Constrained peptides targeting protein−protein interfaces Chiara Cabrele

Department of Biosciences, University of Salzburg, Billrothstraße 11, 5020 Salzburg, Austria

Constrained peptides are useful tools to mimic structural and functional motifs of peptides and

proteins: indeed, while remaining very similar to their natural source, they may be superior for binding

affinity and specificity towards the respective biological targets, as well as for metabolic stability.

Different chemical approaches are exploited to develop constrained peptides reproducing native β-

turns, β-hairpins, two or more helical turns, and extended motifs. Some of these approaches are based

on (i) cyclization of the backbone, (ii) linkage between two side chains or between a side chain and the

backbone, and (iii) replacement of α-amino acids with Cα-alkylated α-amino acids, β- or γ-amino

acids. We have used side-chain−to−side-chain cyclization to develop constrained peptides mimicking

helical motifs involved in protein−protein[1,2] and hormone−receptor[3] interactions, and cyclic β-amino

acids to gain receptor-subtype ligand specificity.[4,5] Examples of biological applications of such

constrained peptides will be given.

References

1.Targeting of a helix-loop-helix transcriptional regulator by a short helical peptide. C. Roschger, S. Neukirchen, B.

Elsässer, M. Schubert, N. Maeding, T. Verwanger, B. Krammer, C. Cabrele ChemBioChem, 12, 1497-1503 (2017).

2. Impact of the amino acid sequence on the conformation of side chain lactam-bridged octapeptides. S. Neukirchen, V. Krieger, C. Roschger, M. Schubert, B. Elsässer, C. Cabrele J. Pept. Sci., 23, 587-596 (2017).

3. Side chain cyclization based on serine residues: synthesis, structure, and activity of a novel cyclic analogue of the

parathyroid hormone fragment 1-11. A. Caporale, M. Sturlese, L. Gesiot, F. Zanta, A. Wittelsberger, C. Cabrele J.

Med. Chem., 53, 8072-8079 (2010).

4. Replacement of Thr32 and Gln34 in the C-terminal neuropeptide Y fragment 25-36 by cis-cyclobutane and

ciscyclopentane β-amino acids shifts selectivity toward the Y4 receptor. L. Berlicki, M. Kaske, R. Gutiérrez-Abad, G.

Bernhardt, O. Illa, R.M. Ortuño, C. Cabrele, A. Buschauer, O. Reiser J. Med. Chem., 56, 8422-8431 (2013). 5. Analogues of neuropeptide Y containing β-aminocyclopropane carboxylic acids are the shortest linear peptides

that are selective for the Y1 receptor. N. Koglin, C. Zorn, R. Beumer, C. Cabrele, C. Bubert, N. Sewald, O. Reiser,

A.G. Beck-Sickinger Angew. Chem. Int. Ed., 42, 202-205 (2003).

Ab initio design of foldamer tertiary structures

D. Mazzier1, S. De2, V. Maurizot2 and I. Huc1

1 Department of Pharmacy, Ludwig-Maximilians-Universität München, 81377 – Munich, Germany

2 CNRS, Institut Européen de Chimie et Biologie, 33600 – Pessac, France

Over the last decades, numerous non-natural foldamer backbones have been developed for the

construction of original, predictable, and well defined molecular architectures.[1] The research was

mainly focused on the design of structures analogous to secondary motifs of biopolymers, such as

helices, sheets and turns.[2] However, most of the functions of biopolymers, especially of proteins,

emerge at the level of their tertiary structures and would not be achieved by an isolated α-helix or β-

sheet. Thus, the design of artificial tertiary folds that comprise several secondary structural elements

represents a major challenge. Tertiary folds based on non-natural monomers remain an unexplored

area and may give access to shapes and functions different to those of peptides and nucleotides.

Due to the stability and the predictability of their folded secondary structures, aromatic oligoamide

foldamers possess a high potential in the field of peptidomimetics.[3] Therefore, the high stability of

aromatic amide helices can be exploited to create complex artificial tertiary structures.

In this context, we focused our attention into the design, the synthesis and the characterization of

oligoamide-quinolines foldamers that have side chains designed to interact with each other to form

helix-turn-helix motifs.[4]

Figure 1. Design of helix-turn-helix motif from primary sequence to tertiary structure

References

1. J. Guichard, I. Huc Chem. Comm. (2011), 47, 5933.

2. R. V. Nair, K. N. Vijayadas, A. Roy, G. J. Sanjayan Eur. J. Org. Chem. (2014), 2014, 7763. 3. I. Huc Eur. J. Org. Chem. (2004), 17.

4. S. De, B. Chi, T. Granier, T. Qi, V. Maurizot, I. Huc Nat. Chem. (2018), 10, 51.

Design, Synthesis, Structural and Biochemical characterization of

AIF(370-394) constrained peptide. Alessandra Monti,1,2 Andrea Caporale,1 Mattia Sturlese,3 Menotti Ruvo,1 Nunzianna Doti1*

1 IBB-CNR, CIRPEB, Università di Napoli “Federico II”, 80134, Napoli, Italy

2 DiSTABiF, Università degli Studi della Campania “Luigi Vanvitelli”, Via Vivaldi 43, 81100,

Caserta, Italy 3 Molecular Modeling Section, Dipartimento di Scienze del Farmaco, Università di Padova, Italy

Apoptosis inducing factor (AIF) is a bifunctional mitochondrial flavoprotein, critical for energy

metabolism and induction of caspase-independent apoptosis in neurons.[1] Upon apoptotic stimuli, the

proteolytic form of AIF, AIF(Δ1–121), moves to nuclei, where it triggers chromatin condensation,

large-scale DNA fragmentation and cell death, by its direct interaction with the cytoplasmic protein

cyclophilin A (CypA).[2] In a previous study, we showed that the β-hairpin region of AIF, spanning

residues 370-394, mediates the protein complex between AIF(Δ1–121) and CypA.[3] A peptide

mimicking this region, hereafter AIF(370-394), inhibits the formation of the complex in vitro and

provides neuroprotection if transfected into neuronal cells, by competing with AIF for the same

interaction site on CypA.[4,5] Data obtained demonstrated that the peptide is a good model for studying

the complex AIF(Δ1-121)/CypA and for developing new inhibitors with potential therapeutic value.

On the basis of these data, to improve the biological activity of the AIF(370-394) peptide, we have

designed, synthesized and structurally characterized AIF peptide analogues, containing structural

constraints to limit the molecule conformational freedom and to induce a more native-like

conformation.

In particular, we have investigated the conformational features of a series of highly constrained bi- and

mono- cyclic analogues containing both disulphide and 1,4-disubstituted 1,2,3-triazole bridges.[6]

Comparative biochemical assays have also been carried out to evaluate the ability of the new

analogues to bind the target protein CypA[3-5] and to obtain structure-activity relationship insights.

