PAULROMMER

SL

Ancona, 20-21 Febbraio 2020

XIX Workshop on Pharmacobiometallics

BOOK OF ABSTRACTS

Dip. di Scienze della Vita e dell'Ambiente Università Politecnica delle Marche Via Brecce Bianche - 60131 Ancona

Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici Via Celso Ulpiani, 27 - 70126 Bari

Comitato Scientifico Prof. Francesco Paolo Fanizzi, UniSalento Prof. Nunzio Denora, UniBA Prof. Giuseppe Falini, UniBO Prof. Alfredo Burini, UniCAM Prof. Graziella Vecchio, UniCT Prof. Lorenza Marvelli, UniFE Prof. Luigi Messori,UniFI Prof. Stefano Banfi, UniInsubria Prof. Luigi Monsù Scolaro, UniME Prof. Diego Tesauro, UniNA Prof. Claudia Pellerito, UniPA Prof. Diego La Mendola, UniPI Prof. Lisa Dalla Via, UniPD Prof. Mauro Ravera, UniPO Prof. Giorgio Pelosi, UniPR Prof. Piersandro Pallavicini, UniPV Prof. Elisabetta Giorgini, UnivPM Prof. Maria Pia Donzello, UniROMA1 Prof. Massimiliano Coletta, UniROMA2 Prof. Fabrizia Fabrizi De Biani, UniSI Prof. Walter Dastru’, UniTO Prof. Silvano Geremia, UniTS

Comitato Organizzatore Elisabetta Giorgini, UnivPM Anna Annibaldi, UnivPM Paola Astolfi, UnivPM Patricia Carloni, UnivPM Carla Conti, UnivPM Roberta Galeazzi, UnivPM Emiliano Laudadio, UnivPM Giovanna Mobbili, UnivPM Valentina Notarstefano, UnivPM Michela Pisani, UnivPM Samuele Rinaldi, UnivPM Simona Sabbatini, UnivPM Pierluigi Stipa, UnivPM

INVITED SPEAKER

Highlights on biomedical and chemical activities and experiments at Elettra – Sincrotrone Trieste Birarda Giovanni,1 Lisa Vaccari,1

1 Elettra Sincrotrone Trieste Strada Statale 14 - km 163,5 in AREA Science Park 34149 Basovizza, Trieste ITALY

Corresponding Author, giovanni.birarda@elettra.eu Elettra Synchrotron is a 3rd generation light source, open to the international user community since 1993, completely upgraded in 2009. The light generated at Elettra, orders of magnitude brighter that the conventional sources, enables to conduct state-of-the-art experiments in physics, material science, chemistry, biology, life sciences, environmental science, medicine, forensic science, and cultural heritage. Currently, 28 beamlines are operating at Elettra, allowing for investigation with all the most important optical techiniques: spectroscopy, spectromicroscopy, imaging, diffraction and scattering. The present contribution will be divided in three parts: one quick introduction to Elettra and its capabilities, and a second short part regarding the activities of my colleagues in the field of Chemical and Biochemical Sciences. The third part will focus more on the scientific accomplishments of the SISSI (Synchrotron Infrared Source for Spectroscopy and Imaging) beamline, the infrared laboratory of Elettra.1 Fourier Transform InfraRed (FTIR) spectroscopy and spectromicroscopy is a label free characterization tool well known for the molecular investigation of chemical samples.2 In the last 30 years, FTIR demonstrated to be a powerfull method for the analysis of biological samples, i.e. cells and tissues.3 In particular, due to the non-damaging nature of IR radiation used in the analyses, it is possible to study even living systems, such as cells, without perturbing them with the probing radiation.4–7 When tissue slices are inspected, using the imaging approach, a massive amount of data can be generated by a single study. In this case, statistical and multivariate approaches are applied to the acquired datasets in order to highlight the, sometimes subtle, differencies within the inspected groups of samples, for example diseased and healthy or controls and treated etc.8 With this approach it is then possible to build predictive models that can be applied to the new aquired data to classify them.

Figure 1. Aerial view of Elettra

Synchrotron and FERMI Free Electron Laser Facilities.

Figure 2. Layout of the three endstations at

SISSI beamline @ Elettra.

1

Thanks to recent technical and scientific improvements it was also possible to circumvent the far-field diffraction barrier of IR microspectroscopy and to reach the spatial resolution down to the nanometer scale. The combination of near-field microscopes with IR and THz sources offers, now, unprecedented opportunities for sample characterization, widening the perspectives in a multitude of fields of sciences by confining vibro-electronic information within few tens of nanometers. Last year a new Near Field Endstation was installed at SISSI beamline and during that period several test measurements have been done on manifold of different samples, some example with be shown in the conclusion of the presentation.9 The introduction of the nano-FTIR enstation allow also to fill a resolution gap that was present between infrared and x-ray based characterization techniques, multiplying the collaboration opportunities between the beamlines, and therefore users, of Elettra in all the fields of science.

References1. Lupi, S. et al. Performance of SISSI, the infrared beamline of the ELETTRA storage ring.

J. Opt. Soc. Am. B 24, 959 (2007). 2. French, R. S. & Online, S. A. History of Infrared Spectroscopy. Encycl. Biophys. 988–988

(2013). doi:10.1007/978-3-642-16712-6_100429 3. Baker, M. J. et al. Using Fourier transform IR spectroscopy to analyze biological

materials. Nat. Protoc. 9, 1771–1791 (2014). 4. Grenci, G. et al. Optimization of microfluidic systems for IRMS long term measurement

of living cells. Microelectron. Eng. 98, 698–702 (2012). 5. Birarda, G. et al. IR-Live: fabrication of a low-cost plastic microfluidic device for infrared

spectromicroscopy of living cells. Lab Chip 16, 1644–1651 (2016). 6. Mitri, E. et al. Time-Resolved FT-IR Microspectroscopy of Protein Aggregation Induced

by Heat-Shock in Live Cells. Anal. Chem. 87, 3670–3677 (2015). 7. Vaccari, L., Birarda, G., Businaro, L., Pacor, S. & Grenci, G. InfraRed

MicroSpectroscopy of living cells in Microfluidic Devices (MD-IRMS): toward a powerful label-free cell-based assay. Anal. Chem. (2012).

8. Toplak, M. et al. Infrared Orange: Connecting Hyperspectral Data with Machine Learning. Synchrotron Radiat. News 30, (2017).

9. Birarda, G. et al. Multi-technique microscopy investigation on bacterial biofilm matrices: a study on Klebsiella pneumoniae clinical strains. Anal. Bioanal. Chem. 411, 7315–7325 (2019).

Figure 3. New nano-FTIR capabilities of SISSI

beamline @ Elettra.

2

ORAL COMMUNICATIONS

Nanoformulations of Pt(IV) prodrugs for cancer therapy

Rosa Maria Iacobazzi,1 Ilaria Arduino,

2 Amalia Azzariti,

1 Letizia Porcelli,

1 Simona Serratì,

1

Antonio Lopalco,2 Valentino Laquintana,

2 Annalisa Cutrignelli,

2 Angela Assunta Lopedota,

2

Nicola Margiotta,3 Massimo Franco,

2 Nunzio Denora.

2

1 IRCCS Istituto Tumori Giovanni Paolo II di Bari, Italia 2 Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari, Italia 3 Dipartimento di Chimica. Università degli Studi di Bari, Italia

Rosa Maria Iacobazzi, e-mail: rosamaria.iacobazzi@gmail.com

Kiteplatin, a Pt(II)-based drug analogous of cisplatin, has been extensively studied in recent years

especially for its efficacy in cisplatin-resistant (ovarian cancer) or oxaliplatin-resistant (colon cancer) cell

lines [1]. In order to reduce systemic toxicity and increase the anti-tumor activity of kiteplatin, a drug

delivery strategy suitable for exploiting both

selective tumor targeting and increase of

systemic circulation time of the drugs, was

explored. Therefore, four Pt(IV) prodrugs

derived from kiteplatin with different

lipophilicity were synthesized and

encapsulated in PLGA-PEG polymer micelles

[2], showing a greater anticancer effect and a

marked reduced toxicity compared to

kiteplatin in vivo, in the Lewis lung

carcinoma (LLC) syngeneic murine model.

With the aim to further increase the

encapsulation efficiency of these Pt(IV)

prodrugs, solid lipid nanoparticles (SLN) are

currently being explored for their loading as

well as for a targeted delivery to the brain.

SLN spherical nanoparticles are composed of

biodegradable and biocompatible excipients and are produced from solid lipids with melting points higher

than body temperature, remaining in a solid state after administration. The SLN containing Pt(IV)

prodrugs were prepared by an hot homogenization technique using cetyl palmitate as lipid matrix and

PEG-modified phospholipids in order to obtain an anti-opsonization coating on SLN surface and to confer

stealth properties. These nanoformulations proved to be stable in aqueous medium, with an average

hydrodynamic diameter <100 nm, a polydispersity index <0.2, a zeta potential between −20 and −25 mV

and a drug encapsulation efficiency in the range of 30-40% (compared to 5.8% obtained with the PLGA-

PEG micelles). In addition, preliminary in vitro cytotoxicity and intracellular uptake studies were

conducted on a human glioblastoma cell model (U87 cells) with promising results.

Riferimenti

[1] Margiotta N, et al. Revisiting [PtCl₂(cis-1,4-DACH)]: an underestimated antitumor drug with potential

application to the treatment of oxaliplatin-refractory colorectal cancer. J. Med. Chem. 2012; 55: 7182-7192.

[2] Margiotta N, et al., Encapsulation of lipophilic kiteplatin Pt(IV) prodrugs in PLGA-PEG micelles. Dalton

Trans. 2016; 45: 13070-13081

Figure 1. Nanoparticles loaded with Pt(IV)

prodrugs of kiteplatin.

3

Design of Platinum-loaded, Selenium-doped Hydroxyapatite Nanoparticles for Potential Application in Bone Tumor Therapy Alessandra Barbanente,1 Robin A. Nadar,2 Lorenzo Degli Esposti3, Barbara Palazzo4, Michele Iafisco3, Jeroen J. J. P. van den Beucken2, Sander C. G. Leeuwenburgh2, Nicola Margiotta1.

1 Dipartimento di Chimica, Università degli Studi di Bari Aldo Moro, Via E.Orabona 4, 70125 Bari, Italy. 2 Department of Dentistry-Regenerative Biomaterials, Radboud university medical center, Philips van Leydenlaan 25, 6525 EX Nijmegen, the Netherlands. 3 Institute of Science and Technology for Ceramics (ISTEC), Via Granarolo 64, 48018 Faenza, Italy. 4Ghimas S.p.A, c/o Distretto Tecnologico High Tech Scarl, Via per Monteroni, 73100 Lecce.

Corresponding Author, e-mail: alessandrabarbanente@libero.it

Bone is a common site for metastases derived from non-osseous tumors with high morbidity. Bone metastases cause uncontrolled bone formation or resorption. Since systemic antitumor chemotherapy can lead to severe side-effects such as nephrotoxicity and neurotoxicity, a strategy to overcome these drawbacks consists in the delivery of cytostatic drugs from locally implanted bone substitute materials. Among bone substitute materials, hydroxyapatite (HA) is well known for its biocompatibility and capability to load a wide variety of therapeutic agents. In this work, we focused on the incorporation of SeO3

2− ions into HA nanocrystals due to their ability to kill cancer cells by a combination of caspase-dependent apoptosis and generation of reactive oxygen species [1]. Usually, the delivery of a single chemotherapeutic drug is insufficient to efficiently kill cancer cells due to tumor heterogeneity. A combination therapy with two or more drugs could offer the opportunity to overcome drug resistance and the lack of tumor specificity. Since Pt-compounds are well known antitumor drugs (i.e. cisplatin, carboplatin, and oxaliplatin), we have designed platinum-loaded, selenium-doped hydroxyapatite nanoparticles. A series of Se-doped HA nanoparticles with different Se concentration has been synthesized and characterized and then loaded with [Pt(dihydrogenpyrophosphate)(cis-1,4-DACH)] (DACH = diaminocyclohexane)[2]. This Pt(II)-pyrophosphate derivative is an analog of [Pt(dihydrogenpyrophosphate)(1R,2R-DACH)], a compound currently under Phase I clinical trials in Texas [3]. The chemotherapeutic activity of the platinum-loaded, selenium-doped hydroxyapatite nanoparticles has been tested in vitro against human prostate or breast cancer cells co-cultured with human mesenchymal stem cells. Our novel nanoparticles demonstrated high anti-cancer selectivity against the tumor cells, which offers ample opportunities for the design of novel biomaterials with potent and selective chemotherapeutic efficacy.

References[1] Yifan Wang et al., ACS Nano, 2016, 22, 9927–9937. [2] A. Barbanente et al. 2020, submitted for publication. [3] www.phosplatin.com

Figure 1. Schematic representation for synthesis of HASe nanoparticles and Pt loaded HASe nanoparticles.

4

Structural Insight and Cytotoxicity of Resveratrol loaded into Hexagonal MgAl Layered Double Hydroxide/BSA Nanocomposites

Cristina Minnelli,1 Emiliano Laudadio,2 Giovanna Mobbili,1 Roberta Galeazzi,1 Mattia Cantarini, 1 Gianni Barucca, 2 Valentina Notarstefano,1 Tatiana Armeni.3

1 Dipartimento di Scienze della Vita e dell’Ambiente (DISVA), Università Politecnica delle Marche, via Brecce Bianche, 60131 Ancona, Italy 2 Dipartimento di Scienze e Ingegneria della Materia, dell’Ambiente e Urbanistica (SIMAU), Università Politecnica delle Marche, via Brecce Bianche, 60131 Ancona, Italy. 3 Dipartimento Scienze Cliniche Specialistiche ed Odontostomatologiche, Università Politecnica delle Marche, via Brecce Bianche, 60131 Ancona, Italy

Corresponding Author, c.minnelli@staff.univpm.it Resveratrol (RES) is a stilbenoid polyphenol found in many plants including grapes and berries. RES biological properties, included antitumor activity, can be compromised by its poor solubility and bioavailability [1]. Nanotechnology-based delivery system can be used to improve RES therapeutic effectiveness. [2] In the present study, Layered Double Hydroxide (LDH), composed of cationic metal atom inner layers, sandwiched between the two intercalated anionic hydroxide layers, were employed to encapsulate RES in order to overcome the above-mentioned usage limits. To evaluate the feasibility of neutral RES complexation with cationic LDH and predict the structure and stability of the supramolecular assembly, we carried out molecular dynamics simulations. The colloidal stability of LDH-RES in physiological environment was reached by coating LDH with Bovine Serum Albumin (BSA). The structural characteristics of the produced nanocomposites were determined by X-ray powder diffraction (XRPD), transmission electron microscopy (TEM), dynamic light scattering (DLS), and attenuated total reflection Fourier transform infrared spectroscopy (ATR–FTIR). The encapsulation efficiency and in vitro RES release, both in the presence and absence of BSA, were also studied. The potential anticancer ability of the LDH and LDH-BSA was evaluated in human lung adenocarcinoma epithelial cell line (A549) resulting in higher activity with respect to bare RES. Overall, the results showed that the nanocomposites are suitable for biomedical applications as delivery agents of RES and that the BSA is an essential component to obtain a stable and efficient LDH-based drug delivery system. References

[1] Amri A, Chaumeil JC, Sfar S, Charrueau CJ. Administration of resveratrol: What formulation solutions to bioavailability limitations? Control. Release, 2012, 158, 182–193.

[2] Intagliata S, Modica MN, Santagati LM, Montenegro L. Strategies to Improve Resveratrol Systemic and Topical Bioavailability: An Update. Antioxidants, 2019, 8, 244.

Figure 1. (A) In silico model of layered double hydroxides (LDH) used in this work and (B)

representation of BSA-coated LDH nanocomposite.

5

In vitro label-free screening of furanodiene delivery and cellular effects: new insights from FTIR and Raman microspectroscopy Valentina Notarstefano,1 Elisabetta Giorgini,1 Marco Parlapiano,2 Lisa Vaccari,3 Massimo Bramucci,4 Luana Quassinti,4 Michela Pisani,2 Paola Astolfi2

1 Department of Life and Environmental Sciences, Università Politecnica delle Marche, Ancona 2 Department of Materials, Environmental Sciences and Urban Planning, Università Politecnica delle Marche, Ancona 3 Elettra Sincrotrone Trieste, SISSI Beamline, Trieste 4 School of Pharmacy, University of Camerino, Camerino

Corresponding Author, e-mail: p.astolfi@univpm.it Furanodiene, a sesquiterpene isolated from Rhizoma curcumae, has an anti-cancer activity in vitro and in vivo, exhibiting anti-proliferation effects on breast, liver and lung cancer cells, anti-angiogenic effects, and synergistic therapeutic effects with many chemotherapeutic agents [1]. Despite the reported evidences of the effective anti-cancer activity of furanodiene, the underlying mechanisms still need to be fully investigated. In this study, the cytotoxic effect of furanodiene (FD) was assessed on the immortalized breast cancer cell line MDA-MB 231. FD was encapsulated in glycerol monooleate (GMO), widely applied for the formation of various liquid crystalline drug formulations, given its nontoxic, biodegradable and biocompatible characteristics [3]. Cells were treated with FD, GMO and the GMO+FD system for 24 and 48 hours, and analyzed, by Fourier Transform InfraRed Microspectroscopy (FTIRM) and Raman MicroSpectroscopy (RMS), with the aim to highlight meaningful spectroscopic signatures attributable to the alterations in the composition and structure of cellular proteins, lipids and nucleic acids. The multivariate (PCA-LDA) and univariate (ANOVA) statistical procedures revealed different effects due to GMO and FD: the comparisons of FD, GMO and GMO+FD groups let discriminate between the real effects of FD and the non-cytotoxic alterations of lipid component induced by GMO. Both FTIRM and Raman let highlight changes mostly affecting lipids and nucleic acids, suggesting the impairment of both metabolism and cell cycle of cells. The treatment exhibited a time-dependent cellular response. RMS, given its high spatial resolution, let evidence the mechanism of GMO internalization/extrusion (Figure 1). In the present study, the vibrational analysis by means of FTRIM and RMS shed new light on the in vitro effects of FD cancer cells, confirming some of the known mechanisms of action of FD. Furthermore, the vibrational approach let evaluate the effectiveness of the use of FD encapsulated in GMO for drug delivery use. Riferimenti

[1] Zhong ZF, Yu HB, Wang CM, Qiang WA, Wang SP, Zhang JM, Yu H, Cui L, Wu T, Li DQ, et al. Furanodiene Induces Extrinsic and Intrinsic Apoptosis in Doxorubicin-Resistant MCF-7 Breast Cancer Cells via NF-κB-Independent Mechanism. Front Pharmacol 2017;8:1–12.

