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Synthetic Peptides Varadero 2015
2st International Symposium on Synthetic Peptides as Pharmaceutical
Products: Autoimmunity, Cancer and Infection Diseases
Scientific Program
April 7th - 10s t 2015
Meliá Marina Hotel, Cuba
Synthetic Peptides Varadero 2015
Synthetic Peptides Varadero 2015
April 7th- 10st 2014
Welcome to Synthetic Peptides Varadero 2015.
The Meeting will cover several topics on Synthetic Peptide Chemistry and their Biological
Application in autoimmunity, cancer, infectious diseases and other disorders. We will
talk about modification of peptides, combinatorial peptide library, peptide structure and
function and macromolecular peptide assemblies, peptide pharmacokinetic studies,
peptide therapeutics and vaccines, peptides in diagnostics and biomaterials, peptide
formulation and delivery, peptide manufacturing and peptides in the clinic.
Synthetic Peptides Varadero 2015
Synthetic Peptides Varadero 2015
Scientific Program: Oral presentations
Tuesday, April 7th
Plenary Lectures
17:00 - 17:45
17:45 - 18:30
Cuban Biotechnology and the Center for Genetic Engineering and
Biotechnology.
Dr. Gerardo Guillen Nieto (Cuba)
Building Collaborative Links between Ireland and Cuba - A Tale of
2 Populations.
Dr. Orla Hardiman (Ireland)
Synthetic Peptides Varadero 2015
Wednesday, April 8th
Scientific Program-Oral session
Chairpersons: Dr. Peter White, Dr. Hilda Garay
08:45 - 09:30
Recent advances in Fmoc solid phase synthesis
Dr. Peter White (UK)
09:30 - 10:15
Protein mimics by molecular scaffolding of cyclic peptides toward
synthetic vaccines and beyond
Prof. Robert Liskamp (UK)
10:15- 10:30
Coffee break
10:30- 11:00
Synthesis of Head-to-Side-Chain Marine Cyclodepsipeptides
Dr. Judit Tulla-Puche (España)
11:00- 11:30
Evaluation of different presentation forms for synthetic peptide vaccines
against Neisseria meningitidis serogroup B
Dr. Hilda Garay (Cuba)
11:30- 12:00
Heberprovac, a GnRH based therapeutic vaccine to treat advanced
prostate cancer
Dr. Jesus Junco (Cuba)
12:00 - 12:30
GHRP-6: The wizardry of a small peptide before worldwide challenging
diseases
Dr. Jorge Berlanga (Cuba)
12:30 - 13:00
Functional characterization of CIGB-552, a novel anticancer peptide
designed from a synthetic ala-library
Dr. Maribel Guerra (Cuba)
Synthetic Peptides Varadero 2015
Thursday, April 9th
Scientific Program-Oral session
Chairpersons: Prof. Robert Liskamp, Dr. Glay Chinea
08:45 - 09:30
Synthesis of Glycoprotein by the ligation method
Dr. Hironobu Hojo (Japan)
09:30 - 10:15
Peptide Self-Assembled Monolayers at Bionic Interfaces
Prof. Shlomo Yitzchaik (Israel)
10:15- 10:30
Coffee break
10:30- 11:00
Identification of a novel target to inhibit Dengue virus infection: use of a
dengue-derived peptide for target validation
Dr. Vivian Huerta (CIGB)
11:00- 11:30
Ration Design of Beta-Hairpin Peptides as Entry Inhibitors of Dengue
Virus
Dr. Glay Chinea (Cuba)
11:30- 12:00
12:00 - 12:30
CIGB-300: A peptide-based drug with perspectives for cancer targeted
therapy
Dr. Silvio Perea (Cuba)
One-bead-One-Compound (OBOC) Combinatorial Peptides Libraries:
design, synthesis and application in the biomedicine.
