazil Email: [email protected], [email protected]

Chagas disease is caused by the protozoan Trypanosoma cruzi and is endemic in regions of Latin America. The drugs used most extensively for the treatment of Chagas disease are benznidazole and nifurtimox. Neither of these drugs has been shown to eradicate infection during the chronic phase when most patients are diagnosed.[1] While the search for the novel drug targets and new lead structures for the treatment of Chagas disease is of critical importance, a complimentary strategy is to utilise and further develop lead structures from nature with proven biological activity. Quinine, an alkaloid derived from the bark of the cinchona tree, is highly effective against parasitic infections despite almost 400 years of use. Quinine is a potent molecule completely inhibiting T. cruzi epimastigote replication in vitro.[2] Interaction with DNA would seem to be a reasonable mechanistic hypothesis. Using the Heck reaction, we have pursued modification of the vinyl group in quinine.[3] Following molecular-docking studies on Trypanosoma cruzi trypanothione reductase (TcTR) (PDB Id: 1GXF) and energy minimisation of the ligands, our quinine analogues have displayed excellent binding affinity.[4] For biological activity, all quinine analogues were first screened against cultured epimastigotes with benznidazole as the benchmark. The most active compounds were subsequently tested against Vero cell cultures infected with intracellular amastigotes. Dose-response curves were obtained from CompuSyn software in combination with Operetta Imaging system and Harmony Software.

References: [1] F.S. Buckner, A.J. Wilson, T.C. White, W.C. Van Voorhis, Antimicrob Agents Chemother 42

(12): 3245-50. PMC 106029, PMID 9835521. [2] S. Sepulveda-Boza, B.K. Cassels, Planta Medica, 62(1996), 98-105. [3] T. Dinio, A.P. Gorka, A. McGinniss, P.D. Roepe, J.B. Morgan, Bioorg Med Chem, 2012,

20(10): 3292-3297. [4] D. Saha, A. Sharma, Med Chem Res, 2014, 21 (12).



P.26. Emulsomes: A Tool for Delivery of anti-leishmanial BNIP Derivatives to Macrophages

Zeynep Islek1, Mustafa Güzel2, Fikrettin Sahin1, Mehmet H. Ucisik3,*

1 Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, Istanbul, Turkey

2 Department of Medical Pharmacology, International School of Medicine, Istanbul Medipol University, Istanbul, Turkey

3 Department of Biomedical Engineering, School of Engineering and Natural Sciences, Istanbul Medipol University, Istanbul, Turkey

Corresponding Author; Phone: +90 216 681 5154; E-mail: [email protected]

Similar to other parasitic diseases, chemotherapy is the most efficient strategy for leishmaniasis. However, the high toxicity of many antiparasitic compounds restricts their utility, and the emergence of drug resistant strains often impairs the lifespan of a given drug.

Among alternative drug candidates, bisnapthalimidopropyl (BNIP) derivatives have been recently shown to have anti-leishmanial activities, which even surpass the standard and most common Amphotericin B therapy [1]. However BNIP derivatives have some drawbacks including low aqueous solubility and toxicity. Addressing these limitations, this study applies two diverse technologies including medical chemistry approach together with the structure-based drug design, and nanotechnological drug delivery approach. The former approach will focus on design of new BNIP derivatives that have higher efficacy and bioavailability, whereas the latter will be used to deliver the drug specifically to the parasite, thereby decreasing the side effects of the chemotherapy, in particular on macrophages.

The delivery of BNIP derivatives into the macrophages will be achieved by encapsulating the active molecule in a lipid- based nanocarrier system, so-called emulsomes [2]. Emulsome is preferred mainly because of its four major features. Firstly, owing a solid lipid core like the solid lipid nanoparticles, emulsome may offer high loading capacities for hydrophobic substances such as BNIP [2,3]. Secondly, composed of only lipids and in the absence of any surfactants, emulsome is highly biocompatible [3]. Thirdly, the solid character of the nanocarrier provides a prolonged drug release profile, which can be controlled, or tuned, by the selection of the lipid composition as well as by surface modifications [4]. Lastly, but most importantly, the natural feature of lipids allows emulsome to accumulate in the organs of the reticuloendothelial system (RES) instead of the kidney, which will not only largely reduce toxicity, but will also improve the anti-leishmaniasis efficacy of the loaded drug, as parazites are also located in the organs of RES.

