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TRANSCRIPT
www.dutchpeptidesymposium.com
Dutch Peptide Symposium2017Eindhoven, June 8Abstract book
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Dear Dutch and international peptide friends and colleagues,
It is a great pleasure to welcome you to Eindhoven, and an honor the laboratory of Chemical Biology
of the TU Eindhoven to host the 25th Dutch Peptide Symposium. Building on the successful meeting
from last year in Lelystad, organized by Pepscan, we have grown even further this year in terms of
participants (around 250) and poster presentations (around 60!). Of course the exciting line-up of
speakers has contributed strongly to this and we would like to take the opportunity to thank them
upfront for their great presentations.
The 2017 DPS has taken a further international outreach via multiple actions. A stronger interaction
with the Belgian Peptide Group Meeting as well as with the EuCheMS and its Division of Chemistry in
Life Sciences has been initiated. The speakers come this year from the USA, UK, Austria, Denmark,
the Netherlands, Germany, Japan, and Belgium, and feature an attractive mix of top academic and
industrial contributions, which you will surely enjoy.
A special word of welcome and thanks goes to our many sponsors. Of course the companies, which
you are warmly advised to visit in the coffee- and lunchroom, are kindly thanked. Additionally, the
TU/e has provided us with the full infrastructure free of any cost, to highlight its great dedication to
science and society. As a result, the DPS can again offer you your attendance free of charge. We
would also like to thank our colleagues at Pepscan who have strongly contributed to the DPS in its
current format and in supporting the organization of this year’s meeting. New, and highly
appreciated, are the Bert L. Schram Awards for Best Posters, generously provided by the ESCOM
Science Foundation, aiming to support peptide science in it broadest sense. This should be yet
another boost to allow young scientist to present and discuss their latest results. Please visit the
attractive posters during the breaks.
We truly hope you will all enjoy the program, and on behalf of the organizing committee we wish you
all an excellent symposium.
Lech Milroy and Luc Brunsveld
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PROGRAM
Time Description
08.30 – 09.30 Display of posters
09.00 – 09.30 Registration, coffee and tea
09.30 – 09.35 Welcome and opening words, Prof Luc Brunsveld, Chairman
09.35 – 10.20 Keynote lecture: Cysteine Arylation to Engineer Peptides and Proteins, Prof Bradley Pentelute (MIT Boston, USA)
10.20 – 10.50 Invited Speaker: Chemical Protein Synthesis – Providing Tailor-Made Proteins for Biology and Medicine, Prof Christian Becker (University Vienna, Austria)
10.50 – 11.15 Coffee and tea break; poster presentation
SESSION Chair Persons: Tilman Hackeng and Jan van Maarseveen
11.15 – 11.45 Invited Speaker: Enzymatic Modification of Biopharmaceuticals, Dr Carsten Behrens (Novo Nordisk, Denmark)
11.45 – 12.05 Selected Presentation: Characterization and Stabilization of the Interaction between 14-3-3 and CFTR, Loes Stevers MSc (Technical University Eindhoven, the Netherlands)
12.05 – 12.25 Selected Presentation: Highly Potent Organometallic Antibacterial Peptides, Dr Bauke Albada (Wageningen University & Research, the Netherlands)
12.25 – 12.55 Invited Speaker: Merrifield Reloaded: Peptide Chemistry Goes Green!, Dipl.-Ing. Sascha Knauer (Sulfotools GmbH, Darmstadt, Germany)
12.55 – 14.00 Lunch break and poster session
SESSION Chair Persons: Roland Brock and Nathaniel Martin
14.00 – 14.45 Keynote lecture: A RaPID Way to Discover Pseudo-natural Peptides and Products for Therapeutic Uses, Prof Hiroaki Suga (Peptidream and University of Tokyo, Japan)
14.45 – 15.15 Invited Speaker: Engineering Hybrid Peptidomimetics for Improved Pain Treatments, Prof Steven Ballet (VU Brussels, Belgium)
15.15 – 15.45 Invited Speaker: Biomade Peptides – Enabling Next-generation Products, Dr Christian Schwarz (Numaferm, Düsseldorf, Germany)
15.45 – 16.10 Coffee and tea break; poster presentation
SESSION Chair Persons: Peter Timmerman and Luc Brunsveld
16.10 – 16.15 Presentation of Bert L Schram Awards for Best Posters, ESCOM Science Foundation
16.15 – 17.00 Keynote lecture: Sugars and Proteins: Towards a Synthetic Biology, Prof Ben Davis (Oxford University, UK)
17.00 – 17.10 Closing words
17.10 – 18.30 Drinks
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KEYNOTE SPEAKERS and INVITED SPEAKERS
Brief biographies & abstracts
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Brief biography Prof Ben Davis
Ben Davis is professor of Chemistry in the Department of Chemistry at the University of Oxford. Ben
Davis got his B.A. (1993) and D.Phil. (1996) from the University of Oxford. He then spent 2 years as a
postdoctoral fellow in the laboratory of Professor Bryan Jones at the University of Toronto, exploring
protein chemistry and biocatalysis. In 1998 he returned to the U.K. to take up a lectureship at the
University of Durham. In the autumn of 2001 he moved to the Dyson Perrins Laboratory, University
of Oxford and received a fellowship at Pembroke College. He was promoted to Full Professor in 2005.
His group’s research centers on the chemical understanding and exploitation of biomolecular
function (Synthetic Biology, Chemical Biology and Chemical Medicine), with an emphasis on
carbohydrates and proteins.
His work has recieved numerous prizes and awards, and he sits on the editorial board of several
prestigeous journals. He is Editor-in Chief of Current Opinion in Chemical Biology and a Senior Editor
for ACS Central Science.
Ben Davis was co-founder of Glycoform, a biotechnology company that from 2002-2011 investigated
the therapeutic potential of synthetic glycoproteins and of Oxford Contrast a company investigating
the use of molecular imaging for brain disease. In 2003 he was named among the top young
innovators in the world by Technology Review, the Massachusetts Institute of Technology (MIT)'s
magazine of innovation.
He was elected to the Royal Society in 2015.
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Brief biography Dr Bradley L. Pentelute
Bradley L. Pentelute, Associate Professor of Chemistry. He is currently the Pfizer-Laubach Career
Development Professor, an Associate Member, Broad Institute of Harvard and MIT, and Member,
Center for Environmental Health Sciences MIT. Since starting his own research group at MIT in 2011
he has been awarded the Amgen Young Scientist Award (2016), Novartis Award in Organic Chemistry
(2016), Alfred P. Sloan Fellowship (2015), NSF CAREER Award (2014), Sontag Distinguished Scientist
Award (2013), Young Chemical Biologist Award, International Chemical Biology Society (2013),
Deshpande Innovation Grant (2013), and the Damon Runyon-Rachleff Innovation Award (2013). He
received his undergraduate degree in Psychology and Chemistry from the University of Southern
California, and his M.S and Ph.D. in Organic Chemistry from the University of Chicago with Prof. Steve
Kent. He was a postdoctoral fellow in the laboratory of Dr. R. John Collier at Harvard Medical School,
Microbiology.
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Brief biography Dr Hiroaki Suga
Hiroaki Suga is a Professor of the Department of Chemistry, Graduate School of Science in the University of Tokyo. He was born in Okayama City, Japan in 1963. He received his Bachelor of Engineering (1986) and Master of Engineering (1989) from Okayama University, and Ph. D. in Chemistry (1994) from the Massachusetts Institute of Technology. After three years of post-doctoral work in Massachusetts General Hospital, he was appointed as a tenure-track Assistant Professor in the Department of Chemistry in the State University of New York at Buffalo (1997) and promoted to the tenured Associate Professor (2002). In 2003, he moved to the Research Center for Advanced Science and Technology in the University of Tokyo as an Associate Professor, and soon after he was promoted to Full Professor. In 2010, he changed his affiliation to the
Department of Chemistry, Graduate School of Science. His research interests are in the field of bioorganic chemistry, chemical biology and biotechnology related to RNA, translation, and peptides. He is the recipient of Akabori Memorial Award 2014, Japanese Peptide Society and Max-Bergmann Gold Medal 2016, etc. He is also a founder of PeptiDream Inc. Tokyo, a publicly traded company, which has many partnerships with pharmaceutical companies in worldwide.
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Sugars & proteins: towards a synthetic biology
Benjamin G. Davis
Department of Chemistry,
University of Oxford
Mansfield Road, Oxford OX1 3TA, UK
E-mail: [email protected]
Our work studies the interplay of biomolecules – proteins, sugars and their modifications.
Synthetic Biology’s development at the start of this century may be compared with Synthetic Organic
Chemistry’s expansion at the start of the last; after decades of isolation, identification, analysis and
functional confirmation the future logical and free-ranging redesign of biomacromolecules offers
tantalizing opportunities. This lecture will cover emerging areas in our group in chemical
manipulation of biomoleclules with an emphasis on new bond-forming and -breaking processes
compatible with biology:
(i) New methods: Despite 90-years-worth of non-specific, chemical modification of proteins, precise
methods in protein chemistry remain rare. The development of efficient, complete, chemo- and
regio-selective methods, applied in benign aqueous systems to redesign and reprogramme the
structure and function of biomolecule both in vitro and in vivo will be presented.
(ii) ‘Synthetic Biologics’ and their applications: biomimicry; functional recapitulation; effector
[drug/agrochemical/gene/radio-dose] delivery; selective protein degradation; inhibitors of pathogen
interactions; non-invasive presymptopmatic disease diagnosis; probes and modulators of in vivo
function.
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Cysteine Arylation to Engineer Peptides and Proteins
Bradley L. Pentelute
MIT, Boston
Here we report a robust bioconjugation method using cysteine arylation. This chemistry enables site-
specific conjugation at cysteine residues within peptides, proteins, and antibodies. Our two
developed approaches use either perfluoroaryl-cysteine SNAr chemistry or organometallic palladium
reagents. This work lead to the discovery of a self-labeling four-residue sequence that enables
regioselective conjugation at only one cysteine residue within an intact antibody containing natural
amino acids. Recently, we discovered a new approach for the native conjugation of complex natural
products such as vancomycin onto peptides and proteins without the introduction of linkers or
chemical handles.
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Chemical Protein Synthesis - Providing Tailor-Made Proteins
for Biology and Medicine
Christian F.W. Becker
University of Vienna, Faculty of Chemistry, Institute of Biological Chemistry, Währinger Str. 38, 1090
Vienna, Austria, Email: [email protected]
The ability to produce proteins in the laboratory and to change their structures and therefore their
properties in a controlled fashion is of crucial importance in basic biological research, in
biotechnology and increasingly in medical applications. I will discuss our latest efforts to use
chemoselective ligation methods to assemble homogeneous, site-specifically modified proteins from
peptides made by solid phase synthesis and from protein segments produced by expression.
Examples that involve the (semi-)synthesis of proteins with specific posttranslational modifications,
including lipidation, glycosylation as well as so far rarely investigated non-enzymatic protein
modifications will be presented. The resulting proteins are unique tools to address open questions
related to specific modifications of amino acid side chains, e.g. in cellular recognition, aging and
neurodegenerative diseases.
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Enzymatic Modification of Biopharmaceuticals
C. Behrens, J. Buchardt, M. Zundel and T.E. Nielsen
Protein and Peptide Chemistry III, Novo Nordisk A/S, Måløv, Denmark
Peptides and proteins are well recognized for being highly specific and potent towards their natural
target, and for having good safety profile in humans [1]. This makes them an excellent choice for the
development of new biopharmaceuticals. However, unmodified peptide and proteins often have
short half-lifes in blood due to renal clearance, proteolytic degradation or receptor specific uptake
[2]. This generally reduces their use to “on demand” or daily administration, and renders them
useless in prophylactic settings.
Modification with half-life extending moieties such as fatty acids [3], polyethylene glycol [4],
polysialic acid and heparosan [5] together comprises efficient ways of increasing the half-life’s of
biopharmaceuticals. For shorter peptides with limited number of reactive groups, half-life extending
moieties can readily be introduced by e.g. acylation of lysine residues or by pH controlled reductive
amination in the N-terminal. However, for larger recombinant protein therapeutics, site selective
chemical modification rapidly becomes challenging.
For the selective modification of large biopharmaceuticals, enzymes have proven to be valuable
tools. Enzymatic transformations are generally milder than their chemical counterparts and more
selective when multiple residues are in scope for modification. This presentation will focus on the
introduction of half-life extending moieties into protein therapeutics using enzyme catalysed
reactions.
[1] K. Fosgerau and T. Hoffmann; Drug Discovery Today 2015, 20 (1), 122-128. [2] S.B. van Witteloostuijn, S.L. Pedersen and K.J. Jensen; ChemMedChem 2016, 11, 2474 – 2495 [3] J. Lau, P. Bloch, L. Schäffer et. al.; J. Med. Chem., 2015, 58 (18), 7370–7380 [4] H.R. Stennicke, M. Kjalke, D.M. Karpf et. al.; Blood, 2013, 121(11):2108-16. [5] C. Behrens, J. Buchardt, M.L. Broberg et. al.; J Thromb. Haemost. 2015, 13, 831.
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Figure 1 | A cartoon representation of a cell expressing mutant CFTR.
Stabilization of the interaction between 14-3-3 and CFTR stimulates
the trafficking of the protein to the plasma membrane.
Characterization and stabilization of the interaction between 14-3-3 and CFTR
Using phosphopeptides to study protein-protein interactions.
L.M. Steversa, C.V. Lama, S.F.R. Leysena, F.A. Meijera, D.S. van Scheppingena, R.M.J.M. de
Vriesa, G.W. Carlileb, L.G. Milroya, D.Y. Thomasb, L. Brunsvelda, C. Ottmanna
Laboratory of Chemical Biology, Department of Biomedical Engineering, TU Eindhoven, The
Netherlands b Cystic Fibrosis Translational Research Centre, Department of Biochemistry,
McGill University, Canada
Cystic fibrosis is a fatal genetic disease, most frequently caused by the retention of the CFTR (cystic
fibrosis transmembrane conductance regulator) mutant protein in the endoplasmic reticulum [1].
The binding of the 14-3-3 protein to the CFTR regulatory (R) domain has been found to enhance CFTR
trafficking to the plasma membrane (see Figure 1) [2]. To define the mechanism of action of this
protein-protein interaction, we have examined the interaction in vitro. The intrinsically disordered
multiphosphorylated R domain contains nine different 14-3-3 binding motifs. Furthermore, the 14-3-
3 protein forms a dimer containing two amphipathic grooves that can potentially bind these
phosphorylated motifs. This results in a number of possible binding mechanisms between these two
proteins. By synthesizing an array of phosphopeptides coding for segments of the CFTR R-domain
and using them in multiple biochemical assays, we showed that the interaction between them is
governed by two binding sites: the key binding site of CFTR (pS768) occupies one groove of the 14-3-
3 dimer, and a weaker, secondary binding
site occupies the other binding groove
[3]. Additionally, we created a High
Throughput Fluorescence Polarization
assay based on a labeled CFTR
phosphopeptide to screen for stabilizers
for the CFTR/14-3-3 interaction. The
found hits show promising results in both
localization and functional cellular CFTR
assays, and X-ray crystallization of the 14-
3-3/CFTR- peptide/compound complex
showed that a totally new stabilization
mechanism for 14-3-3 interactions has
been found which has great potential in
Cystic Fibrosis drug development.
[1] J.M. Rommens et al. Science, 245, 1059 (1989) [2] X. Liang et al. Mol Biol Cell 23, 996 (2012) [3] L.M. Stevers et al. PNAS, 113, E1152 (2016)
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Highly Potent Organometallic-Antibacterial Peptides
Bauke Albada
Wageningen University & Research – Chemical Sciences, Laboratory of Organic Chemistry,
Stippeneng 4, 6708 WE, Wageningen, The Netherlands
Organometallic moieties have shown to be remarkable modulators of the biological activity of bio-
active molecules and peptides [1]. Whereas many organometallics do not tolerate water and
spontaneously ignite when exposed to air, some are tamed to not only tolerate those conditions, but
actually join forces with bioactive peptides in order to assist in the development of novel drugs (the
first organometallic antimicrobial agent successfully passed phase II clinical trials). In this
presentation, I will present the latest solid-phase ánd biocompatible aqueous phase based methods
that allow the once-separated fields of organometallics and bioactive peptides to be joined.
Following that, I will spend the majority of the time on how metallocenoyl groups on moderately
active antibacterial peptides (i) help us to understand the mode-of-action of short antibacterial
peptides [2], and (ii) allow us to obtain the highly potent antibacterial peptides of which each amino
acid residue is more potent than a residue in vancomycin [3]. Importantly, these highly active
antibacterial agents are non-hemolytic and do not negatively affect cell viability of human cancer
cells. Furthermore, remarkable differences in mode-of-action are observed when the metal-ion is
changed from Fe to Ru or Os (all in group 8 of the periodic table). This shows that even a minute
change in the properties of the organometallic fragment dramatically influences the mechanism by
which these membrane-binding peptides operate [4]. I will discuss the differences in mode-of-action,
and correlate these with the properties of the metal-ion in the organometallic fragment.
[1] B. Albada and N. Metzler-Nolte, Organometallic-Peptide Bioconjugates: Synthetic Strategies and Medicinal Applications. Chem. Rev., 2016, 116, 11797–12839. [2] M. Wenzel, A.I. Chiriac, A. Otto, D. Zweytick, C. May, C. Schumacher, R. Gust, H.B. Albada, M. Penkova, U. Kraemer, R. Erdmann, N. Metzler-Nolte, S. Straus, D. Becher, H. Broetz-Oesterhelt, H.-G. Sahl, J.E. Bandow, Small cationic antimicrobial peptides delocalize peripheral membrane proteins. Proc. Natl. Acad. Sci., USA, 2014, 111, E1409–E1418. [3] H.B. Albada, P. Prochnow, S. Bobersky, S. Langklotz, J.E. Bandow, N. Metzler-Nolte, Highly active antibacterial ferrocenoylated or ruthenocenoylated Arg-Trp peptides can be discovered by an L-to-D substitution scan. Chem. Sci., 2014, 5, 4453–4459. [4] M. Wenzel, A.I. Chiriac, C. Schumacher, J.J. Stepanek, J. Kraemer, H.B. Albada, R. Erdmann, C. May, H. Broetz- Oesterhelt, N. Metzler-Nolte, H.-G. Sahl, J.E. Bandow, Studying localization of antimicrobial peptides with metallocene labels. submitted.
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Merrifield Reloaded: Peptide Chemistry Goes Green!
Sascha Knauer, Christina Uth, Harald Kolmar
Sulfotools GmbH, In den Niederwiesen 24a, 64291 Darmstadt(Germany)
The field of synthetic peptides is growing rapidly. These molecules are widely used in biomedical and
physiological research, as pharmaceuticals, or in the cosmetic industry. Today, the market of peptide
pharmaceuticals amounts 15 billion USD. The downside of peptide production is the enormous
amounts of harmful organic solvents such as dichloromethane (DCM) and dimethylformamide (DMF)
which are used in large excess both for solution- and solid-phase peptide synthesis. In terms of
solvent consumption, peptide production processes with an average solvent usage of up to several
tons per kg product, remaining one of the worst chemical processes used today.
Herein, we present the development of a novel concept for the production of peptides based on
Merrifield’s SPPS.[1] Our new protecting group strategy allows for a water-compatible process. The
intrinsic fluorescent properties of our novel protecting group gives an option for the real-time
monitoring of coupling and deprotection efficiency for each reaction step. This would allow reducing
the amount of reaction cycles and, respectively, excess of amino acids and reagents by preventing
unnecessary double or triple coupling steps.
Currently, reversed phase HPLC purification is the method of choice for many peptides. In industry,
this implies a compromise between enormous consumption of acetonitrile used as an eluent and
peptide yield and quality. Merrifield et al. introduced a modified Fmoc-protection group for the
purification of peptides in 1978, but due to the inconvenient purification procedure it was never
generally accepted.[2] With our innovative capping strategy we are able to remove many side
products by simple ion exchange chromatography in water, thus allowing us to reduce the amount of
organic solvents necessary for the purification process. The same system could be used for liquid
waste purification, significantly reducing the amount of hazardous waste.
References [1] R. B. Merrifield, J. Am. Chem. Soc. 1963, 85, 14, 2149–2154. [2] R. B. Merrifield et al., The Journal of Organic Chemistry 1978, 43, 25, 4808-4816.
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A RaPID way to discover pseudo-natural peptides and products for
therapeutic uses
Hiroaki Suga
Department of Chemistry, Graduate School of Science, The University of Tokyo, JST CREST, 113-0033
Tokyo, Japan, and Board of Directors, PeptiDream Inc.
The genetic code is the law of translation, where genetic information encoded in RNA is translated to
amino acid sequence. The code consists of tri-nucleotides, so-called codons, assigning to particular
amino acids. In cells or in ordinary cell-free translation systems originating from prokaryotes, the
usage of amino acids is generally restricted to 20 proteinogenic (standard) kinds, and thus the
expressed peptides are composed of only such building blocks. To overcome this limitation, we
recently devised a new means to reprogram the genetic code, which allows us to express non-
standard peptides containing multiple non-proteinogenic amino acids in vitro. This lecture will
describe the development in the genetic code reprogramming technology that enables us to express
natural product-inspired non-standard peptides and pseudo-natural products. The technology
involves (1) efficient macrocyclization of peptides, (2) incorporation of non-standard amino acids,
such as N-methyl amino acids, and (3) reliable synthesis of libraries with the complexity of more than
a trillion members. When the technology is coupled with an in vitro display system, referred to as
RaPID (Random non-standard Peptide Integrated Discovery) system as a novel “molecular
technology”, the libraries of natural product-inspired macrocycles with a variety ring sizes and
building blocks can be screened (selected) against various drug targets inexpensively, less
laboriously, and very rapidly. This lecture will discuss the most recent development of their
technology and therapeutic applications toward drug discovery innovation.
Y. Iwane; A. Hitomi; H. Murakami; T. Katoh; Y. Goto; H. Suga*, “Expanding the amino acid repertoire of ribosomal
polypeptide synthesis via the artificial division of codon boxes”, Nature Chemistry, 8, 317–325 (2016)
K. Ito; K. Sakai; Y. Suzuki; N. Ozawa; T. Hatta; T. Natsume; K. Matsumoto; H. Suga "Artificial human Met agonists based on
macrocycle scaffolds" Nature Communications, 6, 6373 (2015)
T. Passioura; H. Suga "Reprogramming the genetic code in vitro" Trends in Biochemical Sciences 39, 400-408 (2014).
Y. Tanaka, C.J. Hipolito, A.D. Maturana, K. Ito, T. Kuroda, T. Higuchi. T. Katoh, H.E. Kato, M. Hattori M, K. Kumazaki, T.