References

1. I.F. Sevrioukova. Antioxid Redox Signal (2011) 14, 2545-2579. 2. C. Candé, N. Vahsen,I. Kouranti, E. Schmitt, E. Daugas,C. Spahr, J. Luban, R.T. Kroemer, F. Giordanetto, C.

Garrido, J.M. Penninger and G. Kroemer. Oncogene (2004) 23, 1514-1521.

3. N. Doti, C. Reuther, P.L. Scognamiglio, A. M. Dolga, N. Plesnila, M. Ruvo, and C. Culmsee. Cell Death Dis

(2014) 16, 5:e993.

4. B. Farina, G. Di Sorbo, A. Chambery, A. Caporale, G. Leoni, R. Russo, F. Mascanzoni, D. Raimondo, R.

Fattorusso, M. Ruvo and N. Doti. Sci Rep (2017) 7, 1138.

5. N. Doti and M. Ruvo. Neural Regen Res. (2017) 12, 1428-1429.

6. C.W. Tornøe, C. Christensen and M. Meldal, J. Org. Chem., (2002) 67, 3057.

Conformationally Constrained Analogues of Amyloidogenic

Segments of Islet Amyloid Polypeptide B. Dalla Volta1,2,#, G. Sabatino3, A. Grammatikopoulos2, M. Vignoli1, A.M. Papini3,4, G. Pappalardo3, P. Rovero1,3

A. Kapurniotu2

1 PeptLab & Dept. Neurofarba, Univ. of Florence, Via Ugo Schiff 6, 50019 Sesto Fiorentino,

Italy.

2 Division of Peptide Biochemistry, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354 Freising-Weihenstephan, Germany.

3 CNR-IBB Istituto di Biostrutture e Bioimmagini, Via Paolo Gaifami 18, 95126 Catania, Italy.

4 PeptLab, & Dept. Chemistry, Univ. of Florence, Via della Lastruccia 13, 50019 Sesto

Fiorentino, Italy.

Pancreatic islet amyloid is a characteristic feature of type 2 diabetes and islet amyloid polypeptide

(IAPP) is its major protein component. As the conformational transition of α-helical IAPP

conformations to β-sheet ones underlies IAPP amyloidogenesis[1,2], we designed a series of peptides in

which we attempted to stabilize the α-helical structure in the amyloidogenic IAPP segments IAPP(8-

18) and IAPP(8-28) via i-to-i+4 side chain-to-side chain cyclization. Analogues B1cyclo and B2cyclo

exhibited higher α-helical propensities than their linear precursors while the low solubility of B3cyclo

and B4cyclo prohibited their study. Importantly, significant -helix stabilization was observed in

B5cyclo; by contrast, B6cyclo had a high β-sheet forming propensity and lower α-helical content than

the linear precursor. Most importantly, B6cyclo but not B5cyclo was found to be able to suppress

IAPP amyloid formation and cytotoxicity. Thus, B6cyclo is a promising candidate for the development

of inhibitors of IAPP amyloidogenesis and related cell-damage. Limited proteolysis experiments using

insulin-degrading enzyme (IDE) and/or trypsin to

hydrolyse IAPP either in the absence or in the

presence of B6cyclo were then performed. IDE is a

zinc metalloprotease able to degrade several

different substrates including insulin, β-amyloid

peptides (Aβ) and IAPP. These experiments[3]

coupled with LC-MS analysis allow for

identification of the IAPP region involved in the

interaction with the B6cyclo peptide.

# Current address: Univ. Leipzig, Inst. Biochemistry, 04103 Leipzig, Germany

References

1. R. Kayed, J. Bernhagen, N. Greenfield et al J. Mol. Biol. (1999), 287, 781-796.

2. J. J.Wiltzius, S.A. Sievers, M.R. Sawaya et al Protein Sci. (2009), 18, 1521-1530.

3. V. Villari, R.Tosto, G. Di Natale et al ChemistrySelect 2017, 2, 9122-9129.

Analogues (8-18)

B1cyclo Ac-cyclo(DTQRK)ANFLVH-NH₂

B2cyclo Ac-ATQRLAcyclo(DFLVK)-NH₂

B3cyclo Ac-Cyclo[Nle(ε-N3)-TQR-Pra]ANFLVH-NH₂

B4cyclo Ac-Cyclo[Ala(β-N3)-TQR-Pra]ANFLVH-NH₂

Analogues (8-28)

B5cyclo Ac-cyclo(DTQRK)ANFLVHSSNNFGAILS-NH2

B6cyclo Ac-ATQRLAcyclo(DFLVK)SSNNFGAILS-NH2

Design and Analysis of Peptide Inhibitors of the Interaction

between SAM Domains of Ship2 and the EphA2 Receptor

Flavia Anna Mercurio2, Marian Vincenzi1, Concetta Di Natale2, Luciano Pirone1, Roberta Iannitti1, Daniela Marasco1,2, Emilia Maria Pedone1, Rosanna Palumbo1, and Marilisa Leone1

1Institute of Biostructures and Bioimaging (IBB), CNR, via Mezzocannone 16, 80134-Naples, Italy

2Department of Pharmacy, Research Centre on Bioactive Peptides (CIRPeB), University of Naples “Federico

II”, Via Mezzocannone 16, 80134-Naples, Italy

Ship2 is a lipid phosphatase involved in different diseases including cancer [1]. It contains at

the C-terminus a sterile alpha motif domain (Ship2-Sam) that associates with the Sam domain from the

EphA2 receptor (EphA2-Sam) [2]. This interaction is expected to have pro-oncogenic effects in cancer

cells [3] thus, compound inhibitors of the Ship2-Sam/EphA2-Sam complex may represent innovative

tools in anti-cancer drug discovery.

We designed several peptide sequences encompassing the interacting region of EphA2-Sam

for Ship2-Sam, and performed conformational analyses and interaction assays through a variety of

techniques (CD, NMR, SPR and MST). These studies led to identification of a peptide -named (KRI)3-

in which a positively charged penta-amino acid motif belonging to EphA2-Sam is repeated thrice in

tandem. (KRI)3 gives appreciable binding to Ship2-Sam [4] and NMR experiments indicate that it

mainly targets the negatively charged binding site of Ship2-Sam for EphA2-Sam [4]. The peptide is

also able to induce necrosis of the PC-3 prostate cancer cell line and is more cytotoxic to cancer cells

with respect to normal dermal fibroblasts, as indicated by preliminary in vitro cell-based assays [4].