The development of new active BNIP derivatives and the emulsome-BNIP nanoformulations facilitating the targeted delivery to the macrophages is expected to substantially contribute to the improvements in treating parasitic disease Leishmaniasis in European region as well as worldwide.




References

[1] Tavares J., Quaissi A., Lin P.K.T., Loureiro I., Kaur S., Roy N., Cordeiro-da-Silva A., ChemMedChem (2010), 5, 140-147 [2] Ucisik M.H., Küpcü S., Debreczeny M., Schuster B., Sleytr U.B., Small (2013), 9, 2895- 2904. [3] Ucisik M.H., Küpcü S., Schuster B., Sleytr U.B., J. Nanobiotechnology (2013), 11, 37. [4] Ucisik M.H., Küpcü S., Breitwieser A., Gelbmann N., Schuster B., Sleytr U.B., Colloids Surf. B. (2015),132-139.

Acknowledgement

This study is supported by Tübitak EU-COST project no. 115Z846 and integrated to the COST action CM1307 entitled “Targeted chemotherapy towards diseases caused by endoparasites



P.27. Synergy activities on Neglected tropical diseases drug discovery within FP7 EU context

M.P.Costi1, Rob Leurs2, Jane MacDougall3, Raymond J. Pierce4

NMTrypI, www.nmtrypi.eu1; KINDReD, www.kinderd.eu3, PDE4NPD, www.pde4npd.eu2;

A-ParaDDisE, www.a-paraddise.eu4

The EC has, historically, generously supported research into Neglected Tropical Diseases (NTDs), starting well before the London Declaration of 2012, which put the control or elimination of NTDs at the forefront of global efforts to “chart a new course toward health and sustainability among the world’s poorest communities to a stronger, healthier future”. The development of new drugs is particularly important in these efforts in order to circumvent problems with current treatments, including drug resistance, severe side-effects or exacting dosing schedules. With this aim, four different consortia (NMTrypI, KINDReD, A-ParaDDisE and PDE4NPD), involving more than 50 teams in Europe, Africa, South America Australia and the USA, were funded in the last call related to drug development in neglected infectious diseases; HEALTH.2013.2.3.4-2: Drug development for neglected parasitic diseases. FP7-HEALTH-2013-INNOVATION-1. The projects targeted the major chronic parasitic diseases Leishmaniasis, Chagas Disease, Sleeping Sickness, Schistosomiasis and Malaria. Together, these diseases affect more than a billion people worldwide, cause hundreds of thousands of deaths and are major contributors to the poverty trap in the countries concerned. The projects were given the following objectives:

- To establish a common drug discovery platform that should have the capacity to undertake screening of compound libraries, lead development and

- Testing in relevant animal models as well as toxicology and safety testing of new drug candidates.

It was also requested that a minimum of three parasitic diseases, should be studied. With an expected impact to gather a comprehensive portfolio of drug leads, and develop the most promising of these into drug candidates that can be tested in early clinical trials.

In this call, the Commission particularly emphasized the need for the consortia to work together in a synergistic manner in order to maximise effort and reduce duplications. This aim has been achieved through multiple meetings, teleconferences and bilateral contacts, culminating in a joint meeting in Modena (mid-June 2016). As a result of this meeting, and since three of the four consortia reach termination within the next six months, it is opportune to focus attention on our achievements to date and set out the reasons why the Commission should seek to build on their significant investment and cement these considerable advances by future funding.

The four consortia have used various approaches to develop “hit” and “lead” candidates for drug development. These include: phenotypic screening of extensive or focused compound libraries or natural products, target-based screening (phosphodiesterases, epigenetic enzymes, dihydrofolate reductases etc.) including HTS and in silico screening, repurposing or modifying existing approved drugs,extensive ADMET in a novel high throughput format to pre-select candidates for in vivo studies. In

http://www.kinderd/

http://www.a-paraddise.eu4/



vivo screening in animal disease models including for the most advanced candidates a NHP primate model. As a result of the synergy discussions standardization of criteria for the definition of hit and lead compounds has been achieved, based on published criteria1,2. A number of activities have been carried out. In the present communication we present the shared activities and most important disclosable results including perspectives for results exploitation.