Tsukazaki, R. Ishitani, H. Suga, O. Nureki “Structural basis for the drug extrusion mechanism by a MATE multidrug
transporter” Nature 496, 247-51 (2013).
Y. Yamagishi, I. Shoji, S. Miyagawa, T. Kawakami, T. Katoh, Y. Goto, H. Suga "Natural product-like macrocyclic N-methyl-
peptide inhibitors against a ubiquitin ligase uncovered from a ribosome-expressed de novo library" Chemistry&Biology
18, 1562-1570 (2011).
Y. Goto, T. Katoh, H. Suga “Flexizymes for genetic code reprogramming” Nature Protocols 6, 779-790 (2011)
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Engineering Hybrid Peptidomimetics for Improved Pain Treatments
Steven Ballet,a* Karel Guillemyn,a Charlotte Martin,a Frédéric Simonin,b Barbara Przewlocka,c
Mariana Spetea,d Peter W. Schiller,e and Dirk Tourwéa
a Research Group of Organic Chemistry, Departments of Chemistry and Bioengineering Sciences, Vrije
Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium b University of Strasbourg, CNRS, UMR7242, ESBS, 67412 Illkirch-Graffenstaden, France
c Department of Pain Pharmacology, Institute of Pharmacology, Polish Academy of Sciences, Smetna
12, PL 31-343 Krakow, Poland. d Department of Pharmaceutical Chemistry, Institute of Pharmacy and Center for Molecular
Biosciences (CMBI), University of Innsbruck, Innrain 80-82, A-6020 Innsbruck, Austria e Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montreal, 110
Pines Avenue West, H2W 1R7 QC, Montreal, Canada
To address the different types of pain, different classes of medications, mainly non-steroidal anti-inflammatory drugs and narcotics (opioids), are used. The alleviation or treatment of moderate to severe pain states, in particular, commonly invokes the use of opioids. Unfortunately, their chronic administration induces various undesirable side effects. One strategy to overcome these major side effects and to prolong the antinociceptive efficiency of the applied drugs involves the creation of multifunctional compounds which contain hybridized structures.
Combination of opioid agonist and antagonist pharmacophores in a single chemical entity has been considered and extensively investigated, but opioids have also been combined with non-opioid bioactive neurotransmitters and peptide hormones that are involved in pain perception (e.g. substance P, neurotensin, etc.).[1] Such novel chimeras (also called designed multiple ligands or DMLs), may interact independently with their respective receptors and potentially result in more effective antinociceptive properties. The designed multiple ligands presented in this work include peptide-based opioid-non-opioid dimer analogs, such as for example opioid-neurokinin 1 receptor,[2,3] opioid-nociceptin [4] and opioid-neuropeptide FF DMLs.[5] Some of the prepared ligands demonstrated to be dually effective in both acute and neuropathic pain models. Additionally, compounds with reduced (cross-)tolerance (with morphine) and respiratory depression were unraveled.
[1] Kleczkowska, P. et al. Curr. Pharm Des. 2013, 19, 7435-7450.
[2] Guillemyn, K. et al. Eur. J. Med. Chem. 2015, 92, 64-77.
[3] Betti, C. et al. ACS Med. Chem. Lett. 2015, 6, 1209-1214.
[4] Guillemyn, K. et al. J. Med. Chem. 2016, 59, 3777-3792.
[5] Ballet, S. et al. unpublished
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Biomade Peptides - enabling next-generation products
Christian Schwarz
NUMAFERM GmbH
Underwater adhesives, harmless pesticides or antibiotics that are effective against multi- resistant
microbes - it all sounds utopian, but could already have been a reality for a long time! Because
peptides, "small proteins" of up to 100 amino acids, allow these and numerous other innovations.
And, although already well-researched in laboratories, a launch of "super glue", "Smart Protect" or
"Antibiotic 2.0" has not been previously possible. The leading reason is the cost of the "miracle” raw
materials. The dominant method, chemical synthesis, is extremely costly. The preparation of a
kilogram of peptide costs an average of 1 million €. At the same time, chemical syntheses are not
highly scalable and peptides in large quantities (> kg) are inaccessible. For these reasons, peptides
currently have a very limited role except in pharmaceutical applications.
NUMAFERM has developed a novel patented bioprocess for the production of peptides making their
production up to 1,000 times more cost-effective. With us, the fascinating raw material peptide is
cheaper for pharmaceutics, for the first time, affordable for non- pharmaceutical applications.
NUMAFERM applies and offers the technology for the production of peptides on a mass scale and
supports customers with know-how in the development of marketable peptide-based products. The
broad applicability of peptides enables the development of various markets. We already carry out
projects in the pharmaceutical, cosmetics, adhesives, pesticides and aquaculture industry.
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ABSTRACTS
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IN SEARCH OF NOVEL AMPS FROM WEST AFRICAN SNAILS USING PROTEOMIC TECHNIQUES AND BIOINORGANIC CHEMISTRY
Caleb M. Agbale1,3; Kofi Wiabo-Asabil1, Justice K. Sarfo1, Octavio L. Franco2,3
1. Department of Biochemistry, School of Biological Sciences, College of Agriculture and Natural Sciences, University of Cape Coast, Ghana.
2. Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia, Centro de Análises Proteômicas e Bioquímicas, Universidade Católica de Brasília, Brasília, Brazil.
3. S-Inova, Pos-Graduação em Biotecnologia, Universidade Catolica Dom Bosco, Campo Grande, MS, Brazil.
Novel AMPs are urgently needed to deal with modern health challenges especially due to drug-resistant cancers and bacteria. This is especially needed in many developing countries which are already battling with malaria, HIV/AIDS and tuberculosis as a result of poor health care systems. Based on results of earlier studies that identified two AMPs from gastropods we proposed an indepth proteomic study on edible west African land snails. These slow moving, soil-dwelling gastropods are known to rely extensively on a very complex chemical communication and defense system to ward of prey, withstand soil-borne pathogens and survive in unfavourable environmental conditions. This remarkable resilience is basically due to a limited ability to escape from predators or pursue their prey compared to other larger organisms. Using 3 species of west African snails (Family Achatinidae) as a model organisms our objectives are to use proteomic tools to probe for novel peptides and proteins that are upregulated in response to infection by the drug-resistant bacteria or following induction of cancers. Synthetic analogs of these ‘excretory-secretory’ peptides or proteins will be assessed for antibiofilm, antibacterial or anticancer activity and potential leads modified at their N-terminals with amino terminal Cu(II) and Ni(II) (ATCUN) binding motifs to enhance their potency. Overall, we expect to identify novel AMPs for treatment of drug resistant conditions either singly or in combination with conventional antibiotics. Significantly, this project is expected to produce two MPhil students locally as well as train 10 undergraduate students (five per year) and 3 laboratory technicians in various areas of microbiology, proteomics and bioinformatics. Trials have begun with mytimacin and achacin, two well-characterized AMPs from the African giant snail Achatina fulica. The entire project duration is two years with an estimated cost of USD55,000.00. Keywords: African land snails, anticancer, antibiofilm, antimicrobial, antimicrobial peptides, rational design, drug resistance
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ATCUN MODIFICATION DISPLAY OPPOSING EFFECTS ON THE ALPHA HELICAL ANTIMICROBIAL PEPTIDES CM15 AND CITROPIN 1.1
Mawuli C. Agbale1,3; Isaac Galyuon1, Justice K. Sarfo1, Octavio L. Franco2,3
1. Department of Biochemistry, College of Agriculture and Natural Sciences, School of Biological Sciences, University of Cape Coast, Ghana.
2. Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia, Centro de Análises Proteômicas e Bioquímicas, Universidade Católica de Brasília, Brasília, Brasil.
3. S-Inova, Pos-Graduação em Biotecnologia, Universidade Catolica Dom Bosco, Campo Grande, MS, Brasil.
The importance of antimicrobial peptides (AMPs) in the fight against drug-resistant microbes has become an active area of study. Unlike conventional antibiotics, AMPs employ a complex set of killing mechanisms which enable them to circumvent bacterial resistance. Even more exciting is the application of the amino-terminal copper and nickel (ATCUN) to modulate the bioactivity of AMPs. We tested this hypothesis by synthesizing ATCUN-modified analogs of two α-helical membrane-active peptides CM15 (KWKLFKKIGAVLKVL-NH2) and citropin1.1 (GLFDVIKKVASVIGGL-NH2). Two analogs with the Gly-Gly-His and Val-Iso-His motifs were synthesized for each peptide and their bioactivities tested against a battery of clinically relevant bacteria. We also investigated their antibiofilm activity alone and in combination with various conventional antibiotics. Our data indicates that ATCUN modification improves the bioactivity of CM15 especially against carbapenem-resistant Klebsiella pneumoniase (KPC1825971) and enhances synergy with meropenem in inhibition of biofilm formation by carbapenem-resistant E. coli (KPC1812446). Furthermore, the Val-Iso-His modified analogs of CM15 were more active than the Gly-Gly-His modified analogs. A completely opposing data was observed between ATCUN-modified citropin1.1 analogs and parental citropin1.1. Unexpectedly, ATCUN modification led to a drastic reduction in both antimicrobial and antibiofilm activity indicating that the technique was highly discriminatory. We conclude that effectiveness of this strategy is highly dependent on the type of alpha-helical peptide selected for modification and the sequence of amino acids in the ATCUN motif. Synergism with conventional antibiotics in inhibition of biofilm formation is dependent on the type of antibiotic used in the combination. Based on our study we recommend more structural-activity relationship studies to identify the key features that favor enhancement of activity upon ATCUN modification. This data will help bridge the knowledge gaps in this simple and exciting strategy which potentially could improve the potency of several AMP.
21
Singlet Oxygen-Induced Furan Oxidation for Site-Specific and Chemoselective Peptide Ligation
E. Antonatou1, K. Hoogewijs1, D. Kalaitzakis2, Y. Verleysen1,
G. Vassilikogiannakis2, and A. Madder1
1Department of Organic and Macromolecular Chemistry, Ghent University,
Ghent, Belgium 2Department of Chemistry, University of Crete, Iraklion, Crete, Greece
Site-specific chemical modification of proteins is crucial for understanding protein structure and
interactions as well as providing insights into cellular events [1]. In continuation of previous work
conducted in our lab, where an efficient solid phase-based peptide labeling method was developed
[2], in this study we investigated singlet oxygen (1O2) mediated furan-modified peptide labeling in
physiological aqueous solutions. Furan-containing peptides were subjected to the standard oxidative
conditions (air, light, photosensitiser) so that the reactive electrophilic species were generated.
These reactive intermediates were intercepted by nitrogen nucleophiles to yield stable conjugates.
Incorporation of fluorescent hydrazides through the cascade reaction sequence, led to the efficient
construction of site-selectively labeled fluorescent peptides [3]. The conjugation reactions proceed
smoothly in aqueous solution at neutral pH, without the need for additives such as catalysts or
reducing agents and were found to be compatible with sensitive amino acid residues within the
peptides. In this way, a novel chemoselective ligation methodology has been developed for the facile
construction of peptide-based fluorescent probes.
To further extend the application of our novel methodology, furan peptides were photooxidised and
engaged by hydrazine and hydrazide peptides leading to the efficient formation of peptide-peptide
conjugates at near neutral pH [4]. These results suggest that furan activation meets many, if not all,
requirements for bioorthogonal reactions and has significant potential for use in protein, and more
generally, biomolecule derivatization, thus adding to the toolbox of chemoselective ligation
strategies.
Figure 1. Singlet oxygen-induced labeling and conjugation of furan-containing peptides.
[1] E. M. Sletten and C. R. Bertozzi, Angew. Chemie - Int. Ed.,48, 6974 (2009) [2] A. Deceuninck and A. Ma dder, Chem. Commun. (Camb)., 340 (2009) [3] E. Antonatou, K. Hoogewijs, D. Kalaitzakis, A. Baudot, G. Vassilikogiannakis and A. Madder, Chem. - A Eur. J., 22, 8457 (2016) [4] E. Antonatou,Y. Verleysen
and A. Madder, manuscript in preparation.
22
Bioorthogonal antigens-shedding light on the impact of
posttranslational modifications in rheumatoid arthritis
C. Araman1, L. Pieper1, W. van der Wulp1, B. E. Florea1, R. M. Toes2 and S.I. van Kasteren1
1Leiden Institute of Chemistry,Universiteit Leiden, Gorlaeus Laboratories, Einsteinweg 55,
2333 CC Leiden, The Netherlands 2Department of Rheumatology, Leiden University Medical Centre, Albinusdreef 2,
Leiden 2333 ZA, TheNetherlands
Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that affect the joints. It is caused
by genetic and environmental risk factors. Nevertheless, its etiology remains unknown [1]. The
development of B-cell-mediated immunity against posttranslationally-generated epitopes is considered as
a hallmark of RA. There are two major posttranslational modifications involved in RA
immunopathogenesis, citrullination and carbamylation respectively. Citrullination is the enzymatic
deimination of arginine into citrulline via peptidyl arginine deiminases (PAD), whereas carbamylation
describes the conversion of the e-NH group of lysines into urea derivatives thorough reaction with free
isocyanate, which is generated from urea in vivo. Citrullinated proteins are recognized by anti-
citrullinated protein autoantibodies (ACPA). ACPA response is accepted as an integral part of the disease
process [2]. Recently, Unanue et al. showed that the antigen-processing pathway of citrullinated proteins
and their non-citrullinated counterparts for presentation are distinct from each other [3]. Based on this
and other findings [3-5], we developed a strategy to generate RA-related antigens carrying bioorthogonal
handles. In order to follow proteolytical antigen processing/presentation in APCs, we utilized copper-
catalyzed-azide-alkyne-cycloaddition (CuAAC) [6, 7]. Thus, we envisioned the conjugation of fluorophores
to RA-related bioorthogonal antigens to visualize antigen processing/presentation mechanism and
kinetics in vivo.
Here, we describe the generation of first citrullinated and carbamylated proteinogenic autoantigens
harboring biorthogonal handles. We successfully expressed, purified and characterized these
biorthogonal autoantigens. In addition, neo-epitopes harboring the pathogenic modifications have been
characterized utilizing proteomics and subsequently synthesized via SPPS to use those in antigen
presentation studies.
Furthermore, in vitro and in cellulo experiments showed that our bioorthogonal antigens can be
conjugated to fluorophores via CuAAC and are taken up/processed by primary bone marrow dendritic
cells (BMDCs). The outcome of cellular uptake and proteolytical processing in cellulo will be presented.
Acknowledgements
We gratefully acknowledge the financial support from Dutch Arthritis Foundation (Reumafonds). References 1. Scott, D.L.; Wolfe, F.; Huizinga, T.W. Lancet. 2010, 376 (9746):1094 2. Willemze, A.; Trouw, L. A.; Toes R. E. et al. Nat. Rev. Rheumatol. 2012; 8: 144. 3. Ireland, J. M.; Unanue, E. R. J. Exp. Med. 2011, 208, 2625. 4. Khandpur R.; Carmona-Rivera, C.; Vivekanandan-Giri A, et al. Sci. Transl. Med. 2013, 5:178ra140. 5. Cantagrel, A.; Degboe, Y. Joint Bone Spine 2016, 83, 11. 6. Tornoe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057. 7. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem. Int. Ed. Engl. 2002, 41, 2596.
23
FmocHN
easily made, stable, crystalline
"dipeptide" building blocks, e.g.
R = -'Pr, -CH2Ph, -CH2OtBu -
CH2C02*BU, -(CH2)4NHBOC
O °^N
oxetane modified
bradykinin analogue
(R = CH2Ph)
/. selective cumyl ester deprotection ii. solid-phase peptide synthesis
Solid Phase Synthesis of Oxetane Modified Peptides
J.D. Beadle,1 A. Knuhtsen,2 A. Hoose,2 P. Raubo,3 A. G. Jamieson2 and M. Shipman1 1 Department of Chemistry, University of Warwick, Coventry, UK 2 School of Chemistry, University of Glasgow, Glasgow, UK
3 AstraZeneca, Macclesfield, UK
Despite resurgent interest in the use of peptides as drugs [1], their development is often hampered
by their poor oral bioavailability and short plasma half-lives. As a consequence, there is intense
interest in the discovery of molecules that can mimic the structure and biological function of native
peptides yet possess better drug-like properties. In 2014, we [2] and Carreira [3] simultaneously
reported a novel peptide bond isostere, in which one of the amide C=O bonds of the peptide
backbone is replaced by an oxetane ring. These oxetane modified peptides (OMPs) have the
potential to improve stability to proteases, and to alter the physiochemical properties and
conformation of existing peptides.
In order to assess the impact of oxetane modification on the structure of larger, biologically relevant
peptides, we have developed a practical route towards oxetane modified peptides using solid-phase
peptide synthesis (SPPS). Our strategy involves the synthesis of Fmoc-protected dipeptide building
blocks that are introduced into a growing peptide chain using automated SPPS techniques. Using this
methodology, we have successfully made a number of oxetane modified peptides, including
enkephalin and bradykinin, in high purity and synthetically acceptable yields. With this methodology
in hand, our future work focuses on the secondary structure, physiochemical and biological
properties of oxetane modified peptides, with a view to establishing their potential in drug discovery.
[1] Fosgerau, K.; Hoffmann, T. Drug Discov, Today 2015, 20, 122; (b) Nevola. L.; Giralt, E. Chem. Commun. 2015, 51, 3302; (c) Kaspar, A. A.; Reichert, J. M. Drug Discov. Today 2013, 18, 807. [2] Powell,N. H.; Clarkson,G. J.;Notman,R.; Raubo,P.; Martin, N. G.; ShipmanM. Chem. Commun. 2014, 50, 8797 [3] McLaughlin, M.; Yazaki, R.; Fessard, T. C.; Carreira, E. M. Org. Lett. 2014, 16, 4070
24
MINIMIZING ASPARTIMIDE FORMATION IN FMOC SPPS:
FMOC-ASP(OBNO)-OH
Raymond Behrendt1, Peter White2
1Merck & Cie, Im Laternenacker 5, 8200 Schaffhausen, Switzerland, 2Merck Chemicals, Padge Road,
Beeston NG9 2JR, United Kingdom
The most serious side reaction in Fmoc chemistry is aspartimide formation.[1] It is caused by exposure
of the peptide sequence containing aspartic acid to strong base and is a major problem in the
synthesis of long peptides and sequences containing multiple aspartic acid residues. Aspartimide
formation is particularly pernicious as it leads to racemization of the aspartate residue forming D-
aspartyl peptides which may co-elute with the target peptide.[2]
In our effort to develop a universal solution to standard Fmoc SPPS conditions we recently
introduced Fmoc-Asp(OBno)-OH (see figure), an aspartyl derivative bearing the tributylcarbinol ester
at the ƴ-carboxyl group.[3]
In this poster we applied this derivative to the synthesis of the classic model peptide scorpion toxin II
(VKDGYI) and its sequence variants (VKDNYI and VKDRYI) as well as to the synthesis of Gly2-GLP-2
(teduglutide). We found this new reagent provided a general and simple solution to the problems of
aspartimide formation in Fmoc SPPS.
References
[1] Subirós-Funosas R, El-Faham A, Albericio F. Aspartimide formation in peptide chemistry: occurrence, prevention strategies and the role of N-hydroxylamines. Tetrahedron 2011, 67, 8595. [2] Michels T, Doelling R, Haberkorn U, Mier W. Acid-mediated prevention of aspartimide formation in solid phase peptide synthesis. Org. Lett. 2012, 14, 5218. [3] a) Behrendt R, Huber S, Martí R, White P. New t‐butyl based aspartate protecting groups preventing aspartimide formation in Fmoc SPPS. J. Pept. Sci. 2015, 21, 680. b) Behrendt R, Huber S, White P. Preventing aspartimide formation in Fmoc SPPS of Asp-Gly containing peptides — practical aspects of new trialkylcarbinol based protecting groups. J. Pept. Sci. 2016, 22, 92. [4] Subirós‐Funosas R, El‐Faham A, Albericio F. Use of Oxyma as pH modulatory agent to be used in the prevention of base‐driven side reactions and its effect on 2‐chlorotrityl chloride resin. Peptide Sci. 2012, 98, 89.
25
Bicyclic RGD-peptides with high affinity and selectivity for αvβ3, αvβ5, and α5β1 integrins
D. Bernhagena, L. De Laporteb, P. Timmermana,c
a Pepscan Therapeutics, Zuidersluisweg 2, 8243 RC Lelystad, Netherlands; b DWI – Leibniz Institute for Interactive Materials, Forckenbeckstr. 50, 52056 Aachen, Germany; c Van’t Hoff Institute for Molecular Sciences, University of Amsterdam, Sciencepark 904 XH Amsterdam, Netherlands
The ‘bicyclic peptide’ platform recently attracted considerable interest as a powerful platform for novel therapeutic drugs1 because of their high binding affinities and selectivities in combination with an appreciable proteolytic stability.2 So far, only few cellular proteins have been evaluated for their binding to bicyclic peptides. The integrins represent an interesting target class for novel therapeutic agents because of their significant role in key pathological processes.3 We therefore synthesized libraries of hundreds of different bicyclic peptides, one loop comprising the well-known “RGD”-motif and providing basic integrin-affinity, and second loop consisting of a random sequence (XXX) that allows for controlling the binding selectivity. In order to screen for affinity and selectivity to each integrin (αvβ3, αvβ5, and α5β1) we developed a robust and cost-effective screening assay using a knottin-RGD peptide as a reference binder.4,5 After selection of the best binders for each integrin, we designed 2nd and 3rd generation diversity libraries and thus gradually improved the affinity and selectivity for each integrin. The best IC50 values that we obtained in this way were, for example, 30 nM for αvβ3 (GRGDS: 5 µM, knottin-RGD: 250 nM), and 90 nM for α5β1 (GRGDS: >30 µM, knottin-RGD: 100 nM). We also labeled the best bicyclic integrin-binders with a fluorescent dye and studied their integrin-binding on live cells. Preliminary confocal microscopy data show fast and efficient cellular uptake for some of the labeled bicycles, most likely via an integrin-mediated internalization mechanism. The most potent bicyclic integrin-binders showing highest were also immobilized on 2D hydrogels in order to investigate their effect on cell adhesion, cell proliferation and viability as compared to the conventional RGD-peptides.
1 “Astrazeneca takes Bicycle to work in potential $1B multitarget collaboration”, Bioworld™ Today 2016, 27, 3. 2 P. Li, P. P. Roller, Curr. Top. Med. Chem. 2002, 2, 325–341; V. Baeriswyl, C. Heinis, ChemMedChem 2013, 8, 377–384.
3 M. Barczyk, S. Carracedo, D. Gullberg, Cell Tissue Res. 2010, 339, 269–280; Y. Takada, X. Ye, S. Simon, Genome Biol. 2007, 8, 215.