Dr. Yordanka Masforrol (Cuba)
12:30 - 13:00
Identification and optimization of an Interleukin-15 antagonist peptide
Dr. Yunier Rodríguez (Cuba)
Synthetic Peptides Varadero 2015
Friday, April 10st
Scientific Program-Oral session
Chairpersons:
Dr. Hironobu Hojo, Dr. Luis Javier González
08:45 - 09:15
Quantitation of therapeutic peptide in human plasma by using mass
spectrometry: application to pharmacokinetic studies in clinical trials
Dr. Ania Cabrales (Cuba)
09:15 - 09:45
CIGB-814, a peptide as immunomodulator for the treatment of
arthritis reumatoide
Dr. Noraylis Lorenzo (Cuba)
09:45 - 10:15
CIGB-210: A novel approach to treat HIV infection
Dr. Celia Fernández (Cuba)
10:15 - 10:30
Coffee break
10:30 - 10:50
Peptide formulation strategy for the development of therapeutics.
Dr. Hector Santana (Cuba)
10:50- 11:10
QbD: ¿Reality or Utopia?
Dr. David Diago (Cuba)
Synthetic Peptides Varadero 2015
Poster presentations
Author
Brizaida Oliva
Idania Baladrón
Marlon Cáceres
Yunier Rodríguez
Yanet Támbara
Ania Cabrales
Ayni Rodríguez
Luis J. González
Ania Cabrales
Ever Pérez
Country
Title
Antitumor efficacy, pharmacokinetic and biodistribution
Cuba
studies of the new anticancer peptide cigb-552 in mouse
models
Dose study , safety and efficacy of Heberprovac
Cuba
indications in the treatment of patients with advanced
prostate adenocarcinoma
Antibacterial effect of encapsulated new synthetic
Colombia Peptides against methicillin resistant staphylococcus
Aureus (mrsa)
Evaluation of new variants of CIGB55 in two InterleukinCuba
15-dependent cell lines
Peptide content and Amino acid composition
Cuba
determinations by the EZ:faast amino acid analysis
procedure
Bio-analytical method based on MALDI-MS analysis for
Cuba
the CIGB-300 anti-tumor peptide quantification of in
human plasma
Biochemical characterization and in vitro cytotoxic effect
Cuba
of a polipeptide and protein mix obtained from the
Rophalurus junceus venom
Sequencing by mass spectrometry of the postraslationally
Cuba
modified peptides isolated from Conus princeps
Bio-analytical method development and validation for
Cuba
measurement of ghrp-6 in human plasma. Application to
phase i clinical trial in nine healthy volunteers
Elimination and exchange of trifluoacetate counter-ion in
Cuba
a cationic peptide: improvements in the process
Ania Cabrales
Cuba
Metabolic stability study of antitumor peptide CIGB-552
in Balb/c mice serum
Anna Ramírez
Cuba
Preclinical safety evidences of a novel therapeutic antiHIV peptide
Hilda Garay
Cuba
Noraylis Lorenzo
Cuba
Solid phase synthesis of c-terminal pegylated peptides for
therapeutic applications
Therapeutic effect of an altered peptide ligand derived
from heat-shock protein 60 by suppressing of
inflammatory cytokines secretion in two animal models
of rheumatoid arthritis
Synthetic Peptides Varadero 2015
Alba López
Nanoencapsulation of antimicrobial peptide LA6 with
antibacterial activity against methicillin-resistant
Colombia
Staphylococcus aureus (MRSA) using the methodology of
double emulsion - solvent diffusion
Brayann Martinez
Colombia
Arielis Rodríguez
Cuba
Fidel Morales
Cuba
Synthesis of Chitosan@Peptide nanoparticles with
antimicrobial activity against Escherichia coli O157:H7
P8 peptide - interacting proteins in synovial fluid from
rheumatoid arthritis patients
Solid-phase Synthesis of N-Substituted and TetrazoloLipopeptides and Peptidosteroids by means of
Aminocatalysis-Mediated Ugi Reactions
Synthetic Peptides Varadero 2015
Abstracts
Oral Presentations
Synthetic Peptides Varadero 2015
Synthetic Peptides Varadero 2015
Cuban Biotechnology and the Center for Genetic Engineering and
Biotechnology
Author: Gerardo Guillen Nieto
Center for Genetic Engineering and Biotechnology. La Habana, Cuba.