[2] Milak S, Zimmer A. Glycerol monooleate liquid crystalline phases used in drug delivery systems. I J Pharm 2015;478:569–587.

Figure 1. Typical confocal microscope image of a GMO-treated cell. Raman map recorded on the cell nuclear area. Spectra from different cellular

regions and on a GMO vesicle.

6

Structural and functional insights of nitrosoglutathione reductase from the green microalga Chlamydomonas reinhardtii Simona Fermani1, Andrea Tagliani2, Jacopo Rossi2, Marcello De Mia3, Christophe H. Marchand3,4, Daniele Tedesco2, Gurrieri Libero2, Maria Meloni2, Di Giacinto Nastasia2, Giuseppe Falini1, Paolo Trost2, Stéphane D. Lemaire3, Mirko Zaffagnini2.

1Dipartimento di Chimica “G. Ciamician”, Università di Bologna, Bologna, Italia. 2 Dipartimento di Farmacia e Biotecnologie, FaBiT, Università di Bologna, Bologna, Italia. 3Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, UMR8226 Centre National de la Recherche Scientifique, Sorbonne Université, Institut de Biologie Physico-Chimique, Paris, France. 4Plateforme de Protéomique Institut de Biologie Physico-Chimique, FRC550, CNRS, Paris, France.

Corresponding Author, e-mail: simona.fermani@unibo.it Protein S-nitrosylation plays a fundamental role in plant cell signaling and nitrosoglutathione (GSNO) is considered the major nitrosylating agent [1]. The enzymatic systems controlling GSNO homeostasis are thus crucial to indirectly control the formation of protein S-nitrosothiols. The intracellular level of GSNO is primarily controlled by nitrosoglutathione reductase (GSNOR) that catabolizes GSNO in the presence of NADH [2]. The genome of the microalga Chlamydomonas reinhardtii shows two genes encoding for almost identical GSNOR enzymes. By focusing the attention on gsnor1, we heterologously expressed CrGSNOR1 and determined its kinetic and structural features. These analyses revealed that CrGSNOR1 has a strict specificity towards GSNO and NADH and its three-dimensional crystal structure in both apo and holo (NAD+-CrGSNOR1) forms was determined at a resolution of 1.8 and 2.3 Å, respectively. The enzyme overall folding and the coordination of the structural zinc ion appeared similar to other structurally known GSNORs, while the coordination of the catalytic zinc differs especially in the apo form (Fig. 1). Despite being a Cys-rich protein (15 Cys), CrGSNOR1 only contains two accessible cysteines as determined from the three-dimensional structure and by thiol titration. Unlike Arabidopsis GSNOR, the activity of CrGSNOR1 remained almost unaffected following thiol-based oxidative treatments such as H2O2-dependent oxidation and S-nitrosylation. Based on these findings, we provide functional and structural insights into the response of CrGSNOR1 to cysteine-based modifications. References[1] Airaki, M., Sanchez-Moreno, L., Leterrier, M., Barroso, J. B., Palma, J. M., and Corpas, F. J. (2011) Detection

and quantification of S-nitrosoglutathione (GSNO) in pepper (Capsicum annuum L.) plant organs by LC-ES/MS, Plant Cell Physiol 52, 2006-2015.

[2] Liu, L., Hausladen, A., Zeng, M., Que, L., Heitman, J., and Stamler, J. S. (2001) A metabolic enzyme for S-nitrosothiol conserved from bacteria to humans, Nature 410, 490-494.

Figure 1. 3D crystal structure of apo- CrGSNOR1 from from the microalga

Chlamydomonas reinhardtii

Structural Zn

CatalyticZn

7

Amylin interaction with metal ions: a potential link between type 2 diabetes and Alzheimer disease Antonio Magrì,1 Giovanni Tabbì,1 Francesco Attanasio,1 Giuseppe Di Natale,1 Diego La

Mendola,2 Enrico Rizzarelli1,3 1 Institute of Crystallography, National Research Council (CNR), S.S. Catania, Via P. Gaifami 18, 95126 Catania, Italy 2 Department of Pharmacy, University of Pisa, Via Bonanno Pisano 6, 56126 Pisa, Italy 3 Department of Chemical Sciences, University of Pisa, Via Bonanno Pisano 6, 56126 Pisa, Italy

Corresponding Author, e-mail: lamendola@farm.unipi.it Amylin, also known as islet amyloid polypeptide (IAPP), is a 37-residue peptide hormone produced by the islet β-cells of pancreas. The formation of amylin aggregates is strongly associated with beta-cell degeneration in type 2 diabetes, as demonstrated by more than 95% of patients exhibiting amylin amyloid upon autopsy. Amylin binds to the amylin receptor in the brain and mediates several important brain functions, including regulating glucose metabolism and modulating inflammatory reactions. Noteworthy, plasma amylin concentration was associated with Alzheimer’s disease (AD) incidence. Amylin aggregations are also found colocalized with those formed by amyloid-β peptide (Aβ), a major pathological factor of the AD. Both amylin and amyloid-β are intrinsically disordered proteins (IDPs) that is the absence of a rigid 3D structures. A common conformational variation of these IDPs is the misfolding and the consequent aggregation so to form amyloid plaques. Metal ions play an important role in the aggregation processes of these IDPs and metals dyshomeostasis may be a prominent factor in the pathological development of neurodegenerative diseases. It is widely recognized that metal ions such as copper(II) and zinc(II) have been implicated in the aggregation process of amyloid-β and also of amylin.1,2 Here we reported our experimental studies (thermodynamic and spectroscopic) on the interaction between Cu(II) and Zn(II) with several isoforms of amylin (human, porcine, murine) and their fragments.

References

[1] Magrì A, La Mendola D, Nicoletti VG, Pappalardo G, Rizzarelli E, New Insight in Copper-Ion Binding to Human Islet Amyloid: The Contribution of Metal-Complex Speciation To Reveal the Polypeptide Toxicity. Chem. Eur. J., 2016, 22, 13287–13300. [2] Magrì A, Pietropaolo A, Tabbì G, La Mendola D, Rizzarelli E, From Peptide Fragments to Whole Protein: Copper(II) Load and Coordination Features of IAPP. Chem. Eur. J., 2017, 23, 17898-17902.

8

Carnosine and trehalose-carnosine tunes the activity and expression of

endogenous protection factors and their crosstalk with metal

homeostasis

Francesco Attanasio1, Irina Naletova

2, Valentina Greco

2, Sebastiano Sciuto

2, Enrico

Rizzarelli 1,2

1CNR- Istituto di Cristallografia, Via Paolo Gaifami 18, 95126 Catania, Italy Affiliation of other authors

2Dipartimento di Scienze Chimiche, Università di Catania, Viale A. Doria 6, 95125 Catania, Italy

Enrico Rizzarelli, email: erizza@unict.it

Carnosine (β-alanyl-L-histidine) is a natural dipeptide widely distributed in mammalian tissues

and presented at high concentrations (0.7–2.0mM) in the brain1. As reported previously,

carnosine augmented the secretion and expression of various neurotrophic factors (for example,

BDNF), leading to the induction of neurite growth in SY-SY5Y cells2. Moreover, carnosine

glial release and neuronal utilization in CNS have been described3; carnosine intercepts the

regulatory routes of Cu homeostasis in nervous cells and tissues. Barca et al showed that the

extracellular carnosine exposure influenced intracellular Cu entry and affected the key Cu-

sensing system (SP1 and CTR1)4. On this basis, carnosine, its derivate with trehalose and

potential role of copper ions were investigated in the present study. First of all, we demonstrate

that trehalose-carnosine crosses the cell membrane better than carnosine and its translocation

does not depend on copper ions. On the next step, we analyzed a role of carnosine and its

derivative in the modulation of CREB functions in the normal and in the copper ions deprivation

conditions. Previously, it has been shown that carnosine and copper alone induce CREB

phosphorylation5,6

. Here we found that in culture of PC12 cells trehalose-carnosine stimulates

CREB phosphorylation more than carnosine alone and the level of phospho-CREB depends on

the presence of copper ions in the medium. Moreover, we found that CREB phosphorilation

induce BDNF and VEGF expression.

To compare the influence of trehalose-carnosine and carnosine alone on copper homeostasis, a

measure of the copper transporter CTR1 and transcriptional factor SP1 expression in culture of

PC12 cells was carried out.

Riferimenti

[1] Hipkiss AR et al. Ann. N. Y. Acad. Sci. (1998) 20;854:37-53.

[2] Kadooka K et al. J of Functional Foods (2015) 13;32-37.

[3] Bauer K, Neurochem Res. (2005) 30(10):1339-45.

[4] Barca A et al. Am J Physiol Cell Physiol. (2019) 316(2):C235-C245.

[5] Fujii K et al. Cytotechnology (2017) 69(3):523-527.

[6] Naletova I et al (2019) Cells. (2019) 8(4). pii: E301

9

MS studies of protein metalation: an overview of the last results Alessandro Pratesi,1 Carlotta Zoppi,2 Lara Massai, 2 Chiara Gabbiani, 1 Luigi Messori2

1 Department of Chemistry and Industrial Chemistry, via Giuseppe Moruzzi 13, 56124, Pisa, Italy 2 Laboratory of Metals in Medicine (MetMed), Department of Chemistry “U. Schiff”, University of Florence, via della Lastruccia 3, 50019 Sesto Fiorentino, Italy

Corresponding Author, e-mail: alessandro.pratesi@unipi.it Metal-based drugs have a prominent place in medicine as they are extensively used to treat a wide range of diseases and, in particular, cancer. The broad portfolio of new metal-based therapies progressing through clinical trials demonstrates the potential for new metal-containing compounds in cancer therapy [1]. Cisplatin is still the most widely applied anticancer metallodrug, showing pronounced efficacy most notably in testicular, head-and-neck and ovarian cancer treatment. However, during the last decade has been proved that only 1–10% of cisplatin accumulated in the cell is able to reach nuclear DNA [2]. Probably, this fact is causally linked to the severe side effects seen in CDDP treated patients, inducing the scientific community to deeply investigate the fate of the remaining 90% of cisplatin. In this view, the serum proteins turned out as “alternative targets” leading to a growing interest for the study of these protein metalation processes, their biological implications and lastly for the structure optimization of anticancer metallodrugs. High-resolution mass spectrometry has emerged as an important tool for studying the interaction of anticancer metallodrugs with biomolecules and potential targets on a molecular level. MS provides a wealth of structural and functional information mainly due to its non-destructive nature that even preserves non-covalent interactions and making MS one of the most effective tools of modern metallomics [3]. In the recent years, many efforts have been spent in this way, in order to investigate the protein interactions and better elucidate the mode-of-action not only for the known Pt-based metallodrugs, but also for Auranofin and its analogues as well as for other new metal complexes with potential anticancer properties. Riferimenti [1] Boros E., Dyson P. J., Gasser G., Classification of Metal-Based Drugs according to Their Mechanisms of

Action. Chem. 2020; 6: 41–60. [2] Legin A. A., Schintlmeister A., Jakupec M. A., Galanski M., Lichtscheidl I., Wagner M., Keppler B. K.,

NanoSIMS combined with fluorescence microscopy as a tool for subcellular imaging of isotopically labeled platinum-based anticancer drugs. Chem. Sci. 2014; 5: 3135-3143.

[3] Pratesi A., Cirri D., Fregona D. Ferraro G., Giorgio A., Merlino A., Messori L., Structural Characterization of a Gold/Serum Albumin Complex. Inorg. Chem. 2019; 58: 10616−10619.

[4] Pratesi A., Cirri D., Ciofi L., Messori L., Reactions of Auranofin and Its Pseudohalide Derivatives with Serum Albumin Investigated through ESI-Q-TOF MS. Inorg. Chem. 2018; 57: 10507−10510.

10

Structural and Functional Properties of Hemoglobin complexed with Haptoglobin

Massimiliano Coletta,1 Chiara Ciaccio,1 Alessandra Di Masi,2 Giovanna De Simone,2 Grazia R. Tundo,1 Magda Gioia,1 Fabio Polticelli,2 Paolo Ascenzi2

1Department of Clinical Sciences and Translational Medicine, University of Roma Tor Vergata, Roma 2Department of Sciences, University of Roma Tre, Roma.

Corresponding Author, coletta@med.uniroma2.it Haptoglobin (Hp) belongs to the family of acute-phase proteins, is mainly expressed in the liver and its synthesis is induced by several cytokines during inflammatory processes. Hp acts as a potent immunosuppressor of lymphocyte function and modulates the helper T-cell type 1 and type 2 balance within the body. Hp is the primary hemoglobin (Hb)-binding protein in human plasma. The release of Hb into plasma is a physiological phenomenon, occurring during the hemolysis of senescent erythrocytes and the enucleation of erythroblasts, with potentially severe consequences for health. Any free Hb released from red cell breakdown stably complexes with Hp and is then cleared by the reticuloendothelial system through the CD163 receptor-mediated endocytosis in macrophages, Kupffer cells, and hepatocytes. By acting as a clearance protein, Hp removes the oxidative potential of the iron contained in the Hb molecule. Hp:Hb complexes display heme-based reactivity; in fact they bind several ligands, including O2, CO, and NO and play a role in NO and peroxynitrite scavenging. Beside their anti-inflammatory role, clearing free Hb in the plasma, these complexes are very important macromolecular models, since Hp only binds Hb dimers; therefore, Hp:Hb complexes are the only opportunity to investigate the functional properties of pure Hb α1β1 (or α2β2) dimers without the interference of the tetrameric species. As a whole, this comprehensive structural-functional study has allowed to establish fundamental properties of the Hb dimers, namely (i) in the Hp:Hb complex the structural arrangement of α1β1 (or α2β2) dimers is essentially superimposable to that of these dimeric units in the R-state tetramer, (ii) in the heme Fe(II) species dimers behave exactly as tetramers in the R-state, (iii) in the heme Fe(III) species dimers display an assembly-dependent behaviour, connected to the coordination of H2O with the heme Fe(III). It suggests that while the assembly of isolated α and β subunits in α1β1 (or α2β2) dimers does not alter their tertiary structure, assembly of dimers to tetramers brings about a conformational change which mainly affects the strength of coordination of H2O molecules with the heme Fe(III) atom.

11

Peptide-based soft hydrogels modified with gadolinium complexes as

MRI contrast agents

Carlo Diaferia,1 Enrico Gallo,2 Giancarlo Morelli, Enza Di Gregorio,3 Eliana Gianolio,3

Antonella Accardo1 1 Department of Pharmacy and Interuniversity Research Centre on Bioactive Peptides (CIRPeB),

University of Naples “Federico II”, via Mezzocannone 16, 80134 Naples, Italy. 2 IRCCS SDN, Via E. Gianturco 113, 80143, Napoli, Italy 3 Department of Molecular Biotechnology and Health Science, University of Turin, Via Nizza 52, 10125

Turin, Italy.

Corresponding Author, carlo.diaferia@unina.it

Poly-aromatic peptide sequences are able to self-

assemble into a variety of supramolecular

aggregates such as fibers, hydrogels and tree-like

multi-branched nanostructures.[1] Due to their

biocompatible nature, these peptide nanostructures

have been proposed for several applications in

biology and nanomedicine (tissue engineering,

drug delivery, bioimaging and fabrication of

biosensors).[2] Here we describe the synthesis, the

structural characterization and the relaxometric

behaviour of two novel supramolecular diagnostic

agents for potential applications in magnetic

resonance imaging (MRI). These tools are

obtained for self-assembly of DTPA(Gd)-PEG8-

(FY)3 or DOTA(Gd)-PEG8-(FY)3 peptide

conjugates, in which the Gd-complexes are linked

at the N-terminus of the PEG8-(FY)3 polymer

peptide. This latter was previously found able to form self-supporting and stable soft hydrogels

at a concentration of 1.0 %wt.[3] Analogously, also DTPA(Gd)-PEG8-(FY)3 and DOTA(Gd)-

PEG8-(FY)3 exhibit the trend to gelificate at the same range of concentration. Moreover, the

structural characterization points out that peptide (FY)3 moiety keeps its capability to arrange

into β-sheet structures with an antiparallel orientation of the β-strands. The high relaxivity value

of these nanostructures (~ 12 mM-1·s-1 at 20 MHz) and the very low in vitro cytotoxicity

suggest a potential application as supramolecular diagnostic agents for MRI.

Riferimenti

[1] Ulijn RV, Smith AM, Designing peptide based nanomaterials. Chem. Soc. Rev. 2008; 37: 664-675.

[2] Lampel A, Ulijn RV, Tuttle T, Guiding principles for peptide nanotechnology through directed discovery.

Chem.Soc.Rev. 2018; 47: 3737-3758.

[3] Diaferia C, Balasco N, Sibillano T, Goush M, Adler-Abramovich L, Giannini C, Vitagliano L, Morelli G,

Accardo A, Amyloid‐Like fibrillary morphology originated by tyrosine‐containing aromatic hexapeptides.

Chem-Eur. J. 2018; 7(24): 6804-6817.

Figura 1. Self-assembling peptides,

chemically functionalized with Gd(III)-

complexes, can serve as structural elements

for generate hydrogels for application in

Magnetic Resonance Imaging (MRI)

12

Mn(II)-based lipidic nanovesicles as highly efficient MRI probes Enzo Terreno,1 Gilberto Mulas,1 Gabriele A. Rolla,2 Carlos F.G.C. Geraldes,3 Luc W.E. Starmans,4 Mauro Botta,2 Lorenzo Tei,2

1 Department of Molecular Biotechnology and Health Sciences, University of Torino, Italy 2 Department of Science and Technological Innovation, University of Eastern Piedmont, Italy 3 Department of Life Sciences and Coimbra Chemistry Center, University of Coimbra, Portugal 4 Biomedical Engineering, Eindhoven University of Technology, The Netherlands

Corresponding Author, e-mail: enzo.terreno@unito.it The recent concerns about the potential risks associated with the use of Gd(III)-chelates,1 prompted many researchers working in this field to explore alternative agents. Paramagnetic Mn(II) complexes were the first candidates due to the high biocompatibility and relevance for life of the Mn(II) ion. However, there are only few (and quite old) studies dealing on Mn(II)-based lipophilic nanoparticles2-3 and on the strategies of relaxivity optimization for such systems. Here, three paramagnetic Mn(II) complexes based on amphiphilic derivatives of EDTA and 1,4-DO2A were used for the preparation of lipidic nanoparticles. The structural properties (length and position on the ligand of the aliphatic chains) of amphiphilic Mn(II)-complexes control size and morphology of phospholipid-based self-assembling nanoaggregates, thus forming vesicular liposomes, non-vesicular bicelles or a mixture of both. The identification of the preferred structures adopted by the aggregates formulated with variable amount of the amphiphilic ligands were carried out by cryo-TEM and relaxometric measurements (integrated with ICP-MS data) performed on nanoparticles formulated with the diamagnetic amphiphilic Zn(II) complexes and hydrophilic Gd-based contrast agents or fluorescent dyes. The presence of the hydrophilic probes at the end of the preparation procedure of the nanoparticles is a clear indication of the presence of vesicular aggregates. Finally, a detailed 1H NMR relaxometric analysis was carried out on all systems highlighting an enhanced relaxivity with respect to monomeric analogues. In case of homogeneous systems, the data were also fitted to obtain the relaxometric parameters for comparison with literature data. Riferimenti [1] Yang, L, Krefting, I, Gorovets, A, Marzella, L, Kaiser, J, Boucher, R, Rieves, D, Nephrogenic systemic fibrosis

and class labeling of gadolinium-based contrast agents by the Food and Drug Administration. Radiology. 2012, 265: 248-253.