We thank the European commission for the support of the following projects: NMTrypI, KINDReD, A-ParaDDisE and PDE4NPD.



P.28. Repurposing an old anti-arthritis golden drug, auranofin, and its anticancer GoPI-sugar surrogate for the treatment of

human parasitic diseases: from Leishmania to helminth infections

L. Feng,1 S. Pomel,2 P. Loiseau, 2 D. L. Williams,3 E. Davioud-Charvet 1

1 UMR 7509 CNRS University of Strasbourg, European School of Chemistry, Polymers and Materials (ECPM), F-67087 Strasbourg, France; 2 UMR 8076 CNRS

BioCIS, University of Paris-Sud, 92290 Chatenay-Malabry France; 3 Department of Immunology/Microbiology, Rush University Medical Center, IL 60612 Chicago.


Schistosomiasis (also known as bilharzia)—infection with the helminth parasites in the genus Schistosoma—remains an important infection in many tropical areas, especially Africa. More than 200 million people have schistosomiasis, with 20 million exhibiting severe symptoms. The approved drug auranofin (Ridaura®) targets the seleno-dependent Schistosoma worm thioredoxin-glutathione reductase (TGR) and rapidly kill juvenile and adult Schistosoma mansoni in culture at concentrations achievable in patients (5 μM).1 This phosphine-coordinated gold (I) thiosugar complex was initially developed for the treatment of rheumatoid arthritis, a disorder associated to TrxR overexpression, and reported to inhibit the purified thioredoxin reductase (TrxR) from human placenta.2 In our team, several TrxR inhibitors have already been identified and have shown growth-inhibitory properties on tumor cells and parasites. Among those, the phosphole-containing gold complex {1-phenyl-2,5-di(2-pyridyl)phosphole}AuCl (abbreviated as GoPI) is an irreversible inhibitor of both purified human GR and TrxR.3 GoPI-sugar is a novel 1-thio-β-D-glucopyranose 2,3,4,6-tetraacetatoS-derivative, which was designed from the structure of GoPI and auranofin, for an improved stability and bioavailability upon GoPI. These metal-ligand complexes are of particular interest because of their combined abilities to irreversibly target the dithiol/selenol catalytic pair essential for TrxR activity in addition to bind to DNA, and to kill cancers cells from breast and brain tumors.4

S

O

OAc

OAc

OAcAcO

PAu NN

GoPI-sugar

P EtEtEt

AuS

O

OAc

OAc

OAcAcO

Auranofin

Cl

PAu NN

GoPI

Figure 1. Structures of gold (I)-based complexes as human glutathione and thioredoxin

reductases and Schistosoma mansoni thioredoxin-glutathione reductase.

Recently, auranofin has been shown to inhibit the growth of various parasites5: both