4 D. Bernhagen, L. De Laporte, P. Timmerman, manuscript submitted to Anal. Chem.
5 R. H. Kimura, A. M. Levin, F. V Cochran, J. R. Cochran, Proteins 2009, 77, 359–369.
26
Self-assembled Peptide-nanospheres as artificial enzymes Enantioselective Au-catalyzed transformations
C. B. Bheeter, Sergio Gonell, David A. Poole III, Arnout P. T. Hartendorp and J. N. H. Reek *
University of Amsterdam (UvA), Van ’t Hoff Institute for Molecular Sciences, Homogeneous, Supramolecular and Bio-Inspired Catalysis, Science Park 904, 1098 XH Amsterdam
Recently, we demonstrated that M12L24 self-assembled nanospheres are interesting scaffold for the exploration of transition metal catalysis in confined spaces. The preorganization of 24 gold complexes inside such spheres resulted in extremely high local concentration of catalysts, which in turn resulted in enhanced activity and selectivity in various gold catalyzed transformations.1 In addition, we have developed M12L24 nanosphere with guanidine binding sites for the pre-organization of multiple catalysts that have been functionalized with sulfonate binding sites, and carboxylate containing substrates. The high local concentration of both components within the confined space defined leads to high reaction rates 2. We were wondering if these general strategies can be extended to enantioselective catalytic transformations. We therefore explored the M12L24 nanospheres that are (partly) functionalized with peptide chains to generate chiral cavities around the organometallic complexes. This pre-organization of metal complexes, within the enzyme like peptide environment, indeed leads to efficient transformations with high enantioselectivity and will be presented.
[1] (a) R. G –Doria, J. Hessels, S.H.A.M Leenders, O. TrÖppner, M. Dürr, I.I. Burmazović, and J.N.H. Reek, Angew. Chem. Int. Ed., 2014, 53, 13380. (b) S.H.A.M. Leenders, M. Dürr, I.I. Burmazović, I.I. and J.N.H. Reek, Adv. Synth. Catal., 2016, 358, 1509. [2] Q.Q. Wang, S. Gonell, S.H.A.M. Leenders, M. Dürr, I.I. Burmazović, and J.N.H. Reek, Nat. Chem.,2016, 8, 225.
27
Photochemistry in flow for chemical biology applications
C. Bottecchia, L. Milroy, L. Brunsveld, V. Hessel and T. Noël Department of Chemical Engineering and Chemistry, Micro Flow Chemistry & Process Technology,
Eindhoven University of Technology, Eindhoven, The Netherlands
Two strategies for the selective photocatalytic modification of cysteine residues are presented.
Firstly, a mild and practical method for the preparation of disulfides via visible light induced
photocatalytic aerobic oxidation of thiols is introduced.[1] The method involves the use of TiO2 as a
highly stable and recyclable heterogeneous photocatalyst. The reaction can be substantially
accelerated in a continuous-flow packed-bed reactor, which enables a safe and reliable scale-up of
the reaction conditions.[2] The batch and flow protocol described herein can be applied to a diverse
set of thiol substrates for the preparation of homo- and hetero-dimerized disulfides. Furthermore,
biocompatible reaction conditions (room temperature, visible light, neutral buffer solution, no
additional base) have been developed, which permit the rapid and chemoselective modification of
densely functionalized peptide substrates without recourse to complex purification steps. Secondly, a
visible light-induced perfluoroalkylation of cysteine and cysteine-containing dipeptides is
described.[3] The protocol relies on Ru(bpy)32+ as photocatalyst and inexpensive RFI as coupling
partner and allows the introduction of a variety of perfluoro alkyl groups (C1-C10) and of a CF2COOEt
moiety. The reaction is high yielding (56-94% yield) and fast. Process intensification in a
photomicroreactor resulted in reduced reaction time (5 minutes) and increased yields.
References [1] C. Bottecchia, N. Erdmann, P. Tijssen, L. Milroy, L. Brunsveld, V. Hessel and T. Noël ChemSusChem , 9, 1781-1785 (2016). [2] D. Cambié, C. Bottecchia, N. J. W. Straathof, V. Hessel, T. Noël, Chem. Rev. 116, 10276-10341 (2016). [3] C. Bottecchia, X. Wei, V. Hessel, T. Noёl. J Org Chem, 81, 7301-7307 (2016).
28
A Metal-binding Coiled-Coil Peptide Exhibiting Selective Metal Specificity
A.L. Boyle1, M. Xiao2, N. Crone1, G. Rhys3, P. Voskamp2, N. Pannu2, A. Kros1
1 Supramolecular and Biomaterials Chemistry, Leiden Institute of Chemistry, Leiden University, The Netherlands
2 Biophysical Structural Chemistry, Leiden Institute of Chemistry, Leiden University, The Netherlands 3 School of Chemistry, University of Bristol, United Kingdom
Designing metal-binding sites into simplified peptide scaffolds is a robust, bottom-up, approach to
understanding both fundamental and functional aspects of metalloproteins [1]. One major drawback
however, is that metal specificity is often poor with one scaffold binding a range of different metals
[2, 3].
We have utilised a hyperstable, de novo designed, coiled-coil scaffold containing only the minimum
information required for structural specificity [4], and redesigned it to incorporate metal-binding
functionalities. This approach allowed us to: rationally determine the modifications necessary to
effect metal-binding behaviour; investigate metal-binding affinity and specificity in a controlled
manner; and to evaluate the structural implications of these modifications.
Several constructs were iteratively designed and their metal-binding behaviour was initially
evaluated using circular dichroism (CD) spectroscopy. Further analysis was then performed using
nuclear magnetic resonance (NMR) spectroscopy and analytical ultracentrifugation. We discovered
that it was necessary to include multiple metal-coordinating residues in order to introduce metal-
binding capabilities into the scaffold, and we discovered that a high degree of selectivity for certain
metal ions was observed when compared to other, similar, peptide scaffolds. It is envisaged that, by
determining the structural basis for this selectivity, a range of metal-specific metalloenzymes and
biosensors could be synthesized, emulating natural constructs.
[1] M.L. Zastrow, V.L. Pecoraro, Coordination Chemistry Reviews, 257, 2565 (2013) [2] S. Chakraborty, D.S. Touw, A.F.A. Peacock, V.L. Pecoraro, Journal of the American Chemical
Society, 132, 13240 (2010) [3] K. Suzuki, H. Hiroaki, D. Kohda, H. Nakamura, T. Tanaka, Journal of the American Chemical
Society, 120, 13008 (1998) [4] J.M. Fletcher, A.L. Boyle, M. Bruning, G.J. Bartlett, T.L. Vincent, N.R. Zaccai, C.T. Armstrong,
E.H.C. Bromley, P.J. Booth, R.L. Brady, A.R. Thomson, D.N. Woolfson, ACS Synthetic Biology, 1, 240, (2012)
29
Evaluating designed antileishmanial peptides through their interaction with
model membranes: charge and hydrophobicity effects
Viviane Aparecida Camargo Santana2, Danubia Batista Martins1, Marta Lopes Lima3,4, Andre
G. Tempone4, Marcia Perez dos Santos Cabrera1
1Departamento de Flsica e 2Departamento de Qufmica e Ciencias Ambientais, Universidade Estadual
Paulista, Sao Jose do Rio Preto, SP, Brazil. 3Instituto de Medicina Tropical, Universidade de Sao Paulo, Sao Paulo, SP, Brazil 4Centro de
Parasitologia e Micologia, Instituto Adolfo Lutz, Sao Paulo, SP, Brazil
Some antimicrobial peptides also presente antileishmanial activity whose mechanism of action
involves targeting of the cell membrane. This target is believed to be advantageous due to the
reduced chance of resistance development. Bioactive peptides from natural sources and their
synthetic analogs are investigated to orient structural optimizations that can enhance the
therapeutic action and at the same time minimizing toxic or undesirable side effects. Decoralin is an
undecapeptide, which in the C-terminus amidated form exhibits leishmanicidal activity towards the
promastigote form of the protozoa and reduced cytotoxicity. Previous molecular dynamics studies
comparing the structures of Decoralin and other leishmanicidal peptides suggest that some structural
parameters could be responsible for this activity [1]. Thus, three analogs were designed and
evaluated for their lipid membrane-related effects using vesicles whose composition mimic cell
membranes of the promastigote form and the amastigote infected macrophage. We evaluated the
binding capacity through zeta potential experiments, vesicles aggregation induction in DLS
experiments, lytic activity by monitoring the leakage of a vesicle entrapped fluorescent probe and we
characterized the peptides secondary structure in circular dichroism experiments. We found out that
the analog with the lowest net charge (+3) and medium mean hydrophobicity (0.00) presented the
highest partition coefficient, and was also the more efficient on promastigote cells of L.infantum,
although exhibiting comparable cytotoxicity toward NCTC cells. Accordingly it presented the lowest
threshold concentration in the leakage experiments and strong helical character in the presence of
the membrane mimetic vesicles, except in the promastigote model, where a tendency to 3-sheet
structure was observed. Further studies are under way to evaluate the ability of peptides to
translocate vesicles mimicking macrophage membranes.
[1] M. E. R. Guerra, V. Fadel, V. G. Maltarollo, G. Baldissera, K. M. Honorio, J. R. Ruggiero, M. P. S. Cabrera, Chem Biol Drug Des. DOI: 10.1111/cbdd.12970, (2017).
Support: FAPESP 2012/24259-0; 2014/083727; 2015/17331-5
30
G-quadruplex-templated oligomerization of a pore-forming peptide
L. Cozzoli1. L. Gjonaj1, G. Maglia2 3 4 5 6 7, B. Poolman2, G. Roelfes 1
1 Stratingh Institute for Chemistry, Nijenborgh 4, 9747 AG Groningen, The Netherlands 2 Groningen Biomolecular Sciences and Biotechnology Institute, Nijenborgh 4, 9747 AG Groningen,
The Netherlands
Alamethicin (Alm) is an a-helical antimicrobial peptide, able to self-assemble and form voltage-gated
ion channels in lipid bilayer membranes [1]. Membrane insertion of Alm occurs following the barrel-
stave model, in which channels are formed by assembly of 4-12 monomers to yield parallel bundles
surrounding a central aqueous pore [2]. The study of the ion conductance behaviour of Alm is
complicated due to the detection of multiple conductance levels related to the different association
states of the peptide. In order to control and define the size of the peptide channels, several
strategies have been used where Alm monomers were chemically modified with template molecules,
such as cyclodextrins [3], a leucine zipper motif [4] or a poly-lysine linker [5].
In this study, we developed a new approach to control the oligomerization of an Alm derivative (Alm-
dUL) [6] by employing a DNA G-quadruplex (G-4s) motif. G-4s are composed of planar guanine
tetrads stabilized by monovalent cations, such as K+ and are characterized by high stability, well-
defined conformation and versatility [7]. In our design each Alm monomer is conjugated on the C-
terminus with a 5'-amino-modified DNA strand (5'-GGGTT-3') that in the presence of K+ is able to
assemble in a parallel G-4. The formation of the G-4 is expected to modulate Alm-dUL channels
behaviour by favouring the formation of tetrameric channels and by increasing their stability. The ion
channel forming properties of the G-4-peptide conjugate are studied by single-channel recordings
using the planar lipid bilayer method, and the data are compared to the behaviour of a single strand
DNA-peptide conjugate.
1 G. Andrew Woolley and B. A. Wallace, J. Membr. Biol, 129, 109 (1992). 2 D. S. Cafiso, Annu. Rev. Biophys. Biomol. Struct, 23, 141 (1994). 3 C. U. Hjprringgaard, B. S. Vad, V. V. Matchkov, S. B. Nielsen, T. Vosegaard, N. C. Nielsen, D. E. Otzen and T. Skrydstrup, J. Phys. Chem. B, 116, 7652 (2012). 4 S. Futaki, M. Fukuda, M. Omote, K. Yamauchi, T. Yagami, M. Niwa and Y. Sugiura, J. Am. Chem. Soc., 123, 12127 (2001). 5 H. E. Duclohier, G. Alder, K. Kociolek and M. T. Leplawy, J. Pept. Sci, 9, 776 (2003). 6 P. I. Haris, G. Rard Molle and H. Duclohier, Biophys. J, 86, 248 (2004). 7 J. T. Davis, Angew. Chem. Int. Ed, 43, 668 (2004).
31
Sec-Scan: a new approach for reliable disulfide connection assignment
S. Denisov, I. Dijkgraaf, T. Hackeng, H. Ippel Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, Netherlands
Cysteine-rich peptides are important leads in drug development. Biological activity of these
compounds depends on tertiary structure and correct disulfide bridge connectivities. Known
approaches for assignment of disulfide bond connectivities in cysteine-rich peptides imply laborious
MS methods or NOE analysis by NMR and often yield ambiguous results. Here, a new approach of
disulfide bond connectivity determination using selenocysteine (Sec) scanning (Sec-Scan) and NMR
spectroscopy is demonstrated. This approach involved substitution of single cysteines by
selenocysteines into a chemically synthesized peptide and comparing 13C CP and 1H HP chemical
shift changes of these single Sec mutants from natural abundance 13C-1H HSQC spectra relative to
reference spectra of the native peptide. Our data show that in model peptides and proteins
incorporation of a single selenocysteine leads to the same structure as the corresponding native
form. Substitution of S atoms by Se results in strong upfield shifts (~8 ppm) of the CP atom in Sec
itself, but also induced a through-bond upfield shift (~1 ppm) of the cysteine CP atom that is present
in the mixed Se-S bond. Based on assigned CP chemical shifts alone, this observation allows to
determine which particular cysteine is covalently attached to the Sec residue that was introduced at
a predefined position in the amino acid sequence. Sec-Scan was successfully validated on various-
sized peptide and protein model systems with known structures having two to six cysteine residues
linked in a complex disulfide network even containing multiple neighbouring CC-sequences (Arg-
Vasopressin, Kalata B1, and |i-conotoxin KIIIA). Finally, disulfide connections were established in
Evasin-3, a protein with hitherto unknown structure and disulfide connectivity.
32
Monitoring quality attributes of biotherapeutic products using a Mass
Spectrometry based analytical platform
Perry Derwig1, Jing Fang2, Brooke Koshel2, Robert Birdsall2, Ying Qing Yu2, Min Du2,
Scott J Berger2, Weibin Chen2
1Waters Chromatography BV, Etten Leur, The Netherlands 2Waters Corporation, Milford, MA
The use of mass spectrometry for pharmaceutical analysis in late development and quality control
environments has been discussed with both optimism and concern. The challenges of deploying
High-Resolution-MS (HRMS) methodologies in regulated environments must be weighed against
more established and routine nominal mass detection approaches. The establishment of Multi-
Attribute Method (MAM) based analyses in late stage and QC labs requires more rigorous evaluation
in their suitability.
In this study, in order to assess the applicability of HRMS and nominal mass detection strategies for
attribute monitoring, we have generated a set of monoclonal antibody (mAb) samples under forced
degradation conditions. The ability to monitor intrinsic attributes such as glycosylation profiles and
induced modifications from accelerated stress was evaluated using peptide mapping with optical
detection combined with either high resolution Q-Tof HRMS or quadrupole mass detection
technologies. An integrated data processing software was used in both peptide characterization and
targeted attribute monitoring. Most critical attributes are identified by HRMS and monitored/
quantified by HRMS and mass doctor. Results from these studies have been compiled to enable data-
based discussions of fit-for-purpose MS.
33
Stabilizing the p53 - 14-3-3 Protein-Protein Interaction
Richard G. Doveston1, Ave Kuusk1,2, Sebastian Andrei1, Seppe Leysen1, Hongming Chen2,
Helen Boyd2, Luc Brunsveld1, Christian Ottmann1
1. Laboratory of Chemical Biology, Department of Biomedical Engineering and Institute for Complex
Molecular Systems, Eindhoven University of Technology, The Netherlands.
2. Discovery Sciences, Innovative Medicines and Early Development Biotech Unit, AstraZeneca R&D
Gothenburg, Mölndal 43183, Sweden.
This poster will present the results of recent studies towards the discovery small-molecule stabilisers
of the protein-protein interaction between the tumor suppressor p53 (''the guardian of the
genome'') and 14-3-3 adapter proteins.
p53 is frequently mutated in many human cancers and is therefore an attractive therapeutic target.
Current strategies typically involve restoration of wild-type function or inhibition of the interaction
with MDM2, its key negative regulator. Despite the efficacy of these approaches, the alternate
approach of stabilizing the interaction of p53 with positive regulators such as 14-3-3 proteins, and
thus maintaining or enhancing tumor suppressor activity, has not been explored.
In this poster we present data that demonstrates proof-of-concept for small-molecule stabilization of
the 14-3-3 – p53 PPI as a therapeutic modality. This has been achieved by addressing a previously
identified potential ligand binding pocket (Figure 1) with the natural product fusicoccin A. However,
in a fascinating twist we also show a unique disconnect between biophysical and crystallographic
data in the presence of the stabilizing molecule. Furthermore our results indicate that not all peptide
binding partner mimics behave as you might expect! Both of these twists in the tale could have
important implications for how we study small-molecule PPI stabilization in general.
Figure 1. Binary crystal structure showing a p53-CTD 9mer peptide (coloured according to atom with red residue labels)
bound to 14-3-3σ (grey with black residue labels). The potential ligand binding pocket is identified. (3LW1: wwpdb.org).
34
N-acyl azetine and N-vinylamides as tetrazinophiles for inverse-electron
demand Diels-Alder (IEDDA) ligations
T.C. van den Ende, S.B. Engelsma, G.A. van der Marel, H.S. Overkleeft, and D.V. Filippov
Bio-organic synthesis, Leiden Institute of Chemistry, Leiden University, The Netherlands
Bio-orthogonal chemistry requires selective ligation of reactants without generation of side-products
and reactive by-products in vivo. The ever growing importance of bio-orthogonal chemistry strategies
for labelling proteins fuels the demand for better ligation handles. While the reaction rate of these
ligation partners are very important, size and synthetic accessibility also bring their own advantages.
This has resulted in a wide range of different type of ligations with various reaction partners1,2. On
this poster we compare N-acyl azetine3 with terminal N-vinylamides as click partners for tetrazines
during inverse-electron demand Diels-Alder (IEDDA) ligations.
First, we present the newly optimized reaction conditions for synthesizing of these ligation handles.
Five different ligation handles were synthesized and evaluated in kinetic experiments. Furthermore,
two water-soluble terazinophilic derivatives were subjected to NMR experiments in aqueous media
in order to assess their stability. Finally, the reaction rates were determined experimentally for all
derivatives.
1. Debets, M. F., van Hest, J. C. M. & Rutjes, F. P. J. T. Bioorthogonal labelling of biomolecules: new functional handles and ligation methods. Org. Biomol. Chem. 11, 6439–6455 (2013). 2. Ramil, C. P. & Lin, Q. Bioorthogonal chemistry: strategies and recent developments. Chem. Commun. 49, 11007– 11022 (2013). 3. Engelsma, S. B. et al. Acylazetine as a dienophile in bioorthogonal inverse electron-demand Diels-Alder ligation. Org. Lett. 16, 2744–2747 (2014).
35
Synthetic ADP-ribosylated peptides for biochemical studies
H.A.V. Kistemaker, Q. Liu, J. Voorneveld, G.J. van der Heden van Noort,
G.A. van der Marel, D.V. Filippov
Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands
ADP-ribosylation is a reversible post-translational modification of proteins that entails an enzymatic
transfer of single or multiple adenosine diphosphate ribose (ADPr) units from NAD+ to a nucleophilic
amino acid residue within the polypeptide chain of a protein. When a number of ADPr moieties are
sequentially transferred to a protein the poly-ADPr chain, either linear or branched, is formed. Such,
poly-ADPr plays a pivotal role in DNA-repair and has well established signalling functions.[1]
However, the present level of understanding of the processes where intracellular mono-ADP-
ribosylated proteins are involved is much more limited. Little is known about the exact structure and
stereochemistry of ADPr-protein junction. To facilitate the studies of the proteins involved in the
metabolism and recognition of mono-ADP-ribosylation (MARylation) at the molecular level we
developed a solid-phase synthesis of mono-ADP-ribosylated peptides. I would like to discuss the
synthesis of various mono-ADPr-peptides containing different modification sites making use of pre-
phosphoribosylated amino acid building blocks developed by us.[2, 3] I also wish to demonstrate the
usefulness of these synthetic well-defined peptides in the studies of the molecular biology of ADP-
ribosylation. [2, 4]
[1] K. W. Ryu, D. S. Kim and W. L. Kraus, Chem. Rev. 115, 2453-2481 (2015). [2] H. A. V. Kistemaker, A. P. Nardozza, H. S. Overkleeft, G. A. van der Marel, A. G. Ladurner and D. V. Filippov, Angew. Chem. Int. Ed. Engl. 55, 10634-10638 (2016). [3] a) H. A. V. Kistemaker, G. J. van der Heden van Noort, H. S. Overkleeft, G. A. van der Marel and D. V. Filippov, Organic Lett. 15, 2306-2309 (2013); b) G. J. van der Heden van Noort, M. G. van der Horst, H. S. Overkleeft, G. A. van der Marel and D. V. Filippov, J. Am. Chem. Soc., 132, 5236-5240 (2010). [4] V. Bilan, N. Selevsek, H. A. Kistemaker, J. Abplanalp, R. Feurer, D. V. Filippov and M. O. Hottiger, Mol. Cell. Proteomics, doi: 10.1074/mcp.O116.065623 (2017).
36
Extracellular matrix peptide presentation at biomaterials with different
supramolecular building blocks
R.C. van Gaal1,2, A.B.C. Buskermolen1,2, S. Zaccaria2,3, C.V.C. Bouten1,2, P.Y.W. Dankers1,2,3 1Laboratory for Cell and Tissue Engineering, 2Institute for Complex Molecular Systems, 3Laboratory of
Chemical Biology, Eindhoven University of Technology, Eindhoven, The Netherlands
The development of functional kidney tissue in vitro is of great interest for various purposes, such as
disease modelling, nephrotoxicity assessment of drugs, and renal replacement therapies. At the base
of in vitro kidney tissue engineering lay biomaterials resembling the renal tubular basement
membrane’s extracellular matrix (BM-ECM). An imitated BM-ECM can be achieved by introducing
ECM mimicking peptides at the biomaterial surface.1 Supramolecular biomaterials based on
hydrogen bonding ureido-pyrimidinone (UPy) or bis-urea (BU) functionalities are eminently suitable
for this. The supramolecular nature of the biomaterials allows for a modular approach in which
compounds, such as bioactive peptides, with the same supramolecular motif can be mixed and
matched with various base materials resulting in biomaterials with various chemical properties.2,3
Both the UPy and the BU system are based on hydrogen bonding interactions, however it is unknown
if the two systems have different peptide presenting properties at the surface. In this study we
examined cyclic RGD (cRGD) and linear RGD surface presentation in both systems through
investigation of the primary protein complexes involved in cell-biomaterial interactions, the focal
adhesions, and cell migration.