[email protected]
An extensive product pipeline has been built by the Cuban Biotechnology
Institutions and by the CIGB. Growing under particular environment with
limited resources, the biotechnology is tightly connected with the health
system and is driven by a national collaboration instead of competition. The
urgency for immediate application of scientific results reinforced the need
for expanding collaboration across the sector. Research generated by the
CIGB has developed a number of products with significant impact to
society. An overview of the achievements including the R&D projects with a
strong intellectual property position and the project pipeline comprising
several hepatitis related projects will be introduced to the participants.
Building Collaborative Links between Ireland and Cuba - A Tale of 2
Populations.
Orla Hardiman BSc MB BCh BAO MD FRCPI Academic Director, Trinity
Biomedical Sciences Institute,
Professor of Neurology, Trinity College Dublin, Ireland.
[email protected]
Synthetic Peptides Varadero 2015
Wednesday, April 8th
Recent advances in Fmoc solid phase synthesis
Author: Peter White
Product Manager Novabiochem, Merck Millipore, Merck Chemicals, UK
[email protected]
In the last two decades, the Fmoc/tBu approach has displaced Boc/Bzl as the
method of choice for the solid phase synthesis of peptides, with the vast
majority of peptides made today being synthesised by Fmoc SPPS.
However, despite its popularity, a number of difficulties and challenges still
remain. In my talk I look at these remaining issues and consider some recent
developments from our and others laboratories aimed at solving those
problems. The focus will be on advances in Asp, Cys and His protecting
group chemistry, backbone protection for enhanced solubility of peptides,
and new approaches to convergent synthesis using protected and
unprotected peptides
Synthetic Peptides Varadero 2015
Wednesday, April 8th
Protein mimics by molecular scaffolding of cyclic peptides
toward synthetic vaccines and beyond
Author: Prof. Rob Liskamp
University of Glasgow, UK
[email protected]
In our first generation of protein mimics the discontinuous epitopes on a
scaffold were incorporated as linear peptides. However, accessible epitopes,
mostly present on the surface of a protein, are often loops. Therefore, for the
next generation of protein mimics cyclic peptides are required as adequate
mimics of peptide loops present in proteins. Cyclization of peptides is
believed to lead to increased potency, metabolic stability, bioavailability,
and receptor selectivity. Considerable effort has been spent on efficient
syntheses of cyclic peptides, which were or could be attached to the TAC or
other scaffolds. Thus we have synthesized side chain-to-tail cyclic peptides
containing a thioester handle for native chemical ligation, head-to-tail
cyclized peptides containing an azido functionality for Cu(I)-catalyzed
azide–alkyne cycloaddition (CuAAC), side chain-to-side chain cyclized
peptides also containing an azido functionality. We have found that the
cyclic peptides containing a thioester handle and the side chain-to-side chain
azide functionality containing cyclic peptides are most suitable for the
preparation of protein mimics libraries and selected individual protein
mimics.
Synthetic Peptides Varadero 2015
Wednesday, April 8th
Synthesis of Head-to-Side-Chain Marine Cyclodepsipeptides
Author: Judit Tulla-Puchea,b Marta Pelay-Gimeno, Yésica García-Ramos,
and Fernando Albericioa,b,c
a
Institute for Reseach in Biomedicine Barcelona (IRB Barcelona),
Barcelona, Spain
b
CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and
Nanomedicine,
Barcelona, Spain
c
Department of Organic Chemistry, University of Barcelona, Barcelona,
Spain
[email protected]
Pipecolidepsin and Stellatolide A belong to the family of head-to-side-chain
cyclodepsipeptides. They present a unique structural arrangement: a
macrocyclic region closed through an ester bond between the C-terminus
and a β-hydroxyl group, terminated with a polyketide moiety. This complex
structure makes this their synthesis a real challenge.