[2] Zhao, X, Zhuo, R, Lu, Z, Liu, W, Synthesis, characterization and relaxivity of amphiphilic chelates of DTPA derivates with GdIII, YbIII and MnII. Polyhedron. 1997; 16:2755-2759.

[3] Schwendener, RA, Wüthrich, R, Duewell, S, Wehrli, E, von Schulthess, GK, A pharmacokinetic and MRI study of unilamellar gadolinium-, manganese-, and iron-DTPA-stearate liposomes as organ-specific contrast agents. Invest Radiol. 1990, 25: 922-932.

Figure 1. General scheme illustrating the main results of the work.

13

A Radioactive dinuclear 195mPt-bisphosphonate Complex as Potential

Theranostic Agent for Bone Tumors and Metastases.

Nicola Margiotta,1 Robin A. Nadar,2 Karlijn Codee-van der Schilden,3 Jeroen J. J. P.

van den Beucken,2 Sander C. G. Leeuwenburgh.2 1Dipartimento di Chimica, Università degli Studi di Bari Aldo Moro, Via E. Orabona 4, 70125

Bari, Italy. 2 Department of Dentistry - Regenerative Biomaterials, Radboud University Medical Center, Philips van

Leydenlaan 25, 6525 EX Nijmegen, the Netherlands. 3Nuclear Research & Consultancy Group, Westerduinweg 3,1755 LE Petten, the Netherlands

Corresponding Author, e-mail: nicola.margiotta@uniba.it

Systemic administration of clinically used platinum-based antitumor drugs is associated with

severe side effects and poor therapeutic outcome. By tumor-targeted drug delivery it is possible to

overcome the above described drawbacks. We have recently prepared several dinuclear platinum

complexes containing amino-bisphosphonates as bridging ligands and ethylenediamine (en) as

non-leaving ligand and, in some cases, the the complexes have been loaded in inorganic matrices

such as silica xerogels and hydroxyapatite nanocrystals.[1] In the present work, we aimed to design

a radioactive dinuclear bisphosphonate-functionalized platinum complex, [{195mPt(en)}2(µ-

AHBP-H2)](NO3) (195mPt-BP; AHBP = 2-amino-1-hydroxyethane-1,1-diyl-bisphosphonate),

which combines the diagnostic and therapeutic properties of 195mPt with the bone-seeking

properties of bisphosphonates.[2] Upon systemic administration to mice, the cold Pt-BP complex

exhibited a 4.5-fold higher affinity to bone compared to the precursor [Pt(NO3)2(en)] lacking the

bone-seeking bisphosphonate ligand. Subsequently, the radioactive 195mPt-BP complex was

synthesized using [195mPt(NO3)2(en)] as precursor and injected intravenously into mice. Micro-

SPECT/CT imaging experiments showed the specific accumulation of 195mPt-BP at skeletal sites

with high metabolic activity (Figure). Furthermore, laser ablation-ICP-MS imaging of proximal

tibia sections from treated mice confirmed that 195mPt-BP co-localized with calcium in the

trabeculae. Our results confirm that 195mPt-BP could be used as theranostic agent for the

simultaneous diagnosis and treatment of bone tumors and metastases with high metabolic activity.

References

[1] M. Iafisco, N. Margiotta. J. Inorg. Biochem. 2012, 117, 237-247.

[2] R. A. Nadar, K. Farbod, K. Codee-van der Schilden, L. Schlatt, B. Crone, N. Asokan, A. Curci, M. Brand, M.

Bornhaeuser, M. Iafisco, N. Margiotta, U. Karst, S. Heskamp, O. C. Boerman, J. J. J. P. van den Beucken, S.

C. G. Leeuwenburgh. 2020, submitted for publication.

14

Cytotoxic platinum(II) complexes bearing N-heterocycle rings as novel theranostic agents Isabella Rimoldi,1 Giorgio Facchetti,1 Nicola Ferri,2 Andreea Ionescu,3,4 Rossella Caligiuri,3 Nicolas Godbert,3,4 Loredana Ricciardi,4 Massimo La Deda,3,4 Mauro Ghedini,3,4 Maria Giovanna Lupo,2 Iolinda Aiello3,4 1Università degli Studi di Milano, Dipartimento di Scienze Farmaceutiche, Via Venezian 21, 20133 Milan, Italy.2Università degli Studi di Padova, Dipartimento di Scienze del Farmaco, Via Marzolo 5, 35131, Padua, Italy. 3MAT-InLAB, LASCAMM CR-INSTM, Unità INSTM della Calabria,Dipartimento di Chimica e Tecnologie Chimiche, Università della Calabria, Ponte Pietro Bucci Cubo 14C, 87036 Arcavacata di Rende (CS), Italy.4CNR NANOTEC-Istituto di Nanotecnologia U.O.S. Cosenza, 87036 Arcavacata di Rende (CS), Italy

Corresponding Author, e-mail: Isabella.rimoldi@unimi.it

The discovery of cisplatin and its later approved derivatives started a new era in the bioinorganic medicinal chemistry field but the persistence of severe side-effects along with the emerging of drug resistance evoke the need of a new generation of transition metal-based chemotherapeutics with the aim to overcome these limitations. Given that many transition metal complexes also display interesting photophysical properties, an increasing interest has recently arisen in the development of platinum based theranostic agents, i.e. compounds combining both therapeutic and detection properties in a single entity.1,2 Our research group has synthesised and evaluated for the treatment of triple-negative breast cancer (TNBC) a series of cyclometalated anionic platinum complexes carrying tetrabromocatechol or alizarine as O^O chelating ligands.3 All these complexes resulted emissive in solution and the fluorescence confocal analysis showed their localization in the cytosol of MDA-MB231 cells proving their ability to serve as luminescent probes. By matching these diagnostic imaging properties with the potent cytotoxicity exhibited by the dichloro platinum(II) complex based on 8-aminoquinoline (4.5±µM) and its 5,6,7,8- tetrahydro derivatives on the highly aggressive TNBC cell line,4 a novel series of promising theranostic agents can be developed.

References1. Facchetti, G.; Rimoldi, I. Bioorg. Med. Chem. Lett. 2019, 29, 1257-1263. 2. Jin, G.; He, R.; Liu, Q.; Dong, Y.; Lin, M.; Li, W.; Xu, F. ACS Appl. Mater. & Interf. 2018, 10, 10634-10646. 3. Andreea Ionescu; Rossella Caligiuri; Nicolas Godbert; Loredana Ricciardi; Massimo La Deda; Mauro

Ghedini; Nicola Ferri; Maria Giovanna Lupo; Giorgio Facchetti; Isabella Rimoldi; Aiello, I. Appl. Organomet. Chem. 2020, accepted.

4. Facchetti, G.; Ferri, N.; Lupo, M. G.; Giorgio, L.; Rimoldi, I. Eur. J. Inorg. Chem. 2019, 2019, 3389-3395.

Figure 1. Combined complexes for novel theranostic applications.

15

Cytotoxic effects of 5-Azacytidine on Primary and Stem Cells from Oral Squamous Cell Carcinoma: in vitro FTIRM analysis Chiara Pro1, Alessia Belloni1, Valentina Notarstefano1, Simona Sabbatini2, Carla Conti2, Lisa Vaccari3, Elisabetta Giorgini1

1 Department of Life and Environmental Sciences, Polytechnic University of Marche, Ancona, Italy. 2 Department of Materials, Environmental Science and Urban Planning, Polytechnic University of Marche, Ancona, Italy. 3 SISSI Beamline, Elettra-Sincrotrone Trieste, S.C.p.A., Trieste

Corresponding Author, e-mail: c.pro@pm.univpm.it 5-Azacytidine, a pyrimidine nucleoside analogue, is an antineoplastic agent which impairs DNA methylation inhibiting DNA methyltransferase. It also acts as an antimetabolite of cytidine, mainly incorporated into RNA, damaging RNA metabolism. As gene expression changes are responsible also for cellular differentiation, in this study we exploited FTIRM spectroscopy to in vitro analyze the effects of this drug on different cellular groups, to evaluate how the loss of methylation impacted on gene expression. Primary cells (A) and their corresponding stem cells (B), were obtained from bioptic samples of grade 3 Oral Squamous Cell Carcinoma. Both cellular groups were treated with 5-Azacitidine (3 µg/ml) at two exposure times (24 and 48 hours). All cell samples were in vitro analyzed by using FTIRM spectroscopy. The spectral data were compared with those from the same cell lines

cultured without any treatment (considered as control groups, CTRL) (Table1). The spectral data were submitted to multivariate analysis (PCA) to highlight the spectral differences among groups. For each sample, average spectra were also curve fitted (Gaussian algorithm) to analyze the region of nucleic acids and phosphates (1350-900 cm-1). The following band area ratios were calculated: 968/TR and 939

cm-1/TR (B and Z forms of DNA), 994 cm-1/TR and 913 cm-1/TR2 (RNA), 1171/TR (phosphorylated compounds), 1030 cm-1/TR (carbohydrate) (TR defines the integrated areas of the whole spectral range 1350-900 cm-1). The detected biochemical changes were related mainly to: (1) lipids and carbohydrates metabolism, (i) nucleic acids quantity and integrity, (iii) phosphorylated compounds. In particular, the reduction of the methylated form of DNA induced the transcription, thus causing an increase in the concentration of RNA.

References[1] Martens U.M., Muller A., Florek M. 5-

Azacytidine/Azacitidine. Small molecules in oncology. Springer: 2010; 159-170. 978-3-642-01221-1.

[2] Giorgini E., Sabbatini S., Rocchetti R., Notarstefano V., Rubini C., Conti C., Orilisi G., Mitri G., Bedolla D.E., Vaccari L. In vitro FTIR microspectroscopy analysis of primary oral squamous carcinoma cells treated with cisplatin and 5-fluorouracil: a new spectroscopic approach for studying the drug–cell interaction. Analyst: 2018. 143, 3317-3326.

Table 1. Experimental design of the cellular groups. A: Primary cells; B: Stem cells.

CTRL 5-Azacytidine

A24h A_CTRL_24 A_5AZA_24

48h A_CTRL_48 A_5AZA_48

B24h B_CTRL_24 B_5AZA_24

48h B_CTRL_48 B_5AZA_48

Figure 1. Average spectra.

16

Indazolil-N-arilbenzensolfonammidi e chinazolinil-N-arilbenzensolfonammidi come inibitori della anidrasi carbonica

Emma Baglini,1 Silvia Salerno,1 Elisabetta Barresi,1 Anna Maria Marini,1 Sabrina Taliani,1 Federico Da Settimo,1 Andrea Angeli,2 and Claudiu T. Supuran2, 1 Dipartimento di Farmacia, Via Bonanno, 6, 56126, Pisa, Italy 2 Dipartimento NEUROFARBA, Università̀ degli Studi di Firenze, Via Ugo Schiff, 6, 50019 Sesto Fiorentino Florence, Italy

Emma Baglini, e-mail: emma.baglini@phd.unipi.it Le anidrasi carboniche (CA) sono una superfamiglia di metallo-enzimi che catalizzano l’interconversione tra CO2 e ione bicarbonato. Sono enzimi ubiquitari, coinvolti in diversi processi fisiologici e patologici; per questo sono state sviluppate numerose classi di inibitori delle anidrasi carboniche (CAI), al fine di modularne l’attività. Le CA vengono inibite principalmente da due classi di composti: gli anioni complessanti lo zinco (tra cui molti carbossilati) e le solfonammidi/sulfamati/sulfammidi, che generalmente si legano allo ione Zn2+ del sito attivo dell’enzima. Le solfonammidi primarie rappresentano la classe più importante di CAI, ma allo stesso tempo possiedono una bassa selettività tra le diverse isoforme dell’enzima. Recentemente, le solfonammidi secondarie e terziarie sono state riportate in letteratura come inibitori efficaci e selettivi delle isoforme CA IX e CA XII, correlate allo sviluppo del cancro [2]. Così, sulla base della struttura di CAI potenti descritti in un precedente lavoro [3], caratterizzati da un gruppo solfonammidico primario e da scaffold tetraidroindazolico e tetraidrochinazolico, abbiamo studiato i derivati delle serie 1 e 2, per valutare come la presenza di un gruppo benzensolfonammidico secondario possa influenzare l’attività delle molecole nei confronti di quattro isoforme fisiologicamente rilevanti, l'hCA I, II, IV e IX. Inoltre, gruppi di varia natura (R3 = H, OCH3, Cl, NO2, CH3) sono stati inseriti sull’anello fenilico distale, in modo da modulare l'acidità della solfonammide stessa e studiare come questa influenza l’attività biologica dei composti.

Riferimenti

[1] Supuran C.T., Carbonic anhydrases, Bioorg Med Chem. 2013;21(6),1377-1378 [2] Supuran C. T. How many carbonic anhydrase inhibition mechanisms exist? J Enzyme Inhib Med Chem 2016;

31:345–60 [3] Salerno S., Barresi E., Amendola G., Berrino E., Milite C., Marini A.M., Da Settimo F., Novellino E., Supuran

C.T., Cosconati S., and Taliani S., 4-Substituted Benzenesulfonamides Incorporating Bi/Tricyclic Moieties Act as Potent and Isoform-Selective Carbonic Anhydrase II/IX Inhibitors. J.Med Chem 2018, 61, 5765−5770.

Figura 1. Derivati biciclici a scaffold tetraidroindazolico (1)e tetraidrochinazolico (2)

17

An ESI-MS investigation of the interactions occurring between the gold-based drug Auranofin and a panel of representative proteins

Carlotta Zoppi,1 Alessandro Pratesi,2 Luigi Messori1 1 Laboratory of Metals in Medicine (MetMed), Department of Chemistry “U. Schiff”, University of Florence, via della Lastruccia 3, 50019 Sesto Fiorentino, Italy 2 Department of Chemistry and Industrial Chemistry, via Moruzzi 13, 56124, Pisa, Italy

Corresponding Author, e-mail: carlotta.zoppi@unifi.it

Since the serendipitous discovery of its antiproliferative properties, Cisplatin has been -and is still -widely used in the treatment of various types of cancer. However, its severe side effects and the frequent insurgence of platinum resistance triggered new researches aimed at discovering different metal-based anticancer drugs. Thus, several other Platinum-based compounds, but also ruthenium and gold-based, were synthesized and studied, providing encouraging results. In particular, gold compounds represent a variegate family of promising anticancer agents. Among these, the case of Auranofin (1-Thio-β-D-gluco pyranosate triethylphosphine gold-2,3,4,6-tetraacetate - AF) is of particular interest. AF has been used as an anti-arthritic agent since 1988 but then it was gradually abandoned because of its significant toxicity. In the last years, however, AF has been re-evaluated as an anti-infective, antibacterial and even anticancer agent. The mechanism of action of AF seems to be mainly related to the interaction with proteins, rather than with nucleic acids (which are instead the main targets for Pt-based compounds). AF acts as a prodrug, losing the thiosugar ligand and binding through the AuPEt3+ fragment to the protein sidechains. According to the hard-soft theory, the preferred binding sites of AF are cysteine and selenocysteine residues.[1] In the study of the interactions between proteins and metal-based drugs, high resolution ESI mass spectrometry turns out to be a very powerful and high performing technique [2]. It allows to characterize, at the molecular level, the adduct formed between proteins and metal fragments: the chemical nature of the fragment bound to the biomolecule, the binding stoichiometry and the kinetics of the adduct formation can be evaluated. Therefore, through a robust MS method for the investigation of proteins and their adducts with metal compounds, metalation studies of a series of proteins with AF have been carried out. It has been further confirmed that the fragment that binds to proteins is AuPEt3+; in addition, some interesting trends in the reactivity of this compound have been highlighted.

References[1] Pratesi A, Cirri D., Ciofi L., Messori L., Reactions of Auranofin and Its Pseudohalide Derivatives with

Serum Albumin Investigated through ESI-Q-TOF MS. Inorg. Chem.. 2018; 57:10507−10510 [2] Pratesi A., Zoppi C., Messori L., ESI MS studies highlight the selective interaction of Auranofin with protein

free thiols. Chemistry A European Journal, submitted.

Figure 1. Deconvoluted ESI-Q-TOF mass spectrum of HSA solution 5 ×10-7 M with AF (3:1 metal to protein ratio) in 2 mM AmAc buffer, pH 6.8

HSA + AuPEt3+

HSA + 2(AuPEt3+)

66500 67000 67500

Mass, Da

0%

20%

40%

60%

80%

100%

%In

ten

sit

y(o

f3

.1e

4)

66751.462

67069.155

18

Synthesis, DNA binding studies, and antiproliferative activity of new platinum complexes containing a nucleoamino acid-based ligand Domenica Capasso,1 Domenica Musumeci,2 Sonia Di Gaetano,3 Angela Coppola,2 Giovanni N. Roviello,3 Daniela Montesarchio2

1 Department of Pharmacy, University of Naples Federico II, Naples, Italy; 2 Department of Chemical Sciences, University of Naples Federico II, Naples, Italy; 3 Institute of Biostructures and Bioimaging - CNR, Naples, Italy.