Our investigation showed that focal adhesions adopt a punctuated morphology and increase in
numbers with increasing amounts of (c)RGD incorporation in the corresponding UPy and BU
biomaterials. This behaviour of the focal adhesions was more pronounced on functionalized BU-
biomaterials than the corresponding UPy-materials. In addition, cell migration was reduced on
(c)RGD functionalized materials compared to both pristine UPy and BU materials. However, the BU-
based system showed a markedly greater reduction in cell velocity than observed in the UPy system.
Taken together these results suggest that the BU-system is more effective in presenting ECM
mimicking peptides at the surface, therefore becoming the supramolecular system by choice to be at
the base of an BM-ECM mimic.
References
1 Dankers, P. Y. W. et al. Bioengineering of living renal membranes consisting of hierarchical, bioactive supramolecular
meshes and human tubular cells. Biomaterials 32, 723-733, doi:http://dx.doi.org/10.1016/j.biomaterials.2010.09.020 (2011).
2 Wisse, E. et al. Molecular Recognition in Poly(ε-caprolactone)-Based Thermoplastic Elastomers. Biomacromolecules 7, 3385-3395, doi:10.1021/bm060688t (2006).
3 Dankers, P. Y. W., Harmsen, M. C., Brouwer, L. A., Van Luyn, M. J. A. & Meijer, E. W. A modular and supramolecular approach to bioactive scaffolds for tissue engineering. Nat Mater 4, 568-574, doi:http://www.nature.com/nmat/journal/v4/n7/suppinfo/nmat1418_S1.html (2005).
37
Metal and Polyoxometalate Driven Assembly of Novel Peptide
Astha Guptaa, Sandeep Vermab*
aDepartment of Chemical Engineering and bDepartment of Chemistry
Indian Institute of Technology Kanpur, Kanpur 208016
Uttar Pradesh, INDIA
We present the strategic design of a novel
tripeptide that has the capacity to generate a
carbene on its side chain. This tripeptide was
used for the synthesis of an organometallic
carbene complex by reaction with appropriate
gold and mercury precursors. These complexes
showed the characteristic features of carbene
complexes i.e. disappearance of peak for the
proton where carbene is generated in 1H NMR
and deshielding of the carbenoid carbon peak in
the 13C NMR spectra. Their structures were
elucidated by Density Functional Theory (DFT) calculations. Studies into their anti-cancer potential
have also been performed.
The tripeptide alone showed fibrous morphology, but showed the formation of spherical
nanostructures due to electrostatic interaction with polyanionic phosphotungstate salt. Their
interaction was elucidated by various techniques like FTIR and XRD. Their applications have also been
studied in the present work.
References 1. J. Ferna'ndez-Gallardo, B. T. Elie, M. Sanau and M. Contel, Chem. Commun., 2016, 52, 3155 2. J. Li, Z. Chen, M. Zhou, J. Jing, W. Li, Y. Wang, L. Wu, L. Wang, Y. Wang and M. Lee, Angew. Chem. Int. Ed. 2016, 55, 2592.
38
Sensitive and Precise Measurement of Protein Therapeutics Using LC/Q-TOF
Ning Tang, Martin Haex and David Wong
Agilent Technologies
Monoclonal antibodies (mAbs) are a very important class of biopharmaceutical molecules. As a
protein drug, thorough characterization of the mAb is required in each of the manufacturing steps.
Intact mAb analysis offers rapid assessment on determining the accurate molecular weight of a mAb
product and its degree of heterogeneity, such as post-translational modifications (PTMs), antibody-
drug conjugate (ADC), mAb sequence variations, or degradation products. Quadrupole Time-of-flight
(Q-TOF) LC/MS systems are often used to analyze intact proteins or antibodies due to excellent
resolution at the high mass range1-3. The Agilent 6545XT AdvanceBio LC/Q-TOF system includes
hardware and software features to significantly improve the measurement of biomolecules up to
30,000 m/z. This Application Note describes a seamless workflow using the Agilent 1290 Infinity II
UHPLC system, 6545XT AdvanceBio LC/Q-TOF, and automatic data processing with Agilent
MassHunter BioConfirm software to analyze a variety of mAb products.
39
Fast and Cost Effective Solid Phase Synthesis of High Quality Crude Peptides
Hossain Saneii(*), Mostafa Hatam(*), Farshad Karimi, William Bennett
AAPPTEC, LLC
We have studied the factors that affect the quality of crude peptides prepared by solid phase
methods with the goal of producing crude peptides with increased yield and purity utilizing overall
cost effective procedures. We have found that delay gradient heating during coupling is most
effective in minimizing racemization and accelerating coupling rate. Crude peptides prepared with
delay gradient heating had higher purity and yields compared to peptides prepared at room
temperature, with rapid conventional heating, microwave heating or gradient heating. To reduce
solvent waste, we derived a formula to calculate the amount of wash needed to efficiently remove
piperidine and Fmoc-deprotected byproducts following the Fmoc-deprotection steps. In addition, we
developed an algorithm to predict difficult couplings in a peptide sequence. Utilizing this algorithm,
we were able to successfully predict when double coupling would be required, thus improving
synthesis efficiency and reducing unnecessary and wasteful double coupling steps.
Synthesis and evaluation of novel “scrambled” peptoids
W. Ichinose1, H. R. King1, Paul W. Denny1, S. L. Cobb1 1Department of Chemistry, Durham University, Durham, UK
Peptides are attractive molecules to explore as ligands for a range of target proteins.
However, peptides are inherently unstable in vivo and are rapidly degraded by proteases.
Therefore, it has become increasing important to study peptidomimetics that contain
enhanced in vivo stability. One class of peptidomimetics are peptoids; N-substituted glycine
oligomers. In peptoids each side chain of the amino acid residue has been moved to the
backbone nitrogen atom. This structural change affords considerable stability towards
enzymatic degradation.
Peptoids such as 1 which contain cationic side chain can exert antimicrobial properties via
disruption of the cell membrane.1,2 Understanding this process in more detail will be key to
the future development of peptoid based anti-infectives. Recently, the Fei group
demonstrated that -helix peptides designed by calculation of their amphipathicity showed
differing biological activities.3 We considered that the amphipathicity of peptoids (like 1)
would also affect their biological properties but this has yet to be explored in any detail in the
field of peptoid chemistry. In part this is due to the fact that almost all of the peptoids
reported in the literature have block type sequences and scrambled sequences are rarely
reported.
In the work presented we have synthesized and analyzed a small library of peptoids (2-7)
that have “scrambled” sequences. The structural (CD analysis), physical (logD) and
biological properties of the peptoids were then analysis and compared against conventional
peptoid sequences such as 1.
[1] Y. Luoa, H. L. Bolt, G. A. Eggimann, D. F. McAuley, R. McMullan, T. Curran, S. L. Cobb, F. T.
Lundy, ChemBioChem, 18, 111 (2017) [2] N. P. Chonqsiriwatana, J. A. Patch, A. M. Czyzewski, M. T. Dohm, A. Ivankin, D. Gidalevitz,
R. N. Zuckermann, A. E. Barron, Proc. Natl. Acad. Sci. U.S.A. 105, 2794 (2008) [3] X. Liu, R. Cao, S. Wang, J. Jia, H. Fei, J. Med. Chem. 59, 5238 (2016)
41
Substrate Peptidomimetic Inhibitors (SPIs) of Zinc Dependent Enzymes
A. Mahindra1, P. Watson, A. Echalier8, J. W. R. Schwabe2, A G. Jamieson1
Department of Molecular and Cell Biology , University of Leicester, University Road, LE1 7RH,
United Kingdom
School of Chemistry , Joseph Black Building, University Avenue, Glasgow, G12 8QQ, UK
Substrate peptidomimetic inhibitors (SPIs) of zinc dependent enzymes are excellent tool compounds
for chemical biology and as lead compounds for drug discovery. SPIs benefit from good target
selectivity because they are derived from the native enzyme substrate, however have been
structurally modified to improve binding affinity and physicochemical properties. The development
of SPIs is thus a useful generic strategy to target different zinc dependent enzymes through
modification of the peptide sequence.
One limitation of this approach is that the synthetic preparation of non-native amino acids can be
laborious and expensive. Previously, we reported a robust and scaleable method for the asymmetic
synthesis of amino acids.1 Our current research focuses on the synthesis of Fmoc protected, non-
native amino acids that incorporate a zinc binding motif as the side-chain.
These amino acid building blocks have been incorporated into the histone-4 tail peptide using
microwave assisted SPPS. The resulting SPIs are nM inhibitors of HDAC1 in a fluorescence based
activity assay.2 A crystal structure of this SPI bound to HDAC1 has been solved and reveals the key
interactions for substrate binding.
The same strategy has been used to target the CSN5 zinc-dependent deubiquitylase by recapitulating
the cullin portion of the CSN substrate.
[1] Watson, P. J., Millard, C. J., Riley, A. M., Robertson, N. S., Wright, L. C., Godage, H. Y., Cowley, S. M., Jamieson, A. G., Potter, B. V.L., Schwabe, J. W.R. Insights into the activation mechanism of class I HDAC complexes by inositol phosphates. Nat. Commun., 2016, 7, 11262. [2] Aillard, B., Robertson, N. S., Baldwin, A. R., Robins, S., Jamieson, A. G. Robust asymmetric synthesis of unnatural alkenyl amino acids for conformationally constrained α-helix peptides. Org. Bio. Chem., 2014, 12, 8775 - 8782.
Figure 1. Crystal structure of HDAC1 (grey) / MTA1 corepressor (orange) / IP4 (green) / SPI
Peptide (cyan)
42
A high-resolution NMR study of plectasin – a lipid II targeting defensin
S. Jekhmane1, J. Medeiros Silva1, F. Torres1, B.O.W. Elenbaas1, E.J. Breukink2,
M.H. Weingarth1 1NMR Spectroscopy, Bijvoet Center, Science for Life, Utrecht University, The Netherlands
2Membrane Biochemistry and Biophysics, Bijvoet Center, Science for Life, Utrecht University,
The Netherlands
Defensins are cysteine-rich small molecular weight proteins with high antimicrobial activity against a
broad range of bacteria, viruses and fungi. These peptides are endogenously expressed in
vertebrates, invertebrates and plants, and are part of the innate immune system. Plectasin is a 40
amino acid defensin isolated from the saprophytic ascomycete Pseudoplectania nigrella, that folds
into a cysteine-stabilized alpha-beta conformation [1]. Unlike most antimicrobial peptides, plectasin
does not kill its target by permeabilizing the bacterial cell membrane. It interferes with bacterial
growth by specifically binding to the key bacterial cell-wall precursor lipid II [2].
High-resolution data on the plectasin – lipid II complex is limited and only available in non-
physiological conditions. Lipid II binding modes that require physiological conditions have therefore
not been visualized so far. Herein, we use a high-resolution 1H-detected solid-state NMR approach to
study the structure and the dynamics of the plectasin – lipid II complex in a native-like media.
[1] P.H. Mygind, R.L. Fischer, K.M. Schnorr, M.T. Hansen, C.P. Sönsken, S. Ludvigsen, D. Raventós, S. Buskov, B. Christensen, L. De Maria, O. Taboureau, D. Yaver, S.G. Elvig-Jørgensen, M.V. Sørensen, B.E. Christensen, S. Kjærulff, N. Frimodt-Moller, R.I. Lehrer, M. Zasloff, H. Kristensen, Nature 437, 975 (2005). [2] T. Schneider, T. Kruse, R. Wimmer, I. Wiedemann, V. Sass, U. Pag, A. Jansen, A.K. Nielsen, P.H. Mygind, D.S. Raventós, S. Neve, B. Ravn, A.M.J. Bonvin, L. De Maria, A.S. Andersen, L.K. Gammelgaard, H. Sahl, H. Kristensen.
43
De novo macrocyclic peptide modulators of protein-carbohydrate
interactions
S. A. K. Jongkees1,2, S. Caner3, C. Tysoe4,5, L. van Gijzel1, G. Brayer3, S. Withers3,4,5, H. Suga2 1 Department of Chemical Biology and Drug Discovery, Utrecht Institute of Pharmaceutical Sciences,
Utrecht University 2 Department of Chemistry, Tokyo University, Tokyo, Japan
3 Department of Biochemistry and Molecular Biology, University of British Columbia,
Vancouver, Canada 4 Department of Chemistry, University of British Columbia, Vancouver, Canada
5 Centre for High-Throughput Biology, Michael Smith Laboratories, Vancouver, Canada.
The interactions of proteins with carbohydrates are involved in many biological recognition
processes, and so are of relevance to many diseases including immune disorders, cancers, and
metabolic disorders.[1] The surface where this interaction takes place can be relatively shallow and
difficult to perturb using small molecules, and so can be difficult to target therapeutically. An
alternative approach is to use a new technique for mRNA display with genetic code reprogramming
called the RaPID system[2] to find macrocyclic peptide ligands, which are able to form larger
interacting surfaces and so are more likely to bind tightly and selectively.
Presented here is a proof-of-principle work on the development of small macrocyclic peptide ligands
for human pancreatic α-amylase (HPA), a starch digestive enzyme of relevance for treatment of type
2 diabetes.[3] Existing inhibitors of the starch digestion pathway can help prevent a postprandial
blood glucose spike, but currently suffer from side effects that severely hamper compliance. A HPA-
selective inhibitor should overcome these side-effects. Peptide ligands discovered using the RaPID
system showed low nanomolar inhibition for both HPA and the closely related salivary variant, with
50-fold selectivity over the subsequent enzyme in the starch digestion pathway. X-ray co-crystal
structures rationalise the tight binding of the two different interacting motifs, and are providing the
basis for structure-guided optimisation.
[1] G. W. Hart, R. J. Copeland, Cell 2010, 143, 672–676. [2] T. Passioura, H. Suga, Chem. Commun. 2017, 53, 1931–1940. [3] S. A. K. Jongkees, S. Caner, C. Tysoe, G. D. Brayer, S. G. Withers, H. Suga, Cell Chem. Biol. 2017, 24, 1–10.
44
Optimized synthesis of therapeutic MK2 inhibitor cell penetrating peptide:
reduced synthesis time using parallel automated synthesis
A. Kennedy, D. Martinez, C.N. Ramos-Colón, J.P. Cain
Gyros Protein Technologies, Tucson, Arizona, U.S.A.
Peptide based drug discovery and research is increasingly at the forefront in addressing new
therapeutic challenges due to their high target selectivity and potency with low toxicity. Automated
solid phase peptide synthesis (SPPS) increases reliability, efficiency, and crude purity for peptides in
drug discovery and development. For example, introducing heat (>50°C) in the SPPS of linear
peptides has shown improved results with shorter coupling cycles and higher crude purity.
The MK2 inhibitor, MMI-0100, is a cell penetrating peptide that is currently undergoing clinical trials
for fibrotic and inflammatory lung disease [1,2]. Previously, the optimized synthesis of a MMI-0100
analog (AAVGLQRALAKARAQRAAARAY) was done using an automated system with a four-day
method that consisted of double and triple couplings with the highest purities around 50%. In this
study, different conditions and methods for the synthesis of the MMI-0100 analog using parallel
synthesis were assessed. The full synthesis was completed in less than 24 h with improved crude
purities.
References [1] Komalavilas, Am J Respir Crit Care Med, vol. 193, p. A1349, 2016. [2] "Moerae Matrix - Scientific Platform," Moerae Matrix, 2017. [Online]. Available: http://moeraematrix.com/. [Accessed March 2017].
45
Temporal Controlled Membrane Fusion as a Tool to
Deliver Cell Impermeable Drugs
Li Kong, Jian Yang, Frederick Campbell and Alexander Kros*
Leiden University
Membrane fusion results in the transport and mixing of (bio)molecules across otherwise
impermeable barriers. In our group, we have reported a new method for direct drug delivery into the
cytosol of live cells in vitro and in vivo utilizing targeted membrane fusion between liposomes and
live cells. A pair of complementary coiled-coil lipopeptides was embedded in the lipid bilayer of
liposomes and cell membranes respectively, resulting in targeted membrane fusion with concomitant
release of liposome encapsulated cargo. To further regulate the fusion process, we applied steric
shielding and rapid, photoinduced deshielding method to temporal control the whole process.
Furthermore, the photo-activated membrane fusion could be used as an ideal method to deliver cell
membrane impermeable drug.
46
Studying the role of PPARγ phosphorylation via enzymatical
protein semi-synthesis
C.V. Lam, L.G. Milroy, L. Brunsveld
Eindhoven University of Technology
Post-translational modifications (PTMs) have been shown to play an important role in gene
expression. One particular example is the phosphorylation1 of serine 273 (S273) in a transcription
factor called peroxisome-proliferator activated receptor γ (PPARγ). Phosphorylation of this serine in
the PPARγ ligand binding domain (LBD) leads to selective gene regulation and ultimately leads to the
symptoms of diabetes type 2. However, the same kinase that phosphorylates S273 also
phosphorylates threonine 296, and it is unclear what the exact roles are of each phosphorylation and
how they lead to altered gene activity. To unravel the role of each modification, a protein semi-
synthetical route will be followed in which part of the protein domain will be expressed in E. coli and
the part with S273/T296 will be synthesized via FMOC solid phase peptide synthesis (SPPS). Then,
traceless ligation of both parts with an engineered enzyme2 allows for formation of the complete and
phosphorylated PPARγ LBD. (see fig)
1. Choi, J. H. et al. Anti-diabetic drugs inhibit obesity-linked phosphorylation of PPARgamma by Cdk5. Nature 466, 451– 456 (2010). 2. Schmidt, M., Toplak, A., Quaedflieg, P. J. & Nuijens, T. Enzyme-mediated ligation technologies for peptides and proteins. Curr. Opin. Chem. Biol. 38, 1–7 (2017).
Expression FMOC SPPS
His- SUMO PPARγ
279-505 -strHis
protease
PPARγ
279-505
Omniligase
pS273
PPARγ LBD
pS273
PPARγ
234-278
47
A Ligand Directed Divergent Synthesis Approach
to Establish Orthogonal Biological Modulators
Yen-Chun Lee1,2, Sumersing Patil1,2, Slava Ziegler1, Kamal Kumar1 and Herbert Waldmann1,2
1 Abteilung Chemische Biologie, Max-Planck-Institut für Molekulare Physiologie, Dortmund, Germany 2 Fakultät für Chemie und Chemische Biologie, Technische Universität Dortmund, Dortmund, Germany
In chemical biology and drug discovery, the development of novel methods for efficient synthesis of structurally divergent molecular scaffolds holds immense importance. Gold catalyzed enyne cycloisomerizations have been demonstrated as a powerful tool to access a wide range of complex molecules owing to the tunable nature of gold complexes with ligand and reaction condition [1]. Here we present a “Ligand directed divergent scaffold synthesis” (LDS) approach that targets synthesis of structurally distinct molecular scaffolds by means of a single mode of catalysis on common substrates. In this strategy, when oxindole derived 1,6-enynes were treated with gold complexes, the fate of the common bicyclic gold carbene intermediate could be steered by ligand variations of gold(I) cation, and selectively led to three structurally distinct scaffolds, the spirooxindoles, quinolones, and the df-oxindoles. Further biological studies in cell-based assays revealed small molecules based on three different scaffolds displaying orthogonal modulation in the activities of hedgehog signaling pathway, autophagy and cellular proliferation [2].
[1] R. Dorel, A. M. Echavarren, Chemical Reviews 2015, 115, 9028-9072. [2] Y.-C. Lee, S. Patil, C. Golz, C. Strohmann, S. Ziegler, K. Kumar, H. Waldmann, Nature Communications 2017, 8, 14043.
48
Synthesis of ADP ribosylated asparagine for structural studies on
ADPr binding proteins
Q. Liu, H. S. Overkleeft, G. A. van der Marel, D. V. Filippov
Bioorganic synthesis, Leiden Institute of Chemistry, Leiden University
Leiden, The Netherlands
Post-translational modification of proteins by (ADP-ribosyl)ation regulates many cellular pathways that are critical for genome stability, including DNA repair, chromatin structure, mitosis and apoptosis1. In contrast to the limited amino acid specificities of most PTMs, nearly all chemically reactive amino acid side chains have been reported as targets of ADPr2. However, little is known about the selectivity and exact mechanism of the amino acids it targets on cellular proteins. It is widely accepted that proteins, called macro-domains, capable to bind ADPr are important for biological functioning of this post-translational modification3. ADP-ribosylated peptides are known to be a powerful tool to gather the information on macro domain binding. Unfortunately, one of the reportedly most widespread modification site ADPr-Asp is labile and easy to migrate to 2'-OH of ribose. The possible way to tackle this problem is use amide instead of the ester linkage to stabilize the glycosydic bond while having the synthetic modification approximately isosteric to the native ADPr-Asp. This means the synthesis of ADPr-Asn. In this poster, I demonstrated the coupling of protected Asn with ribose moiety and the subsequent construction of diphosphate linkage. The deprotected and purified final ADPr-Asn might prove useful for the structural studies on macro domain proteins. 1 D. Slade, M. S. Dunstan, E. Barkauskaite, R. Weston, P. Lafite, N. Dixon, M. Ahel, D. Leys and I. Ahel, Nature, 2011, 477, 616-620. 2 C. M. Daniels, S. E. Ong and A. K. Leung, Mol Cell, 2015, 58, 911-924. 3 H. A. Kistemaker, A. P. Nardozza, H. S. Overkleeft, G. A. van der Marel, A. G. Ladurner and D. V. Filippov, Angew Chem Int Ed Engl, 2016, 55, 10634-10638.
49
Enhanced Peptide Purification via Novel
Orthogonal, Doped Reverse Phase Chromatography
Jürgen Machielse1, †Prof. Dr. Kinkel2, Andrea Wild1, Tim O`Mara3,
1. Zeochem AG, Uetikon am See, Switzerland
2. Former Prof. at TU Georg-Simon-Ohm, Nuremberg
3. Itochu Chemicals America Inc.
Peptides are important API’s for modern pharmaceuticals and have to be produced in industrial scale
with increasing demand on separation costs.
RPC is the well-established chromatographic mode within the available tool-box of methods and
procedures for the purification of this class of compounds in industrial scale production.
The paper will describe the beneficial use of Doped Reversed Phase packings in the repulsive-
attractive mode compared to non-doped RP packings on crude peptides. The novel orthogonal
Doped Reversed Phase materials combine the dual action of strong IEX groups (acidic or basic) and
Reversed Phase ligands like octyl chains on the packing surface.
Usually, Mixed-Mode packings are applied under conditions which add the retention power of IEX
groups by electrostatic attractive forces to the retention received from hydrophobic surface groups
to the solutes. Both species, e. g. the peptide and the IEX group carry opposite charges.