Pipecolidepsin A bears the unprecedented and extraordinary bulky
AHDMHA residue at the branching point, which makes the construction of
the extremely hindered ester bond the major synthetic challenge to
overcome. On the other hand, Stellatolide A is one of the few complex βOMe-Tyr-containing peptides whose synthesis has been attempted. These
kinds of peptide are not stable in moderate concentrations of acid, thus
leading to the elimination of MeOH from the β-MeOTyr residue to give
didehydrotyrosine. In addition, when attempting the synthesis of complex
depsipeptides, careful attention needs to be paid to transacylation, nitrile
formation from Asn/Gln residues, and both pyro-diMeGln and
diketopiperazine (DKP) formation.
The successful solid-phase synthetic strategies that resulted in the two
synthetic and active cyclodepsipeptides will be discussed.
Synthetic Peptides Varadero 2015
Wednesday, April 8th
Evaluation of different presentation forms for synthetic peptide
vaccines against Neisseria meningitidis serogroup B
Author: Hilda E. Garay
Center for Genetic Engineering and Biotechnology, Havana, Cuba
[email protected]
Recent advances in the field of peptide synthesis have aroused renewed
interest in the use of peptides for therapeutic or prophylactic vaccines.
Synthetic peptides exhibit significant advantages as vaccine immunogens,
being easily manufactured, chemically defined molecules with an excellent
safety record that induce highly specific immune responses and have
undemanding storage requirements. Synthetic peptides are usually poorly
immunogenic, but this problem can be overcome using adequate
presentation systems, including (i) branched peptide structures such as
multiple antigen peptides (MAPs), (ii) peptides conjugated to a carrier
protein and (iii) polymerized peptides. MAPs are in general more
immunogenic than monomeric peptides or peptide-carrier protein
conjugates. In addition, although MAPs are immunogenic by themselves,
they can be conjugated to protein carriers to induce even higher antibody
levels. In the present work we studied the immunogenicity of several
presentation forms for two peptide-based vaccine candidates against
Neisseria meningitidis serogroup B (MenB). The first candidate, a peptide
mimetic of the MenB capsular polysaccharide, was synthesized either as a
monomer or as MAPs, which were then conjugated to the P64k carrier
protein by means of different conjugation methods. A separate, nonconjugated MAP was also synthesized, containing the mimetic peptide and a
T-cell epitope derived from tetanus toxoid. The second vaccine candidate
was actually a series of peptides derived from surface loop 4 of the class 1
outer membrane protein of MenB, consisting of the loop synthesized as a
single peptide, as a cyclic peptide, as a polymerized-cyclic peptide, or as
MAPs containing different copies of the B-cell epitope of loop 4 and a T-cell
epitope derived either from tetanus toxoid or the P64k carrier protein.
Finally, all peptide presentation forms were evaluated in terms of their
immunogenicity in mice.
Synthetic Peptides Varadero 2015
Wednesday, April 8th
Heberprovac, a GnRH based therapeutic vaccine to treat advanced
prostate cancer
Authors: Junco Ja Fuentes Fa, Rodríguez R, Calzada La, Baladrón Ib,
Pimentel Ea, Basulto Ra, Reyes Ob, Garay Hb and Guillén Gb.
a
Centro de Ingeniería Genética y Biotecnología Camagüey. Carretera
Circunvalación Norte Apdo Postal 387 Camaguey, Cuba
b
Centro de Ingeniería Genética y Biotecnología de la Habana. Ave 31 entre
158 y 190 Cubanacan Playa. Apdo Postal 6072. La Habana, Cuba.