the bloodstream and procyclic stages of Trypanosoma brucei, the malarial parasite Plasmodium falciparum, to kill larval worms of Echinococcus granulosus in vitro, and also the promastigote stage of Leishmania infantum. Consequently, screening of parasites to identify the most sensitive organism to GoPI-sugar, compared to auranofin, has been undertaken in our team. Selected data will be presented. 1 Kuntz et al. Thioredoxin glutathione reductase from Schistosoma mansoni: an essential parasite enzyme and a key drug target. PLoS Med 2007, 4, e206. 2 Gromer et al. Human placenta thioredoxin reductase. Isolation of the selenoenzyme, steady state kinetics, and inhibition by therapeutic gold compounds. J Biol Chem. 1998, 273:20096-101. 3 a) Deponte et al., Mechanistic studies on a novel, highly potent gold-phosphole inhibitor of human glutathione reductase. J. Biol. Chem. 2005, 280:20628-37; b) Urig et al., Undressing of phosphine gold(I) complexes as irreversible inhibitors of human disulfide reductases. Angew Chem Int Ed Engl. 2006, 45:1881-6. 4 a) Viry et al., A sugar-modified phosphole gold complex with antiproliferative properties acting as a thioredoxin reductase inhibitor in MCF-7 cells. ChemMedChem 2008, 3:1667-70; b) Jortzik et al., Antiglioma activity of GoPI-sugar, a novel gold(I)-phosphole inhibitor: chemical synthesis, mechanistic studies, and effectiveness in vivo. Biochim Biophys Acta 2014, 1844:1415-26. 5 a) Lobanov, A.V., Gromer, S., Salinas, G. & Gladyshev, V.N. Selenium metabolism in Trypanosoma: characterization of selenoproteomes and identification of a kinetoplastida-specific selenoprotein. Nucleic Acids Res. 2006, 34, 4012–4024; b) Sannella, A.R. et al. New uses for old drugs. Auranofin, a clinically established antiarthritic metallodrug, exhibits potent antimalarial effects in vitro: Mechanistic and pharmacological implications. FEBS Lett. 2008, 582, 844–847; c) Bonilla, M. et al. Platyhelminth mitochondrial and cytosolic redox homeostasis is controlled by a single thioredoxin glutathione reductase and dependent on selenium and glutathione. J. Biol. Chem. 2008, 283, 17898–17907; d) Ilari, A. et al. A gold-containing drug against parasitic polyamine metabolism: the X-ray structure of trypanothione reductase from Leishmania infantum in complex with auranofin reveals a dual mechanism of enzyme inhibition. Amino Acids 2012, 42, 803–811.



LIST OF PARTICIPANTS



Name Institution E-mail address

Alunda J. M. Universidad Complutense de Madrid, Spain [email protected]

André-Barrès C. CNRS-Université Paul Sabatier, France [email protected]

Baltas M. CNRS-Université Paul Sabatier, France [email protected]

Bautista L. Universidad Complutense de Madrid, Spain [email protected]

Bermejo M. Vitakoras Pharma S.L., Spain [email protected]

Biedermann D. The Czech Academy of Science, Czech Republic [email protected]

Boije af Gennäs G. University of Helsinki, Finland [email protected]

Bolás F. Universidad Complutense de Madrid, Spain [email protected]

Botta M. Università di Siena, Italy [email protected]

Caffrey C. R. University of California San Diego, USA [email protected]

Caljon G. University of Antwerp, Belgium [email protected]

Calogeropoulou T. National Hellenic Research Foundation, Greece

[email protected]

Campillo N. E. Centro de Investigaciones Biológicas, CSIC, Spain [email protected]

Castro G. Polytechnic Institute of Porto, Portugal [email protected]

Cordeiro-Da Silva A. I3S, Institute for Molecular and Cella Biology (IBMC), Portugal

[email protected]

Corral M. J. Universidad Complutense de Madrid, Spain [email protected]

Costi M. P. Università degli Studi di Modena e Reggio Emilia, Italy

[email protected]




















Couvreur P. Université Paris-Saclay, France [email protected]

Dardonville C. Medicinal Chemistry Institute, CSIC, Spain [email protected]

Davioud-Charvet E. CNRS - Université de Strasbourg, France [email protected]

De Koning H. P. University of Glasgow, United Kingdom [email protected]

Dea Ayuela M.A. Universidad CEU-Cardenal Herrera, Spain [email protected]

Descoteaux A. INRS-Institut Armand-Frappier, Canada [email protected]

Doligalska M. J. University of Warsaw, Poland [email protected]

Ebiloma G. U. University of Glasgow, United Kingdom [email protected]

Espuelas M. S. Universidad de Navarra, Spain [email protected]

Fernández García R. Universidad Complutense de Madrid, Spain [email protected]

Gamarro F.