In 2014, ETH-Zürich (Prof. Dr. M. Morbidelli et al) in cooperation of Zeochem AG have introduced the
repulsive – attractive mode for peptide separation.
It can be shown that in the majority of all cases tested so far, improved selectivities and increased
resolution at decreased retention time and solvent consumption can be obtained.
[1]
R. Khalaf et al, Journal of Chromatography A 1397 (2015) 11-18
[2] R. Khalaf et al, Journal of Chromatography A 1407 (2015) 169-175
50
Cellular high-resolution studies of Nisin-Lipid II complexes
J. Medeiros-Silva1, S. Jekhmane1, R. Cox2, F. Torres1, E. Breukink2, M. Weingarth1 1 NMR spectroscopy, Bijvoet Center for Molecular Research, Utrecht University, Utrecht,
The Netherlands 2 Membrane Biochemistry and Biophysics, Bijvoet Center for Molecular Research, Utrecht University,
Utrecht, The Netherlands
Antimicrobial peptides (AMPs) are excellent candidates for the development of new antibiotics
with therapeutic applications. Particularly, AMPs that target Lipid II – a key molecule involved in the
synthesis of the bacterial cell wall – are extremely robust and less prone to resistance development
from bacteria[1].
Nisin is a lantibiotic AMP that effectively bind to Lipid II forming a pore-complex in the cellular
membrane, causing the bacteria to ultimately burst[2]. Therefore, the molecular determinants that
result in such complexes are of major interest. High resolution information on the Nisin-lipid II
complex is however scarce and totally absent regarding native-like conditions.
We present a cutting-edge solid-state NMR approach that allowed studying the Nisin-Lipid II
complex with atomic resolution in native cell membranes. These studies were also performed in
liposomes that provided high-resolution assignments, stoichiometry for the complex and respective
dynamics for the Nisin residues, on the micro-second and nano-second time-scale.
These studies will lead to a structural elucidation of Nisin-Lipid II complex and, ultimately, a
rational understanding of how the differences of the native chemical environment across bacterial
strains could modulate antibiotic binding.
[1] Oppedijk et al, Biochimica et Biophysica Acta 1858 (2016) 947–957
[2] Breukink et al, Nature Structural & Molecular Biology 11, 963 - 967 (2004)
51
Selenocysteine Chemistry and Total Chemical Synthesis Applied for Accessing
Human Selenoproteins
Norman Metanis1 1 Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel
Once considered a toxic element, selenium is now known as an essential element for life. For
example, in humans it is incorporated in proteins known as selenoproteins containing the 21st
encoded amino acid selenocysteine (Sec). The differences between selenium and sulfur in their redox
potentials, pKas, and nucleophilicities and electrophilicities give selenium interesting chemistry. For
example, Sec can be used as a tool for chemical protein synthesis, and allowing for site selective
modifications. It can be also incorporated into protein sequences to enhance oxidative protein
folding. This lecture will discuss our recent studies on chemical protein synthesis using Sec and
selective deselenization reactions, which convert Sec into Ala or Ser.
These advances in chemical protein synthesis bring us closer to accessing naturally occurring
selenoproteins, especially human selenoproteins that still await functional characterization.
[1.] Dery, L.; Reddy, P. S.; Dery, S.; Mousa, R.; Ktorza, O.; Talhami, A. and Metanis, N. Chem. Sci., DOI: 10.1039/c6sc04123j (2017).
[2.] Reddy, P. S.; Dery, S. and Metanis, N. Angew. Chem. Int. Ed., 55, 992–995 (2016). [3.] Dery, S.; Reddy, P. S.; Dery, L.; Mousa, R.; Dardashti Notis, R. and Metanis, N. Chem. Sci., 6, 6207-6212 (2015). [4.] Metanis, N. and Hilvert D. Chem. Sci., 6, 322-325 (2015). [5.] Metanis, N. and Hilvert D. Curr. Open. Chem. Biol., 22, 27-34 (2014). [6.] Metanis N. and Hilvert D. Angew. Chem. Int. Ed., 51, 5585-5588 (2012)
52
Purification and molecular docking study of an ACE-inhibitory peptide from
Kluyveromyces marxianus protein hydrolysates
Mahta Mirzaeia, Saeed Mirdamadib*, Mohamad Reza Ehsanic*, Mahmoud Aminlarid
aDepartment of food science and technology, Shahr-e-Qods Branch, Islamic Azad University,
Tehran, Iran. bDepartment of Biotechnology, Iranian Research Organization for Science & Technology (IROST),
Tehran, Iran. cDepartment of Food Science and Technology, Science and Research Branch, Islamic Azad University,
Tehran, Iran.
Hypertension is one of the risk factors for cardiovascular diseases and the renin–angiotensin system
plays an important role in the regulation of blood pressure. There has been some evidence that
proteins are potentially excellent sources of antihypertensive peptides, and enzymatic hydrolysis is
an effective method to release them from the protein structure. Kluyveromyces marxianus protein
was used as a source of food proteins for producing peptides with possible ACE-inhibitory activity.
Protein hydrolysates were prepared by trypsin and chymotrypsin enzymes. Hydrolysates obtained by
5 h, exhibited the highest ACE-inhibitory activities. After fractionation using ultrafiltration and
reverse phase high performance liquid chromatography (RP-HPLC) techniques, a new peptide (LL-9,
MW=1180 Da) with the amino acid sequence of Leu-Pro-Glu-Ser-Val-His-Leu-Asp-Lys was identified
and showed significant ACE inhibitory activity (IC50=0.027 mg/ml). The molecular docking studies
revealed that the ACE inhibitory activity of purified peptide is due to interaction with the S1 (Thr345)
and S2 (Tyr520, Lys511, Gln281) pockets of ACE. This study confirmed that the yeast protein
hydrolysate of K. marxianus and its purified peptides may be beneficial as food additives and
pharmaceutical agents and can potentially replace the antihypertensive agents with chemical origin.
Key words: K. marxianus; Bioactive peptides; ACE inhibitory; Protein hydrolysate; Molecular docking
53
Development of a novel epitope-based diagnostic assay for systemic sclerosis
Gianluca Moroncinia, Matteo Mozzicafreddob, Massimiliano Cucciolonib, Antonella Griecoa,
Chiara Paolinia, Cecilia Tonninia, Sara Salvuccia, Enrico Avvedimentoc, Ada Funarod,
Armando Gabriellia a) Department of Molecular and Clinical Sciences, Università Politecnica Marche, Ancona, Italy
b) School of Biosciences and Veterinary Medicine, University of Camerino, Camerino, Italy
c) Department of Molecular Medicine, Università Federico II, Napoli, Italy
d) Department of Medical Sciences, University of Torino, Torino, Italy
Background Our previous study1 described the conformational PDGFRa epitope of VHPAM-VK16F4 agonistic autoantibody, cloned from memory B cells of a SSc patient, that can induce fibrosis in vivo9. The same study showed that peptides composing this epitope may be specifically recognized by serum IgG of patients with systemic sclerosis (SSc), but not of controls.
Objectives i. To identify the immunodominant peptide within the discontinuous PDGFR epitope of profibrotic autoantibodies; ii. to identify further immunodominant epitopes recognized by agonistic autoantibodies; iii. to use these immunodominant peptides to develop an epitope- based assay for diagnosis of SSc and classification of SSc clinical subtypes.
Methods i.The large PDGFRa peptide library used for epitope mapping of monoclonal anti-PDGFRa antibodies1 was screened with 25 SSc (12 limited, 13 diffuse) and 25 sex- and age-matched healthy control (HC) serum samples. ii. A smaller PDGFRa peptide library containing only the top 20 conformational binders plus 20 linear and 20 conformational controls was synthesized. 60 conformational and linear peptides of a cognate protein forming a molecular complex with PDGFRa were included in the array. 20 scrambled peptides were added as negative controls. This library was screened with the same 50 serum samples. iii. A third library was synthesized, retaining the top cognate protein peptide binders, and 15 chimeric PDGFRa/cognate protein peptides, chosen among the best binders, with some nonbinding controls. This library was tested as before. Libraries were synthesized by Pepscan Presto, Netherlands. Statistical analysis was performed by Wilcoxon-Mann- Whitney test. Correlations between serological results and clinical status were made.
Results i. An immunodominant peptide discriminating between SSc and HC serum samples was identified in the first library. ii. This was confirmed by the second library, which highlighted also one immunodominant epitope from the cognate protein. Statistical analysis identified two cohorts of SSc samples (reactive vs nonreactive, the latter undistinguishable from HC) each composed by limited and diffuse SSc subtypes. iii. The third peptide library identified the chimeric peptide recognized exclusively by the reactive SSc serum samples, which were taken from patients with active disease regardless of limited vs diffuse classification, whereas the nonreactive SSc samples were taken from subjects with less active, non progressive disease.
Conclusions We developed a conformational epitope-based assay detecting SSc-specific serum agonistic autoantibodies. The preliminary results suggest that this novel array may identify SSc patients with active disease, regardless of the canonical classification criteria. We propose this assay for prospective screening of large cohorts of patients affected by, or suspected for, SSc, to validate it as a tool for disease activity assessment and/or early diagnosis.
References 1 Moroncini G et al. Epitope specificity determines pathogenicity and detectability of anti- PDGFRa autoantibodies in systemic sclerosis. Arthritis and Rheumatology. 2015; 67(7):1891-1903. 2 Luchetti MM et al. Induction of Scleroderma Fibrosis in Skin-Humanized Mice by Administration of Anti-Platelet- Derived Growth Factor Receptor Agonistic Autoantibodies. Arthritis & Rheumatology. 2016; 68(9):2263-73.
54
Peptide Design and Utility Based on Chemical Features of Amino Acid Side Chains
A. Norouzy1, Z. Azizi2, W.M. Nau3
1 Industrial and Environmental Biotechnology department, National Institute of Genetic
Engineering and Biotechnology (NIGEB), Tehran, Iran 2 Department of Molecular Medicine, School of Advanced Technology in Medicine, Tehran
University of Medical Sciences, Tehran, Iran 3 Department of Life Sciences and Chemistry, Jacobs University Bremen, Germany
Peptides sequence determines their special feature. The interaction between the side chains
themselves within a peptide sequence should be considered for designing a peptide.
For unraveling the intramolecular charge repulsion affecting peptide dynamics, we used homo
hexapeptides made of acidic amino acids (Asp6, Glu6), basic amino acids (Lys6, Arg6), and His6.
Peptides were given positive or negative charges by altering pH. For measuring their flexibility, the
peptides were labeled with 5-Fluorotryptophan and Dbo at N- and C-terminus respectively (Figure
below).[1] Our –fluorescence based− measurements [1, 2] surprisingly revealed that there is no
charge repulsion between negative charges on acidic side chains; while, it does exist between
positive charges on basic side chains.
On the other hand, attraction between positively charged peptides and negative charges on the cell
membrane helps poly arginine peptides passing through the cell membrane.[3] Based on this
mechanism, protamine, a highly positively charged peptide, was assayed in live cells.[4] The cells had
been preloaded with p-sulfonatocalix[4]arene/lucigenin (CX4/LCG) complex. After passing through
the cell membrane, the protamine displaced the LCG dye from CX4 to form CX4/protamine complex.
The free lucigenin recovered its fluorescence which was an indicator for the assay.
Our research is continuing based on amino acid side chain charges and other characteristics for
designing cyclic cell-penetrating peptides, and epitope peptides for binding to human leukocytes
antigens (HLA).
5-Fluorotryptophan at the N-terminus of a peptide schematically quenches an exited Dbo molecule at the C-terminus
References [1] A. Norouzy, K.I. Assaf, S. Zhang, M.H. Jacob, W.M. Nau. Coulomb repulsion in short polypeptides. J Phys Chem B, 2015;119:33-43. [2] M.H. Jacob, R.N. Dsouza, I. Ghosh, A. Norouzy, T. Schwarzlose, W.M. Nau. Diffusion-enhanced Förster resonance energy transfer and the effects of external quenchers and the donor quantum yield. J Phys Chem B, 2013;117:185- 198. [3] H.D. Herce, A.E. Garcia, M.C. Cardoso. Fundamental molecular mechanism for the cellular uptake of guanidinium-rich molecules. J Am Chem Soc, 2014;136:17459-17467. [4] A. Norouzy, Z. Azizi, W.M. Nau. Indicator displacement assays inside live cells. Angew Chem Int Ed 2015;54:792-795.
55
Targeting the Ubiquitin – Proteasome Pathway Chemical Ubiquitination
Huib Ovaa
Leiden University Medical Center, the Netherlands Einthovenweg 20, 2333 ZC Leiden, the Netherlands
h.ovaa at lumc.nl
The complexity of the ubiquitylation machinery makes it difficult to construct ubiquitylated proteins
and polypeptides in vitro for research purposes. Frequently, the required combination of ligases is
either not known or difficult to obtain in recombinant form. We have developed chemical strategies
that allow the construction of virtually any ubiquitin or ubiquitin-like conjugate, including ubiquitin
chains. These techniques allow the design of novel assay reagents.
Active site directed probes for deubiquitylating enzymes (DUBs) can report DUB activity in complex
samples containing multiple DUB activities. The latest generation of active site directed probes, is
based on alkynes. We recently discovered the alkyne as a reactive moiety by serendipity. Alkynes
have so far been considered to be unreactive but nevertheless cysteine proteases can trigger a
chemical reaction not known in the textbooks. The advantage of alkyne-based probes lies in their
stability and ease of use. More recently, we designed a next generation of specific active site directed
probes with substrate context that allow monitoring the activity of deubiquitylating enzymes (DUBs)
in lysates with increased sensitivity. Also the development of probes that report ligase activity will be
discussed.
References Zhang X, Smits AH, van Tilburg GB, Jansen PW, Makowski MM, Ovaa H, Vermeulen M. An Interaction Landscape of Ubiquitin Signaling. Mol Cell. 2017 Mar 2;65(5):941-955.e8. doi: 10.1016/j.molcel.2017.01.004.PMID:28190767
Mevissen TE, Kulathu Y, Mulder MP, Geurink PP, Maslen SL, Gersch M, Elliott PR, Burke JE, van Tol BD, Akutsu M, El Oualid F, Kawasaki M, Freund SM, Ovaa H, Komander D. Molecular basis of Lys11-polyubiquitin specificity in the deubiquitinase Cezanne. Nature. 2016 Oct 20;538(7625):402-405. doi: 10.1038/nature19836. PMID:27732584
Mulder MP, Witting K, Berlin I, Pruneda JN, Wu KP, Chang JG, Merkx R, Bialas J, Groettrup M, Vertegaal AC, Schulman BA, Komander D, Neefjes J, El Oualid F, Ovaa H. A cascading activity-based probe sequentially targets E1-E2-E3 ubiquitin enzymes. Nat Chem Biol. 2016 Jul;12(7):523-30. doi: 10.1038/nchembio.2084.
Flierman D, van der Heden van Noort GJ, Ekkebus R, Geurink PP, Mevissen TE, Hospenthal MK, Komander D, Ovaa H. Non-hydrolyzable Diubiquitin Probes Reveal Linkage-Specific Reactivity of Deubiquitylating Enzymes Mediated by S2 Pockets. Cell Chem Biol. 2016 Apr 21;23(4):472-82. doi: 10.1016/j.chembiol.2016.03.009. Ekkebus R, van Kasteren SI, Kulathu Y, Scholten A, Berlin I, Geurink PP, de Jong A, Goerdayal S, Neefjes J, Heck AJ, Komander D, Ovaa H. On terminal alkynes that can react with active-site cysteine nucleophiles in proteases. J Am Chem Soc. 2013 Feb 27;135(8):2867-70. doi: 10.1021/ja309802n. PMID:23387960 Geurink PP, El Oualid F, Jonker A, Hameed DS, Ovaa H. A general chemical ligation approach towards isopeptide-linked ubiquitin and ubiquitin-like assay reagents. Chembiochem. 2012 Jan 23;13(2):293-7. doi: 10.1002/cbic.201100706. No abstract available. PMID:22213387 El Oualid F, Merkx R, Ekkebus R, Hameed DS, Smit JJ, de Jong A, Hilkmann H, Sixma TK, Ovaa H. Chemical synthesis of ubiquitin, ubiquitin-based probes, and diubiquitin. Angew Chem Int Ed Engl. 2010 Dec 27;49(52):10149-53. doi: 10.1002/anie.201005995. No abstract available. PMID:21117055
56
Novel Highly Stereoselective Synthesis of Carbon-11 Labeled Small Peptides
Pekošak, Aleksandra1,2; Filp, Ulrike1; Rotstein, Benjamin H.2; Collier, Thomas L.2; Windhorst,
Albert D.1; Vasdev, Neil2; Poot, Alex J.1 1Department of Radiology and Nuclear Medicine, VU University Medical Center, Amsterdam; e-mail:
[email protected] 2Division of Nuclear Medicine and Molecular Imaging, Massachusetts General Hospital, Boston, USA
& Department of Radiology, Harvard Medical School, Boston, USA
Objectives: Radiolabeled peptides have become indispensable PET tracers for tumor imaging. To date, peptides are labeled by the addition of a prosthetic group or a chelated radiometal, each of which may negatively influence pharmacokinetics and receptor affinity of the probe.
[1] To overcome current limitations, isotopologue
labeling in a highly regio- and stereoselective manner would enable rapid preclinical assessment of the peptide. Here, we describe a broadly applicable Schiff base alkylation with [
11C]benzyl iodide ([
11C]1)
[2] in the presence of
a chiral phase-transfer catalyst (PTC), as a novel, direct and highly stereoselective method for native peptide labeling with carbon-11.
Methods: Precursors for
11C-labeling and all reference compounds for analysis were synthesized using multi-
step solution-phase peptide synthesis. The 11
C-labeling was achieved via stereoselective PTC alkylation at the α-carbon of the N-terminal glycine Schiff base with [
11C]1. Asymmetric induction was investigated by screening a
panel of 10 quaternary ammonium salts as PTC, as well as the influence of temperature, reaction time, stirring, solvent, precursor and catalyst amounts and base concentration.
Figure 1: A: Achieved stereoselective alkylations with [
11C]1. B: Matched/mismatched catalyst pair.
Results: In total 10 precursors and 20 reference compounds were synthesized in 5-7 steps in sufficient yield (4-50%) and high purity (>95%). [
11C]1 was prepared in a 3-step one-pot Grignard reaction from cyclotron-
produced [11
C]CO2 (Fig 1A).[2]
Subsequently, the influence of the substrate peptidic backbone on stereoselectivity was studied on dipeptides [
11C]2a-2h, identifying matched/mismatched catalyst pairs (Fig 1B)
for N-terminal Phe peptides. Further, the highly stereoselective 11
C-benzylation employing cinchonine PTC (Fig 1B, blue) as an optimal catalyst was achieved for peptide [
11C]3 (Fig 1A, right), an essential pharmacophore for
somatostatin receptors binding. Finally, adjacent amino acid residue control was confirmed by alkylation of the isomeric tetrapetide precursor with neighboring L-Trp in a de of 75±4% using cinchonidine PTC (Fig 1B, red).
[3]
Conclusions: A powerful stereoselective
11C-labeling of peptides was developed, and has since been extended
to various N-terminal peptides using other 11
C-alkyl halides. Furthermore, a fully automated procedure resulted in the first native
11C-labeled enantiomerically pure tetrapeptide [
11C]3.
[3]
Acknowledgements: A.P. is a Fulbright Visiting Researcher and a Marie-Curie fellow (RADIOMI). References:
[1]M. Fani et al., Eur. J. Nucl. Med. Mol. Imaging, 2012.
[2]A. Pekošak et al., J. Label Compd. Radiopharm. 2015.
[3]A. Pekošak et al., EurJOC, 2017.
57
Multipurpose pluribodies
Elko Peterse1, Angela el Hebieshy2, Dmitri V. Filippov1, Ferenc A. Scheeren3, Ton N.
Schumacher4, Fred Falkenburg5, Huib Ovaa2* and Hermen S. Overkleeft1* 1 Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands
2 Department of Chemical Immunology, Leiden University Medical Center (LUMC), Leiden,
The Netherlands 3 Department of Oncology, The Netherlands Cancer institute (NKI), Amsterdam, The Netherlands
4 Division of Immunology, The Netherlands Cancer institute (NKI), Amsterdam, The Netherlands 5 Department of Hematology, Leiden University Medical Center (LUMC), Leiden, The Netherlands
Our research focuses on selectively targeting T-cell malignancies using antibodies. To combat the
heterogeneity present in these malignancies, we’re aiming to selectively target a combination of cell-
surface proteins specific to tumor cells. To ensure high avidity binding when all ligands are expressed,
single-chain variable fragments (scFvs) will be used that display reduced affinity for its respective
antigen. These scFvs will be assembled on a synthetic scaffold based on Gramicidin S to create a
multivalent display. The use of bioorthogonal chemistry for the coupling will allow a variety of scFVs
to be displayed with relative ease.
58
Self-assembling nanobodies for nanomedicine
J. Pille, S. van Lith, W. Leenders, J.C.M van Hest
TU Eindhoven
Recombinant heavy-chain antibody fragments (VHHs), also called nanobodies, are promising tools in
the field of targeted nanomedicine. 7D12 is a VHH against the epidermal growth factor receptor
(EGFR) that is overexpressed in various cancers and has been evaluated as an effective cancer
targeting VHH in multiple studies. The small size of VHHs (15-20 kDa) results in a low circulation half-
life. Incorporation of VHHs in supramolecular structures overcomes the limited circulation time in
blood by increasing the hydrodynamic radius. It simultaneously allows the incorporation of different
VHHs and/or other targeting moieties and active components into one structure, creating
multispecificity and versatility for therapy and diagnosis. Here, we produced well-defined 7D12-
containing nanoparticles by utilizing thermo-responsive diblock elastin-like peptides that reversibly
self-assemble into micellar structures. The resulting particles have a hydrodynamic radius of 24.3 nm
+ 0.9 nm and retain full EGFR-binding capacity. We present proof of principle of the usability of such
particles by controlled incorporation of a photosensitizer and show that the nanoparticles elicit
EGFR-specific photoimmunotherapy. We propose that this approach allows for the controlled
incorporation of various groups, improving therapy and diagnosis with targeted nanomedicine.
59
Using cation-exchange resin as a new method of labelling carboxylgroups with 18O
Halina Plociennik, Piotr Stefanowicz
Faculty of Chemistry, University of Wroclaw, Wroclaw, Poland
We have developed a new universal method which allows labelling of Fmoc protected amino acids
and peptides. This method can find practical application in labelling active pharmacological
substances which contain carboxyl groups. The isotopologues obtained in this way will can be applied
as standards in quantitative analysis by LC-MS. The new method is based on using catalytic
properties of cation-exchange resin.