[email protected]
GnRH-based vaccines represent a promising anti-hormonal treatment
alternative in prostate cancer, because they can reduce serum testosterone to
castrating levels, avoid the "hot flushes” produced by GnRH analogues and
can be administered in acute and complicated forms of prostate cancer. The
present study assesses the application of Heberprovac, a GnRH based
vaccine candidate for patients suffering from advanced prostate cancer.
The main objective of the trial was to evaluate the safety and efficacy of this
vaccine candidate in 4 levels of dosage.
To this aim, 56 patients affected by advanced prostate cancer diagnosed by
biopsy, were included. As result, since the first immunizations, the patients
exhibited anti GnRH antibodies and in turn, testosterone levels reduction. In
concordance with the hormonal and immunological response the patients
exhibit a decrease of both; the number of obstructive symptoms as well as
the severity of them. There was also a normalization of the prostatic specific
antigen (PSA) in the 80% of the patients after they finished the last
immunization and the clinical evaluation demonstrated the significant
reduction of the primary tumor from grades III/IV to I/II in all the patients
that respond biochemically.
Regarding the safety, more adverse effects of the vaccine were those
expected according to the mechanism of action of the vaccine. No severe
adverse events were found in any of the patients. Majority of them were
related with the current vaccination and only the 4.4% of the adverse
effects were grade III. As conclusion the vaccine proved to be safe and
effective in the 4 dose levels.
Synthetic Peptides Varadero 2015
Wednesday, April 8th
GHRP-6: The wizardry of a small peptide before worldwide
challenging diseases
Authors: Dr. Jorge Berlanga Acosta
[email protected]
GHRP-6 is a synthetic, GH-secretagogue hexapeptide with a substantial
safety profile. Experiments conducted by our group have shown that a
single pre-conditioning or multiple GHRP-6 administrations were able to
amplify cellular cyto-protective mechanisms during hepatic and cardiac
ischemia / reperfusion episodes in which surgically-relevant times were
used. In the rats' hepatic ischemia study, CIGB500 largely prevented
hepatocytes death and transformed the placebo sub-massive necrotic pattern
into a hepatocytes' individual one – all likely due to attenuation of proinflammatory mediators release and by enhancing cellular anti-ROS
defenses. In a porcine model of acute transmural myocardial infarction,
necrosis was reduced by more than 70% as compared to placebo-treated
pigs. Further studies using the doxorubicin (DX) induced dilated
myocardiopathy rat model proved that GHRP-6 prevents heart failure and
other toxic systemic complications when concomitantly administered to
(DX). Independent studies demonstrated that the peptide could restore
myocardial damages and failure when therapeutically administered. In both
models CIGB 500 significantly reduced lethality. We have also shown that
GHRP-6 is endowed with anti-fibrotic capabilities as demonstrated in
animal models of of liver cirrhosis and skin hypertrophic scars. Different set
of genes related with cell survival and tissue repair appear to be controlled
by GHRP-6 irrespective to the target tissue. We envision a broad
pharmacological usefulness for the agent.
Synthetic Peptides Varadero 2015
Wednesday, April 8th
Functional characterization of cigb-552, a novel anticancer peptide
designed from a synthetic ala-library
Authors: Maribel G Vallespí2, Brizaida Oliva Arguellez2, Julio R Fernández
Massó1, Yolanda Gomez2, Soledad Astrada4, Mariela Bollati-Fogolín4,
Hilda Garay3, and Osvaldo Reyes3
1
Department of Genomic; 2Pharmaceutical Department, 3Synthetic Peptide
Group, Chemistry-Physics Department, Center for Genetic Engineering and
Biotechnology, P.O.Box 6162 Habana 10600, Cuba. 5Cell Biology Unit,
Institute Pasteur of Montevideo, Mataojo 2020 11400 Montevideo,
Uruguay.