Instituto de Parasitología y Biomedicina López-Neyra, CSIC, Spain

[email protected]

Gandhi H. University College Cork, Ireland [email protected]

García-Sosa A.T. University of Tartu, Estonia [email protected]

Gemma S. Università di Siena, Italy [email protected]

Gil C. Centro de Investigaciones Biológicas, CSIC, Spain [email protected]

Gold D. The Hebrew University of Jerusalem, Israel [email protected]

Golenser J. The Hebrew University of Jerusalem, Israel [email protected]

Gomes-Alves A. G. I3S, Institute for Molecular and Cella Biology (IBMC), Portugal

[email protected]





















González Sánchez M. E. Universidad Complutense de Madrid, Spain [email protected]

González-Álvarez I. Vitakoras Pharma S.L., Spain [email protected]

González-Álvarez M. Vitakoras Pharma S.L., Spain [email protected]

Gul S. Fraunhofer IME-SP, Germany [email protected]

Gutiérrez A. Universidad Complutense de Madrid, Spain [email protected]

Harrington J. M. Merial Inc., USA [email protected]

Heby O. Umeå University, Sweden [email protected]

Hendrickx S. University of Antwerp, Belgium [email protected]

Hervás P. Veterindustria, Spain [email protected]

Horn M. The Czech Academy of Sciences, Czech Republic

[email protected]

Jaffe C. The Hebrew University of Jerusalem-Hadassah, Israel

[email protected]

Jiménez-Antón M. D. Universidad Complutense de Madrid, Spain [email protected]

Kivrak A. Yuzuncu Yil University, Turkey [email protected]

Krauth-Siegel R. L. Biochemie-Zentrum der Universität Heidelberg, Germany

[email protected]

Leontovyč A. The Czech Academy of Sciences, Czech Republic

[email protected]

Leurs R. Vrije Universiteit Amsterdam, The Netherlands

[email protected]

Loiseau P. M. Université Paris-Sud, France [email protected]

Lopes F. Universidade de Lisboa, Portugal [email protected]






















López Medrano F. Hospital 12 de Octubre Madrid, Spain [email protected]

Maes L. University of Antwerp, Belgium [email protected]

Mangalagiu I. I. Alexandru Ioan Cuza University of Iaşi, Romania

[email protected]

Maran U. University of Tartu, Estonia [email protected]

Martins R. Polytechnic Institute of Porto, Portugal [email protected]

Mukherjee B. University of Geneva, Switzerland [email protected]

Natto M. J. University of Glasgow, United Kingdom [email protected]

Olías-Molero A. I. Universidad Complutense de Madrid, Spain [email protected]

Opperdoes F. De Duve Institute, Belgium [email protected]

Otero-Espinar F. J. Universidad de Santiago de Compostela, Spain [email protected]

Palmieri N. Instiute of Parasitology-Vetmeduni Vienna, Austria

[email protected]

Pereira S. I. Polytechnic Institute of Porto, Portugal [email protected]

Peric M. University of Zagreb, Croatia [email protected]

Persson L. Lund University, Sweden [email protected]

Peterlin Mašič L. University of Ljubljana, Slovenia [email protected]

Pfister K. LMU München, Germany [email protected]

Pomel S. Université Paris-Sud, France [email protected]

Quirynen L. M. M. Janssen Pharmaceutica, Belgium [email protected]






















Roussis V. National and Kapodistrian University of Athens, Greece

[email protected]

Sarlauskas J. Vilnius University, Lithuania [email protected]

Schmidt T. J. Universität Münster, Germany [email protected]

Sebastián V. Centro de Investigaciones Biológicas, CSIC, Spain [email protected]

Seifert K. London School of hygiene & tropical medicine, United Kingdom

[email protected]

Selzer P. M. Boehringer Ingelheim Animal Health, Germany

[email protected]

Serrano D. R. Universidad Complutense de Madrid, Spain [email protected]

Šolmajer T. National Institute of Chemistry, Slovenia [email protected]

Stevanović S. Center for Multidisciplinary Science Vinča, Serbia

[email protected]

Tomás A. M. I3S, Institute for Molecular and Cella Biology (IBMC), Portugal

[email protected]

Torrado J. J. Universidad Complutense de Madrid, Spain [email protected]

Ucisik M. H. Istanbul Medipol University, Turkey [email protected]

Valladares B. Universidad de las Palmas, Spain [email protected]

Vasilache V. Alexandru Ioan Cuza University of Iaşi, Romania

[email protected]

Vivancos V. Universidad Miguel Hernández de Elche, Spain

[email protected]


















NOTES

3rd COST ACTION CM1307 CONFERENCE ● SOCEPA ● SEFIG

3rd cost action cm1307 conference socepa sefig · 3rd cost action cm1307 conference socepa sefig,...

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