Corresponding Author, e-mail: domenica.capasso@unina.it Cisplatin is one of the most active chemotherapeutic agents in use for the treatment of a variety of malignancies, especially testicular and ovarian carcinoma,1 even if its clinical utility is restricted by both toxicological and especially tumour resistance considerations.1 Therefore, much attention has been focused on the development of new Pt complexes with improved pharmacological properties and broader anticancer activity.1 Most researches on platinum-based complexes included, as Pt-ligands, bioactive molecules such as peptides, carbohydrates, nucleic acids, amino acids,2 nucleosides,3 and natural products, in order to obtain compounds with enhanced activity thanks to the synergistic effects of the Pt and the bioactive ligand.4 Considering the capability of platinum ion to bind both nucleobases and amino acids, our idea consisted in the design and synthesis of new Pt(II) complexes incorporating as ligands nucleoamino acids, constituted by nucleobases linked to an amino acid through a suitable linker, and thus intrinsically containing various potential platinum binding sites. Here we report our investigation on the Pt-complexation reaction of the nucleoamino acid DAP(T)-OH, constituted by the 2,3-diaminopropanoic acid (DAP-OH) linked to the thymine nucleobase by a carboxymethylene linker. The obtained platinum-complexes were characterized by NMR, MS and UV-vis techniques, and their ability to bind various DNA model systems was investigated by CD. Furthermore, their biological activity in terms of antiproliferative effects towards several human cancer cell lines was evaluated.

References

[1] Dasari S and Tchounwou PB, Cisplatin in cancer therapy: molecular mechanisms of action. Eur J Pharmacol. 2014; 740: 364-78.

[2] Riccardi C1st, Capasso D1st, et al., Synthesis, DNA binding studies, and antiproliferative activity of novel Pt(II)-complexes with an L-alanyl-based ligand. J Inorg Biochem. 2019; 203:110868.

[3] D’Errico S, et al., Synthesis and Pharmacological Evaluation of Modified Adenosines Joined to Mono-Functional Platinum Moieties. Molecules. 2014; 19: 9339-53.

[4] R. Cincinelli, et al., Design, modeling, synthesis and biological activity evaluation of camptothecin-linked platinum anticancer agents. Eur. J. Med. Chem. 2013; 63: 387-400.

Figure 1. New Pt-complexes from the reaction of

the thyminyl DAP-based nucleoamino acid

NH

N

O

O

CH3

H

O

NH

O

OHNH

2

K2PtCl4

CH3COCH3/H2O

DAP(T)-OH

NewPt-complexes

NMR, MScharacterization

DNA-binding studies

antiproliferative activity

19

NAMI-A: repurposing of an old molecule for new challenges Damiano Cirri,1 Luigi Messori1

1 Laboratory of Metals in Medicine (MetMed), Department of Chemistry “U. Schiff”, University of Florence, via della Lastruccia 3, 50019 Sesto Fiorentino, Italy.

Corresponding Author, e-mail: damiano.cirri@unifi.it Since the discovery of their anticancer properties, ruthenium-based compounds have attracted a growing attention in the field of medicinal chemistry as possible alternatives to currently available Platinum anticancer drugs. In this frame, many Ru(II) and Ru(III) complexes bearing different kind of ligands were investigated, some of these with very appreciable results [1,2]. Indeed, four ruthenium compounds, i.e. TLD1433, NAMI-A, KP1019 and KP1339 are nowadays in various stages of clinical trials. Among these, NAMI-A (Fig.1) is undoubtedly the most promising compound, being this the only Ru based compound to have reached phase II clinical trials for the treatment of a tumoral disease (i.e. non-small cell lung cancer) [1,2].

Fig.1: NAMI-A molecular structure. In this presentation, the history of ruthenium anticancer candidates will be briefly resumed (with a particular attention for structure activity relationships), starting from pioneering work developed in the early 80’ from E. Alessio and B. Keppler to the latest results. References

[1] Alessio E, Thirty Years of the Drug Candidate NAMI‐A and the Myths in the Field of Ruthenium Anticancer Compounds: A Personal Perspective. Eur. J. Inorg. Chem. 2017; 1549-1560.

[2] Thota S, Rodrigues DA, Crans DC, Barriero EJ, Ru(II) Compounds: Next-Generation Anticancer Metallotherapeutics?. J. Med. Chem. 2018; 61: 5805–5821.

20

Synthesis and characterization of new water-soluble organometallic complexes of the type [PtL(1-C2H4OMe)(N^N)] (L = NH3, DMSO; N^N = dinitrogen ligand) with potential antitumor activity Michele Benedetti,1 Federica De Castro,1 Erika Stefano,2 Danilo Migoni,1 Giulia M.C. Sanzia,1 Antonella Muscella,3 Santo Marsigliante,2 Francesco P. Fanizzi.1

1 Laboratorio di Chimica Generale ed Inorganica DiSTeBA, Università del Salento. 2 Laboratorio di Fisiologia cellulare DiSTeBA, Università del Salento. 3 Laboratorio di Patologia cellulare DiSTeBA, Università del Salento.

Corresponding Author, e-mail: michele.benedetti@unisalento.it Since the discovery of the antitumor activity of cisplatin, many studies have been devoted to understanding the relation between structure of platinum compounds and their antitumor activity.[1] Several investigations on cisplatin analogues demonstrated that their effectiveness could be greatly improved by substituting the labile chlorido ligands with other leaving groups, giving a 2nd generation platinum drugs (e.g. carboplatin, nedaplatin, etc.).[1] A 3rd generation could be obtained by replacement of both ammonia and chlorido ligands of cisplatin with different ligands (e.g. oxaliplatin, heptaplatin, etc.).[1] The here studied new organometallic compounds belong to this last category. We synthesized complex precursors of type [PtCl(1-C2H4OMe)(N^N)], 3, by using a previously reported procedure consisting in the reaction of a dinitrogen ligand with the Zeise’s salt in basic methanol. [2-3] Reaction of the neutral complex 3 with excess NH3 or DMSO in water solution, gave the final [Pt(NH3)(1-C2H4OMe)(N^N)]+, 4’, or [Pt(DMSO)(1-C2H4OMe)(N^N)]+, 4’’, complexes, respectively. The citotoxic activity of such complexes were studied in comparison with cisplatin. Preliminary experiments on different cell lines showed a relevant cytotoxic activity for complex 4’’ differently from 4’, when N^N = 1,10-phenanthroline. [1] Johnstone T.C., Suntharalingam K., Lippard S.J.. The Next Generation of Platinum Drugs: Targeted Pt(II)

Agents, Nanoparticle Delivery, and Pt(IV) Prodrugs. Chem Rev. 2016, 116, 3436-3486. [2] Benedetti M., Fanizzi F. P., Maresca L., Natile G. The unexpected reactivity of Zeise’s anion in strong basic

medium discloses new substitution patterns at the platinum centre. Chem. Commun. 2006, 1118-1120. [3] Benedetti M., Barone C.R., De Pinto S., De Castro F., Natile G., Fanizzi F.P.. Cationic olefin complexes of

platinum(II): Aspects of availability and reactivity. Inorg. Chim. Acta 2018, 470, 172-180.

Figure 1. Schematic representation of the formation of the here studed [PtL(1-C2H4OMe)(N^N)]+, 4, (L = NH3, DMSO; N^N =

dinitrogen ligand) complexes starting from [PtCl(2-C2H4)(N^N)]+, 1, and [PtCl(1-C2H4OMe)(N^N)], 3, species. The decomposition [PtCl2(N^N)], 2, product is also shown.

21

Bionic synthesis of a magnetic calcite skeletal structure through living foraminifera Giulia Magnabosco,1 Simona Fermani,1 Vittorio Morandi,2 Jonathan Erez,3 Giuseppe Falini,1

1 Dipartimento di Chimica ‘‘Giacomo Ciamician’’, Alma Mater Studiorum-Universita` di Bologna, Bologna, Italy. 2 National Research Council, Institute for Nanostructured Materials (ISMN), Bologna, Italy. 3 Institute of Earth Sciences, The Hebrew University of Jerusalem, Edmond Safra Campus, Jerusalem,

Israel. Corresponding Author, e-mail: giuseppe.falini@unibo.it

The peculiar functional properties of calcium carbonate biominerals, such as shells, echinoderm spines and brittle stars, have stimulated both fundamental research in biomineralization and in vitro bio-inspired synthetic processes in material science. As a consequence of the latter, bio-materials having different/additional properties with respect to the natural ones have been produced by cell-free laboratory activities. Despite this effort, the production of materials having functional properties even similar to the natural ones remains elusive. We have demonstrated that new nano-composite materials can be prepared in vivo exploiting the special mineralization pathway of the foraminifera Amphistrigina lessoni, a calcifying organism producing a calcitic skeleton by vacuolization of seawater. Accordingly, a bionic skeleton possessing a chamber entrapping magnetic nanoparticles has been obtained growing the organism in seawater containing such nano-particles. Such a bionic synthetic approach differs and goes beyond the biologically inspired synthetic processes and the biosynthesis of nano-particles by bacteria. It represents the first research in which a bionic calcified tissue has been prepared in vivo.[1] This represents a new powerful tool for the preparation of nano-materials exploiting the capability of organisms to control the calcification pathway with an accuracy that is unparalleled in laboratory cell free synthetic processes. References [1] Magnabosco, G., Hauzer, H., Fermani, S., Calvaresi, M., Corticelli, F., Christian, M., Albonetti, C., Morandi,

V., Erez, J. and Falini, G. Bionics synthesis of magnetic calcite skeletal structure through living foraminifera. Materials Horizons, 2019, 6, 1862-1867

Figure 1. Camera picture of the foraminifera Amphistrigina lesson.

22

Ionomic study: metabolomic analysis of Xylella fastidiosa subsp. pauca-infected, non infected and treated olive groves of Apulia, Italy Laura Del Coco1, Chiara Roberta Girelli1, Federica Angilè1, Danilo Migoni1, Marco Scortichini2, Francesco Paolo Fanizzi1

1 Dipartimento di Scienze e Tecnologie Biologiche e Ambientali, Università del Salento, Via Prov.le Lecce-Monteroni, 73100 Lecce, Italia 2 Consiglio per la Ricerca in Agricoltura e l’Analisi dell’Economia Agraria, Centro di ricerca per l’Olivicoltura, Frutticoltura e Agrumicoltura, Via di Fioranello, 52, 00134, Roma, Italia

Corresponding Author, e-mail: laura.delcoco@unisalento.it Xylella fastidiosa subsp. pauca is associated with the olive quick decline syndrome in Salento [1]. First outbreak of the disease was noticed in the Gallipoli district, from where, subsequently, it reached nearby areas [2]. So far, no specific study was undertaken to verify if an abnormal availability of some micronutrients in the soil and/or in the leaves might be associated with this outbreak. In this study, ionomic profiles, associated to multivariate statistical analyses, were used to understand plant ionome modifications by infection of X. fastidiosa. For this pourpose, infected plants, (NTR, not treated with Dentamet®, a Zn and Cu citrate preparation) in comparison with treated (TR) and not infected plants (NI), located in different geographical districts of Apulian and Lucanian regions were studied. Both soil and olive leaf samples of Cellina, Ogliarola salentina, Leccino and Coratina cultivars (for a total of ~125 samples) were analyzed for macro and micronutrients content by using ICP-AES spectroscopy analysis. The present investigation and previous experimental data [4] corroborate the observed decrease of Mo in soil and a low bioavailability of Cu and Mo in the leaves of X. fastidiosa infected plants. Moreover, a high relative content of Ca and Mg and a low relative content of Na were found in NI olive leaf samples. A high relative content of Zn and Cu and a low relative content of Na was observed for TR with respect to NTR samples. The high relative amount of Zn and Cu could probably be due to the specific treatment with Dentamet®, while an increment of Na in infected samples (NTR) was already observed in the case of tobacco leaves infected by X. fastidiosa subsp. fastidiosa. A reduced Cu content in the infected leaves was already recorded for X. fastidiosa. subsp. multiplex as well as for other bacterial phytopathogens [3]. The role of Cu in relation to X. fastidiosa infection is discussed [4]. Riferimenti[1] Cariddi, M. Saponari, D. Boscia, A. De Stradis, G. Loconsole, F. Nigro, F. Porcelli, O. Potere, G.P. Martelli;

Journal of Plant Pathology 2014, 96, 425 [2] G. Strona , C.J. Carstens, P. Beck; Scientific Reports 2017, 7, 71 [3] J.E. Oliver, P.A. Cobine, L. De La Fuente, Phytopathology 2015, 105, 855 [4] M. Scortichini, D. Migoni, F. Angilè, L. Del Coco, C.R. Girelli, L. Zampella, F. Mastrobuoni, F. P. Fanizzi

Phytopathologia Mediterranea 2019, 59, 39

Figura 1. PLS-DA scoreplot

obtained for olive leaf samples.

23

Anticancer Metal based Drugs and Omics Sciences: some Reflections Luigi Messori

Department of Chemistry “Ugo Schiff”, University of Florence, Florence, Italy Corresponding Author, e-mail: luigi.messori@unifi.it

Metal based drugs form today an important class of anticancer agents. Indeed, three platinum drugs, i.e. cisplatin, carboplatin and oxaliplatin, are clinically approved worldwide for cancer treatment and are part of about half of the current chemotherapeutic regimens in clinical use. Several other Pt and non-Pt experimental metal based drugs are undergoing intense preclinical and clinical investigations with encouraging results. In spite of that, the molecular mechanisms of anticancer metal based drugs are not fully understood and, in several cases, largely unknown and unexplored. Difficulties of these mechanistic studies mostly arise from the extreme complexity of the biological systems as well as from the broad and poorly selective reactivity of metal based drugs, so that the classical one drug- one target paradigm does not hold anymore. However, modern Omics technologies offer potent weapons to handle such large complexity and give us the chance to shed some light on the underlying molecular mechanisms. Accordingly, we report here some examples of the successful application of Omics technologies to the analysis of the molecular mechanisms of selected anticancer metal based drugs. For instance, classical proteomics experiments have been exploited to clarify the molecular mechanisms of a variety of gold compounds as well as of some Pt drugs. In turn, metabolomics measurements provide insight into the alterations in the metabolites’ patterns induced by treatments and on the associated metabolic alterations of the cell. Metallomics, an omics technology still under development, permits to perform a detailed analysis of all metal containing species that are present inside treated cells, with an emphasis on metallodrug-protein adducts, and may turn very helpful for target search and identification. Results deriving from the application of the various Omics technologies to cellular models must then be interpreted with the aid of bioinformatics tools and integrated with the current knowledge of the numerous biological processes taking place inside cells, that are organized in a large variety of metabolic and signaling pathways. Thus, the talk will try to give an overview of the “state of art” on these issues offering hints for future research. .

24

Strategies for Understanding the Mode of Action of Anticancer Gold Complexes Lara, Massai,1 Damiano Cirri,1 Carlotta Zoppi,1 Tania Gamberi,2 Luigi Messori1

1 Department of Chemistry “Ugo Schiff”, University of Florence, Florence, Italy 2 Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Florence, Italy

Corresponding Author, e-mail: lara.massai@unifi.it

Gold compounds form a new attractive class of cytotoxic metal compounds of potential application as anticancer agents. Notably, the mode of action of cytotoxic gold compounds appears to be deeply distinct from that of the widely used anticancer Pt drugs and, basically, DNA independent. More recently, a variety of gold derivatives has been tested as potential antitumor agents, including organogold derivatives, analogues of Auranofin and gold-N-heterocyclic carbenes (NHC)[1]. Based on the great structural variety of the used ligands and their role in controlling the reactivity of the gold centre, a single mode of action or pharmacological profile is unlikely to exist. Thus, the identification of the cellular processes affected by the various gold compounds as well as of their respective target proteins is the goal of this work. In this study, mass spectrometry (MS) methods were used to investigate the binding of various gold(I)/gold(III) compounds with proteins, elucidate the interaction mechanisms and describe the resulting adducts. An additional technique, UV-vis spectrophotometry, further offered the opportunity to monitor continuously, in ‘‘real time’, the various metallodrug–protein species. Moreover, a classical proteomic approach has been used, to gain a deeper insight into perturbations in protein expression induced by gold drugs treatment in a selected cancer cell line[2]. A few overlapping mechanisms were identified. Notably, it emerges that the affected proteins were found to belong - in most cases- to metabolic (redox) processes and/or to the proteasome system implying that the severe cellular damage induced by gold compounds predominantly originates at these two distinct levels. However, though the detailed mechanisms of action remain unclear, the inhibition of the seleno-enzyme thioredoxin reductase (TrxR) seems to be a common mechanistic trait to explain, at least partially, the cytotoxic actions of several gold(I) and gold(III) complexes. References [1] Marzo T, Massai L, Pratesi A, Stefanini M, Cirri D, Magherini F, Becatti M, Landini I, Nobili S, Mini E, Crociani O, Arcangeli A, Pillozzi S, Gamberi T, Messori L, Replacement of the Thiosugar of Auranofin with Iodide Enhances the Anticancer Potency in a Mouse Model of Ovarian Cancer. ACS Med Chem Lett. 2019;10: 656-660 [2] Magherini F, Fiaschi T, Valocchia E, Becatti M, Pratesi A, Marzo T, Massai L, Gabbiani C, Landini I, Nobili S, Mini E, Messori L, Modesti A, Gamberi T, Molecular and cellular basis for the cytotoxicity of two gold(I)-N-heterocyclic carbene complexes in A2780 human ovarian cancer cells: a comparative proteomic study. Oncotarget. 2018; 9: 28042-28068

Figure 1. IC50 TrxR inhibition of various gold(I) complexes

Auranofin

Au(PEt3)Cl

Au(PEt3)Br

Au(PEt3)I

[Au(PEt3)2]Cl

0

5

10

15

20

IC50

Trx

R

25

Interaction of cisplatin with oxidized human Atox1

Maria Incoronata Nardella,1 Antonio Rosato,1 Benny D. Belviso,2 Rocco Caliandro,2 Giovanni Natile,1 Fabio Arnesano1 1 Department of Chemistry, University of Bari, via E. Orabona, 4, 70125 Bari, Italy 2 Institute of Crystallography, CNR, Bari, Italy

Corresponding Author, e-mail: m.incoronatanardella@gmail.com Cancer cells treated with cisplatin are characterized by an increase in free Cu(I) levels and high oxidative stress. Under these conditions, the Cu(I) chaperone Atox1, which is involved in cisplatin binding and resistance, gets oxidized, i.e. a disulfide bond is formed between the two cysteine residues of the metal binding site, CxxC. We investigated whether the oxidized form of Atox1 is still able to interact with cisplatin. The metallodrug was found to target methionine residues on the surface of the protein and to promote disulfide bond cleavage in the presence of reduced glutathione (GSH) [1]. The results indicate that the depletion of both Cu(I) and GSH may reduce the Atox1-mediated cisplatin resistance. To monitor the distribution of cisplatin in cancer cells, a drug derivative was used that could be labeled with a fluorophore by a click chemistry reaction catalyzed by Cu(I).