[email protected]
Abstract
Novel therapeutic peptides are increasingly making their way into clinical
application. The cationic and amphipathic properties of antimicrobial
peptides allow them to cross biological membranes in a non-disruptive way
without apparent toxicity increasing drug bioavailability. The antimicrobial
peptides (AMP) which are toxic to bacteria but not to the mammalian cells
are potential sources of new antitumor agents.
Objectives:
Studies on the structure-function relationship of the
antimicrobial peptide LALF32-51 have identified which amino acids are
associated with the binding capacity to LPS and which amino acids are
essential for the cytotoxic activity. Taking the abovementioned into account,
a new anticancer peptide (CIGB-552), was designed, which is a derivative of
an anti-LPS peptide.
Methods: We have done a synthetic ALA-library from de 32-51 region of
the Limulus anti-LPS factor protein and obtained a novel synthetic peptide
(CIGB-552) that has lost its LPS binding capacity and is a powerful in vitro
and in vivo cytotoxic agent.
Results: We documented that analogous peptides from non-cyclic region
32–51 of the LALF possess the ability to inhibit tumor cell proliferation by a
mechanism that does not imply to bind LPS. Further, we have demonstrated
that CIGB-552-mediated stabilization of COMMD1 promotes RelA(p65)
ubiquitination and subsequent repression of the NF-κB activity, an event
linked to the apoptosis of the cancer cells.
Conclusion: Our study is the first in describing the antitumor effect induced
by systemic administration of a peptide that targets COMMD1 for
stabilization. Moreover, our data reinforce the perspectives of CIGB-552 for
cancer targeted therapy.
Synthetic Peptides Varadero 2015
Thursday, April 9st
Synthesis of Glycoprotein by the ligation method
Author: Hironobu Hojo
Institute for Protein Research, Osaka University, Suita, Osaka 565-0871,
Japan
[email protected]
Glycosylation is a common post-translational modification of proteins.
These glycan chains are engaged in various biological processes, such as cell
attachment, cell-cell interaction and viral infection. However, the detail of
their function is still in vague in most cases, which is mainly due to the
difficulty in obtaining homogeneous glycoprotein from natural sources. As
the glycan chain is constructed by various glycosyl transferases, its structure
is highly heterogeneous, making glycoprotein as highly heterogeneous
mixture called “glycoform”. In addition, E-coli expression system, useful
for protein production, can not express glycoprotein. These problems result
in the lack of pure glycoprotein, essential for functional and structural
analyses. To overcome these problems, we have been developing a facile
method for glycoprotein synthesis, which can yield homogeneous
glycoproteins, based on the thioester method [1]. The thioester method uses
the direct aminolysis of the N- and S-protected peptide alkyl thioester by the
other segment using silver ions as the activator. We recently found that the
reaction also proceeds in the absence of silver ions, when more reactive aryl
thioesters are used. Using these activation methods, we developed the onepot ligation method composed of three segments as shown in the Scheme
shown below [2]. In this presentation, I will talk about the usefulness of the
one-pot method by the chemoenzymatic synthesis of the extracellular
immunoglobulin domain of TIM-3 carrying N-linked complex type glycan
as well as the chemical synthesis of human interleukin 2, carrying core 1 Olinked carbohydrate.
Scheme One-pot synthesis by the thioester method.
References
1. Hojo, H., Aimoto, S. (1991) Bull. Chem. Soc. Jpn., 64, 111-117.
2. Asahina, Y., Kamitori, S., Takao, T., Nishi, N., and Hojo, H. (2013) Ang. Chem.
Int. Ed., 52, 9733-9787.
Synthetic Peptides Varadero 2015
Thursday, April 9st
Peptide Self-Assembled Monolayers at Bionic Interfaces
Author: Shlomo Yitzchaik
Institute of Chemistry and The Nanoscience and Nanotechnology Center,
The Hebrew University of Jerusalem, Israel
[email protected]
Figure 1. Enzyme gated ion mobility through peptide monolayer: AFM topography and EIS
In this contribution we describe the effectiveness of peptide monolayers as
bioelectronic interfaces in neuroelectronic hybrids and medical diagnosis.