Currently there are several methods which employ acid catalysis for labelling with oxygen-18 isotope.
A peptide is dissolved in acidic H218O solution, and thereby peptide is slowly labelled (11 days or 85h
if temperature of mixture is increased).[1,2] The other method variant proposed by Haaf and
Schlosser significantly decreases the necessary time of labelling reaction.[3] Our previously published
method utilizes a mixture of dioxane with H218O (19:1, v:v) saturated with hydrogen chloride. This
procedure allows decrease the cost of labelling simultaneously reducing the time.[4] However,
working with dry hydrogen chloride is inconvenient, especially on micro scale. The greatest
advantage of the method proposed here is the replacement of dry hydrogen chloride with cation-
exchange resin. It simplifies the experimental procedure and completely eliminates the problem with
back-exchange due to water vapor in atmosphere, because the cation-exchange resin can be quickly
and efficiently separated from the labelling mixture, thus immediately stopping the exchange
reaction. The exchange resins are widely used as catalysts of many reactions in industry. They are
easily available, cheap and easy to work with.
[1] R. Niels, E.H. Witkowska et al., Anal. Chem., 81(7), 2804-2809 (2009). [2] H. Jiang, A. Ramos, X. Yao, Anal. Chem, 82, 336-342 (2010). [3] E. Haaf, A. Schlosser, Anal. Chem., 84, 304-311 (2012). [4] M. Modzel, H. Płóciennik, A. Kluczyk, P. Stefanowicz, Z. Szewczuk, J. Pept. Sci., 20, 896-900 (2014).
60
A novel integrin-targeting peptide attenuates pancreatic tumor growth in
vivo
Praneeth R. Kuninty1, Tushar N. Satav1, Gert Storm1, Jai Prakash1,2’* 1 Section: Targeted Therapeutics, Department of Biomaterials, Science and Technology, MIRA
Institute for Biomedical Technology and Technical Medicine, University of Twente, The Netherlands; 2ScarTec Therapeutics BV, University of Twente, Enschede, The Netherlands
*Presenting author: [email protected]
Pancreatic tumor is characterized by high stromal tissue, mainly comprised of cancer-associated
fibroblasts and extracellular matrix. Tumor stroma supports tumor cells to proliferate and
metastasize, thereby leading to enhanced tumor growth. We have recently identified integrin alpha5
(ITGA5) as a novel target overexpressed in tumor stroma in patients and shows its significance for
prognosis and therapy. To target ITGA5, we recently designed a novel 7 amino acid peptide (so-called
AV3) against ITGA5 and investigated its specific binding to ITGA5, cells and examined therapeutic
effects in vitro and in vivo.
The specific binding of the peptide to ITGA5 was examined with microscale thermophoresis. These
data showed Kd value of <100nM. In addition, the binding to another integrin alpha receptor (ITGA4)
did not show any binding. Furthermore, the binding to stromal cells (human pancreatic stellate cells)
was confirmed using flow cytometric analyses and fluorescent microscopy. Knocking down of the
ITGA5 led to the significantly reduced binding to the cells. Then, we examined whether the peptide
blocks ITGA5 and inhibits cells phenotype. Cells were activated with human recombinant TGF-beta
and co-incubated with increasing concentration of AV3. We found that AV3 at 10-20 uM strongly
inhibited the activation of these cells, as determined by immunostaining and western blot analyses of
different markers. In addition, AV3 also inhibited the contraction of these cells embedded in a
collagen matrix.
Then, we examined our peptide in vivo in a human pancreatic xenograft tumor model. Intriguingly,
treatment of the tumors with the peptide either injected intratumorally or intraperitoneally twice a
week significantly reduced the tumor growth.
In conclusion, this study reveals that inhibition of ITGA5 using a novel peptide AV3 is in potential to
inhibit pancreatic tumor growth in vivo.
61
Polyoxometalates as Inorganic Amino Acids for Biomedical Applications
Ken Princena, Anna M. Kaczmareka, Rik Van Deuna, Annemieke Madderb, Kristof Van Heckea aDepartment of Inorganic and Physical Chemistry, Ghent University,
Krijgslaan 281-S3, B-9000, Ghent, Belgium bDepartment of Organic and Macromolecular Chemistry, Ghent University, Krijgslaan 281-S4, B-9000
Ghent, Belgium [email protected]
This research encompasses the organic functionalization of inorganic clusters, namely polyoxometalates1
of the Anderson arche-type (APOMs). In regard, (K5.5H1.5)[SbW6O24].6H2O, (Na3)[CrMo6O24H6].8H2O and
[(n-C4H9)4N]4[Mo8O26] were chosen based on miscellaneous biomedical factors2 and ease of
manipulability. These APOMs contain reactive, surface-based, triple-bridged (μ3) oxygen atoms that can
be interchanged, under reflux conditions, with the oxygens of a tripodant tris-ligand. In one part of the
study the APOMs were first grafted simply with tris(hydroxymethyl)aminomethane, specifically the single
and double sided compounds respectively (C16H36N)3{Cr(OH)3Mo6O18[(OCH2)3CNH2]}·12H2O (fig. 1) and
[N(C4H9)4]3[MnMo6O18{(OCH2)3CNH2}2] (fig. 1b). Subsequently manipulated to form activated esters (e.g.
(C16H36N)3[MnMo6O24(C12H15N2O5)2]) through the formation of amide bonds, termed post-
functionalization and to be conjugated to a L-di-Phe peptide. The other part of the study examines the
grafting of a tris-ligand bearing a chloride functionality, (C16H36N)3[MnMo6O18{ (OCH2)3C3H3NOCl}2] (fig.
2c), in a pre-functionalized manner towards an SN2 reaction with glutathione. Finally, the latter was
transformed to bear the organic azide handle (C16H36N)3-[MnMo6O18{(OCH2)3C3H3N4O}2] (fig. 2d)
envisaging a click reaction with a biological relevant peptide.3 The APOMs and ligands were structurally
characterized by single crystal X-ray diffraction, elucidating new crystal structures. Conjugate products
were investigated with NMR, elemental analysis and RPLC-ESI-MS.
1. M. T. Pope, A. Müller, Angew. Chem., Int. Ed. Engl, 30, 34 (1991). 2. P. A. Lorenzo-Luis, P. Gili, Recent Res Dev Inorg Chem, 2, 185 (2000). 3. C. Yvon, A. J. Surman, M. Hutin, J. Alex, B. O. Smith, D.-L. Long, L. Cronin, Angewandte Chemie International Edition 53, 3336 (2014).
Fig. 1 Some functionalized APOMs: (a) single sided tris(hydroxymethyl)aminomethane, (b) double sided tris(hydroxymethyl)aminomethane, (c) chloride-functionalized, (d) azide-functionalized
62
Antimicrobial peptides as cosmetic ingredients to deter cutaneous pathogens
M. Rahnamaeian1, A. Vilcinskas1,2 1 Department of Bioresources, Fraunhofer Institute for Molecular Biology and Applied Ecology,
Giessen, Germany 2 Institute for Insect Biotechnology, Justus Liebig University of Giessen, Giessen, Germany
The diverse activity spectrum of antimicrobial peptides (AMPs) against microbial pathogens makes
them attractive candidates for development of a new generation of antibiotics. Although there are
reservations regarding the systemic administration of AMPs because they might be allergic or
sensitive to endogenous peptidases, these peptides seem ideal for topical application against
cutaneous fungal and bacterial pathogens in formulations such as lotions, creams, shampoos and
wound dressings, and could therefore be valuable products for the cosmetic industry [1]. Among
AMPs, the relatively short ones (< 20 amino acids) combine optimal antimicrobial activity with
inexpensive chemical synthesis and are therefore more compatible with large-scale production and
the modifications required to ensure stability, low toxicity and microbial specificity. Proof-of-concept
for the application of AMPs as novel anti-infectives has already been provided in clinical trials and we
considered, here, the anti-infective properties of short AMPs lacking disulfide bonds, which are active
against dermatologically-important microflora. To follow such an approach for using AMPs as
cosmetic ingredients, we need to consider certain facts. For instance, skin pathogens can modulate
the efficacy of AMPs via secretion of effector molecules [2] as well as induction of resistance
development. Also, cationic AMPs are sensitive to pH [3], so the pH and ionic strength of the carrier
matrix should be optimized to achieve the greatest efficacy. On the other hand, many AMPs can
interact to maximize their antimicrobial potency [4], and this promotes the activity of AMPs at low
concentrations. In addition, the organometallic derivation of AMPs has the potential to increase the
potency of AMPs [5], and provide metal-specific modes of action that delay the acquisition of
resistance by bacterial populations. However, AMPs may need specific modifications in the amino
acid sequence to meet the therapeutic criteria such as high antimicrobial activity and low hemolytic
or cytotoxic effects [6]. [1] M. Rahnamaeian and A. Vilcinskas, Appl Microbiol Biotechnol, 99, 8847 (2015) [2] M. Swidergall et al, Antimicrob Agents Chemother, 57, 3917 (2013) [3] W.F. Walkenhorst et al, Antimicrob Agents Chemother, 57, 3312 (2013) [4] M. Rahnamaeian et al, Proc Biol Sci, 282, 20150293 (2015) [5] G. Gasser and N. Metzler-Nolte, Curr Opin Chem Biol, 16, 84 (2012) [6] M. Tonk et al, Appl Microbiol Biotechnol, 100, 7397 (2016)
63
Tricyclic Peptides via Templated Tandem CLIPS/CuAAC Cyclizations
G.J.J. Richelle1, S. Ori1, H. Hiemstra1, J.H. van Maarseveen1, P. Timmerman1,2
1 Van ’t Hoff Institute for Molecular Sciences (HIMS), University of Amsterdam, The Netherlands 2 Pepscan Therapeutics, Lelystad, The Netherlands
Multicyclic peptides provide a very attractive molecular format for the design of novel therapeutics,1
Therefore, novel routes for synthesis and HTS-screening of this fascinating class of compounds are
desperately needed. A decade ago, we launched a novel scaffold-assisted peptide-cyclization
technology platform, termed “CLIPS”, to generate in a one-steps procedure a new class of bicyclic
peptides able to act as potent inhibitors of hitherto undruggable therapeutics targets.2,3
Following this, we now present a next-generation technology that combines both the CLIPS and
CuAAC technology in order to create a novel class of tricyclic peptides. We present the chemical
synthesis of four different types of CLIPS/CuAAC-scaffold and show how each of these scaffold
behaves in the two-steps, one-pot synthesis of CLIPS/CuAAC-tricycles.
Scheme 1: tandem CLIPS/CuAAC reactions of a linear peptide onto a T4-scaffold to generate tricyclic peptides
1 Craik, D.J.; Fairlie, D.P.; Liras, S.; Price, D. Chem. Biol. Drug. Des., 2013, 81, 136–147.
2 Heinis, C.; Rutherford, T.; Freund, S.; Winter, F.; Nat. Chem. Biol., 2009, 5, 502–507.
3 Timmerman, P.; Beld, J.; Pruijk, W.C.; Meloen, R.H. Chembiochem, 2005, 6, 821–824.
64
Use of subtilisin variants for assembling peptide head-to-tail macrocycles
Marcel Schmidt1,2, Ana Toplak1, Peter J.L.M. Quaedflieg1, Jan H. van Maarseveen2,
Timo Nuijens1
1 EnzyPep B.V., Brightlands Campus, Geleen. 2Van’t Hoff Institute of Molecular Sciences, University of Amsterdam.
Peptide macrocycles represent an extremely diverse class of molecules that are attracting increased
attention as drug leads and prospective pharmaceuticals.[1] The increasing number of macrocyclic
peptides being investigated as therapeutics mandates efficient and cost-effective routes that enable
their synthesis at large scale. However, both lab scale synthesis and efficient large scale manufacture
of peptide macrocycles using traditional synthetic methods is still a major challenge.[2] Due to their
excellent stereo- and chemo-selectivity, the use of enzymes for the head-to-tail cyclization of
peptides represents a promising and scalable strategy and provides an elegant link between
chemistry and biology.[3] We have recently developed several subtilisin variants that are capable of
forming a peptide bond between a C-terminal activated (Cam-) ester segment and the N-terminus of
an acyl acceptor segment with high efficiency.[4]
In this study, the peptide ligase omniligase-1 has been used as a tool for the cyclization of several
head-to-tail (multi)cyclic peptides, including the cyclotide MCoTI-II. Cyclization and oxidative folding
of the cyclotide MCoTI-II was efficiently performed in a one-pot reaction on gram scale. The native
cyclotide was isolated and the correct disulfide bonding pattern was confirmed by NMR structure
determination. Furthermore, compatibility of chemo-enzymatic peptide synthesis (CEPS) using
omniligase-1 with methods such as chemical ligation of peptides onto scaffolds (CLIPS) was
successfully demonstrated by synthesizing a kinase-inhibitor derived tricyclic peptide. Our studies
indicate that the minimal ring size for omniligase-1 mediated cyclization is 11 amino acids; the
cyclization of peptides longer than 12 amino acids proceeds with remarkable efficiency. In addition,
several macrocycles containing non-peptidic backbones (e.g. polyethylene glycol), isopeptide bonds
(using amino acid side-chain attachment) as well as D-amino acids could be efficiently cyclized.
In conclusion, the efficiency of omniligase-1 (>95 % cyclization yield on average) combined with its
broad substrate scope allows traceless ligation and makes CEPS technology a flexible and generally
applicable tool, which provides a viable and economically attractive route for the synthesis of
cyclotides and other head-to-tail cyclic peptides.
[1] A. Zorzi, K. Deyle, C. Heinis, Curr. Opin. Chem. Biol. 2017, 38, 24–29. [2] C. J. White, A. K. Yudin, Nat. Chem. 2011, 3, 509–524. [3] M. Schmidt, A. Toplak, P. J. L. M. Quaedflieg, T. Nuijens, Curr. Opin. Chem. Biol. 2017, 38, 1–7. [4] A. Toplak, T. Nuijens, P. J. L. M. Quaedflieg, B. Wu, D. B. Janssen, Adv. Synth. Catal. 2016, 358, 2140–2147. [5] M. Schmidt, A. Toplak, P.J.L.M. Quaedflieg, H. Ippel, G.J.J. Richelle T.M. Hackeng, J.H. van Maarseveen, T. Nuijens, Adv. Synth. Catal., 2017, accepted.
65
The nanoFleming: High-throughput screening for antimicrobial peptides
S. Schmitt1, M. Montalbán-López2, D. Peterhoff3, R. Wagner3, O.P. Kuipers2,
S. Panke1, M. Held1 1ETH Zürich, Department of Biosystems Science and Engineering, Basel, Switzerland
2University of Groningen, Department of Genetics, Groningen, The Netherlands 3University of Regensburg, Institute of Medical Microbiology and Hygiene, Regensburg, Germany
The number of multidrug resistant pathogens is growing and novel antibiotic substances are
desperately needed in order to fight infectious diseases in the future.
We employ lantibiotics – a subclass of ribosomally synthesized and posttranslationally modified
antimicrobial peptides – as a template for the design of novel antibiotics. Lantibiotics can be
dissected into subunits mediating different for the pathogen detrimental effects (e.g. membrane
piercing or cell wall destabilizing). We shuffled these subunits and generated thousands of novel,
putative active, chimeric peptides which were subsequently analyzed in order to isolate variants
displaying modulated or improved antimicrobial
activity. Fig. 1 nanoFleming assay. Growth-inhibition of NLR-embedded
sensor colonies (green) in the presence of a candidate colony
(red).
In order to cope with large sample-amounts, we
developed a miniaturized assay based on “Fleming’s
approach” of antimicrobial activity assessment at
rates of 105 variants per day (nanoFleming). As
compartments during growth and analysis, small
hydrogel reactors (nanoliter reactors = NLRs)1–3 were used as carriers for producer (secreting the
peptide-candidates) and sensor cells (indicating the bioactivity of the peptides simultaneous to their
biosynthesis). The NLRs contained on average one candidate but were highly overpopulated with
sensor cells (~100 cells) and were incubated in a hydrophobic environment in order to prevent
peptide leakage and crosstalk. After incubation, biomass levels are visualized using a fluorescent dye
and NLRs are analyzed. Clearance of a NLR from sensor cells is indicative for the co-localization of a
candidate putatively secreting a highly active peptide. The positive events were sorted out by large
particle flow cytometry, candidates were recovered from the NLRs, and subjected to secondary
analysis.
We subjected a library of 104 peptide variants to the nanoFleming assay and isolated 150 new-to-
nature antimicrobial peptides, some of which display activity profiles exceeding the potency of their
natural templates. Furthermore, we extracted design rules for novel antimicrobial peptides that may
facilitate a semi-rational design of additional variants in the future.
[1] Walser, M. et al. Isolation of monoclonal microcarriers colonized by fluorescent E. coli. Cytometry. A 73, 788–798 (2008). [2] Walser, M. et al. Novel method for high-throughput colony PCR screening in nanoliter-reactors. Nucleic Acids Res. 37, e57 (2009). [3] Meyer, A. et al. Optimization of a whole-cell biocatalyst by employing genetically encoded product sensors inside nanolitre reactors. Nat. Chem. 7, 673–678 (2015).
66
Quantification of peptide hormone hepcidin for a better diagnosis of anemia
E.M.H. Schmitz1234, N.M Leijten134, D. van de Kerkhof13, M.A.C. Broeren12, J.L.J. van Dongen14, L. Brunsveld14, V. Scharnhorst134
1. Expert Center Clinical Chemistry Eindhoven, The Netherlands 2. Máxima Medical Center Veldhoven, Clinical Laboratory, The Netherlands
3. Catharina Hospital Eindhoven, Clinical Laboratory, The Netherlands 4. Eindhoven University of Technology, Department of Biomedical Engineering, Laboratory of
Chemical Biology and Institute for Complex Molecular Systems, The Netherlands Peptide hormone hepcidin is the key regulator of iron homeostasis (1). Since its discovery in 2001, it has been suggested as a promising diagnostic marker for iron-related disorders. However, accurate and reproducible quantification is challenging (2). Reference values for different populations and added value in diagnosis are thus still unclear. A promising technique for accurate quantification is UPLC-MS/MS (ultra performance liquid chromatography – tandem mass spectromerty). Therefore, we developed a UPLC-MS/MS assay for quantification of hepcidin.
First we made hepcidin, consisting of 25 amino acids, using solid phase peptide synthesis. Also, an isotopically labeled hepcidin was synthesized containing two [13C9,
15N]-phenylalanines, to serve as an internal standard during UPLC-MS/MS analysis. The four disulfide bridges were formed by incubation with glutathione to obtain the correctly folded 3D structure. Calibrators and quality control samples for UPLC-MS/MS analysis were made by spiking the synthesized hepcidin (1-250 ng/mL) into rabbit serum, which showed to be a suitable matrix (3). Samples were prepared using solid phase extraction (SPE) before analysis. The developed method was analytically validated and patient samples were collected to measure hepcidin levels.
The UPLC-MS/MS assay had excellent linearity (R2 = 0.9999) and reproducibility (between-run error ≤ 5.7%). Sensitivity was good with a limit of quantification of 1.0 ng/mL. Correlation of our UPLC-MS/MS assay with a commercially available hepcidin ELISA was good (R2 = 0.81). Median hepcidin concentrations in plasma of patients with iron deficiency anemia (IDA), normal subjects, and patients with anemia of chronic disease (ACD) were 5.2, 12 and 47 ng/mL, respectively.
In conclusion, we developed a robust and reproducible UPLC-MS/MS assay for quantification of hepcidin. Different hepcidin levels were found in IDA and ACD patients and normal subjects. In the near future, the diagnostic value of this hepcidin assay will be established in a clinical study that includes patients with an anemia of unknown origin. References
1. Ganz T. Hepcidin and iron regulation, 10 years later. Blood. 2011 Apr 28;117(17):4425–33. 2. Konz T, Montes-Bayón M, Vaulont S. Hepcidin quantification: methods and utility in diagnosis. Metallomics. 2014 Aug
20;6(9):1583–90. 3. Li H, Rose MJ, Tran L, Zhang J, Miranda LP, James CA, et al. Development of a method for the sensitive and
quantitative determination of hepcidin in human serum using LC-MS/MS. J Pharmacol Toxicol Methods. 2009 Jun;59(3):171–80.
67
Controlling Nuclear Receptor activity via 14-3-3 protein-protein interactions
E. Sijbesma1,2, I. van de Gevel1, L.G. Milroy1,2, C. Ottmann1,2, L. Brunsveld1,2
1 Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of
Technology, the Netherlands 2 Institute for Complex Molecular Systems (ICMS), Eindhoven University of Technology, the
Netherlands
Protein-protein interactions (PPIs) are an important yet challenging target class in drug discovery [1].
In recent years a number of inhibitors have been developed that target unfavorable interactions.
Despite the potentially enormous therapeutic benefit, the opposite strategy of PPI stabilization has
not been systematically pursued. An example of relevant PPIs that would greatly benefit from
stabilization is the interaction between the adaptor protein 14-3-3 and Nuclear Receptor (NR)
proteins.
The NR superfamily of transcription factors are currently one of the highest impact drug
targets but most developments are limited to small molecules binding in the ligand binding pocket,
associated with problems related to specificity and resistance. We investigate PPI stabilization as an
alternative approach. One of the cellular binding partners is 14-3-3, a hub protein that docks on to
phosphorylated residues of its partner proteins, thereby influencing their activity, localization or
dimerization behavior.
The therapeutic benefit of 14-3-3 PPI stabilization by a natural product was illustrated for the
Estrogen Receptor, an important breast-cancer target [2]. By applying a fragment-based drug
discovery approach combined with x-ray crystallography we now discovered a series of new chemical
starting points for stabilization of the binary complex between 14-3-3 and an ER-derived
phospopeptide. Additionally, we investigated interaction motifs for 14-3-3 proteins in other Nuclear
Receptors and identified a strong binding peptide motif in the DNA-binding domain of the Estrogen-
Related Receptor. We now aim for the elucidation of structural details of this interaction and present
a peptide-based approach combining enzymatic ligation and native chemical ligation to synthesize
this entire phosphorylated domain.
In short, our research focuses on molecular details of the interactions between Nuclear Receptors
and 14-3-3 proteins. In this poster we present an overview of structural details and an outlook to
study the therapeutic potential on stabilizing these interactions.
[1] M.R. Arkin & J.A. Wells. Small-molecule inhibitors of protein-protein interactions: progressing towards the dream. Nature Reviews Drug Discovery 3, 301–317 (2004). [2] I.J. De Vries-van Leeuwen, D. da Costa Pereira, K.D. Flach, S.R. Piersma, C. Haase, D. Bier, Z. Yalcin, R. Michalides, K.A. Feenstra, C.R. Jiménez, T.F.A. de Greef, L. Brunsveld, C. Ottmann, W. Zwart, A.H. de Boer. Interaction of 14-3-3 proteins with the estrogen receptor alpha F domain provides a drug target interface. Proceedings of the National Academy of Sciences of the USA 110, 8894-8899 (2013).