Riferimento[1] Nardella M. I., Rosato A., Belviso B. D., Caliandro R., Natile G., and Arnesano F. Oxidation of Human Copper

Chaperone Atox1 and Disulfide Bond Cleavage by Cisplatin and Glutathione. Int. J. Mol. Sci., 2019, 4390, 20(18).

Figura 1. Schematic model of Atox1 oxidation and interaction with cisplatin.

26

The opposite effect of platinum drugs and zinc ions on copper trafficking: a structural and mechanistic study Maria Incoronata Nardella,1 Alessia Lasorsa,1 Antonio Rosato,1 Valentina Mirabelli,2 Rocco Caliandro,2 Giovanni Natile,1 Patrick J. Farmer,3 Fabio Arnesano1

1 Department of Chemistry, University of Bari, via E. Orabona, 4, 70125 Bari, Italy 2 Institute of Crystallography, CNR, Bari, Italy 3 Department of Chemistry and Biochemistry, Baylor University, Waco, TX 76798, USA

Corresponding Author, e-mail: fabio.arnesano@uniba.it The anticancer drug cisplatin is able to interfere with the mechanism of rapid Cu(I) exchange between the Cu chaperone Atox1 and the first domain of Menkes ATPase (Mnk1). The heterodimeric complex formed by the two proteins interacting both with Cu(I) and cisplatin was characterized by NMR spectroscopy and X-ray diffraction, thus providing the structural basis for the inhibition of copper trafficking by the metallodrug [1]. The Atox1-Cu(I)-Mnk1 heterodimer also interacts with Zn(II) ions and a Zn(II) complex with the thiomaltol ligand (a chelator that increases Zn bioavailability). Unlike cisplatin, Zn(II) promotes the transfer of Cu(I) to Mnk1, by stabilizing the Atox1-Zn(II)-Atox1 homodimer.

Riferimento[1] Lasorsa A., Nardella M.I., Rosato A., Mirabelli V., Caliandro R., Caliandro R., Natile G., Arnesano F.

Mechanistic and Structural Basis for Inhibition of Copper Trafficking by Platinum Anticancer Drugs. J. Am. Chem. Soc., 2019, 141(30):12109-12120.

Figura 1. Proposed mechanism for the inhibition of the rapid exchange of Cu(I) between Atox1 and Mnk1 by cisplatin.

27

The challenge to understand the precise mechanisms of action of approved platinum anticancer drugs Tiziano Marzo,1 Alessandro Pratesi2, Damiano Cirri,3 Lara Massai,3 Jürgen Gailer,4 Luigi Messori3

1 Department of Pharmacy, University of Pisa, via Bonanno Pisano 6, 56126, Pisa, Italy 2 Department of Chemistry and Industrial Chemistry, via Moruzzi 13, 56124, Pisa, Italy 3 Laboratory of Metals in Medicine (MetMed), Department of Chemistry “U. Schiff”, University of Florence, via della Lastruccia 3, 50019 Sesto Fiorentino, Italy 4 Department of Chemistry, University of Calgary, 2500 University Drive NW, Calgary, AB T2N 1N4, Canada

Corresponding Author, e-mail: tiziano.marzo@unipi.it Since the first approval of cisplatin for cancer treatment in 1978, huge research efforts have been made to design innovative and improved metal based anticancer drugs. However, after cisplatin, only other two platinum drugs, i.e. carboplatin and oxaliplatin, were approved worldwide. Despite platinum-based chemotherapy is widely used as an essential weapon to combat cancers and there is a substantial consensus on the mechanism of action relying on the DNA interaction; to date, the general mechanistic picture is still rather incomplete. Thus, it is extremely important to shed light into the transport and metabolic pathways and mechanisms of action of these compounds. It is now recognized that therapeutic as well as acute or chronic toxic effects and resistance phenomena related to platinum drugs treatments, are not only a consequence of covalent adducts formation between platinum complexes and DNA, but also with other biological targets including RNA and proteins. Here, through a few relevant examples, we will discuss the limits of the knowledge and the opportunities that the advance in the investigational techniques give us to better understand the overall mechanisms of action of established platinum based antineoplastic agents [1, 2]. Also, this is important since the field is currently experiencing a paradigm shift: formerly, after the discovery of an effective compound, the primary mechanism of action was then investigated, while today, the knowledge of the mechanism of action is increasingly used to drive the drug-discovery process [3]. Acknowledgments

TM thanks Beneficentia Stiftung (Vaduz) project code: BEN 2019/48 and University of Pisa (Rating Ateneo 2019) for the financial support. Riferimenti

[1] Massai L, Pratesi A, Gailer J, Marzo T, Messori L, The Cisplatin/Serum Albumin System: a Reappraisal. Inorg. Chim. Acta. 2019; 495: 118983.

[2] Bruno PM, Liu Y, Park GY, Murai J, Koch CE, Eisen TJ, Pritchard JR, Pommier Y, Lippard SJ, Hemann MT, A subset of platinum-containing chemotherapeutic agents kill cells by inducing ribosome biogenesis stress rather than by engaging a DNA damage response. Nat Med. 2017; 23: 461–471.

[3] Boros E, Dyson PJ, Gasser G, Classification of Metal-Based Drugs according to Their Mechanisms of Action. Chem. 2019; https://doi.org/10.1016/j.chempr.2019.10.013.

28

Bifunctional cisplatin-based Pt(IV) complexes containing non-steroidal anti-inflammatory drugs (NSAIDs) Elisabetta Gabano,1 Beatrice Rangone,1 Elena Perin,1 Mauro Ravera,1 Domenico Osella1

1 Dipartimento di Scienze e Innovazione Tecnologica, Università del Piemonte Orientale, Viale T. Michel 11, 15121 Alessandria, Italy

Corresponding Author, e-mail: elisabetta.gabano@uniupo.it Chronic inflammation plays an important role in several human cancers. Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used as analgesics, anti-inflammatories and antipyretics. Their primary targets are cyclooxygenase (COX) isoforms, COX-1 and COX-2, responsible for the formation of biological mediators of inflammation. Since an enhanced COX-2 expression is found in some tumors, it appears to play a role in carcinogenesis. For this reason, COX inhibitors, including NSAIDs and COX-2 selective inhibitors, are suggested for cancer prevention (especially for colon adenocarcinoma). NSAIDs show modest tumor growth inhibition when employed as a single agent; however, they have been tested as adjuvant agents in some chemotherapeutic protocols. For this reason, cisplatin and the NSAIDs ketoprofen and naproxen were combined in bifunctional Pt(IV) complexes (Figure 1), that were tested on a panel of human tumor cell lines, including the very chemoresistant malignant pleural mesothelioma [1]. The Pt(IV) compounds showed good antiproliferative activity in comparison with cisplatin and free NSAIDs. The activity observed can be explained in terms of lipophilicity of the compounds, that in turn promotes increased cellular accumulation (synergistic accumulation). A quick Pt(IV) Pt(II) reduction then generates the active cisplatin metabolite, while releasing the NSAIDs. The NSAID adjuvant action seems to be scarcely dependent on cyclooxygenase-2 (COX-2) expression in the tumor cells under investigation, suggesting that a COX-2-independent mechanism plays a role. This mechanism seems to rely on the activation of the NSAID activated gene, NAG-1 (a member of the transforming growth factor beta, TGF-, superfamily), which has proapoptotic and anti-tumorigenic activities. The whole data, however, suggest that most of the observed biological effects are related to the cisplatin metabolite.

References

[1] Ravera M, Zanellato I, Gabano E, Perin E, Rangone B, Coppola M, Osella D, Antiproliferative Activity of Pt(IV) Conjugates Containing the Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) Ketoprofen and Naproxen. Int. J. Mol. Sci. 2019; 20: 3074-3091.

Funding: This research has been carried out within the HERMES project, “Progetto finanziato attraverso l’offerta di indennizzo ai residenti di Casale Monferrato deceduti o affetti da mesotelioma” (Project funded by offer of compensation to the residents of Casale Monferrato died or suffering from mesothelioma.

Figure 1. Bifunctional Pt(IV) complexes.

29

POSTER COMMUNICATIONS

Synthesis, characterization, and in vitro cytotoxicity of platinum(IV) dehydrogenated oxaliplatin derivatives. Alessandra Barbanente,1 Katia Micoli,1 Paride Papadia,2 Nicoletta Ditaranto,1 Valentina Gandin,3 Giovanni Natile,1 Nicola Margiotta.1

1 Dipartimento di Chimica, Università degli Studi di Bari Aldo Moro, Via Orabona 4, 70125 Bari, Italy. 2 Department of Biological and Environmental Sciences and Technologies (DISTeBA), University of Salento, 73100 Lecce, Italy. 3 Dipartimento di Scienze Farmaceutiche e Farmacologiche. Università di Padova. Via Marzolo 5, 35131 Padova.

Corresponding Author, e-mail: alessandrabarbanente@libero.it

Platinum(IV) complexes have been extensively investigated in recent years as prodrugs of the clinically used antitumor drugs cisplatin and oxaliplatin.[1] We have recently prepared Pt(II) complexes with the racemic trans-1,2-diamine-4-cyclohexene (DACHEX) ligand.[2] DACHEX possesses a double bond in the diaminocyclohexane ligand present in oxaliplatin, used specifically for the treatment of colorectal cancer. The unsaturation present in DACHEX constrains the structure of the diamine ring in a more rigid half chair conformation and adds unprecedented π-electrons to the potential lipophilic interactions, opening the door to a further functionalization of the DACH ligand via electrophilic addition. In this work, six Pt(IV) complexes with the carrier ligand DACHEX have been synthesized: cis,trans,cis-[Pt(OXA)(OH)2(DACHEX)] (1), cis,trans,cis-[Pt(OXA)(AcO)2(DACHEX)] (AcO = acetate) (2), cis,trans,cis-[Pt(OXA)(BzO)2(DACHEX)] (BzO = benzoate) (3), cis,trans,cis-[Pt(OXA)Cl2(DACHEX)] (4), cis,trans,cis-[Pt(OXA)(AcO)(Cl)(DACHEX)] (5), cis,trans,cis-[Pt(OXA)(OH)Cl(DACHEX)] (6). All complexes were characterized by multinuclear NMR, X-ray photoelectron spectroscopy (XPS) to determine Pt chemical oxidation state, and also by cyclic

voltammetry (CV) to evaluate the possibility of reduction in

vivo. Moreover, the six compounds were tested in vitro on human A431 and 2008 carcinoma cell lines, showing to be much more effective than the reference compounds cisplatin and oxaliplatin.

References

[1] M. D. Hall, T.W. Hambley. Coord. Chem. Rev., 2002, 232, 49-67. [2] P. Papadia, V. Gandin, A. Barbanente, A.G. Ruello, C. Marzano, K. Micoli, J.D. Hoeschele, G. Natile, N. Margiotta. RSC Advances, 2019, 9, 32448-32452.

Figure 1. Sketches of the new six Pt(IV) prodrugs oxaliplatin analogues.

33

Copper Coordination Compounds Conjugated to Gold Nanoparticles as Innovative Anticancer Drugs: Structural Investigation Carried Out by Synchrotron Radiation-Induced Techniques.

Chiara Battocchio1, Irene Schiesaro1, Iole Venditti1, Maura Pellei2, Carlo Santini2, Luca Bagnarelli2, Giovanna Iucci1, and Carlo Meneghini1

1Dept. of Science, University of Roma Tre, Viale G. Marconi 446, Rome, Italy. Irene.schiesaro@uniroma3.it

2School of Science and Technology, Chemistry Division, University of Camerino, Camerino (MC), Italy.

Chiara Battocchio, e-mail: chiara.battocchio@uniroma3.it In recent years, the biomedical research of new metal-based anticancer drugs alternative to Pt(II) based complexes, has been extended to Au, Ru, and Cu metals. In particular, novel Cu-based antitumor agents have been studied according to the view that endogenous metals may be less toxic toward normal cells with respect to cancer ones. The synthesis strategy utilizes ligands having soft donor atoms such as aromatic sp2 hybridized nitrogen of pyrazolyl derivatives but these coordination compounds have low solubility in aqueous medium, making necessary to design specific approach for drug delivery. Conjugating the copper complexes with hydrophilic gold nanoparticles appears a promising way, suitable to improve their solubility and stability in water, and consequently raising their bioavailability. Furthermore, these complexes seem to provide slow and controlled release of copper complexes, making them suitable for targeted therapies. In this context, we investigated the molecular and electronic structure of a selection of Cu(II)-coordination compounds as pristine or bonded to Au NPs, exploiting SR-XPS and Cu-K edge XAFS spectroscopy in order to understand the electronic state and local coordination chemistry around Cu as a function of molecular complexes. The Cu K-edge XAFS data were analysed in the near edge (XANES) and extended (EXAFS) regions. The pristine ligands were also studied for sake of comparison. The combined use of complementary probes (XPS, XANES, EXAFS) is providing an accurate and reliable understanding of local coordination chemistry and electronic structure of Cu(II)-coordination compounds which appear elementary building blocks suitable to realize nanoassemblies by conjugation with hydrophilic AuNPs, along the route already successfully tested by some of us on model systems. Here the results of multidisciplinary characterization of Cu(II)-coordination compounds and preliminary results on coordination compound/AuNPs interactions will be presented, discussing the further steps for the research.

34

Potassium cation self-assembles Guanosine quadruplexes: a new drug delivery system ?

Alessia Belloni, Alessia Pepe, Paolo Moretti, Paolo Mariani

Department of Life and Environmental Sciences, Università Politecnica delle Marche. Corresponding Author, a.belloni@pm.univpm.it

Guanosine derivatives (Fig 1A), as Guanosine monophosphate (GMP), self-assemble in specific stable structures: the basic building blocks are quartets of guanosine (Fig 1B) formed through non-canonical Hoogsteen hydrogen bonds. In the presence of potassium ions, G-quartets self-assemble each other to form long 4-stranded helices called quadruplexes (Fig 1C). G-quadruplexes show an extended polymorphism, with the formation of liquid crystalline columnar structures and hydrogels able to trap large amount of water. Self-assembled guanosine hydrogels show very peculiar properties as softness, assembly/disassembly, reversibility, adaptability to environmental changes (such as pH, temperature, enzymatic activity, ionic strength), responsiveness and self-healing. They have been suggested as novel biomaterials for drug delivery applications. In this work, the structural properties of guanosine hydrogels (prepared by mixing GMP and guanosine), have been characterized by SAXS and AFM, both with and without model proteins and intercalating dyes. Moreover, the possibility to enhance the stability of hydrogels has been considered, by exploiting the self-synthesizing properties of GMP induced by EDC, a non-enzymatic polymerizing compound able to produce a phosphodiester bond between two GMP molecules. Results show the formation of stable hydrogels, formed by a 3D networks of G/GMP quadruplexes (Fig 1 D-E-F), even in the presence of high concentration of dyes and proteins. Furthermore, the quadruplex flexibility is observed to strongly depend on EDC activity. The control of quadruplex rigidity, and then of the hydrogel stability, opens new possibilities for biomedical and biotechnological applications.

References[1] Dash J, Patil AJ, Das RN, Dowdall FL, Mann S. Supramolecular hydrogels derived from silver ion-mediated

self-assembly of 5’-guanosine monophosphate. Soft Matter (2011) 7:8120–8126. [2] Carducci F, Yoneda JS, Itri R, Mariani P. On the structural stability of guanosine- based supramolecular

hydrogels. Soft Matter (2018) 14: 2938–2948. [3] Nava G, Carducci F, Itri R, Sakamoto Yoneda J, Bellini T, Mariani P. Quadruplex knots as network nodes:

nano-partitioning of guanosine derivates in supramolecular hydrogels. Soft Matter (2019) 15: 2315.

Fig. 1: A Guanosine (Gua) and Guanosine5’-monophosphate (GMP); B G/GMP-quartets; C G/GMP-quadruplexes; D AFM image of hydrogel; E with knots; Fadhesiveness of Gua-doped G-quadruplexes.

' D W �

µ

D)

A B C

E F

35

Mechanistic aspects of the binding to biosubstrates of Au(I)L2/Ag(I)L2 where L = NHC-anthracenyl ligand Francesca Binacchi,1 Maria Giulia Fabbrini,2 Damiano Cirri,2 Alessandro Pratesi,2 Tiziano Marzo,3 Luigi Messori,2 Chiara Gabbiani,1 Tarita Biver,3,1

1 Department of Chemistry and Industrial Chemistry, University of Pisa, Via G. Moruzzi 13, 56124 Pisa, Italy. 2 Laboratory of Metals in Medicine (MetMed), Department of Chemistry “U. Schiff”, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Italy. 3 Department of Pharmacy, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy.

Corresponding Author, e-mail: francescabinacchi@hotmail.com In 1929, Forestier identified the utility of gold in the treatment of rheumatoid arthritis (RA); later, the well-known orally active gold(I)–phosphine–thiolate complex, Auranofin, was developed to treat RA in a clinical setting [1]. There have recently been extensive studies on the therapeutic applications of gold complexes as anticancer agents [2,3]. Most previously reported anticancer gold(I) complexes contain a thiolate group and/or a phosphine ligand but the NHC ligand has emerged as a promising ligand [4]. Also silver(I)-NHC systems have been tested, and recent reports underline an antitumor activity comparable and, in some cases, greater than that of gold carbenes [5]. We report here on some of our recent results on the biological activity of the Au(I)/Ag(I) – NHC –anthracenyl fluorescent complexes shown in the Figure. The solution behavior of these complexes and some mechanistic details on their interaction with biosubstrates as DNA, RNA, G-quadruplex and BSA were analysed. To this aim ESI-MS experiments, spectrophotometric and spectrofluorometric titrations, melting assays and viscometric tests were done. The results enlighten differences in the binding features depending on the metal center.

Riferimenti

[1] Che C.-M., R. Sun, W.-Y., Therapeutic applications of gold complexes: lipophilic gold (III) cations and gold (I) complexes for anti-cancer treatment. Chemical Communications. 2011; 47, 9554–9560.

[2] Nobili S., Mini E., Landini I., Gabbiani C., Casini A., Messori L., Gold compounds as anticancer agents: chemistry, cellular pharmacology, and preclinical studies. Medicinal Research Reviews. 2010; 30, 550–580.

[3] Romero-Canelon I., Sadler P. J., Next-generation metal anticancer complexes: multitargeting via redox modulation. Inorganic Chemistry. 2013; 52, 12276–12291.