Signal transduction schemes covering bio-cues mediated field potentials
recording and small molecules sensing via molecular dipoles modulated
surface potentials will be appraised. A new sensing strategy, chargetransport gating through peptide monolayers, will be described
systematically for monitoring cellular signaling pathways. Kinasemediated phosphorylation plays a major role in regulating signaling
pathways in cells. Abnormal phosphorylation has been reported to facilitate
cancer development. Here we refer to selectivity data in the kinasepromoted phosphorylation of peptide substrates, using the kinases PKA,
PKC, and CaMK2. Peptide substrates for each kinase were investigated
using electrochemical impedance spectroscopy (EIS), cyclic voltammetry
and square wave voltammetry (SWV) methods. EIS based sensor shows a
remarkably high sensitivity and sequence specificity. These studies
suggests that the enhanced activities and kinase-selectivity are related to the
monolayer's packing motifs. Nanoscopic studies demonstrate a distinct
disordering of the monolayer following phosphorylation, which can explain
the high sensitivity for transducing the biocatalytic event into a measurable
impedimetric signal. An enhanced sensitivity is demonstrated with the
SWV amperometric sensor that allows screening of new kinase-inhibitors as
anticancer drug candidates.
Synthetic Peptides Varadero 2015
Thursday, April 9st
Identification of a novel target to inhibit Dengue virus infection: use
of a dengue-derived peptide for target validation
Authors: Vivian Huerta1*, Yassel Ramos1, Patricia Toledo1, Alexis Yero1,
Dianne Pupo1, Sebastien Gallien2, Alejandro Martín1, Noralvis Fleitas1,
Gabriel Márquez1, Mónica Sarría1, Osmany Guirola1, Luis J. González1,
Bruno Domon2, Osvaldo Reyes1, Hilda Garay1, Ania cabrales1 and Glay
Chinea1.
1
Center for Genetic Engineering and Biotechnology. Apdo 6162. Habana
10600, Cuba.
2
Luxembourg Clinical Proteomics center (LCP), Centre de Recherche
Public de la Santé, 1 B rue Thomas Edison, L-1445 Strassen,
Luxembourg.
[email protected]
The four serotypes of dengue virus (DENV1-4) are able to infect a large
variety of cell lines of human and non-human origin. However, while it has
been well demonstrated that DENV enters these cells by receptor-mediated
endocytosis, the identity of the endocytic receptor implicated in this step has
remained elusive. The identification of cell surface molecules that mediate
virus entry to target cells is an importance piece of information since such
knowledge can greatly influence the development of effective strategies to
neutralize infection making the receptor a target for drug development.
Because a wealth of data indicates that domain III (DIII) of the envelope
protein of DENV is pivotal for the interaction of the virus with cell surface
molecules, we used affinity chromatography with a recombinant
preparation of DIII of DENV2 as a ligand in order to identify proteins
interacting in vivo with the virus. Structural and functional relation among
the identified proteins suggested a potential role for the low density
lipoprotein receptor-related protein (LRP1) as endocytic receptor for DENV
in mammalian cells. Here we present experimental evidences on the direct
interaction of LRP1 with DENV. Based on the structure of DIII, we designed
and synthesized a number of structurally constrained peptides which display
antiviral activity in vitro with IC50 in the micromolar range of concentration.
The inhibitory peptides bind to a recombinant LRP1 fragment and the
antiviral activity correlates with the binding strength. Our results contribute
to a better understanding of the interactions with host factors that establish
DENV during infection to humans and give foundation for further
development of more potent inhibitory peptides to control DENV infection.