68
Supramolecular antimicrobial “nanobullets” for intracellular bacteria targeting
J. Song1,2, P. K.H. Fransen1,3, M. H. Bakker1,3, S. Zaccaria1,3, P.Y.W. Dankers1,2,3
1 Institute for Complex Molecular Systems, Eindhoven University of Technology, the Netherlands 2 Laboratory for Cell and Tissue Engineering, Eindhoven University of Technology, the Netherlands
3 Laboratory for Chemical Biology, Eindhoven University of Technology, the Netherlands
Bacterial infections are common in human beings. Although endless effort has been spent to combat
infections, even more complicated and persistent infections, e.g. intracellular infections [1], are still
emerging. With these infections, the bacteria shield themselves from the host immune system and
antibiotic treatment. This self-protection regime results into antibacterial resistance [2], and further
induces chronic and recurrent infections.
To address this problem, intracellular delivery of high doses of antibacterial drugs has been proposed
as an effective strategy. Among the antibacterial drugs, antimicrobial peptides (AMPs) attract more
attention due to their broad ranges of activity and less possibility of inducing antibacterial resistance
[3]. Therefore, the aim of this study was to develop an efficient AMPs delivery system to target
intracellular bacteria.
To achieve this strategy, we introduced supramolecular ureidopyrimidinone (UPy) system as drug
carrier to transport AMPs into phagocytic (THP-1 derived macrophages) and non-phagocytic cells
(Human Kidney 2 cell line, HK-2 cells). We prepared the peptide Lasioglossin-III (Lasio-III) through a
solid phase peptide synthesis method and coupled Lasio-III onto the UPy molecules in liquid phase.
Together with UPy molecules with different functional groups, we obtained UPy-AMP containing
nanobullets with multi functions through a mix-and-match method in aqueous phase. With
incubation of Lasio III free nanobulltes with THP-1 derived macrophage and HK-2 cells, we found that
internalization of UPy-based aggregates by mammalian cells was associated with the charge
properties of the nanobullets. More specifically, the internalization of nanobulltes by THP-1 derived
macrophage was independent of the charge properties of the nanobullets, whereas nanobullets
containing positively charged molecules affiliated their internalization by HK-2 cells. This result
indicated that cell targeting uptake of nanobullets was achieved by simple adjustment of the
components of the nanobullets. The cellular uptake of UPy-AMP containing nanobullets will be
performed, followed by antibacterial test.
[1] S.M. Lehar et al. Nature 527(7578), 2015, 323-328. [2] E. Briones et al. Journal of controlled release, 125(3), 2008, 210-227. [3] J. Yeom et al. Biomaterials, 104, 2016, 43-51.
69
Scaffold-assisted Synthesis of Multicyclic Peptides via an Orthogonal Ligation Strategy
D.E. Streefkerk, J.H. van Maarseveen, P. Timmerman
Synthetic Organic Chemistry, Van ’t Hoff Institute for Molecular Sciences, University of Amsterdam
Compared to their linear analogues, cyclic peptides are potential drug candidates due to their conformational rigidity and enhanced metabolic stability. For the mimicry protein-protein interactions, a single ‘peptide loop’ is often not enough. As a result, there is a need for robust methodology towards multicyclic peptides. In the past, CLIPS chemistry has been used to synthesize a bicyclic peptide onto a central scaffold 1,2,3 (fig 1), but increased complexity is not possible using this technique due to the formation of a complex mixture of isomers (fig 2).
Figure 1: Synthesis of a bicyclic peptide via the CLiPS reaction
Figure 2: Synthesis of a tricyclic peptide via CLiPS, yielding up to 6 isomers
My research focusses on increasing the complexity to three peptide loops, using CLIPS chemistry in combination with an orthogonal ligation strategy: i.e. oxime ligation4 (fig 3). This pathway can yield a maximum of two isomers, but with careful selection of the scaffold and the appropriate complimentary reactivity in the peptide, a single isomeric product can be obtained.5 Peptides bearing a side-chain ketone-functionalized unnatural amino acid were investigated in combination with an amino-oxy group containing scaffold. The resulting tricyclic peptides showed undesired E/Z isomers of the oxime linkage. Our recent efforts are focused on incorporating an aldehyde reactivity in either the peptide or scaffold, which should circumvent this isomer formation.
Figure 3: The use of orthogonal ligation strategies CLiPS and oxime ligation to form tricyclic peptides
References 1. Timmerman, P.; Beld, J.; Puijk, W. C.; Meloen, R. H. ChemBioChem 2005, 6, 821-824. 2. Smeenk, L. E. J. Double-CLIPS Technology for the Mimicry of Structurally Complex Antibody Binding Sites on Proteins, Ph.D. Dissertation, University of Amsterdam, Faculty of Science, Van 't Hoff Institute for Molecular Sciences, Amsterdam, 2013. 3. Smeenk, L. E. J.; Dailly, N.; Hiemstra, H.; van Maarseveen, J. H.; Timmerman, P. Org. Lett. 2012, 14, 1194-1197. 4. Dirksen, A.; Hackeng, T. M.; Dawson, P. E. Angew. Chem. Int. Ed. 2006, 45, 7581-7584. 5. J.H.van Maarseveen; G.J.J. Richelle; D.E.Streefkerk; P. Timmerman; Multicyclic peptides and methods for their Preparation, Eur. Pat. Appl. EP16202466, December 6th 2016.
70
Engineering and Expanding the Genetic Code for In Vivo Incorporation of
Noncanonical Amino Acids into Biologically Active Ribosomal Peptides
Rashed Al Toma 1, Linda Sukmarini 1, Maria Lopatniuk 2, Stefan Grätz 1, Maksym Myronovskyi 2, Andriy Luzhetskyy 2 Nediljko Budisa 1 and Roderich Süssmuth 1
1Institut für Chemie, Technische Universität Berlin, Straße des 17. Juni 124, 10623 Berlin (Germany),
Fax: +49-(0)30-314-79651, E. mail: [email protected] 2Helmholtz Institute for Pharmaceutical Research, Saarland Campus, Building C2, 66123 Saarbrücken,
Germany
Due to their wide structural diversity and remarkable stability combined with a broad
spectrum of bioactivities, ribosomally synthesized and post-translationally modified peptides (RiPPs)
are of great interest for the development and use as drugs. Reprogramming protein translation in
living cells with an expanded amino acids repertoire was allowed by the recent developments in
genetic code engineering and expansion. Structure alteration of in vivo-produced biologically active
RiPPs can be achieved by employing these strategies in a rational designed manner. This includes the
application of selective-pressure incorporation (SPI) and stop codon suppression (SCS) approaches
for the incorporation of isostructural and orthogonal noncanonical amino acids (ncAAs) into the core
peptides of the target RiPPs. By using Streptomyces lividans as a heterologous expression platform
we were able to utilize the SPI and the SCS approaches for generating new unnatural derivatives of
the class III lanthipeptide labyrinthopeptin LabA1 and LabA2. Various isostructural analogues of Pro,
Trp and Met were incorporated into LabA1 and LabA2 by using the SPI approach with newly
generated auxotrophic S. lividans strains. The application of the SCS approach for the incorporation
of orthogonal ncAAs, e.g.: Alk (Nε-Alloc-L-lysine), required the usage of an S. lividans strain that
contains an orthogonal system of aminoacyl-tRNA synthetases: tRNA pair (aaRS:tRNA) for amber stop
codon suppression. We have also succeeded to incorporate various isostructural and orthogonal
ncAAs into the core peptides of the class II lanthipeptide lichenicidin Bliα and Bliβ,[1,2] and the class II
lasso peptide capistruin.[3] The tested amino acids include also surrogates of Pro, Trp and Met in
addition to many orthogonal ncAAs. This was achieved by applying the SPI and the SCS approaches in
Escherichia coli. Bio-orthogonal chemistry (olefin metathesis and click reactions) was also applied on
some of the newly generated congeners as post-biosynthetic modifications in term of further
increasing their structural diversity. The successful incorporation of all the above mentioned
examples was proved by means of high resolution ESI-HPLC-MS analysis.
[1] F. Oldach, R. S. Altoma, A. Kuthning, T. Caetano, S. Mendo, N. Budisa, R. D. Süssmuth, Angew. Chem. Int. Ed. Engl. 2012, 51, 415-418. [2] A. Kuthning, P. Durkin, S. Oehm, M. G. Hoesl, N. Budisa, R. D. Süssmuth, Sci Rep 2016, 6, 33447. [3] R. S. Altoma, A. Kuthning, M. P. Exner, A. Denisiuk, J. Ziegler, N. Budisa, R. D. Süssmuth, ChemBioChem 2015, 16, 503- 509.
71
MODELING OF DIPEPTIDES CONTAINING POTENTIAL BIOLOGICALLY ACTIVE (S)-α-PROPARGYLGYCINE, DISCLOSURE OF POSSIBLE BIOLOGICAL
PROPERTIES AND TARGETED SYNTHESIS
A.H. Tsaturyana,b, T.H. Sargsyana,b, M.Yu. Danghyana, Yu.M. Danghyana, S.M. Jamgaryana, E.A. Gyulumyana, А.М. Hovhannisyanb, L.A. Hayriyana,b, A.S. Saghyana,b
aScientific and Production Center “Armbiotechnology” NAS RA, 14 Gyurjyan Str. 0056, Yerevan, Armenia: E-mail: [email protected]
b Yerevan State University, Republic of Armenia, Yerevan, 0025, 1 Alex Manoogian
Considering a wide spectrum of biological properties of peptides, we found it actual to study by “Pass-online” software the range of possible biological activities of dipeptides, containing (S)-α-propargylglycine. “Pass-online” is a subsidiary tool to evaluate the general biological potential of organic drug-like molecules [1]. Table.
Table
Some possible biological activities of peptides
Peptides
An
ticon
ulsan
t
Pro
tein
-
disu
lfide
red
uctase
(glu
tathio
ne
)
inh
ibito
r
Mu
cositis
treatm
en
t
C
ystathio
nin
e
gamm
a-lyase
inh
ibito
r
Pa Pi Pa Pi Pa Pi Pa Pi
Boc-glycyl-(S)-propargylglycine (5) 0.846 0.005 0.722 0.012 0,574 0,052 0,291 0,006
Glycyl-proparglycine (12) 0,667 0,012 0,849 0,005 0,876 0,008 0,885 0,002
Boc-(S)-alanyl-(S)-propargylglycyl (6) 0,675 0,011 0,662 0,026 0,501 0,073 - -
(S)-alanyl-(S)-propargylglycyl (13) - - 0,862 0,005 0,911 0,006 0,918 0,002
Boc-(S)-alanyl-(S)-α-benzylpropargylglycyl (7 0,797 0,005 0,488 0,074 - - - -
(S)-alanyl-(S)-α-benzylpropargylglycyl (14) 0,677 0,011 0,730 0,016 0,724 0,021 - - Pa (probability “to be active”) estimates the probability of belonging of the test compound to the subclass of active compounds based on the similarity of the structure with those molecules that are most typical in this subset. Pi (probability “to be inactive”) estimates the probability of belonging of the test compound to the subclass of inactive compounds. The research results are expressed by (Pa) and (Pi) values in the range from 0 to 1.
According to the PASS predictions, the greatest probability of activity being anticipated with dipeptides (S)-alanyl-(S)-propargylglycyl (13), Glycyl-proparglycine (12). Synthesis of the selected peptides was carried out by the method of [2], scheme.
Scheme
This work was supported by the RA MES State Committee of Science, in the frames of the research project № 15T-21215. References 1. Veselovsky A.V., Ivanov A.S. // Design. Current Drug Targets – Infectious Disorders., 2003, v.3, issue 1, p. 33. 2. Dangyan Yu.M, Sargsyan T.H., Jamgaryan S.M., Gyulumyan E.A., Panosyan H., Saghyan A.S. // Chem. Journal of Armenia, 2010, v. 63, № 1, p. 95-100.
72
Cucurbit[8]uril Enhanced Peptides to Surrogate Protein-Protein Interactions
within a Multi-Component Complex of 14-3-3 and Estrogen Receptor α
Pim J. de Vink1, Lech.-G. Milroy1, Christian Ottmann1 and Luc Brunsveld*1
1 Department of Biomedical Engineering, Eindhoven University of Technology, The Netherlands.
Peptides have proven to be excellent tools to study protein-protein interactions (PPI), since they closely resemble the primary native structure of native sequence. However due to their reduced size compared to proteins, information on secondary, ternary structure and quaternary structure is often lost.
Supramolecular recognition of amino acids through host-guest chemistry has great potential to control biomolecular assemblies1. Supramolecular host molecules can be used as a tool to supplement, surrogate and extend the native interactions within a multicomponent complex. For examples, cucurbit[8]uril (Q8) recognizes phenylalanine-glycine-glycine (FGG) tripeptide motif on the N-terminus of peptides an proteins2, thus allowing for bio-orthogonal control over protein dimerization, immobilization and enzyme activity3
. Here we describe the development of a Q8-based supramolecular system, with the 14-3-3 scaffold protein and two synthetic peptides derived from estrogen receptor α (ERα) in order to recapture quaternary interactions. Upon addition of Q8, a well-defined multi component complex is formed (Figure 1)4.
Q8-induced dimerization of the ERα epitope increases its affinity towards 14-3-3. Also the affinity toward Q8 is enhanced in the presence of 14-3-3. The induced binary bivalence as was confirmed via various biophysical assays, fluorescence, scattering data, and flow-field-fractionation. In addition, the final structure is confirmed to atomistic resolution by protein X-ray crystallography which reveals the structural basis for the supramolecular chemically-induced switch between the mono- and bivalent modes of interaction thus shaping the way for future synthetic supramolecular signalling systems.
References 1 Barrow, S.J.; Kasera, S.; Rowland, M.J.; del Barrio, J. ; Scherman, O. A. Chem. Rev. 2015, 115, 12320 2 Heitmann, L.M.; Taylor, A.B.; Hart, P.J.; Urbach, A.R., J. Am. Chem. Soc. 2006, 128, 12574–12581. 3 Bosmans, R.P.G.; Briels, J.M.; Milroy, L.-G.; de Greef, T.F.A.; Merkx, M.; Brunsveld, L. Angew. Chem. Int. Ed. 2016, 55, 8899–8903. 4 De Vink, P.J.; Briels, J.M.; Schrader, T.; Milroy, L.-G.; Brunsveld, L.; Ottmann, C., Under review. 2017
Figure 1. A) Schematic representation of multicomponent supramolecular protein assembly based on the simultaneous scaffolding of peptides via the Q8 and 14-3-3 platform, surrogating quaternary structure interactions. B) Protein crystal structure of 14-3-3 in complex with Q8 and FGG-ERα peptides.
A)
B)
73
Structural interface between LRRK2 and 14-3-3 protein Rens M.J.M. de Vries1, Loes M. Stevers1, Richard G. Doveston1, Lech-Gustav Milroy1, Luc
Brunsveld1 and Christian Ottmann1,2
1Laboratory of Chemical Biology, Department of Biomedical Engineering and Institute for Complex
Molecular Systems, Eindhoven University of Technology, PO Box 513, 5600 MB Eindhoven, The
Netherlands
2Department of Chemistry, University of Duisburg-Essen, 45141 Essen, Germany
In 2004 there was a breakthrough in Parkinson’s disease (PD) research when the leucine-rich repeat
kinase 2 (LRRK2) was discovered and mutations in this protein were linked to sporadic and familial
PD.1,2 The 14-3-3 protein is known to be an important protein interaction partner of LRRK2, i.a.
modulating its kinase activity.3,4 Many pathogenic mutations in LRRK2, either directly or indirectly,
disrupt or weaken the interaction between 14-3-3 and LRRK2, causing an enhanced LRRK2 kinase
activity.3,5 This results in increased ubiquitination of LRRK2, accumulation of LRRK2 into inclusion
bodies and reduction in neurite length.4 Therefore, this interaction is of notable interest as a
potential drug target for the treatment of PD. However, LRRK2 has multiple sites that, upon
phosphorylation, can bind to 14-3-3, thus rendering the interaction relatively complex.5,6 Using
peptide fragments derived from LRRK2, the binding affinities of the individual and combined binding
sites were quantified. Finally, we used crystallography to characterize the multivalent interaction
between these two proteins. These results contribute to the discovery of small-molecules that
stabilize this protein-protein interaction, which have the potential as a promising approach for PD
therapy.
1. Paisán-Ruíz, C. et al. Cloning of the gene containing mutations that cause PARK8-linked Parkinson’s disease. Neuron 44, 595–600 (2004). 2. Zimprich, A. et al. Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology. Neuron 44, 601–607 (2004). 3. Muda, K. et al. Parkinson-related LRRK2 mutation R1441C/G/H impairs PKA phosphorylation of LRRK2 and disrupts its interaction with 14-3-3. Proc. Natl. Acad. Sci. U. S. A. 111, E34-43 (2014). 4. Lavalley, N. J., Slone, S. R., Ding, H., West, A. B. & Yacoubian, T. A. 14-3-3 Proteins regulate mutant LRRK2 kinase activity and neurite shortening. Hum. Mol. Genet. 25, 109–122 (2016). 5. Nichols, R. J. et al. 14-3-3 binding to LRRK2 is disrupted by multiple Parkinson’s disease-associated mutations and regulates cytoplasmic localization. Biochem. J. 430, 393–404 (2010). 6. Dzamko, N. et al. Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(910)/Ser(935), disruption of 14-3-3 binding and altered cytoplasmic localization. Biochem. J. 430, 405–413 (2010).
74
Structural interface between LRRK2 and 14-3-3 protein
Rens M.J.M. de Vries1, Loes M. Stevers1, Richard G. Doveston1, Lech-Gustav Milroy1, Luc
Brunsveld1 and Christian Ottmann1,2
1Laboratory of Chemical Biology, Department of Biomedical Engineering and Institute for Complex
Molecular Systems, Eindhoven University of Technology, PO Box 513, 5600 MB Eindhoven,
The Netherlands
2Department of Chemistry, University of Duisburg-Essen, 45141 Essen, Germany
In 2004 there was a breakthrough in Parkinson’s disease (PD) research when the leucine-rich repeat
kinase 2 (LRRK2) was discovered and mutations in this protein were linked to sporadic and familial
PD.1,2 The 14-3-3 protein is known to be an important protein interaction partner of LRRK2, i.a.
modulating its kinase activity.3,4 Many pathogenic mutations in LRRK2, either directly or indirectly,
disrupt or weaken the interaction between 14-3-3 and LRRK2, causing an enhanced LRRK2 kinase
activity.3,5 This results in increased ubiquitination of LRRK2, accumulation of LRRK2 into inclusion
bodies and reduction in neurite length.4 Therefore, this interaction is of notable interest as a
potential drug target for the treatment of PD. However, LRRK2 has multiple sites that, upon
phosphorylation, can bind to 14-3-3, thus rendering the interaction relatively complex.5,6 Using
peptide fragments derived from LRRK2, the binding affinities of the individual and combined binding
sites were quantified. Finally, we used crystallography to characterize the multivalent interaction
between these two proteins. These results contribute to the discovery of small-molecules that
stabilize this protein-protein interaction, which have the potential as a promising approach for PD
therapy.
1. Paisán-Ruíz, C. et al. Cloning of the gene containing mutations that cause PARK8-linked Parkinson’s disease. Neuron 44, 595–600 (2004). 2. Zimprich, A. et al. Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology. Neuron 44, 601–607 (2004). 3. Muda, K. et al. Parkinson-related LRRK2 mutation R1441C/G/H impairs PKA phosphorylation of LRRK2 and disrupts its interaction with 14-3-3. Proc. Natl. Acad. Sci. U. S. A. 111, E34-43 (2014). 4. Lavalley, N. J., Slone, S. R., Ding, H., West, A. B. & Yacoubian, T. A. 14-3-3 Proteins regulate mutant LRRK2 kinase activity and neurite shortening. Hum. Mol. Genet. 25, 109–122 (2016). 5. Nichols, R. J. et al. 14-3-3 binding to LRRK2 is disrupted by multiple Parkinson’s disease-associated mutations and regulates cytoplasmic localization. Biochem. J. 430, 393–404 (2010). 6. Dzamko, N. et al. Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(910)/Ser(935), disruption of 14- 3-3 binding and altered cytoplasmic localization. Biochem. J. 430, 405–413 (2010).
75
ISOBARIC LABELING OF PEPTIDES BY COMBINATION OF 18O ENZYMATIC
LABELING AND CHEMICAL MODIFICATION WITH PYRYLIUM SALT
Mateusz Waliczek, Monika Kijewska, Dorota Gaszczyk, Piotr Stefanowicz,
Zbigniew Szewczuk
Faculty of Chemistry, University of Wroclaw, Wroclaw, Poland
Enzymatic labeling using heavy isotopes including 18O has been commonly used technique in
comparative proteomics. Despite the great utility of this method, the quantitative analysis of
proteins and peptides using 18O labeling approach has some disadvantages including the back-
exchange of labeled oxygen. Additionally, some problems related to the peak overlapping during
mass spectrometry analysis, have been reported [1].
Pyrylium salts react with primary amines, especially these sterically unhindered e.g. -amine group of
lysine, giving pyridinium salts. Recently, we reported new ionization enhancer based on 2,4,6-
triphenylpyridinium scaffold allowing for sensitive analysis of peptides by mass spectrometry [2]. To
overcome above described problems we proposed the modified procedure consisted of H218O and
H2O enzymatic digestion followed by modification of peptides using quadruply 13C labeled and
unlabeled 2,4,6-triphenylpyrylium salts. The combination of these samples results in obtaining of
isobaric peptides. MS/MS experiment reveals the abundant and protonated 2,4,6-
triphenylpyrydinium diagnostic ions, which can serve in quantitation analysis.
trypsin
H2 16O
H2 18O
Ph Ph
Ph
O+
MS
13C
18Otrypsin
18O
2+
0
[%]
m/z
743.38
308.141+
309.151+
312.151+
313.161+
m/z0
[%]
MS/MS
13CPh
Ph PhO+
16O
12C
13C16O18O12C
16O
18O
The modified peptides are not recognized by trypsin, thus do not subject to back-exchange. The
quantitative analysis is based only on comparison of singly charged diagnostic ions (shifted by 4Da),
which reduces the problem of peak overlapping. Application of MS/MS mode improves the signal to
noise ratio, which in combination with ionisation enhancement effect resulting from modification
using pyrylium salts allow for highly sensitive analysis of peptides by tandem mass spectrometry.