References

1.A. Suwa, T. Kurama, T. Shimokawa Expert Opin Ther Targets, 14, 727-737 (2010)

2.M. Leone, J. Cellitti, and M. Pellecchia Biochemistry, 47, 12721-28 (2008)

3.G. Zhuang, S. Hunter, Y. Hwang, J. Chen J Biol Chem, 282, 2683-2694 (2007)

4.F.A. Mercurio, C. Di Natale, L. Pirone, R. Iannitti, D. Marasco, E.M. Pedone, R. Palumbo, M. Leone Sci Rep,

7(1):17474 (2017)

Phage-encoded bicyclic peptide inhibitors of a tumor-associated

serine protease

Alessandro Angelini1* and Christian Heinis2

1 Department of Molecular Sciences and Nanosystems, Ca' Foscari University of Venice,

Via Torino 155, Venezia Mestre 30172, Italy (alessandro.angelini@unive.it) 2 Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne (EPFL),

CH-1015 Lausanne, Switzerland

Bicyclic peptides are small (<2 kDa) and highly constrained molecules that combine multiple favourable

properties such as good binding affinity, exquisite selectivity, high proteolytic stability and low toxicity that

make them an attractive format for the development of therapeutics1. Combinatorial libraries of billion of

different bicyclic peptide binders can be generated and screened by using a phage display-based

methodology2. By applying this approach, we identified bicyclic peptide inhibitors of human urokinase-type

plasminogen activator (uPA), a serine protease implicated in tumor growth and invasion3. Selected bicyclic

peptides appear to have properties typical of proteins explaining their good binding affinity and exquisite

specificity4. Their binding affinity and specificity can be further tuned by varying i) the length of the peptide

loops and ii) the nature and the size of the chemical linker connecting them5,6

. Importantly, their half-life

could be extended to several days by fusing them to an antibody Fc domain or non-covalently appended it to

serum albumin7,8

. In vivo studies revealed that long-lived bicyclic peptides can stay fully intact and

functional for >24 hrs in mice, overcoming a limitation faced by many peptide leads8,9

. Finally, when tested

in tumor-bearing mice, bicyclic peptides diffused deeply into tissues reaching high nanomolar concentrations

in wide areas of solid tumors8,9

. Given these encouraging qualities, in vitro evolved bicyclic peptides offer a

promising format for the development of next generation peptide therapeutics.

References

1. Phage selection of cyclic peptides for application in research and drug development. Deyle K., Kong X.D. and

C. Heinis. Accounts of Chemical Research, 50, 1866-74 (2017)

2. Phage-encoded combinatorial chemical libraries based on bicyclic peptides. Heinis C., Rutherford T., Freund S.

and G. Winter. Nature Chemical Biology. 5, 502-7 (2009)

3. Multifaceted role of the urokinase-type plasminogen activator (uPA) and its receptor (uPAR): diagnostic

prognostic, and therapeutic applications. Mahmood N., Mihalcioiu C. and S.A. Rabbani. Frontiers in Oncology, 8,

24 (2018)

4. Bicyclic peptide inhibitor reveals large contact interface with a protease target. Angelini A., Cendron L., Chen S., Touati J., Winter G., Zanotti G. and C. Heinis. ACS Chemical Biology, 7, 817–21 (2012)

5. Phage display libraries of differently sized bicyclic peptides. Rentero Rebollo I., Angelini A. and C. Heinis.

MedChemComm, 4, 145-50 (2013)

6. Peptide ligands stabilized by small molecules. Chen S., Bertoldo D., Angelini A., Pojer F. and C. Heinis.

Angewandte Chemie International Edition, 53, 1602-6 (2014)

7. Chemical macrocyclization of peptides fused to antibody Fc fragments. Angelini A., Diderich P., Morales-

Sanfrutos J., Thurnheer S., Hacker D., Menin L. and C. Heinis. Bioconjugate Chemistry, 23, 1856-63 (2012) 8. Bicyclization and tethering to albumin yields long-acting peptide antagonists. Angelini A., Morales-Sanfrutos J.,

Diderich P., Chen S. and C. Heinis. Journal of Medicinal Chemistry, 55, 10187-97 (2012)

9. Bicyclic peptides conjugated to an albumin-binding tag diffuse efficiently into solid tumors. Pollaro L.,

Raghunathan S., Morales-Sanfrutos J., Angelini A., Kontos S. and C. Heinis. Molecular Cancer Therapeutics. 14,

151-61 (2015)

II° Convegno Società Italiana Peptidi

Diego Brancaccio,1* Donatella Diana,2 Salvatore Di Maro,3 Francesco Saverio Di Leva,1 Stefano Tomassi,3 Roberto Fattorusso,3 Luigi Russo,3 Stefania Scala,4 Anna Maria Trotta,4

Luigi Portella,4 Ettore Novellino,1 Luciana Marinelli,1 and Alfonso Carotenuto,1

1 Dipartimento di Farmacia, Università di Napoli Federico II, 80131, Naples, Italy.

2 Istituto di Biostrutture e Bioimmagini,

C.N.R., 80134 Naples, Italy. 3

Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche, Università

degli Studi della Campania “Luigi Vanvitelli”, 81100 Caserta, Italy. 4

Molecular Immunology and Immunoregulation,

Istituto Nazionale Tumori "Fondazione G. Pascale", IRCCS-Napoli, 80131 Naples, Italy.

G Protein-Coupled Receptors (GPCRs) family are well-established drug targets within

pharmaceutical intervention, and to date about 50% of the marketed drugs exert their activity by

modulating distinct members of this class of transmembrane signal pathway. Among GPCRs,

peptides-binding receptors play a crucial role in many pathological and physiological pathways.1,2 The

assessment of the receptor-bound conformation of a peptidic ligand within a membrane receptor such

as a GPCR is of great impact for a rational drug design of more potent analogues. In this work, we

applied ligand-based NMR methods to study the interaction of heptapeptides, derived from the C-X-C

Motif Chemokine 12 (CXCL12), and the C-X-C Chemokine Receptor Type 4 (CXCR4) located on the

cytosolic membrane of CCRF-CEM cells. This study represents the first structural investigation on the

binding mode of a peptide to a GPCR directly on a living cell. As model for our investigation, we

selected CXCR4 since it represents an important potential therapeutic target for various severe

diseases involving immune system, including cancer.23 The results obtained in the field of

CXCL12/CXCR4 are proofs of concept, although important information for researchers dealing with

the CXCR4 field arise. General application of the presented NMR methodologies is possible and

surely may help to boost the development of new therapeutic agents targeting GPCRs.

References

1.Peptide-Binding G Protein-Coupled Receptors: New Opportunities for Drug Design. Gurrath, M. Curr Med Chem,

8, 1605–1648 (2001).

2.Peptide Ligand Recognition by G Protein-Coupled Receptors. Krumm, B. E.; Grisshammer, R. Front. Pharmacol.

6, (2015).

3.The Intricate Role of CXCR4 in Cancer. Chatterjee, S.; Behnam Azad, B.; Nimmagadda, S. Adv. Cancer Res. 124, 31–82 (2014).