[4] Hussaini S. Y., Haque R. A., Razali M. R., Recent progress in silver (I)-, gold (I)/(III) and palladium (II)-N-heterocyclic carbene complexes: A review towards biological perspectives. Journal of Organometallic Chemistry. 2019; 882, 96–111.

[5] Li Y., Liu G.-F., Tan C.-P., Ji L.-N., Mao Z.-W., Antitumor properties and mechanisms of mitochondria-targeted Ag (I) and Au (I) complexes containing N-heterocyclic carbenes derived from cyclophanes. Metallomics. 2014; 6, 1460–1468.

Figura 1. Molecular structure of the Ag(I)/Au(I)-NHC-anthracenyl complexes.

36

Divalent metal ions influence cathechins and flavonoids’ encapsulation in liposomal nanovectors Mattia Cantarini,1 Emiliano Laudadio2, Cristina Minnelli1, Giovanna Mobbili1, Roberta Galeazzi1

1 DISVA, Università Politecnica delle Marche, via Brecce Bianche, 60131, Ancona 2 SIMAU, Università Politecnica delle Marche, via Brecce Bianche, 60131, Ancona

Corresponding Author, e-mail: m.cantarini@pm.univpm.it; r.galeazzi@univpm.it Cathechins and flavonoids are associated to numerous health benefits. Epigallocatechin 3-Gallate (EGCG), from green tea extracts, and morelloflavone (MF), from Garcinia dulcis, are two of the most representatives’ compounds for their antioxidant and known therapeutic effects. In this research study, we investigated by full atoms Molecular Dynamics simulations, how EGCG and MF interact with lipid bilayers and we found out divalent metal cations influence on their encapsulation degree in neutral liposomes. In particular, we observed that EGCGs naturally bind to the hydrophilic regions of phospholipids, positioning themselves mostly at the interface between water and lipid phases. The presence of divalent cations clearly influences the EGCG molecules’ absorption and the total effect depends strongly on the metal nature and concentration. Beside, for MF, we observed a high stability of the intermolecular MFs aggregates in water that strongly penalizes the flavonoid’s interaction with the lipid polar heads. However, metal cations can influence MF's liposomial penetration, even if they are not able to promote completely its absorption inside the bilayer. For both compounds, the increase of penetration is more marked in presence of magnesium chloride, whilst calcium chloride showed the opposite effect.

References

[1] Minnelli, C., Moretti, P., Fulgenzi, G., Mariani P., Laudadio E., Armeni T., Galeazzi, R., Mobbili, G., A Poloxamer-407 modified liposome encapsulating epigallocatechin-3-gallate in the presence of magnesium: Characterization and protective effect against oxidative damage, International Journal of Pharmaceutics, 2018,552(1-2), 225-234.

[2] Laudadio E., Mobbili G., Minnelli C., Massaccesi L., Galeazzi R., Salts influence cathechins and flavonoids encapsulation in liposomes: a molecular dynamics investigation, Molecular informatics, 2017, 36 (11), 1700059.

Figure 1. Morelloflavone penetration as an intermolecular cluster at 0.15 M

MgCl2; the lipid bilayers are represented as transparent surface model, magnesium ions as cyan spheres and MFs in ball and stick representation; the

focus highlights the intermolecular interactions between MF molecules.

37

Photoactivated aqueous nanocolloids of Platicur: NMR metabolomic study of treated HeLa cancer cells Federica De Castro,1 Viviana Vergaro,1,2 Michele Benedetti,1 Francesca Baldassarre,1,2 Laura Del Coco, 1 Maria Michela Dell’Anna,3 Piero Mastrorilli,3 Giuseppe Ciccarella, 1,2 Francesco Paolo Fanizzi.1

1 Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento & UdR INSTM di Lecce, Via Monteroni, I-73100 Lecce, Italy 2 CNR NANOTEC - Istituto di Nanotecnologia c/o Campus Ecotekne, Università del Salento, Via Monteroni, 73100 Lecce, Italy 3DICATECh, Politecnico di Bari, via Orabona 4, 70125, Bari, Italy

Corresponding Author, e-mail: fp.fanizzi@unisalento.it Platicur is a cis-diamine-platinum(II) complex linked to curcumin. Previous studies have shown that the photoactivation of Platicur, with visible light, is responsible for the production of active species of platinum(II) and a photoactive curcumin. The active species formed act synergistically causing an interesting anticancer activity. [1] In this work, we tried to improve the antitumor activity of photoactivated Platicur by using a nano delivery system. With this aim, Platicur's aqueous chitosan nanocolloids were synthesized, Figure 1. The preliminary in vitro biological assays on HeLa tumor cell line have demonstrated a significantly higher cytotoxicity (a significantly lower IC50 dose) of the photoactivated nanocolloids with Platicur, compared to the photoactivated Platicur not encapsulated in the nanocolloids. The use of a metabolomic approach based on NMR spectroscopy in the research of the mechanism of action or for the evaluation of the tumor response to the anticancer metal drugs is a new tool of recent growth. The side effects induced by metal based drugs, the prediction of the response to treatment and the key information on the mechanism of action (for known and new compounds) could be easily obtained using NMR-based metabolomics. [2, 3] For this reason, the 1H NMR spectroscopy coupled with multivariate statistical analysis was used to characterize the metabolic variations of intracellular metabolites and the compositional changes of the corresponding culture media of HeLa cells treated with photoactivated Platicur nanocolloids. The metabolomic analysis allowed to highlight specific metabolic differences between the cells treated with photoactivated Platicur nanocoloids compared to non-photoactivated ones. The NMR has once again proved to be a valid tool for the study of the mechanism of action of metal-based drugs. References

[1] Mitra K., Gautam S., Kondaiah P., Chakravarty A. R.. The cis-Diammineplatinum(II) Complex of Curcumin: A Dual Action DNA Crosslinking and Photochemotherapeutic Agent. Angew Chem Int Ed Engl. 2015; 54 (47): 13989-13893.

[2] De Castro F., Benedetti M., Antonaci G., Del Coco L., De Pascali A. S., Muscella A., Marsigliante S., Fanizzi P. F.. Response of Cisplatin Resistant Skov-3 Cells to [Pt(O,O′-Acac)(γ-Acac)(DMS)] Treatment Revealed by a Metabolomic 1H-NMR Study. Molecules. 2018, 23 (9): 2301/1-2301/21.

[3] De Castro F., Benedetti M., Del Coco L., Fanizzi P. F.. NMR-Based Metabolomics in Metal-Based Drug Research. Molecules. 2019, 24(12):2240/1-2240/14.

Figure 1. Chitosan-nanocolloids of Platicur

38

Cysteine-Functionalized Self-Assembling Peptides on Gold: the Role of Head and Tail Giovanna Iucci,1 Valeria Secchi,1 Iole Venditti,1 Monica Dettin,2 Annj Zamuner,2 Stefania De Rosa,3,4 Luca Tortora,1,3 Valeria Secchi,1 Chiara Battocchio,1

1 Department of Science, Roma Tre University, Via della Vasca Navale 79, 00146 Rome, Italy 2 Department of Industrial Engineering, University of Padua, Via Marzolo, 9, Padua, 35131, Italy 3 Surface Analysis Laboratory INFN Roma Tre, via della Vasca Navale 84, 00146, Rome, Italy

4 Department of Mathematics and Physics, Roma Tre University, via della Vasca Navale 84, 00146, Rome

Corresponding Author, e-mail: giovanna.iucci@uniroma3.it Molecular self-assembling consists in the spontaneous aggregation of molecules in a well-defined structure, guided by non-covalent bonds. Self-assembling is pervasive in nature and has recently been proposed as a biomimetic strategy in tissue engineering. In this context, our goal is to design and test innovative but simple chemical strategies to effectively modify surfaces, exploiting small modifications in the bioactive molecules, such as the introduction of L-cysteine (Cys) as a terminal residue in self-assembling peptides (SAP). These peptides are capable of organizing spontaneously in nanostructures both in solution and in thin films on surfaces of biocompatible materials. The insertion of a cysteine residue in the amino-terminal (Cys-SAP), carboxy-terminal (SAP-cys) position or in both positions (cys-SAP-Cys) in the peptide backbone allows the covalent anchoring of the self-assembling peptides on the gold surface. Thin films of SAP covalently immobilized on gold surfaces were investigated using ATR-FTIR (Attenuated Total Reflection Fourier Transform InfraRed), SR-XPS (Synchrotron Radiation-induced X-ray Photoelectron Spectroscopy), NEXAFS (Near Edge X-ray Absorption) Fine Structure spectroscopy) and TOF-SIMS (Time of Flight Secondary Ion Mass Spectrometry) in order to determine the structure and thickness of the peptide overlayer and the molecular order and the orientation of the peptide chains with respect to the gold surface. The ultimate goal of our research is to achieve effective chemical functionalization of surfaces by anchoring biomolecules in order to create innovative bioactive materials for applications in the field of tissue engineering.

Riferimenti[1] Secchi V, Franchi S, Santi M, Dettin M, Zamuner A, Iucci G, Battocchio C. Self-assembling behavior of

Cysteine-Modified Oligopeptides on Gold: an XPS and NEXAFS Study. J. Phys. Chem. 2018; 122: 6236-6239

[2] Secchi V, Iucci G, Dettin M, Zamuner A, De Rosa S, Tortora L, Battocchio C. Cysteine-Modified Self-Assembling Peptides on Gold: the Role of Head and Tail. Langmuir 2019; 35: 16593-16604

Figura 1. Structure of Cys-SAP on gold.

39

Metal ions effect on stabilization of a lipophilic edaravone derivative in liposome for ophthalmic uses. Emiliano Laudadio,1 Cristina Minnelli,2 Mattia Cantarini,2 Giovanna Mobbili2, Roberta Galeazzi2, Pierluigi Stipa1 1 Dipartimento di Scienze e Ingegneria della Materia, dell'Ambiente ed Urbanistica (SIMAU), Università Politecnica delle Marche, Ancona 2 Dipartimento di Scienze della Vita e dell’Ambiente (DiSVA), Università Politecnica delle Marche, Ancona

Corresponding Author: e.laudadio@univpm.it This study concerns the design of an innovative product, which could be used as part of a nanovector drug delivery system in ocular drops [1], able to deal effectively and for a long time with free radicals at the ocular surface. Such objective is of great interest in the pharmaceutical field because free radicals are responsible for the processes of aging and cell death of the ocular tissues. The innovation of our research involves the functionalization of the liposome binding the new antioxidant molecule directly to the lipid molecule which thus becomes itself a constituent of the liposomal nanovector [2] and not only incorporated inside. Among the pool of known antioxidant molecules, we chosen edaravone (EDR), which is known to have high antioxidant activity acting as radical scavenger. Thus, an aliphatic chain of 18 carbons (C18) have been added to the molecule, in order to increase its lipophilicity. We used an in silico approach based on molecular dynamics (MD) simulations to predict the EDR-C18 conformational behaviour and stability in lipid bilayer system with mixed composition. We found that edaravone derivative presents a high conformational freedom within the lipidic medium, without affecting the physical properties of the liposomal vector. The main aim is to design a liposomal vector in which the EDR-C18 can be inserted to protect its surface shielding free radicals outside the lipid system. Thus, we investigated the effect of metal ions on the formulation, in order to promote the positioning of EDR-C18 polar head within the aqueous layer. In details, we carried out MD simulations adding NaCl, KCl, CaCl2 and MgCl2. Indeed, the salt played an important role in stabilizing of EDR derivative within the bilayer; comparing the resulting simulated models in absence and in presence of NaCl, we found a greater cluster aggregation for the functionalized molecule in the latter models, due to the Na+ ions, which interacted directly in a -cation association with EDR-C18 aromatic moiety. Such interaction was even more evident in the presence of CaCl2. Since we found that the divalent cations Ca++ are maintained broadly more close to the heads of EDR-C18, compared to the Na+ ions. As a consequence, it results a greater stabilization of the complex, and that hence induced a more ordered cluster organization of the antioxidant compound within the bilayers leaflets. References[1] Patel A, Cholkar K, Agrahari V, Mitra AK. Ocular drug delivery systems: An overview. World J Pharmacol.

2013;2(2):47-64. [2] Mufamadi M. S., Pillay V., Choonara Y. E., Du Toit L. C., Modi G., Naidoo D., Ndesendo V. M. K., J.Drug

Delivery, 2011, 1-19; Kulkarni P.R., Yaday J.D., Vaidya K.A., Int.J.Curr.Pharm.Res., 2011, 3, 10-16.

Figure 1. POPC-EDRC18 system. The figure shows Ca++ and Cl- ions (blue and gold VdW spheres respectively), POPC (transparent surface), EDRC18 (in green sticks, hydrogen atoms hidden), and water (transparent red and white VdW spheres).

40

An insight on the BLBC anticancer activity of azolate phosphane gold(I) compounds.

Lorenzo Luciani,1 Rossana Galassi,1 Alfredo Burini,1 Augusto Amici,2 Cristina Marchini,2 Junbiao Wang,2 Silvia Vincenzetti,3 Stefania Pucciarelli,3 Giulio Lupidi3 1 School of Science and Technology, University of Camerino, Via Sant’Agostino 1, Camerino, I-62032, Italy 2 School of Drugs and Health Products Sciences, University of Camerino, Piazza dei Costanti, 4, Camerino, I-62032, Italy 3 School of Biosciences and Veterinary Medicine, University of Camerino, Via Gentile III da Varano, Camerino, I-62032, Italy

Corresponding Author, e-mail: lorenzo.luciani@unicam.it Azolate phosphane gold(I) compounds, in example 4,5-dichloro-imidazolate-1H-gold(I)-(triphenylphosphane) 4,5-dicyano-imidazolate-1H-gold(I)-(triphenylphosphane) and 3,5-dinitro-pyrazolate-1H-gold(I)-(triphenylphosphane), have been found to be cytotoxic on the regards of several cancer lines and also against cisplatin or Multi-Drug resistant cancer cells.[1] SKBR3, A17 and MDA-MB231 cells are breast cancers models corresponding to different phenotypic features and then different therapy. Herein, MTT tests on SKBR3, MDA-MB231 and A17 cells with the three selected azolate phosphane gold compounds were performed. They show IC50 values ranging from 5.49 ±0.04 M for the most active compound to 28.66 ± 0.02 M for the least successful match of cell line and gold(I) compound. Moreover, as an ongoing study on the inhibitory effect of the tested gold(I) compounds,[2] the residual enzymatic activity of dihydrofolate reductase from E. coli (DHFR) has been measured in cells treated with methotrexate (MTX, which is a DHFR’s classic inhibitor), or with these gold complexes within 12 hours upon treatment in comparison with control cells. The activity of DHFR was reduced in A17 cells from 4,5-dicyano-imidazolate-1yl-gold(I)-(triphenylphosphane) and 4,5-dichloro-imidazolate-1yl-gold(I)-(triphenylphosphane) to 64% and 51%, respectively; interestingly this effect was higher than the one exerted by MTX (Figure 1). Noteworthy, despite the strong cytotoxic activity of gold complexes in human MDA cells, their effect did not result in a strong inhibition of DHFR activity. To gain additional information, fluorimetric studies on the binding of these gold complexes with calf thymus DNA (ct-DNA), bovine serum albumin (BSA) and apotransferrin (ATF) remarked, as expected, a weaker interaction with DNA with respect to an appreciable linking to both proteins, affording to a partial explanation for the cellular toxicity. References

[1] Galassi, R. et al Synthesis and characterization of azolate gold(i) phosphane complexes as thioredoxin reductase inhibiting antitumor agents, Dalton Trans, (2012) 41 (17), pp. 5307-5318. DOI: 10.1039/c2dt11781a

[2] Pucciarelli, S. et al Studies on the Interaction between Poly-Phosphane Gold(I) Complexes and Dihydrofolate Reductase: An Interplay with Nicotinamide Adenine Dinucleotide Cofactor. Int. J. Mol. Sci., 2019 20, 1802; DOI:10.3390/ijms20071802

Figure 1. Plot of DHFR residual enzymatic activity measured in breast cancer cell lines after treatment

with methotrexate and gold complexes 4,5-dicyano-imidazolate-1yl-gold(I)-(triphenylphosphane) and

4,5-dichloro-imidazolate-1yl-gold(I)-(triphenylphosphane)

41

Renium(I) complexes of the type [(phen)Re(CO)3(L)]: role of the ligand L on

antiproliferative activity and drug resistance.

Lorenza Marvelli,1 Paola Bergamini,1 Mariafrancesca Hyeraci,2 Lisa Dalla Via2 1 Department of Chemical and Pharmaceutical Sciences, University of Ferrara, Italy 2 Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Italy

The request for cancer therapies progressively more effective and selective looks towards

organometallic compounds as promising anti-cancer drug candidates. In this context, rhenium

compounds can display interesting multimodal properties, forming complexes with suitable

characteristics as luminescent probes for cell

imaging or as radio-imaging and

pharmaceutical agents; the radioactive 186/188Re compounds are presently used in

clinics for cancer treatment.

So far, cytotoxicity data have been reported

almost exclusively for Re(I) organometallic

complexes, containing the chemically robust

and easily accessible fac-{Re(CO)3} moiety,

whose properties can be modulated

introducing appropriate ligands in the three

residual coordination positions. Cytotoxicity

equalling or exceeding that of the leader anti-

cancer drug cisplatin was observed for several

compounds of this type.

With the aim of investigating this aspect of rhenium chemistry, we chose [(phen)Re(CO)3L]0/+ as

a base-structure with phenanthroline as chromophoric moiety, while the ligands L = chloride (1)

or methanol (2) moiety, present in the starting complexes, have been easily replaced with

molecules containing an O-donor group. In this way we introduced betaine and carnitine

(provided of a quaternary nitrogen which could favour an interaction with the DNA poly-anion)

and some carboxylate R-COO- (e.g. dichloroacetic acid (DCA) or salicylic acid).

All the Re-derivatives were obtained in good yield and characterized by NMR and IR

spectroscopy, elemental analysis, mass spectrometry and X-ray analysis for some derivatives.

Preliminary tests revealed a good luminescence efficiency, a relatively high value of the life time

and a large Stokes shift, desirable characteristics for luminescent markers.

The antiproliferative activity was assayed on three human tumour cell lines: HeLa (cervix

adenocarcinoma), A2780 (ovarian carcinoma) and A2780cis (cisplatin-resistant ovarian

carcinoma). A detectable antiproliferative activity, characterised by GI50 values ranging from

2.27 µM to 23.8 µM, has been found for all the derivatives, and the complex

[(phen)Re(CO)3(MeOH)]NO3 (2) has been identified as the most active on all cell lines.