[1] X. Yao, C. Alfonso, C. Fenselau, J. Proteome Res., 2, 147 (2003) [2] M. Waliczek, M. Kijewska, M. Rudowska, B. Setner, Sci Rep., 6, 37220, (2016)
76
Development of a peptide macrocycle FXII inhibitor for safe anticoagulation therapy
J. Wilbs1, S.J. Middendorp1, R. Prince2, A. Angelillo-Scherrer2 and C. Heinis1
1ISIC, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland 2Department of Clinical Research, University of Bern, Bern, Switzerland
Coagulation factor XII (FXII) is a plasma protease that has emerged in recent years as a potential
target to treat or prevent pathological thrombosis, to inhibit blood clot formation in medical devices
and to treat the swelling disorder hereditary angioedema.[1] Inhibiting FXII has been shown to reduce
thrombosis without increasing the bleeding risk, a major side-effect of currently used anticoagulants.
Until recently, several protein-based FXII inhibitors were developed, out of which at least one is in
clinical trial, but no high affinity small molecule inhibitor has been reported.[2,3] In our laboratory we
have generated a potent and highly selective FXII inhibitor based on a macrocyclic peptide format
(MW <2000 kDa). Recently, we had improved the potency and stability of the inhibitor using
unnatural amino acid incorporation.[4,5] After further optimization, the final peptide shows inhibitory
affinity in the picomolar range and selectivity with a high stability in plasma. The inhibitor prolonged
the FXII-activated intrinsic coagulation in human, mouse and rabbit plasma (EC2X human = 1 µM).
Pharmacokinetic studies in mouse and rabbit showed that the peptide was active in vivo and no
signs of toxicity or abnormal bleeding were observed. In a FeCl3-induced thrombosis model in mice
the peptide could reduce thrombus formation substantially. Our results suggest that FXII inhibition
by a peptide macrocycle can potentially offer a safe anticoagulation therapy.
Figure: Blood clot formation in artery 10 min after FeCl3 application
References
[1] J. I. Weitz, Thromb. Res. 2016, 141, S40–S45. [2] M. Larsson, V. Rayzman, M. W. Nolte, K. F. Nickel, J. Bjorkqvist, A. Jamsa, M. P. Hardy, M. Fries, S. Schmidbauer, P. Hedenqvist, et al., Sci. Transl. Med. 2014, 6, 222. [3] F. May, J. Krupka, M. Fries, I. Thielmann, I. Pragst, T. Weimer, C. Panousis, B. Nieswandt, G. Stoll, G. Dickneite, et al., Br. J. Haematol. 2016, 173, 769–778. [4] J. Wilbs, S. J. Middendorp, C. Heinis, Chembiochem 2016, 17, 2299–2303. [5] S. J. Middendorp, J. Wilbs, C. Quarroz, S. Calzavarini, A. Angelillo-scherrer, C. Heinis, J. Med. Chem. 2017, 60, 1151– 1158
77
Models of helical proteins based on the aromatic disulfide chemistry
Grzegorz Wołczański1, Marta Cal2, Piotr Stefanowicz1, Mateusz Waliczek1, Marek Lisowski1
1Chemistry and Stereochemistry of Peptides and Proteins Research Group, Faculty of Chemistry,
University of Wrocław, Poland 2Institute of Organic and Biomolecular Chemistry, Georg-August-Universität Göttingen, Germany
The template assisted proteins (TASP molecules) are the artificial protein models that allow studying
complex biochemical processes on simplified models. Mutter has demonstrated, that short,
amphipathic sequences attached to a cyclic template (e.g. cyclic peptide) formed stable helical
conformations and can be considered as models of tetrahelical bundles [1]. In further studies the
template was modified and currently many TASP systems have been described. Such systems are
usually synthesizing by stepwise construction of the template with following attaching of peptide
chains. The employing of reversible reactions (disulfide metathesis, oximes or hydrazones formation,
metal complexes) gives opportunities to generate dynamic combinatorial libraries of TASP molecules
[2].
In our previous studies we reported formation of tetrahelical bundles by coordination of Cu2+ ions by
polypeptides containing amino hydroxamic acid moieties [3]. In this case, the degree of
oligomerization has forced by geometry of copper(II) metallacrown. Therefore, in this work we
present a new template for TASP molecules, which is based on an aromatic system containing two
sulfhydryl groups in meta positions. This motif was utilized previously by Otto group for designing
mechanosensitive self-replication systems [4]. However in our research, in contrast to mentioned
study, we focused on designing systems dominated by intramolecular interactions of peptide chains,
which result in formation of soluble, protein-like molecules stabilized by leucine’s interactions in a
hydrophobic core.
[1] M. Mutter, E. Altmann, K. - H. Altmann, R. Hersperger, P. Koziej, K. Nebel, G. Tuchsecherer, S. Vuilleumier, H. - U. Gremlich, K. Müller, K., Helv. Chim. Acta, 1988, 71, 835-847. [2] Liton Roy, Martin A. Case, J. Phys. Chem. B, 2011, 115, 2454-2464; P. T. Corbett, J. Leclaire, L. Vial, K. R. West, J.-L. Wietor, J. K. M. Sanders, S. Otto, Chem. Rev., 2006, 106, 3652-3711 [3] a) M. Cal, M. Jaremko, Ł. Jaremko, P. Stefanowicz, J. Pept. Sci., 2013, 19, 9-15; b) M. Cal, A. Kotynia, Ł. Jaremko, M. Jaremko, M. Lisowski, M. Cebo, J. Brasuń, P. Stefanowicz, Dalton Trans., 2015, 44, 11172-11181. [4] a) M. Colomb-Delsuc, E. Mattia, J. W. Sadownik, S. Otto, Nat. Commun., 2015, 6, 7427; J. W. Sadownik, E. Mattia, P. Nowak, S. Otto, Nature Chemistry, 2016, 8, 264-269; E. Mattia, A. Pal, G. Leonetti, S. Otto, Synlett, 2017, 28, 103-107
78
The conjugated tautomerism in dehydropeptides
Grzegorz Wołczański1, Marek Lisowski1
1Chemistry and Stereochemistry of Peptides and Proteins Research Group, Faculty of Chemistry,
University of Wrocław, Poland
On the one hand, dehydroamino acids belong to the α,β-unsaturated carbonyl compounds, and on
the other, the α-amine group results in the imines/enamines [1] or N-acyl imines/enamides
tautomerism [2] in those systems. We expected that a basic catalysis would allow to obtain
isotopologues of dehydropeptides labeled at the β-position of dehydroamino acid residues.
However, during incubation of model peptides containing ΔPhe in 1% TEA-D2O mixture, we observed
unusual fast proton-deuterium exchange (HDX) at α-position of adjacent glycine residues. Then, we
examined a series of analogues containing various α,β-dehydroamino acid residues (ΔPhe, ΔTyr,
ΔPhe(4-NO2), ΔTyr(OBn), ΔLeu, ΔAbu), using mass spectrometry, NMR spectroscopy and UV
absorption spectroscopy. We found that electron withdrawing group in ΔPhe(4-NO2) facilitates HDX
on adjacent glycine residues, while ΔTyr containing peptide undergoes HDX slowest. These
differences may be explained by ability to stabilization of negative charge at β-position of
α,β-dehydroamino acid residue. We propose that deprotonation of ΔXaa’s amide group facilitates
N-acylimine/enamide tautomerisation with delocalization of negative charge, what promotes
enolization of i-1 residue through the creation of conjugated double bonds in a transitional state, as
well as enolization of i+1 residue involving additionally enol form of amide bond in the conjugated
bonds system – similarly to influence of Schiff base formation [3]. This hypothesis was confirmed
with cyclo(Gly-ΔPhe)3, which undegoes very fast proton deuterium exchange in comparison to linear
peptides. Moreover, its absorption spectrum presents wide continuum of absorption in the presence
of TBD (1,5,7-triazabicyclo[4.4.0]dek-5-en). The cyclodehydropeptide is the first example of a new
class of compounds, which may be considered as macrocycles with switchable aromaticity. In our
opinion, polymers based on XaaΔXaa motif should be carefully examined as potential conductive
polymers with a wide range of possibilities for structural modifications. Summarizing, according to
our best knowledge, we described the first observation of polyene form of peptide main chain, which
is facilitated by conjugated tautomerism enabled by double bond of dehydroamino acid residues.
[1] S.-P. Lu, A.H. Lewin, Tetrahedron, 1998, 54, 15097-15104 [2] C. Cativiela, J. I. García, J. A. Mayoral, L. Salvatella, J. Mol. Struct., 1996, 368, 57-66 [3] J. Crugeiras, A. Rios, E. Riveiros, Y. L. Amyes, J. P. Richard, J. Am. Chem. Soc., 2008, 130, 2041-2050
79
DEVELOPMENT OF AN ALBUMIN-BINDING LIGAND
FOR PROLONGING THE PLASMA HALF-LIFE
OF PEPTIDE THERAPEUTICS
A. Zorzi, C. Heinis
Laboratory of Therapeutic Proteins and Peptides (LPPT)
Institute of Chemical Sciences and Engineering (ISIC)
EPFL, Lausanne, Switzerland
Peptide therapeutics applied intravenously are rapidly cleared from the blood circulation by renal filtration. The short half-life prevents their application to diseases that require drug exposure of several hours or days (1). An attractive strategy to hamper filtration of peptides in the kidneys is to tether them non-covalently to a long-lived serum protein such as human albumin (2). Several albumin-binding ligands based on peptides or small molecules were developed but they suffer from relatively low affinities for human albumin as well as a poor solubility in physiological buffers, reducing their potential application to peptide therapeutics.
To overcome these limitations, a chimeric peptide-fatty acid albumin-binding ligand with low nanomolar affinity for human, rat and rabbit albumin, a high solubility and a small size suitable for automated synthesis of complex conjugates was successfully developed. Peptides conjugated to the tag retained their bioactivity and displayed around a 25-fold increase in half-life in rats and rabbits.
References (1) Kontermann, R. E. (2016). Expert Opin. Biol. Ther., 16, 903-915. (2) Pollaro, L. and Heinis, C. (2010). Med. Chem. Commun., 1, 319-324.
Schematic structure of the albumin-binding ligand (tag) (left panel) and pharmacokinetics of a therapeutic peptide and its
conjugated format in rat plasma upon i.v. injection (right panel).
80
Endogenous Peptides Stability Addressed by Fluorescence
Correlation Spectroscopy
C. R. Zuconelli1, V. Krykliva1, M. J. Adjobo-Hermans1, R. Brock1 1 Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University
Medical Center, Nijmegen.
It is generally assumed that inside a cell a free peptide is proteolytically degraded within seconds.
Nevertheless, quite paradoxically in the past 10 years it has been shown that some peptides of the
intracellular peptidome are present inside cells in concentrations in which they influence intracellular
signal transduction processes [1]. Moreover, the demonstration that free peptides added to the
intracellular milieu can regulate cellular functions mediated by protein interactions suggests new
putative roles for these molecules in gene regulation, metabolism, cell signaling, and protein
targeting. In this scenario, a large number of intracellular peptides were identified by peptidomics
and it was demonstrated that peptides isolated from rat brain tissue were capable of altering G
protein-coupled receptor signal transduction when introduced into several different cell lines [2].
The aim of this study is to reveal the stability of peptides which were isolated in previous
peptidomics assays. In addition, the influence of terminal capping on the stability of the endogenous
peptides is addressed.
In order to directly address differences in proteolytic sensitivity, the endogenous peptides AGH, FE3,
FE2 and VFD7 were fluorescently labeled using carboxyfluorescein as the fluorophore conjugated via
an aminododecanoic acid (Ado) linker. The amidated, non-amidated, carboxylated, non-carboxylated
and retro-inverso version of the peptides were incubated with HeLa cell lysate, which serve as a
source of proteolytic activity, and samples were collected over time. The proteolytic degradation of
the peptides was followed by Fluorescence Correlation Spectroscopy (FCS). Carboxyfluorescein had a
half maximal residence time <1 h. The FCS data analysis shown that small differences in peptide
structure result in large differences in proteolytic stability. As for the L-peptides with long residence
times, fluorescence remained homogenously distributed within the cells for the retro-inverso
peptides as well. Surprisingly, amidation and carboxylation had little or no effect on the sensitivity of
peptides to proteolytic degradation. In contrast, with a minor modification of two amino acids in the
N-terminus and C-terminus, major differences in peptide stability were found. Although the peptides
isolated by peptidomics were considered to be stable, large differences in their stability were
observed ranging from 2 h to more than 24 h. Remarkably, the data also indicate that partially
degradation of the peptides can occur. Overall the results demonstrate that the peptides varied
considerably in their proteolytic stability even if terminally capped. Contrary to expected, the amidation
of the peptides used in this study had little influence on their stability.
[1] L. C. Russo, A. F. Asega, L. Castro, P. Negraes, L. Cruz, F. C. Gozzo, H. Ulrich, A. C.M. Camargo, V. Rioli and E. S. Ferro. Proteomics, 12, 2641 (2012) [2] F. M. Cunha, D. A. Berti , Z. S. Ferreira , C. F. Klitzke, R. P. Markus and E. S. Ferro. J. Biol. Chem., 283, 24448 (2008)
81
LIST OF ATTENDEES DUTCH PEPTIDE SYMPOSIUM 2017
Surname Name Company
Sura Abbood Leicester University
Bauke Albada Wageningen UR
Can Araman University of Leiden
Friso Assema, van Friso Food Research
Steven Ballet Vrije Universiteit Brussel
Jonathan Beadle University of Warwick
Christian Becker University of Vienna
Michèle Beelen Biosolve
Carsten Behrens Novo Nordisk A/S
Willemien Benckhuijsen Leiden University Medical Center
Dominik Bernhagen Pepscan Therapeutics
Charles Beromeo Bheeter University of Amsterdam
Lucas Beroske Utrecht University
Christian Birr Orpechem Peptide Chemicals GmbH
Sjef Boeren Wageningen UR
Cecilia Bottecchia Technical University Eindhoven
Jeroen Bouwhuis Pepscan Therapeutics
Aimee Boyle Leiden University
Luc Brunsveld Technical University Eindhoven
Roland Brock Radboud University
Carlo Brouwer CBMR Scientific BV
Matthew Burton NLO European Patent Attorneys
Marcia Cabrera, Dos Santos UNESP - Sᾶo Paulo State University
Anish Chakkumkal Janssen Pharmaceuticals
Christophe Chambard Bachem
James Churchill Biotage GB Ltd
Jan Cordewener Wageningen UR
Ruud Cox Universiteit Utrecht
Liliana Cozzoli University of Groningen
Niek Crone Leiden University
Duco Dalen, van Radboud UMC
Maarten Danial Intravacc
Klaas Decoene Ghent University
Karel Decroos Corden Pharma Brussels SA
Stepan Denisov Maastricht University
Perry Derwig Waters
Huiwen Ding Radboud University
Evert Dijk, van Pepscan Therapeutics
Natasja Dolezal Leiden University Medical Center
Richard Doveston Technical University Eindhoven
Jan Wouter Drijfhout Leiden University Medical Center
Rik Duin, van ISA Pharmaceuticals B.V.
82
Surname Name Company
Johan Elgersma ESCOM Science Foundation
Luc Elst, Vander Imcyse SA
Elena Egorova Leiden University
Thomas Ende van den Leiden University
Eva Magdalena Estirado Technical University Eindhoven
Bart Faber Biomedical Primate Research Center
Lorraine Fathers Leiden University Medical Center
Omar Paulino da Silva Filho Radboud UMC
Dmitry Filipenko Russian Peptide
Ivan Filipenko Russian Peptide
Dmitri Filippov Leiden University
Gabriele Fischer S.C.J. Research
Jaap Flohil Foldyne Research International
Ronald Gaal van Technical University Eindhoven
Yongzhi Gao Utrecht University
Marta Pelay Gimeno Vrije Universiteit Amsterdam
Ravi Girdhar Gyros Protein Technologies/PTI
Adrian Glas Peptide Specialty Laboratories
Michel Goldbach EnzyPep B.V.
Jolanda Golde, van Maastricht University & Enabling Technologies BV
Michael Goldflam Pepscan Therapeutics
Stefanos Guido Senn Christof Senn Laboratories
Maurice Grube Karolinska Institute and Science for Life Laboratory
Smita Gunnoo Ghent University
Dat Guy Utrecht University
Tilman Hackeng Universiteit Maastricht
Martin Haex Agilent Technologies
Matthijs Haren, van University of Utrecht
Alastair Hay Almac
Lars Bo Hansen Zealand Pharma A/S
Peter t Hart Max Plank Institute for Molecular Physiology
Mohamed El Hasnaoui Gyros Protein Technologies/PTI
Mostafa Hatam AAPPTec
Lars Heide, van der University of Amsterdam
Fabian Hensbergen Leiden University Medical Center
Simone Hendrikse Technical University Eindhoven
Tanja La Cour Hoekstra Delta Patents
Tim Hogervorst Leiden University
Pascal Hommen Max Planck Institute for Molecular Physiology Dortmund
Peter Hoogerhout Intravacc
Patrick Houts, van Bruker
Gang Huang University of Groningen
Wataru Ichinose Durham University
Hans Ippel Maastricht University
Miranda Jekhmane Utrecht University
83
Surname Name Company
Håvard Jenssen Roskilde University
Zhi-Jun Jia MPI-Dortmund
Hennie Jong, de S.C.J. Research
Niels Jong, de Nilesk
Wim Jongen BLSF B.V.
Seino Jongkees Utrecht University
Jan-Elo Jørgensen ISD Immunotech ApS
Hacer Karatas Max Planck Institute for Molecular Physiology Dortmund
Khawla Kasar Leicester University
Peter Keizer, de Erasmus MC
Andrew Kennedy Gyros Protein Technologies/PTI FAS
Sebastian Kiehstaller Vrije Universiteit Amsterdam
Laurens Kleijn Utrecht University
Sascha Knauer Sulfotools GmbH
Li Kong Leiden University
Spela Korat VU Medical Center
John Kruijtzer Utrecht University
P.R. Kuninty University of Twente
Karina Laar, van Agilent Technologies
Chan Vinh Lam Eindhoven University of Technology
Helmus Langemheen, van de Mercachem
Yen-Chun Lee Max Plank Institute of Dortmund
Minglong Liu Utrecht University
Qiang Liu Leiden University
Dennis Löwik Radboud UMC
Jan Maarseveen van University of Amsterdam
Jurgen Machielse Zeochem AG
Hanaa Al-Mahmoodi Leicester University
Taryn March Leiden University Medical Center
Nathaniel Martin University of Utrecht
Niall McLoughlin Vrije Universteit Amsterdam
Dennis Meeuwsen ChemConnection B.V.
Femke Meijer Eindhoven University of Technology
Ghislaine Meiners ISA Pharmaceuticals B.V.
Beatrice Menz Springer International Publishing Basel
Karsten Meyenberg Corden Pharma
Lech Milroy Technical University Eindhoven
Wim Mol Pepscan
Steven Moss Isomerase Therapeutics Ltd
Monique Mulder Leiden University Medical Center
Carolin Mueller Vrije Universiteit Amsterdam
Daan Muilwijk Biomarin Nederland B.V.
Bjoern Niebel Ablynx NV
Thomas E. Nielsen Novo Nordisk A/S
Timo Nuijens EnzyPep
84
Surname Name Company
Cami Talavera Ormeño Leiden University Medical Center
Ana Toplak EnzyPep B.V.
Danny Oevelen, van Agilent Technologies
Sjonni van Oosterwijk Actu-All chemicals
Coen Oude, de Pepscan Presto B.V.
Huib Ovaa Leiden University Medical Center
Shubhendu Palei University of Muenster
Martijn Patist Vrije Universiteit Medical Center
Aleksandra Pekosak Vrije Universiteit Medical Center
Brad Pentelute Massachusetts Institute of Technology
Cristina Chamorro Perez Institute for Life Sciences & Chemistry
Julia Piccoli Sao Paulo State University/Oxford University
Saravanan Pillai Exiexpressions
Jan Pille Technical University Eindhoven
Halina Plociennik University of Wroclaw
Tobias Postma Cannabiszorg
Ken Princen Ghent University
Peter Quaedflieg EnzyPep B.V.
Mohammad Rahnamaeian Fraunhofer Inst. For Molecular Biology & Applied Ecology
Dietrich Rein BASF SE
Gaston Richelle University of Amsterdam
Dirk Rijkers Utrecht University
Rick Rink Lanthio Pharma
Marcus Rothe Intavis
Jan Rooy, de AHV/Qleans
Rusul Al Rubaay University of Leicester
Tatevik Sargsyan SPC "Armbiotechnology" NAS RA
Nada al Sader Leiden University Medical Center
Wim Schaaper Pepscan Therapeutics
Marcel Scheepstra Mercachem
Christof Senn Christof Senn Laboratories
Jie Shi Utrecht University
Eline Sijbesma Eindhoven University of Technology
João Silva University of Utrecht
Anton Terwisscha van Scheltinga Leiden University Medical Center
Marcel Schmidt EnzyPep B.V.
Steven Schmitt ETH Zürich
Ellen Schmitz Technical University Eindhoven
Elizabeth Schram ESCOM Science Foundation
Christian Schwarz Numaferm
Mengjie Shen Leiden University
Jie Shi Utrecht University
Joᾶo Silva Utrecht University
Mike Smeenk Radboud University
Jiankang Song Technical University Eindhoven
85
Surname Name Company
Hiroaki Suga University of Tokyo
Loes Stevers Technical University Eindhoven
Dieuwertje Streefkerk University of Amsterdam
Gertjan Streefland AHV int.
Xianbin Su Nanjing Tech University
Dennis Suijlen Maastricht University
Peter Timmerman Pepscan
Rashed Al Toma TU Berlin
Avetis Tsaturyan SPC "Armbiotechnology" NAS RA
Raisa Veizaj Radboud UMC
Remco Venema Waters
Martijn Verdoes Radboud UMC
Yentl Verleysen Ghent University
Sanne Verhoork Liverpool John Moores University
Thomas De Vijlder Janssen
Pim Vink, de Technical University Eindhoven
Rowin Visser, de Enzypep
Rowin Vos LPS BV
Mateusz Waliczek University of Wroclaw
Kerstin Wallraven Vrije Universiteit Amsterdam
Marthe Walvoort University of Groningen
Kim Mulder Waters
Nicola Wade Utrecht University
Lee Webster Isomerase Therapeutics Ltd
Mathias Wendt Vrije Universiteit Amsterdam
Charlotte Wesseling Universiteit Utrecht
Peter White Merck Chemicals UK
Danny Willigen, van Leiden University Medical Center
Grzegorz Wolczanski University of Wroclaw
Irene Wuethrich ETH Zürich
Bo-Tao Xin Leiden University Medical Center
Liubov Yakovlieva University of Groningen
Ryoji Yoshisada Utrecht University
Cristiane Zuconelli Radboud UMC