Peptide Decorated Gold Nanostructures for high effective SERRS

Detection of Colorectal Cancer Cells

F. Biscaglia *a, S. Rajendran,b C. Benna,b G. Bocchinfuso,c P. Conflitti,c L. Litti,a R. Sommaggio,b D. Nitti,b A.

Rosato,b A. Palleschi,c S. Mocellin,b M. Meneghettia and M. Gobbo a

a University of Padova, Dept. of Chemical Sciences, Padova, Italy;

b University of Padova, Dept. of Oncological and Surgical Sciences, Padova, Italy;

c University of Rome Tor Vergata , Dept. of Chemical Sciences & Technologies, Roma, Italy;

Due to their optical properties, facile surface chemistry and biocompatibility, Gold Nanoparticles are

increasingly utilized for biotechnological applications including imaging, diagnostics and therapy. In

order to fully exploit their potentialities, they need to be directed to the target using biomolecular

targeting agents such as antibodies, peptides or other small molecules. Recently, the dodecapeptide

YHWYGYTPQNVI (GE11) was identified as a specific ligand for the Epidermal Growth Factor

Receptor (EGFR), frequently overexpressed in many types of cancer (1). According to this knowledge,

the present study aims to demonstrate that Gold Nanostructures properly decorated with the EGFR

binding peptide GE11 (2) can effectively target and detect different types of colorectal tumour cells

overexpressing EGFR.

Gold Nanostructures were prepared from naked gold nanoparticles obtained by laser ablation in water

and encoded with a SERRS reporter (3) to achieve very intense Surface-Enhanced Raman Resonance

Scattering (SERRS) signals. Nanostructures were then functionalized with the targeting peptide and

PEG polymers, as antifouling agents, in different arrangements to investigate how they affect the

receptor recognition. Peptides were directly linked to gold nanoparticles, exploiting an extra cysteine

residue, or through a thiolated PEG chain. The Gold Nanostructures targeting activity on colorectal

cancer cells, expressing or not EGFR, was quantified by recording the SERRS signals cell by cell.

Nanoaggregates covered with PEG alone or with Cetuximab, an anti-EGFR antibody already used in

the clinical settings, were used as negative and positive control, respectively. (2) The overall results

show that the presentation of the targeting peptide on the nanostructures surface plays the pivotal role

for the high selective and sensitive association with the EGF Receptor.

References

1. Identification and characterization of a novel peptide ligand of epidermal growth factor receptor for targeted

delivery of therapeutics, Z. Li et all, FASEB J. 19, 1978-1985 (2005).

2. Enhanced EGFR Targeting Activity of Plasmonic Nanostructures with Engineered GE11 Peptide, F.Biscaglia et

all, Adv. Healthcare Mater, 6, 1700596 (2017).

3. Plasmonic Nanostructures for SERRS Multiplexed Identification of Tumor-Associated Antigens, M. Meneghetti et all Small, 8, 3733-3738 (2012).

Peptidomimetics of suppressor of cytokine signaling 1 and 3:

new therapeutics in cancer-inflammation diseases

S. La Manna1, G. Morelli1 and D. Marasco1

1 University of Naples “Federico II”, Department of Pharmacy, 80134, Naples, Italy.

The suppressor of cytokine signaling (SOCS) proteins are negative regulators of the JAK/STAT pathway [1]. Many

studies outline that they can play crucial roles in a variety of diseases including cancers with acute inflammation [2].

Only two members of this family, SOCS1 and SOCS3, contain a unique small kinase inhibitory region (KIR) that is

directly involved in inhibition of JAKs.

We investigated the abilities of the peptidomimetic called PS5[3] to mimic SOCS1 biological functions in cellular

vascular smooth muscle cells and to test new compounds following medicinal chemistry rules to improve the

affinity and cellular stability of PS5 a new binding assay based on a Surface Plasmon Resonance (SPR) experiment

was set up. Several analogues of PS5 were analyzed through SPR, Circular Dichroism (CD) and Nuclear Magnetic

Resonance (NMR) spectroscopies [4 ].

Furthermore, recent studies showed that recombinant SOCS3 was able to prevent triple negative breast cancer

(TNBC) growth and metastasis by suppressing inflammatory cytokines [5]. To develop new potential therapeutics in

TNBC, we designed and characterized, by CD and SPR spectroscopies, several SOCS3’mimetics derived from the

N-terminal region of SOCS3 encompasses KIR and extended SH2 domain (ESS) domain that interface the complex

with JAK2. The activity of the most promising sequence, KIRESS, was further investigated in vivo in mouse

xenografts of MDA-MB-231-luci tumours as model of human TNBC subtype. This peptide demonstrated capable

to eliminate pulmonary metastasis and showed a significant reduction of primary tumour growth [6].

Overall data could be considered as a promising scaffold to develop higher-affinity, -stability compounds active in

cancer-inflammation diseases.

References

1. B. Carow, M. E. Rottenberg, Front. Immunol. (2014), 5, 58. 2. A. Yoshimura, H. Yasukawa, Immunity (2012), 36, 2.

3. N. Doti, P. L. Scognamiglio, S. Madonna, C. Scarponi, M. Ruvo, G. Perretta, C. Albanesi, D. Marasco,

Biochem. J. (2012), 443, 231.

4. S. La Manna, L. Lopez-Sanz, M. Leone, P. Brandi, P. L. Scognamiglio, G. Morelli, E. Novellino, C. Gomez-

Guerrero, D. Marasco, Biopolymers (2017).

5. G. Kim, M. Ouzounova, A. A. Quraishi, A.Davis, N.Tawakkol, S. G. Clouthier, F. Malik, A. K. Paulson, R. C.

D'Angelo, S. Korkaya, T. L. Baker, E. S. Esen, A. Prat, S. Liu, C. G. Kleer, D. G.Thomas, M. S. Wicha, H.

Korkaya, Oncogene (2015), 34, 6. 6. S. La Manna, L. Eunmi, M. Ouzounova, C. Di Natale, E. Novellino, A. Merlino, H. Korkaya and D. Marasco,

Int. J. Cancer. submitted.

Functional characterization of a selective avb5 antagonist

Domenica Capasso1, Sonia Di Gaetano2, Annarita Del Gatto2, Luigi Russo3, Roberto Fattorusso3, Daniela Comegna2, Michele Saviano4 and Laura Zaccaro2

1Dipartimento di Farmacia, Università di Napoli Federico II, 80134 Naples (Italy)

2Istituto di Biostrutture e Bioimmagini, CNR, 80134 Naples (Italy)

3Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche Seconda Università degli Studi Napoli

81100, Caserta (Italy) 4

Istituto di Cristallografia, CNR, 70126 Bari (Italy)

Integrins are cell surface receptors involved in cell-cell and cell-extracellular matrix adhesion,

recognizing the RGD triad found in many extracellular matrix proteins. Their overexpression is

associated with different pathologies such as inflammation, psoriasis, wound-healing and, in

particular, cancer and tumor angiogenesis thus contributing to drug resistance and tumor recurrence1.