Interestingly, all the tested Re-derivatives demonstrated a comparable cytotoxicity on both

cisplatin-sensitive and cisplatin-resistant cells. This result indicates that the new complexes

overcome the resistance phenomenon, suggesting that their antiproliferative effect could be

related to a different intracellular mechanism from cisplatin. In this connection, the biological

events responsible for the antiproliferative effect were deeply investigated.

References

[1] Hostachy S., Policar C., Delsuc N. Re(I) carbonyl complexes: Multimodal platforms for inorganic chemical

biology. Coordination Chemistry Reviews. 2017; 351: 172–188.

[2] Varma R.R., Pursuwani B.H., Suresh E., Bhatt B.S, Patel M.N. Single crystal, DNA interaction and

cytotoxicity studies of rhenium(I) organometallic compounds. Journal of Molecular Structure. 2020; 1200,

127068.

Figure 1. General formula and a molecular

structure of Re(I) organometallic complexes.

42

Metal Complexes of Cyclodextrins modulate the protein aggregation Valentina Oliveri,1 Graziella Vecchio1

1 Dipartimento di Scienze Chimiche, Università degli Studi di Catania, A. Doria 6, 95125, Catania, Italia Corresponding Author, e-mail: valentina.oliveri@unict.it

A number of human diseases, including Alzheimer’s disease (AD) and Parkinson's disease (PD), is suspected to be directly related to protein aggregation. Amyloid protein aggregates and oligomeric intermediates of Aβ and α-synuclein are observed in AD and PD and considered to be mediators of cellular toxicity. Hence, these proteins have seen as leading, and most compelling targets and numerous efforts to identify chemical entities able to modulate protein aggregation pathways have been made.1 Cyclodextrins (CDs), cyclic oligosaccharides, reduce Aβ fibrillogenesis and Aβ‐induced toxicity. Nevertheless, the binding affinity between CDs and Aβ is low to make them effective inhibitors.2 Therefore, CDs have been functionalized by us to obtain more active systems in inhibiting protein aggregation. We found that the conjugation with porphyrins and quinolines improves the antiaggregant ability and the neuroprotective effects of CDs.3,4 On the other hand, transition metal complexes have also been designed and investigated as a class of chemical modulators against protein aggregation.5 In particular, Pt(II) and Co(III) complexes have shown to coordinate Aβ and perturb the intramolecular and intermolecular interactions of Aβ peptides. 5 This context inspired us to evaluate metal complexes of CD derivatives as inhibitors of Aβ and α-synuclein aggregation. In particular, we found that platinum(II) complexes of aminocyclodextrins can modulate the protein aggregation at micromolar concentrations, as demonstrated by several techniques, including circular dichroism and dynamic light scattering measurements. The data suggest that platinum complexes act at the early stages of the protein aggregation suppressing the fibrillogenesis. The obtained results highlight the potential of metal complexes of CD derivatives as active agents in amyloid-related pathologies.

References

[1] Oliveri V. Toward the discovery and development of effective modulators of α-synuclein amyloid aggregation. European journal of medicinal chemistry. 2019; 167:10-36.

[2] Oliveri V, Vecchio G. Cyclodextrins as protective agents of protein aggregation: an overview. Chemistry–An Asian Journal. 2016; 11: 1648-1657.

[3] Oliveri V, Zimbone S, Giuffrida ML, Bellia F, Tomasello MF, Vecchio G. Porphyrin Cyclodextrin Conjugates Modulate Amyloid Beta Peptide Aggregation and Cytotoxicity. Chemistry-A European Journal. 2018; 24: 6349-6353.

[4] Oliveri V, Bellia, F, Vecchio G. Cyclodextrin Nanoparticles Bearing 8‐Hydroxyquinoline Ligands as Multifunctional Biomaterials. Chemistry-A European Journal. 2017; 23: 4442-4449.

[5] Suh JM, Kim G, Kang J, Lim MH. Strategies Employing Transition Metal Complexes To Modulate Amyloid‑β Aggregation. Inorganic Chemistry. 2019; 58: 8-17.

43

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� � � � � � � � � k � PdD� px--PdsEx. y� , dEEePyA� . -� i G:g� , hdAeA�� E. , � � dyp� � o � � b � xy� Aes. yp� pePx2dEx2e� � i . EE. f � � �

�

44

Manganese complexes for multimodal PET/MRI imaging

Micol Pasquali1, Alessandra Boschi

2, Petra Martini

1, Lorenza Marvelli

2, Erika Marzola

2, Juan

Esposito1, Adriano Duatti

2

1 National Institute of Nuclear Physics, National Laboratories of Legnaro (INFN – LNL), Legnaro (PD) Italy

2 Department of Chemical and Pharmaceutical Sciences, University of Ferrara, Italy

Corresponding Author, e-mail: psqmcl@unife.it

Hybrid imaging has been emerging as a new important tool for improving the diagnostic power of

nuclear imaging through the combination of Positron Emission Tomography (PET) and Magnetic

Resonance Imaging (MRI). This approach exploits the intrinsic differences between the two

imaging modalities, which adopt different

physical processes to collect the diagnostic

information and, therefore, generate images

fundamentally different in nature. Typically,

MRI allows for obtaining anatomical images of

organ tissues, whereas PET is able to deeply

penetrate the inner cellular structure and collect

molecular-type information. Thus, by

combining images having such a diverse

diagnostic content, a deeper understanding of

the clinical picture could be obtained. However,

it should be noted that there exists an

inescapable diagnostic mismatch between PET

and MRI originating from the diversity between

the chemical composition of the paramagnetic contrast and radioactive agents used with these

imaging modalities, respectively. This makes hard to achieve a true molecular fusion between

information from nuclear and magnetic resonance signals.

The aim of the ongoing research program METRICS (acronym for Multimodal pET/mRI imaging

with Cyclotron-produced 51/52

Mn iSotopes), funded by the INFN fifth National Scientific

Committee (CSN5), is to develop an experimental strategy to attain a genuine molecular fusion

between PET and MRI. This strategy is based on the simple hypothesis that this result can be

achieved when the contrast and the radioactive agents share the same chemical structure. Since the

transition element manganese possesses stable paramagnetic isotopes as well as a couple of

positron-emitting radioisotopes, namely 52

Mn and 51

Mn, it turns out that it constitutes an ideal

candidate for pursuing the scope of the METRICS project. Clearly, the use of chemically identical

cold and radioactive Mn compounds may afford a perfect matching between the biomolecular

properties of the two imaging probes.

With the aim of obtaining stable Mn (II) complexes with paramagnetic properties, the reactivity

of the Mn2 +

ion towards various classes of bidentate ligands has been investigated. Therefore, a

series of Mn(II) complexes were prepared, in aqueous conditions, with different bidentate ligands

with the goal to identify the most robust ligand arrangement, capable of stabilizing the Mn2+

ion a

biological environment. Along with stability, the pharmacokinetic properties of these complexes

are currently under analysis, in order to isolate derivatives that could be able to mimic the behavior

of perfusion imaging agents widely employed in nuclear scintigraphy. The isolated compounds

were characterized by HPLC chromatography, IR spectroscopy, elemental analysis and mass

spectrometry.

[1] Costa R. O. et al., A new mixed-valence Mn(II)Mn(III) compound with catalase and superoxide dismutase activities. Front. Chem. 2018; 6, 1–18 Costa, R. O. et al. A

new mixed-valence Mn(II)Mn(III) compound with catalase and superoxide dismutase activities. Front. Chem. 6, 1–18 (2018).

[2] Lewis C., Graves S. et al., 52Mn Production for PET/MRI Tracking Of Human Stem Cells Expressing Divalent Metal Transporter 1 (DMT1). Theranostics. 2015. 5(3),

227-239.

Fig 1. Mn has isotopes with positron-emitting nuclear

properties and with paramagnetic properties useful for

hybrid PET/MRI analisys

45

Chemistry and antitumor investigations of new Cu(I) complexes of

bis(pyrazol-1-yl)acetate ligands functionalized with an NMDA

receptor antagonist Maura Pellei,1 Carlo Santini,1 Luca Bagnarelli,1 Lorenzo Luciani, 1 Fabio Del Bello,2 Wilma Quaglia, 2 Cristina Marzano,3 Valentina Gandin3

1 School of Science and Technology, Chemistry Division, University of Camerino, Italy 2 School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, Italy 3 Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Italy

Corresponding Author, e-mail: maura.pellei@unicam.it Copper complexes are coming out as metal-based drug candidates for the treatment of cancer, due to their wide structural variability, biologically accessible redox properties and bioavailability [1]. They display broader spectra of activities and lower toxicity, thereby providing the possibility of circumventing the problems encountered by clinically approved platinum drugs. In the search for Cu-based anticancer agents, over the last decades our attention has been focused on the design and synthesis of copper complexes of bis(azol-1-yl)carboxylate heteroscorpionate ligands of general formula [HC(COOH)(az)2], with az = pyrazole or 1,2,4-triazole [2,3]. Here we report copper(I) complexes of bis(pyrazol-1-yl)carboxylic acid (LH), bis(3,5-dimethylpyrazol-1-yl)carboxylic acid (L2H) as well as of bis(pyrazol-1-yl)acetates conjugated with with the NMDA receptor antagonist 6,6-diphenyl-1,4-dioxan-2-yl)methanamine, named NMDA-ANT for briefness (LNMDA and L2NMDA) and phosphane ligands (triphenylphosphine and 1,3,5-triaza-7-phosphaadamantane, PTA) were synthesized. The selection of an NMDA antagonist for the coupling with LH and L2H was suggested by the observation that NMDA receptors are expressed an play a role in different types of cancer models. All the new complexes showed significant antitumor activity on a panel of human tumor cell lines of different histology and cisplatin sensitive or resistant or with Multi-Drug Resistant phenotype, being significantly more active than cisplatin even in 3D spheroids of H157 and BxPC3 cancer cells. Finally, morphological analysis revealed that the most representative complex [(L2NMDA)Cu(PTA)2]PF6 induced a massive swelling of the endoplasmic reticulum (ER) membrane, which is a clear sign of ER stress [4].

References

[1] Santini C, Pellei M, Gandin V, Porchia M, Tisato F, Marzano C. Advances in Copper Complexes as Anticancer Agents. Chem Rev. 2014; 114 (1): 815-862.

[2] Pellei M, Gandin V, Marchiò L, Marzano C, Bagnarelli L, Santini C. Syntheses and Biological Studies of Cu(II) Complexes Bearing Bis(pyrazol-1-yl)- and Bis(triazol-1-yl)-acetato Heteroscorpionate Ligands. Molecules. 2019; 24 (9), 1761.

[3] Morelli M B, Amantini C, Santoni G, Pellei M, Santini C, Cimarelli C, Marcantoni E, Petrini M, Del Bello F, Giorgioni G, Piergentili A and Quaglia W. Novel antitumor copper(II) complexes rationally designed to act through synergistic mechanisms of action, due to the presence of an NMDA receptor ligand and copper in the same chemical entity. New Journal Chemistry, 42 (2018) 11878-11887.

[4] Pellei M, Bagnarelli L, Luciani L, Del Bello F, Giorgioni G, Piergentili A, Quaglia W, De Franco M, Gandin V, Marzano C, Santini C. Synthesis and antitumor mechanism investigations on human pancreatic cancer cells of new Cu(I) complexes of bis(pyrazol-1-yl)acetate ligands functionalized with an NMDA receptor antagonist. Dalton Trans. 2020, submitted.

Figure 1. Chemical structure of ligands

46

From tetrahedrical homoleptic phosphino copper (I) complexes [CuP4]+ to low coordinated species: effect on the antiproliferative activity. Marina Porchia,1 Francesco Tisato,1 Cristina Marzano2, Valentina Gandin2, Michele De Franco2

1 CNR - ICMATE, Corso Stati Uniti 4, 35127 Padova, Italy

2 Department of Pharmaceutical and Pharmacological Sciences, University of Padova, via Marzolo 5, 35131 Padova, Italy

Corresponding Author, e-mail: marina.porchia@cnr.it Several tetrahedrical homoleptic phosphino copper(I) complexes of the type [CuP4]+ (P = tertiary phosphine) have shown a high antitumor activity in vitro and in vivo together with the ability of overcoming platinum and multidrug resistance.[1] Solution studies at micromolar concentration of lead [CuP4]+ compounds, by means of ESI(+)MS and XAS-EXAFS, evidenced the following speciation process: [CuP4]+

→ [CuP3]+ → [CuP2]+

→ [CuP]+ [2-3] where such coordinative vacant CuP4-n (n = 1-3) assemblies could be the entities responsible for the biological activity. Hence we thought to directly synthesize a series of low coordinated Cu(I) complexes by using appropriate phosphino ligands whose steric hindrance was suitable to stabilize di- and tri-coordinated species. Both hydrophilic (Fig. 1) [4] and lipophilic ligands [(C6H5)3P, (p-FC6H4)3P, (p-MeC6H4)3P, (p-OMeC6H4)3P)] were considered in order to modulate the solubility of the final complexes. In this study we report on the synthesis of [CuP2]+-type derivatives endowed with antiproliferative activity and able to overcome oxaliplatin and multidrug resistance. From preliminary biological data in 3D cell culture systems it comes out that complexes containing lipophilic ligands are more effective at inhibiting cancer cell proliferation. [1] Tisato F., Porchia M. ,Santini C., Gandin V., Marzano C. Phosphine-copper(I) complexes as anticancer

agents: design, synthesis and physicochemical characterization. Part I. in “Copper(I) Chemistry of Phosphines, Functionalized Phosphines and Phosphorus Heterocycles” Ed. Maravanji S. Balakrishna, Elsevier: 2019; 3: 61-82. ISBN: 978-0-12-815052-8

[2] Quaretti M., Porchia M., Tisato F., Trapananti A., Aquilanti G., Damjanovic M., Marchiò L., Giorgetti M., Tegoni M. Elucidation of thermodynamic stability and structure in aqueous solution of the [Cu(PTA)4]+ complex (PTA = aminophosphine 1,3,5-triaza-7-phosphaadamantane). J. Inorg. Biochem. 2018; 188 : 50-61.

[3] Tisato F., Marzano C., Peruzzo V., Tegoni M., Giorgetti M., Damjanovic M., Trapananti A., Bagno A., Santini C., Pellei M., Porchia M. , Gandin V. Insights into the cytotoxic activity of the phosphane copper(I) complex [Cu(thp)4][PF6] J. Inorg. Biochem. 2016; 165:80-91

[4] Starosta R., Florek M., Krol J., Puchalska M., Kochel A. Copper(I) iodide complexes containing new aliphatic aminophosphine ligands and diimines—luminescent properties and antibacterial activity. New J. Chem. 2010; 34: 1441–1449

Fig. 1. Hydrophilic phosphino ligands utilized in this study.

PN

O

N

O

N

O

PN

N

N

N

N

N

CH3

CH3

CH3

47

Synthesis, Cytotoxicity, and Preliminary Mechanistic Investigation of Platinum(IV) Anticancer Prodrugs Conjugated with Poly(ADP-ribose) Polymerase Inhibitors Mauro Ravera,1 Elisabetta Gabano,1 Beatrice Rangone,1 Domenico Osella,1 Cecilia Balzano2, Giulia Pinton2, Laura Moro,2

1 Dipartimento di Scienze e Tecnologie Avanzate, Università del Piemonte Orientale, Viale Michel 11, 15121 Alessandria (Italy) 2 Dipartimento di Scienze del Farmaco, Università del Piemonte Orientale, Via Bovio, 6, 28100 Novara (Italy)

Corresponding Author, mauro.ravera@uniupo.it There is a strong interest for Pt(IV) derivatives, obtained from cisplatin or other Pt(II) complexes by addition of two axial ligands via chemical oxidation and then conjugation. These compounds act as prodrugs being reduced (ideally) only in the hypoxic tumor microenvironment, and thus producing in situ cytotoxic cisplatin and the two axial ligands. These axial ligands can consist in one or two molecules of a second drug, synergistic or adjuvant with respect to cisplatin. The second drug can be a Poly(ADP-ribose) polymerase inhibitor (PARPi); inhibition of PARP-1, one of the member of the PARP family of enzymes, drives cytotoxicity in tumor cells lacking functional BRCA1 or BRCA2, genes involved in the homologous-recombination DNA repair. Importantly, PARPi and Pt drugs proved to have synergistic effects. Since PARPs use nicotinamide dinucleotide (NAD) as a substrate, most of the PARPi so far developed are structural analogues of NAD and are thought to compete with NAD itself at the level of the catalytic domain. Various benzamide derivatives, including cyclic conformationally constrained molecules, are shown to be PARPi, and 3-substitution with electron-donating groups generally increases the potency. In this work a series of six dual-action Pt(IV) prodrugs, either containing 3-aminobenzamide conjugated via its amide group to a Pt(IV) synthon with a spacer of various length or containing its analogue 3-aminobenzoic acid directly coordinated to the metal center, were synthesized, characterized and tested in vitro against malignant pleural mesothelioma cell lines. Funding: This research has been carried out within the HERMES project and funded by offer of compensation to the residents of Casale Monferrato died or suffering from mesothelioma (Progetto finanziato attraverso l’offerta di

indennizzo ai residenti di Casale Monferrato deceduti o affetti da mesotelioma).

Figure 1. Sketch of the complexes under investigation.

48

A Focal Plane Array – Fourier Transform-Infrared (FPA-FTIR)

spectroscopic study on drugs penetration in a model spheroid treated with

cisplatin derivatives

Simona Sabbatini1, Carla Conti1, Chiaro Pro2, Alessia Belloni2, Valentina Notarstefano2,

Maurizio Sabbatini3, Aldo Arrais3, Flavia Caprì3, Ilaria Zanellato3, Domenico Osella3 and

Elisabetta Giorgini2

1. Dipartimento di Scienze e Ingegneria della Materia, dell’Ambiente ed Urbanistica (SIMAU), Università

Politecnica delle Marche, via Brecce Bianche, 60131 Ancona, Italy.

2. Dipartimento di Scienze della Vita e dell’Ambiente (DISVA), Università Politecnica delle Marche, via

Brecce Bianche, 60131 Ancona, Italy.

3. Dipartimento di Scienze e Innovazione Tecnologica (DISIT), Università del Piemonte Orientale, Alessandria,

Italy.

Corresponding Author, e-mail: s.sabbatini@staff.univpm.it

Focal Plane Array – Fourier Transform-Infrared (FPA-FTIR) spectroscopy has found application

in exploring the numerous and complex molecular changes occurring in tumoral tissue [1].