For these reasons, integrins are considered interesting targets in the development of new anticancer

and anti-inflammatory molecules. We reported the design and characterization of RGDechi, a chimeric

peptide containing a cyclic RGD motif linked to an echistatin C-terminal fragment, able to recognize

selectively αvβ3 integrin both in vitro and in vivo 2. A computational analysis, combined with the

NMR data, provided a three-dimensional model of the RGDechi-αvβ3 complex elucidating the

molecular requirements for the peptide specific recognition of the receptor3. On the basis of the

a widely studied member of integrin receptor family involved in the process of liver metastasis and

epithelial-metastatic transition 4. In this work we performed a deeply functional characterization of the

integrin, investigating its effect on cell adhesion, proliferation and invasion.

References

1. Zaccaro L. et al. Current Topics in Biochemical Research 2007, 9, 45-56.

2. Comegna D et al. Journal Medicinal Chemistry 2017, 60 (23), 9874-9884.

3. Farina B, et al. Chemistry 2016, 22, 681 – 693.

4. Nejjari M, et al. Hepatology. 2002, 36 (2), 418-26

The mechanism of action of the GKY20 antimicrobial peptide

Pompea Del Vecchio1, Eugenio Notomista1, Valeria Cafaro1, Katia Pane1, Antonio Grimaldi1, Roland Winter2,

Luigi Petraccone1, Rosario Oliva1*

1University of Naples Federico II, Naples 80126, Italy

2Technische Universität Dortmund, Dortmund 44227, Germany

Antimicrobial peptides (AMPs) are membrane-active peptides with a broad spectrum of

activity against different pathogenic organisms (e.g. bacteria, viruses, cancer cells).1 The emergence of

resistance to antibiotics in bacteria has catalyzed the attention of the scientific community on AMPs as

drug of the future. In fact, it is believed that AMPs, interacting in a non-specific way with the lipid

matrix of the membrane, could circumvent the problem of resistance.2 For their application as drugs, it

is mandatory to reveal their mechanism of action. Some models have been proposed which may, in

part, explain the mechanism of action.1 Unfortunately, despite extensive studies, most details are still

unknown. As complicating factors, the mechanism strongly depends on the lipid composition and

molecular properties of both lipids and peptide.3

In this study, we report a comprehensive physico-chemical study of the interaction of a human

thrombin derived peptide, GKY20,4 with liposomes composed by POPC and POPC/POPG (8/2

mol/mol) as models of an eukaryotic and bacterial membrane, respectively. The reported fluorescence

and circular dichroism data suggest that the peptide is able to interact with both model membranes

with high affinity, adopting a helical structure upon binding. The percentage of α-helices, obtained

from the deconvolution of the CD spectra, is significant less with POPC (13%) compared to

POPC/POPG (46%). Upon the interaction with the bacterial model membrane, atomic force

microscopy (AFM) and differential scanning calorimetry (DSC) experiments revealed the formation of

lipid domains enriched in POPG and peptide molecules. Further, the AFM data showed that beyond a

certain threshold concentration, complete disruption of the membrane takes place through lipid

extraction via formation of micelles. Overall, the data presented support a description based on a

carpet-like mechanism of action for the GKY20 peptide.

References

1. Role of lipids in the interaction of antimicrobial peptides with membranes. V. Teixeira et al., Prog. Lipids Res.

51, 149-177 (2012).

2. Antimicrobial peptides: linking partition, activity and highmembrane-bound concentrations. M. N. Melo et al.,

Nature Rev. Microbiol. 7, 245-250 (2009). 3. Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by α-helical

antimicrobial and cell non-selective membrane-lytic peptides. Y. Shai, Biochim. Biophys. Acta 1462, 55-70

(1999).

4. Structure-activity studies and therapeutic potential of host defense peptides of human thrombin. G. Kasetty,

Antimicrob. Agents Chemotherapy, 55, 2880-2890 (2011).

Mild reactions for peptide-cotton bond formation: preparation of

biocompatible, antimicrobial fabrics

C. Peggion1, M. Carraro1, M. Stefani1, F. Formaggio1, S. Oancea2

1Department of Chemistry, University of Padova, 35131 – Padova, Italy

2Department of Agricultural Sciences and Food Engineering, University of Sibiu, 550012 – Sibiu, Romania

The need to develop new biocompatible fabrics for a variety of applications is greatly promoting

academic and industrial research. In this connection, we prepared cotton-based, antibacterial textiles

characterized by the covalent attachment of short peptides.

Peptide covalent grafting on the cotton surface was achieved in different ways, but always exploiting

the naturally occurring hydroxyl groups of cellulose. In search for green and mild reactions, we

exploited also chemoselective ligations through an oxime or a thioazolidine ring in aqueous solution.

With this last approach we were able to link an octapeptide, derived from the N-terminal domain of a

dermaseptin 1S mutant, known for its antimicrobial properties. In this example, we used the chemo-

enzymatic, TEMPO-mediated oxidation of the hydroxyl groups into aldehydes by means of laccase in

mild acidic aqueous conditions. The subsequent reaction with the β-aminothiol of a Cys-peptide gave a

stable covalent bond (Figure 1).

Figure 1. Laccase mediated cotton oxidation and chemoselective reaction with a Cy-peptide

We characterized our cotton-peptide samples by means of FT-IR, UV-Vis and XPS and determined

their antimicrobial activity against Staphylococcus aureus and Escherichia coli. Interestingly, some of

the materials gave promising results against the Gram positive strain, responsible for most hospital-

acquired infections.

References

1. A.P Gomes, J. Mano, F., J. A. Queiroz, I. C. Gouveia Carbohyd. Polym. (2015), 127, 451.

2. A. Orlandin, F. Formaggio, A. Toffoletti, C. Peggion J. Pept. Sci. (2014), 20, 547.

II° Convegno Società Italiana Peptidi

Derivatives of the frog skin peptide esculentin-1a kill Pseudomonas aeruginosa biofilm

on soft contact lenses and preserve their bactericidal activity upon conjugation to the

lens surface

Bruno Casciaro*, Debarun Dutta, Maria Rosa Loffredo, Alison M McDermott, Mark DP Willcox , Maria Luisa

Mangoni

Laboratory affiliated to Pasteur Italia Fondazione Cenci Bolognetti, Department of Biochemical Sciences, Sapienza

University of Rome, Rome, Italy 00185

Antimicrobial peptides (AMPs) represent a class of molecules with interesting biological properties,

including a broad spectrum of action ranging from fungi to Gram-positive and Gram-negative bacteria.

Among the latter, one of the most dangerous microorganisms is Pseudomonas aeruginosa, a motile

bacterium capable of colonizing biological and abiotic surfaces, such as contact lenses (CLs), forming

biofilm communities that are difficult to eradicate. This increases the risks of CL wearers of

developing microbial keratitis. Therefore, novel strategies and compounds to reduce the onset of CL-

associated ocular infections are needed. It has been demonstrated that the frog-skin AMP Esc(1-21)

and its diastereomer Esc(1-21)-1c have potent anti-Pseudomonal activity against the free and biofilm

form of this pathogen. Here we first evaluated the ability of both peptides to kill P. aeruginosa biofilm

formed on soft CLs by microbiological assays and scanning electron microscopy analysis. It turned out

that both peptides have the ability to eradicate Pseudomonas biofilms with the diastereomer showing a

stronger potency (up to 85% killing vs no killing of the all-L peptide at 4 μM for some bacterial

strains). Furthermore, we covalently immobilized both peptides to soft CLs. The peptides-immobilized

CLs were found to cause more than four log reduction in the number of bacterial cells within 20

minutes and to reduce bacterial adhesion to the CL surface (77%–97% reduction) in 24 hours without

any harmful effect on mammalian cells. In addition peptides immobilization to CLs did not affect the

lens properties. These promising results suggest the potential use of peptides tethered to medical

devices for the prevention and treatment of CL‐associated P. aeruginosa keratitis [1].

References

1. Casciaro B, Dutta D, Loffredo MR, et al. Esculentin-1a derived peptides kill Pseudomonas aeruginosa biofilm

on soft contact lenses and retain antibacterial activity upon immobilization to the lens surface. Biopolymers. 2017; e23074. https://doi.org/10.1002/bip.23074

Novel peptide-based nanomaterials with antimicrobial activity

Lucia Lombardi1, Annarita Falanga1, Yejiao Shi2, Giancarlo Morelli1, Helena S. Azevedo2 and Stefania Galdiero*1

1 Department of Pharmacy, School of Medicine, University of Naples Federico II, via Mezzocannone 16, 80134, Naples, Italy

2 School of Engineering & Materials Science, Queen Mary University of London, Mile End Road, London E1 4NS, UK

3 Department of Biological Sciences, University of Naples Federico II, Cupa Nuova Cintia, 21, 80126 Naples, Italy

4 Department of Experimental Medicine, Division of Microbiology, Università of Campania Luigi Vanvitelli, Via De Crecchio 7, 80138, Naples, Italy

The extensive use of antibiotics in both human and animal healthcare has resulted in the development of bacterial resistance towards these drugs. Great attention has been recently devoted to the possibility to build self-assembling peptide nanostructures with intrinsic antibacterial activity.1,2 Here we present a versatile self-assembling nanostructures with antimicrobial activity due to the presence of antimicrobial peptides (AMPs) on the surface. For this purpose, we synthetized two different peptide sequences: one shorter, which aids in the self-assembling process, and a longer one containing two moieties (the one serving for the assembly and the one serving as antimicrobial agent). As a proof of concept, an active analogue of the antimicrobial peptide myxinidin3 was developed. The self-assembled nanostructures were characterized by fluorescence spectroscopy to determine the critical aggregation concentration, circular dichroism, NMR, DLS and transmission electron microscopy for a structural study, biological activity tests to evaluate the eukaryotic cell toxicity and the capability to inhibit the growth or promote the eradication of bacterial and fungal biofilms. This study showed that our peptide nanostructures are promising tools against biofilm formation at low concentration especially against fungal biofilm.

References 1. Stimuli-responsive self-assembling peptides made from antibacterial peptides. Y. Liu, Y. Yang, C. Wang, X.

Zhao Nanoscale, 5, 6413-6421 (2013). 2. Ultrashort cationic naphthalene-derived self-assembled peptides as antimicrobial nanomaterials. G. Laverty, A.

P. McCloskey, B. F. Gilmore, D. S. Jones, J. Zhou, B. Xu Biomacromolecules, 15, 3429-3439 (2014). 3. Structure-Activity Relations of Myxinidin, an Antibacterial Peptide Derived from the Epidermal Mucus of

Hagfish. M. Cantisani, M. Leone, E. Mignogna, K. Kampanaraki, A. Falanga, G. Morelli, M. Galdiero, S. Galdiero Antimicrobial Agents and Chemotherapy, 57, 5665–5673 (2013)

From peptides to peptidomimetics: spectroscopic studies of potential antimicrobial agents

Annalisa Bortolotti1, Valerio Santucci1, Paolo Calligari1, Gianfranco Bocchinfuso1, Mohini Mohan Konai2, Jayanta Haldar2, Lorenzo Stella1

1 Department of Chemical Science and Technologies, University of Rome Tor Vergata, 00133 Rome, Italy 2 Antimicrobial Research Laboratory, New Chemistry Unit, Jawaharlal Nehru Centre for Advanced Scientific

Research, Jakkur, Bengaluru 560064, Karnataka, India

Bacteria are showing increasing resistance against available antibiotics. Naturally occurring antimicrobial peptides (AMPs) are promising molecules to fight multi-resistant microbes. Notwithstanding the potential of AMPs in the fight against drug resistance, their therapeutic application presents some limitations, such as their susceptibility to proteolytic degradation and high manufacturing cost. To address these issues, recently several research efforts were devoted to the development of synthetic peptidomimetics of AMPs. However, the rational design of protease-resistant peptidomimetic molecules with the same properties of AMPs, is complicated by the fact that multiple equilibria (self-assembly, water-membrane partition, insertion in the bilayer, pore formation, etc.) influence the membrane-perturbing activity, and any modification in molecular properties perturbs all of them to different extents. Spectroscopic approaches, in particular fluorescence methods, are extremely powerful in the characterization of the behavior of AMPs or peptidomimetics in interaction with lipid bilayers [1]. By using these techniques, together with molecular dynamics simulations, we studied the mechanism of action of an antimicrobial norspermidine-based peptidomimetic (Nor Trp) characterized by a cationic charge and an aliphatic tail, and centered on norspermidine, an easily available and inexpensive compound that allows easy functionalization. This peptidomimetic exhibited a strong activity and very low toxicity [2]. We characterized aggregation, membrane binding, insertion in the membrane, pore formation and bilayer perturbation. Overall, our findings provide a clear picture of the interaction of Nor Trp with lipid bilayers, and of its mechanism of membrane perturbation.

References 1. Fluorescence spectroscopy and molecular dynamics simulations in studies on the mechanism of membrane

destabilization by antimicrobial peptides, G. Bocchinfuso, Cell. Mol. Life Sci. 68, 2281–2301 (2011). 2. Structure−Activity Relationship of Amino Acid Tunable Lipidated Norspermidine Conjugates: Disrupting

Biofilms with Potent Activity against Bacterial Persisters, M.M. Konai, Bioconjug. Chem. 26, 2442−2453 (2015).