We have used the high potentiality of this technique, to analyze at cellular level, the

macromolecular changes occurring in Malignant Pleural Mesothelioma (MPM) spheroids, a 3D

cellular organization mimicking in vivo solid tumours [2]. We have evaluated the molecular effects

due to treatments with cisplatin and Pt(IV)-OA, a new compound Pt(IV)-based derivative, having

-OH and acetate as axial ligand. Meaningful molecular markers of induced toxicity such as

phosphatidyl-inositol, arachidonic acid and DNA integrity, were explored in terms of distribution

and presence in spheroid cryo-sections. Linear

discriminant analysis (LDA) performed on total

spectra of cells showed, for control groups (CTRL),

a segregation along LD2 axis of spectral profile

between outer and inner part of spheroid. On the

contrary, cisplatin experimental group (cisPt)

displayed a faintly segregation. Furthermore, the

scores plot clearly showed a segregation along LD1

axis of CTRL (IN and OUT) groups and cisPt (IN

and OUT) ones. These signals confirmed that

cisplatin affected the outer and inner part of cell in

a similar way and attested its good permeability and

its pharmacological effect. However, the treatment

with Pt(IV)-OA was more invasive: spectra from

PtIV experimental group were completely different

from all the others and were located in the negative

part of LD2 axis (Fig.1). Concerning spectral

profiles, phosphatidyl-inositol (band at 1080 cm-1) and arachidonic acid (band at 3062 cm-1) are

widely involved in metabolic pathways and defensive cellular response. Their spectral trends

suggested a toxic effect of both treatments, even if the reduction of arachidonic acid was more

evident in Pt(IV) experimental group. The vibrational bands of apoptosis (1040 and 1120 cm-1),

associated with the increase in amino acids residues and the decrease of polysaccharides, were

evident with both treatments. We suppose that Pt(IV)-OA could be more aggressive on tumoral

cells, because it induced biomolecular changes into spheroid cells affecting their morphological

integrity.

References

[1] Tosi et al. Microimaging FT-IR of head and neck tumors. V. Odontogenic cystic lesions. Vibrational

Spectroscopy. 2011; 57: 140–147

[2] Minchinton AI and Tannock IF. Drug penetration in solid tumours. Nat Rev Cancer. 2006; 6: 583–592.

.

Figura 1. LD1 and LD2 represent the first and

second linear discriminant functions obtained by the

canonical variables scores of PCA-LDA.

49

Cytotoxicity of new organometallic Pt(II)-complexes containing 1,10-

phenantroline Erika Stefàno,1 Graziana Assalve,1 Antonella Muscella,2 Federica De Castro,3 Danilo Migoni,3 Michele Benedetti,3 Francesco P. Fanizzi,3 Santo Marsigliante.1

1 Laboratorio di Fisiologia cellulare DiSTeBA, Università del Salento. 2 Laboratorio di Patologia cellulare DiSTeBA, Università del Salento. 3 Laboratorio di Chimica Generale e Inorganica DiSTeBA, Università del Salento.

Corresponding Author, e-mail: fp.fanizzi@unisalento.it

Among the emerging anti-cancer compounds, phenanthroline derivatives are of high interest. In contrast to cisplatin, phenanthrolines and their metal complexes are potentially intercalant molecules that can interact with DNA by aromatic π-stacking between base pairs.[1] In this study, two new organometallic Pt(II)-complexes containing 1,10-phenantroline (phen), [Pt(phen)(DMSO)(η1CH2CH2OMe)]+, 1, [Pt(phen)(NH3)(η1-CH2CH2OMe)]+, 2, have been taken into consideration in order to evaluate their cytotoxicity in different human cancer cell lines. In addition, maximal intracellular uptake (MIU) was assayed by ICP-AES after incubation of cells with 100 μM 1 and 2 for 0.5-12 hours. Ten different human cancer cell lines (Caco-2, Caki-1, HeLa, Hep-G2, MCF-7, MG-63, SH-SY5Y, Skov-3, ZL-34 and ZL-55) were treated with 1, 2 and cisplatin at increasing concentrations (0.1-200 μM) from 12 to 72 hours to assess their effect on cell viability. While 2 did not show significantly greater cytotoxic effects than cisplatin in any cell line, 1 proved to be highly effective in almost all cell lines and mainly in the first 12-24 hours of treatment (Figure 1). The greater effects were observed in neuroblastoma cells SH-SY5Y (IC50 (12-

24 h) between 8.23 ± 1.11 μM and 19.8 ± 3.26 μM) and ovarian adenocarcinoma cells SKOV-3 (IC50 (12-24 h) between 39.8 ± 3.56 μM and 92.13 ± 7.81 μM). ICP-AES in SH-SY5Y and SKOV-3 demonstrated a high intracellular uptake of compound (1) (MIUSH-SY5Y 430.5 ± 40.1 ng Pt/mg protein; MIUSKOV-3 497.6 ± 59.5 ng Pt/mg protein) compared to cisplatin (MIUSH-SY5Y 155.9 ± 31.4 ng Pt/mg protein; MIUSKOV-3 30 ± 10.2 ng Pt/mg protein). Total Pt concentration of compound 2 (MIUSH-SY5Y 300 ± 39.2 ng Pt/mg protein; MIUSKOV-3 140.4 ± 46.3 ng Pt/mg protein) was also higher than cisplatin despite not having significantly greater cytotoxic effects. Further studies are needed in order to evaluate the mechanism of action of both 1 and 2 compounds and therefore understand why compound 1 is more toxic than compound 2. Finally, it is desirable to use healthy cell lines corresponding to the tumor lines used here in order to verify any cellular specificity towards cancer cells of the two compounds. [1] Johnstone T.C., Suntharalingam K., Lippard S.J.. The Next Generation of Platinum Drugs: Targeted Pt(II)

Agents, Nanoparticle Delivery, and Pt(IV) Prodrugs. Chem Rev. 2016, 116, 3436-3486.

Figure 1. In abscissa are shown the IC50 obtained with

[Pt(phen)(DMSO)(η1-CH2CH2OMe)]+, 1, with [Pt(phen)(NH3)(η1-CH2CH2OMe)]+, 2, and with cisplatin

in ten human cancer cell lines at the incubation times reported in ordinate (12, 24, 48 and 72 hours).

50

Silver(I) Bis-Carbene Complexes with antimicrobial properties Diego Tesauro1, Filomena Rossi1, Stefano D’Errico1, Gennaro Piccialli1 Mario Varcamonti2, Anna Zanfardino2, Michela Di Napoli 2, Giuseppe Felice Mangiatordi3, Michele Saviano3.

1 Department of Pharmacy and Interuniversity Research Centre on Bioactive Peptides (CIRPeB), University of Naples “Federico II”, via Mezzocannone 16, 80134 Naples, Italy. 2 Department of Biology University of Naples “Federico II”, via Cynthia 80143, Napoli, Italy 3 Istituto di Cristallografia, CNR Bari Italy

Corresponding Author, diego.tesauro@unina.it The evolution of antimicrobial resistance to most drugs push researchers to develop novel, more potent and multimodal alternatives with least antibiotic effects on human body. Silver has been proven best for its broad spectrum of antimicrobial activity including those resistant to antibiotic drugs. Among silver complexes silver(I)-N-Heterocyclic carbene (Ag(I)-NHC) complexes have strong efficacy in vitro as well as in vivo showing efficacy against many Gram-negative as well as Gram-positive bacterial strains [1-2]. In this study we have selected three bis carbene silver complexes bearing planar ligand (Figure 1) by means of different steric hindrance and rigidity. Both free ligands and their silver complexes were synthesized and purified adapting well assessed procedures. After crystallization all compounds were identified by 1HNMR and ESI MS. Silver complexes were tested on different bacterial strains: Escherichia coli, Pseudomonas

aeruginosa and Staphylococcus aureus. Their activities were compared with free ligands, silver oxide and monocarbene complexes. Preliminary data demonstrate that selected Ag-complexes exhibit antimicrobial activity. Further studies will be carried out in order to set up minimum inhibitory concentrations (MICs) to choose the most promising candidate. This last step will be crucial to support the design of new antimicrobial compounds.

References

[1] Siddappa A Patil, Shivaputra A Patil, Renukadevi Patil, Rangappa S Keri, Srinivasa Budagumpi, Geetha R Balakrishna and Matthias Tacke: "N-heterocyclic carbene metal complexes as bio-organometallic antimicrobial and anticancer drugs" Future Med. Chem. 2015, 7(10), 1305–1333.

[2] Mariagrazia Napoli, Carmela Saturnino, Elena Immacolata Cianciulli, Mario Varcamonti, Anna Zanfardino, Giuseppina Tommonaro, Pasquale Longo Silver(I) N-heterocyclic carbene complexes: Synthesis, characterization and antibacterial activity J. Organomet Chem 2013, 725, 46-53.

Figura 1. Molecular structure of bis carbene silver complexes

51

Gold nanorods - copper(I) complexes conjugates as anticancer drug-delivery system Iole Venditti,1 Chiara Battocchio,1 Luca Tortora,1 Giovanna Iucci,1 Martina Marsotto,1 Marina Porchia,2 Francesco Tisato,2 Maura Pellei,3 Carlo Santini3

1 Sciences Dept. Roma Tre University of Rome, Italy 2 ICMATE, National Research Council (CNR), Padua, Italy 3 School of Science and Technology, Chemistry Division, University of Camerino, Italy

Corresponding Author, e-mail: iole.venditti@uniroma3.it Gold nanorods (AuNRs) are successfully employed in drug delivery, biosensors, and biotechnologies [1,2]. Their wide success is due to their unique chemical properties, biocompatibility, easy, cheap and versatile synthesis, schematically reported in Figure 1. In this framework, AuNRs were synthetized with the aim to obtain strongly hydrophilic nanomaterials, suitable for drug delivery. AuNRs were investigated by UV-Vis-NIR, FT-IR and HR-XPS spectrocopies, confirming nanosize, with typical surface plasmon resonance (SPR) bands at 550 nm and 900 nm, and surface functionalization by ascorbic acid (AA) and cetyl trimethyl ammonium bromide (CTAB). Therefore, AuNRs were used as drug delivery system for copper (I)-based anti-tumor complex namely [Cu(PTA)4]+[BF4]- (PTA = 1,3,5-triaza-7-phosphadamantane) [3-5]. The loading procedures and efficiency of Cu(I) complexes on the AuNRs surface were optimized in order to control their bioavailability and release.

References

[1] Park K., Drummy L. F., Wadams R. C., Koerner H., Nepal D., Fabris L., Vaia R. A. Growth Mechanism of Gold Nanorods. Chem. Mater. 2013; 25: 555-563

[2] Venditti I. Engineered gold-based nanomaterials: morphologies and functionalities in biomedical applications. A mini review. Bioengineering. 2019; 6(2): 53

[3] Gandin V., Tisato F., Dolmella A., Pellei M., Santini C., Giorgetti M., Marzano C., Porchia M. In Vitro and in Vivo Anticancer Activity of Copper(I) Complexes with Homoscorpionate Tridentate Tris(pyrazolyl)borate and Auxiliary Monodentate Phosphine Ligands. J. Med. Chem. 2014, 57 (11): 4745-4760.

[4] M. Porchia, F. Benetollo, F. Refosco, F. Tisato, C. Marzano, V. Gandin. Synthesis and structural characterization of copper(I) complexes bearing N-methyl-1,3,5-triaza-7-phosphaadamantane (mPTA): Cytotoxic activity evaluation of a series of water soluble Cu(I) derivatives containing PTA, PTAH and mPTA ligands. J. Inorg. Biochem. 2009; 103 (12): 1644

[5] Fratoddi I., Venditti I., Battocchio C., Carlini L., Porchia M., Tisato F., Bondino F., Magnano E., Pellei M., Santini C. Highly hydrophilic gold nanoparticles as carrier for anticancer copper(I) complexes: loading and release studies for biomedical applications. Nanomaterials. 2019; 9: 772

Acknowledgements: We are grateful to CIRCMSB (Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici). The Grant of Excellence Departments, MIUR (ARTICOLO 1, COMMI 314 – 337 LEGGE 232/2016), is gratefully acknowledged by authors of Roma Tre University.

Figure 1. Scheme of AuNRs synthesis.

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INDICENDICE

Accardo, A. 12

Aiello, I. 15

Amici, A. 41

Angeli, A. 17

Angilè, F. 23

Arduino, I. 3

Arrais, A. 49

Armeni, T. 5

Arnesano, F. 26 27

Ascenzi, P. 11

Assalve, G. 50

Astolfi, P.

6 44

Attanasio, F. 8 9

Azzariti, A. 3

Baglini, E. 17

Bagnarelli, L. 34 46

Baldassarre, F. 38

Balzano, C. 48

Barbanente, A. 4 33

Barresi, E. 17

Barucca, G. 5

Battocchio, C. 34 39 52

Belloni, A. 16 35 49

Belviso, B.D. 26

Benedetti, M. 21 38 50

Bergamini, P. 42

Binacchi, F. 36

Birarda, G. 1

Biver, T. 36

Bonacucina, G. 44

Boschi, A. 45

Botta, M. 13

Bramucci, M. 6

Burini, A. 41

Caliandro, R. 26 27

Caligiuri, R. 15

Cantarini, M. 5 37 40

Capasso, D. 19

Caprì, F. 49

Ciaccio, C. 11

Ciccarella, G. 38

Cirri, D. 20 25 28 36

Codee-van der Schilden, K.

14

Coletta, M. 11

Conti, C. 16 49

Coppola, A. 19

Cutrignelli, A. 3

Da Settimo, F. 17

Dalla Via, L. 42

De Castro, F. 21 38 50

De Franco, M. 47

De Mia, M. 7

De Rosa, S. 39

De Simone, G. 11

Degli Esposti, L. 4

Del Bello, F. 46

Del Coco, L. 23 38

Denora, N. 3

Dell'Anna M. M. 38

D'Errico, S. 51

Dettin, M. 39

Di Gaetano, S. 19

Di Giacinto, A. 7

Di Gregorio, E. 12

Di Masi, A. 11

Di Napoli, M. 51

Di Natale, G. 8

Di Taranto, N. 33

Diaferia, C. 12

Duatti, A. 45

Erez, J. 22

Esposito, J. 45

Fabbrini, M.G. 36

Facchetti, G. 15

Falini, G 7 22

Fanizzi, F.P. 21 23 38 50

Farmer, J.P. 27

Fermani, S. 7 22

Ferri, N. 15

Franco, M. 3

Gabano, E. 29 48

Gabbiani, C. 10 36

Gailer, J. 28

Galassi, R. 41

Galeazzi, R. 5 37 40

Gallo, E. 12

Gamberi, T. 25

Gandin, V. 33 46 47

Geraldes, C.F.G.C. 13

Ghedini, M. 15

Gianolio, E. 12

Gioia, M. 11

Giorgini, E. 6 16 44 49

Girelli, C.R. 23

Godbert, N. 15

Greco, V. 9

Gurrieri, L. 7

Hieraci, M. 42

Iacobazzi, R.M. 3

Iafisco, M. 4

Ionescu, A. 15

Iucci, G. 34 39 52

La Deda, M. 15

La Mendola, D. 8

Laquintana, V. 3

Lasorsa, A. 27

Laudadio, E. 5 37 40

Leeuwenburgh, S.C.G. 4 14

Lemaire, S.D. 7

Logrippo, S. 44

Lopalco, A. 3

Lopedota, A.A. 3

Luciani, L. 41 46

Lupidi, G. 41

Lupo, M.G. 15

Magnabosco, G. 22

Magrì, A. 8

Mangiatordi, G.F. 51

Marchand, C.H. 7

Marchini, C. 41

Margiotta N. 3 4 14 33

Mariani, P. 35

Marini, A.M. 17

Marsigliante S. 21 50

Marsotto, M. 52

Martini, P. 45

Marvelli, L. 42 45

Marzano, C. 46 47

Marzo, T. 28 36

Marzola, E. 45

Massai, L. 10 25 28

Mastrorilli, P. 38

Meloni, M. 7

Meneghini, C. 34

Messori, L. 10 18 20 24 25 28 36

Micoli, K. 33

Migoni, D. 21 23 50

Minnelli, C. 5 37 40

Mirabelli, V. 27

Mobbili, G. 5 37 40

Montesarchio, D. 19

Morandi, V. 22

Morelli, G. 12

Moretti, P. 35

Moro, L. 48

Mulas, G. 13

Muscella, A. 21 50

Musumeci, D. 19

Nadar, R.A. 4 14

Naletova, I. 9

Nardella, M.I. 26 27

Natile, G. 26 27 33

Notarstefano, V. 5 6 16 49

Oliveri, V. 43

Osella, D. 29 48 49

Palazzo, B. 4

Papadia, P. 33

Parlapiano, M. 6 44

Pasquali, M. 45

Pellei, M. 34 46 52

Pepe, A. 35

Perin, E. 29

Perinelli, D.R. 44

Piccialli, G. 51

Pinton, G. 48

Pisani, M. 6 44

Polticelli, F. 11

Porcelli, L. 3

Porchia, M. 47 52

Pratesi, A. 10 18 28 36

Pro, C. 16 49

Pucciarelli, S. 41

Quaglia, W. 46

Quassinti, L. Rangone, B.

6

29

48

Ravera, M. 29 48

Ricciardi, l. 15

Rimoldi, I. 15

Rizzarelli, E. 8 9

Rolla, G.A. 13

Rosato, A. 26 27

Rossi, F. 51

Rossi, J. 7

Roviello, G.N. 19

Quassinti, L. 6

Sabbatini, M. 49

Sabbatini, S. 16 49

Salerno, S. 17

Santini, C. 34 46 52

Sanzia, G.M.C. 21

Saviano, M. 51

Schiesaro, I. 34

Scortichini, M. 23

Sciuto, S. 9

Secchi, V. 39

Serrati, S. 3

Starmans, L.W.E. 13

Stefano, E. 21 50

Stipa, P. 40

Supuran, C.T. 17

Tabbì, G. 8

Tagliani, A. 7

Taliani, S. 17

Tedesco, D. 7

Tei, L. 13

Terreno, E. 13

Tesauro, D. 51

Tisato, F. 47 52

Tortora, L. 39 52

Trost, P. 7

Tundo, G.R. 11

van den Beucken, J.J.J.P.

4 14

Vaccari, L. 1 6 16

Varcamonti, M. 51

Vecchio, G. 43

Venditti, I. 34 39 52

Vergaro, V. 38

Vincenzetti, S. 41

Wang, J. 41

Zaffagnini, M. 7

Zamuner,A 39

Zanellato, I. 49

Zanfardino, A. 51

Zoppi, C. 10 18 25