Synthesis and characterization of Glucosylated Peptides: toward selective plasmapheresis-based treatment of Multiple Sclerosis

A. Mazzoleni1,2, C. Testa2, S. Francesconi2, S. Lavielle1, P. Rovero2, J.-M. Mallet1, A. M. Papini2

1 Laboratoire des Biomolécules, UMR 7203, Département de chimie, École Normale Supérieure, PSL Research University, Sorbonne Universités, UPMC Univ. Paris 06, CNRS, 24 rue Lhomond,

75005 Paris, France. 2 Interdepartmental Laboratory of Peptide and Protein Chemistry and Biology, Dipartimento di

Chimica ‘Ugo Schiff’ and Department NEUROFARBA, University of Florence, Via Della Lastruccia 13 and Via Ugo Schiff 6, 50019 Sesto Fiorentino, Italy.

Multiple Sclerosis (MS) is a neurodegenerative disease presumably involving in some forms an antibody-mediated mechanism in the damage of myelin sheath surrounding the axons in the central nervous system. Since the aetiology is still unknown and the therapies are feeble, isolating the specific autoantibodies is a major goal to understand and treat this complex pathology. Previous studies already assessed that the glucosylation of the H. Influenzae adhesin protein HMW1 on asparagine residues in consensus sequons (N-X-T/S) is necessary for protein secretion and efficient adherence to host cells, hence vital to assure infectivity of the bacterium.1 Recently, the presence of N-Glucosyl epitopes in H. influenzae C-terminal adhesin fragment HMW1ct (1205-1526) was proven to be essential for the identification of the highest affinity antibodies in MS, showing a very specific recognition. Hence, hyperglucosylated HMW1ct is the first example of an N-glucosylated antigen that can be considered a relevant candidate for triggering pathogenic antibodies in MS.2

From a fundamental point of view, finding the minimal epitope recognized by antibodies and understanding the impact of glucosylation on adhesin sequons is a crucial task. To do so, a collection of adhesin-derived sequences was synthesized through solid-phase peptide synthesis and analysed by CD and NMR techniques. These peptides contain differently glucosylated consensus sequences that were able to recognize antibodies in patients’ sera. Moreover, these putative antigens were loaded onto polymeric scaffolds with the aim to obtain multivalent macromolecules ideally able to snare selectively circulating antibodies. Indeed, the development of a tentacle-like, antigen-decorated polymer is a promising stratagem for the isolation and characterization of reactive antibodies found in the sera of patients suffering from autoimmune diseases. For this scope, the achievement of synthetically accessible, peptide-based microarchitectures may provide a great step forward both in the treatment and in the comprehension of MS.

References 1. J. Gross, et al., J. Biol. Chem. (2008) 283, 26010 2. M. T. C. Walvoort, C. Testa et al., Sci. Rep. (2016) 6, 39430

In vitro and in vivo evaluation of D-BMAP18 peptide for the treatment of pulmonary infections in cystic fibrosis

Margherita Degasperi1 Mario Mardirossian1, Marco Scocchi1.

1 Department of Life Sciences, University of Trieste, Via L. Giorgieri 5, 34127 Trieste, Italy.

Patients with cystic fibrosis require pharmacological treatment against chronic lung infections due to antibiotic resistant pathogens. Antimicrobial peptides are among the leading compound for developing new drugs because of the broad spectrum of activity and the slow rate in acquire resistance. In order to obtain a peptide stable in the pulmonary environment we truncated and modified the natural antimicrobial peptide BMAP27, obtaining the all-D peptide D-BMAP18 [1]. It is an α-helical peptide which has a MIC90= 16 μg/ml against Pseudomonas aeruginosa CF-isolates [2] and has a good activity against their biofilm. We tested with success the bactericidal activity of D-BMAP18 toward P. aeruginosa in CF- sputum by co-treatments with DNase and NaCl. We also tested its in vitro and in vivo toxicity observing a non-negligible toxicity when it was intratracheally administrated to mice [3], a side effect that may be linked to this route of administration. Preliminary test on a systemic infection model in Galleria mellonella (wax moth) are in progress to verify this hypothesis. In order to improve its efficacy and to minimize undesired side effects, we are also trying new routes of administration in mice and the design of a modified in situ activable D-BMAP18 prodrug. The final aim is to obtain a safe drug for the treatment of pulmonary infections in cystic fibrosis.

References 1.Pompilio A, Scocchi M, Pomponio S, Guida F, Di Primio A, et al. (2011) Antibacterial and anti-biofilm effects of

cathelicidin peptides against pathogens isolated from cystic fibrosis patients. Peptides 32: 1807-1814. 2.Mardirossian M, Pompilio A, Crocetta V, De Nicola S, Guida F, Degasperi M, Gennaro R, Di Bonaventura G,

Scocchi M. (2016). In vitro and in vivo evaluation of BMAP-derived peptides for the treatment of cystic fibrosis-related pulmonary infections. Amino acids 48(9):2253-2260 .

3.Mardirossian M, Pompilio A, Degasperi M, Runti G, Pacor S, Di Bonaventura G, Scocchi M (2017) D-BMAP18 antimicrobial peptide is active in vitro, resists to pulmonary proteases but loses ots activity in a murine model of P. aeruginosa lung infection

Use of peptide mimetics of proteins to characterise immune response in different pathologies: a powerful approach

Lorenzo Altamore1, Chiara Testa1, Feliciana Real1, Giuseppina Sabatino1, Annunziata Lapolla2, Carlo Selmi3, Fabio Borri1,4, Michael Przybylski4, Joussef Hayek5, Paolo Rovero1, Anna Maria Papini1

1. Interdepartmental Laboratory of Peptide and Protein Chemistry and Biology, Dip. di Chimica Ugo Schiff’ and Dipartimento di Neuroscienze, Area del Farmaco e Salute del Bambino (NEUROFARBA), University of Florence, Sesto Fiorentino, Italy

2. Dipartimento di Scienze Mediche e Chirurgiche, Cattedra di Malattie del Metabolismo, University of Padova, Padova, Italy

3. Division of Internal Medicine, San Paolo School of Medicine, University of Milan, Italy 4. Steinbeis Centre for Biopolymer Analysis and Biomedical Mass Spectrometry, Marktstraße

29, 65428 Rüsselsheim am Main, Germany 5. Child Neuropsychiatry Unit, University Hospital AOUS of Siena, I-53100 Siena, Italy

Peptides are a powerful tool for the identification of antibodies and for the study of chemical and biological mechanisms involved in several disorders. We present the synthesis and the screening of different libraries of peptides related to some specific diseases for in-depth studies of these pathological conditions. In particular: Type 1 Diabetes: our data show a cross-reactivity of T1D and LADA autoantibodies in patient sera against two hGAD-deriving peptides and one Coxsackievirus-deriving peptide1. These results suggest a possible correlation between viral infections and the onset of T1D. Psoriasis: we preliminarily highlighted a specific interaction between autoantibodies in psoriatic arthritis and rheumatoid arthritis patient sera and the endogenous antimicrobial peptide LL372. Fabry disease: through affinity measures we successfully identified a promising alpha-galactosidase epitope involved in the autoimmune mechanism which lead to the Fabry disease3. Rett syndrome: our preliminary data show a correlation between hyperglucosylated adhesin and Rett syndrome, suggesting a plausible involvement of Haemophilus influenzae in its pathogenesis4.

References 1. Kaufman, J. Clin. Invest. (1992), 283-292; 4. Papini, Nat. Sci. Rep. (2016); 2. Lande, Nat. Comm. (2014), 5621; 3. Przybylski, ChemMedChem (2018);

Photocurrent Generation in Supramolecular DNA-Inspired

Nanoarchitectures on Gold Surface: from 2D to 3D Gatto E.a*, Schriever M.b, Cesaroni S.a, Mazzuca C.a, M. Venanzi,a De Zotti M.c

a Department of Chemical Science and Technlogies, University of Rome Tor Vergata, Rome, Italy bTechnical University of Applied Science, Wildau, Germany

cInstitute of Biomolecular Chemistry, Padova Unit, CNR, Department of Chemistry, University of Padova, Padova, Italy

Fabrication of ordered three-dimensional nanostructures on surface is an ongoing challenge in the field

of materials science. In particular, molecular order and perfect positioning of different redox centers

are of fundamental importance in building-up artificial photosynthetic systems, to control the direction

of the electronic flow. Different systems with all subunits covalently linked have been reported in the

literature, but the synthetic effort to obtain these multicomponent molecules is considerable. In Nature

all supramolecular architectures are built up by non-covalent interactions, between them hydrogen

bonds. In particular, DNA molecules are engineered using pair up of nucleobases. In this

communication, we describe preliminary studies on photocurrent generating supramolecular

components, built with an unprecedented approach: we have used the thymine-adenine DNA base

pairs approach to construct supramolecular films in 3D, composed of different 2D layers. To this end,

we have engineered two types of photocurrent generating films on gold surfaces. Film 1 and 2 consists

of multilayered systems where the light absorbing group (ZnTPP chromophore) is noncovalently

linked to a gold surface through thymine-adenine hydrogen bond. These films are assembled by

consecutive deposition of each layer. In film 1, two components are used: adenine linked to a lipoic

acid molecule (Lipo-A) to covalently bind the gold surface, and ZnTPP linked to a thymine molecule

(T-ZnTPP). Film 2 has an additional noncovalently linked layer: an undecapeptide analogue of the

Thricogin GA IV peptide, in which the two extremities were functionalized with thymine and adenine,

for binding of, respectively, Lipo-A and T-ZnTPP. In this work the photocurrent generation properties

of film 1 and 2 are studied by electrochemical and spectroscopic techniques. The importance of

molecular organization for the optimization of the photoconversion efficiency will be emphasized.

References

[1] From supramolecular chemistry towards constitutional dynamic chemistry and adaptive chemistry. Jean-Marie Lehn Chemical Society Reviews, 36, 151-160 (2007).

Structural metal ion recruitment influences folding mechanism and self-association propensity of high homologous proteins

G. D’Abrosca1, G. Malgieri1, L. Pirone2, A. Toto3, L. Russo1, M. F. M. Sciacca4, V. Sivo1, I. Baglivo1, F. Russo1, M. Coletta5, P. V. Pedone1, C. Isernia1, M. De Stefano1, S. Gianni3, E. M. Pedone2, D. Milardi4 and R. Fattorusso1

1 Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, University of Campania “Luigi Vanvitelli”, Via Vivaldi 43, 81100

2 Institute of Biostructures and Bioimaging, CNR, Via Mezzocannone 16, 80134 Naples, Italy 3 Department of Biochemical Sciences “Alessandro Rossi Fanelli”, University of Rome “La

Sapienza”, Piazzale Aldo Moro 5, 00185, Roma, Italy 4 Institute of Biostructures and Bioimaging, CNR, Viale A. Doria 6, 95125 Catania, Italy. 5 Department of Clinical Sciences and Translational Medicine, University of Rome “Tor Vergata”, Via

Montpellier 1, 00133, Roma, Italy

Using three isostructural proteins of the prokaryotic zinc finger family as model systems (Ml452-151 lacking zinc binding and Ml153-149 and Ros87 that bind a structural metal ion), we propose the study of the structural metal ion influence on proteins structure and function, folding mechanism and self-association propensities1,2. The prokaryotic zinc finger domain3 shows a βββαα globular fold that, while including a ββα motif similar to the eukaryotic domain, is stabilized by an extensive hydrophobic core of 15 amino acids and uses different combinations of amino acids to coordinate the structural metal ion when present4. We will discuss how the recruitment of the structural metal can modify the folding pathway of these relatively small domains, control conformational accessibility to aggregation-prone states and change aggregation kinetics. While these model domains have little direct disease-relevance, our findings should be of broad general interest as many disease-relevant proteins bind metal ions, which could similarly influence their structures, folding pathways and aggregation.

References 1. M. Palmieri, G. Malgieri, L. Russo, I. Baglivo, S. Esposito, F. Netti, A. Del Gatto, I. de Paola, L. Zaccaro, P.V.

Pedone, C. Isernia, D. Milardi, R. Fattorusso, J Am Chem Soc, vol. 135, 2013, pp. 5220-5228. 2. G. Malgieri, G. D’Abrosca, L. Pirone, A. Toto, M. Palmieri, L. Russo, M. F. M. Sciacca, R. Tatè, V. Sivo, I.

Baglivo, R. Majewska, M. Coletta, P. V. Pedone, C. Isernia, M. De Stefano, S. Gianni, E. M. Pedone, D. Milardi and R. Fattorusso, Chemical Science, Issue 13, 2018, pp. 3290–3298

3. G. Malgieri, L. Russo, S. Esposito, I. Baglivo, L. Zaccaro, E.M. Pedone, B. Di Blasio, C. Isernia, P.V. Pedone, R. Fattorusso, PNAS, vol. 104, Issue 44, 2007, pp. 17341-17346

4. G. D'Abrosca, L. Russo, M. Palmieri, I. Baglivo, F. Netti, I. de Paola, L. Zaccaro, B. Farina, R. Iacovino, P. V. Pedone, C. Isernia, R. Fattorusso and G. Malgieri, J Inorg Biochem, 2016, 161, 91-98