Synthetic Peptides Varadero 2015
Thursday, April 9st
Ration Design of Beta-Hairpin Peptides as Entry Inhibitors of Dengue
Virus
Authors: Glay Chinea, Vivian Huerta, Noralvis Fleitas, Patricia Toledo,
Alejandro Martín, Viviana Falcón, Dianne Pupo, Yamilé Vidal, Osmany
Guirola, Mónica Sarría, Osvaldo Reyes, Hilda Garay, Yassel Ramos and
Luis Javier González
Centre for Genetic Engineering and Biotechnology, Havana, Cuba
[email protected]
Beta-Hairpins (BH) are common structural motifs of proteins often involved
in protein-protein interactions. Here we show the design of structurally
constrained synthetic peptides based on the BH “FG” from domain III (DIII)
of the envelope protein of Dengue virus (DV), which lead to the ultimate
development of peptides showing potent antiviral activity. DIII is involved
in key virus/host-cell interactions and it is target of potent anti-DV
neutralizing antibodies (Abs). Proteomics analysis indicates that DIII
interact with serum proteins and putative membrane receptors.
First we demonstrate that designed BH peptides mimic efficiently a
functional surface patch of DIII; peptides bind neutralizing antibodies and
DIII binding host-proteins. Moreover, BH peptides inhibit viral infection
but the dominant epitope recognized by type specific neutralizing mAbs and
the structural determinants for antiviral activity seems to be topologically
distinct. We further explore the impact of several structural properties of
designed BH peptides on its biological activity, including BH sequence
space, structural propensity of amino acids, secondary structure content,
topology, loop length, turn type, linearity or cyclization, supramolecular
structure, etc. BH peptide variants exhibiting high potency and avidity for
receptor were designed by a combined approach regarded to optimization of
putative receptor binding surface and folding of peptides. We also show that
antiviral activity of peptides correlates with putative receptor binding
properties.
Synthetic Peptides Varadero 2015
Thursday, April 9st
CIGB-300: A peptide-based drug with perspectives for cancer targeted
therapy
Authors: Silvio E. Perea, Idania Baladrón, Yanelda Garcia, Yasser Perera,
Hernán Farina, Fernando R Benavent, Leila R Martins, Joao T Barata, ,
Carmen Valenzuela, Pedro López Saura, Hilda Garay, Osvaldo Reyes,
Margarita Solares, Lidia Gonzalez, Idrian Garcia, Yanelda Garcia, Jorge L
Soriano, Noyde Batista.
Center for Genetic Engineering and Biotechnology, Havana 10600, Cuba.
“Hermanos Ameijeiras Hospital, Havana, Cuba. Gynecobstetric Hospital,
Havana, Cuba. ELEA Laboratories, Buenos Aires. Argentina. National
University of Quilmes, Buenos Aires. Argentina. Institute of Molecular
Medicine, Lisbon. Portugal
[email protected]
CIGB-300 is a first-in-class and hypothesis-driven peptide initially
discovered by screening a peptide phage display library using a CK2
phosphoaceptor site. Previous in vivo findings in solid tumor cell lines
indicated that CIGB-300 preferentially binds to B23/NPM protein and
inhibits its CK2-mediated phosphorylation leading to apoptosis.
Interestingly, in blood cancer cells like Chronic Lymphocytic Leukemia
with higher sensitivity toward CIGB-300, it prevented the phosphorylation
of the direct CK2 target residue Ser129 on Akt/PKB and also downregulated
Ser380 PTEN phosphorylation. Accordingly, phosphorylation of PI3K
downstream targets Akt/PKB, PKCδ and GSK-3β decreased in a dosedependent manner after CIGB-300 treatment. In line with this, CIGB-300
reduced the tumor growth in a CLL animal model. Importantly, CIGB-300
has also showed both antiangiogenic and antimetastasic effect in animal
models in separated experimental settings. This peptide is being explored as
a therapeutic candidate and different Phase 1 clinical studies have already
shown safety, tolerability and pharmacokinetic of CIGB-300 using either
local or systemic administration. Importantly, an intravenous doseescalation trial of CIGB-300 in patients with Acute Leukemia is currently in
progress. Recently, in a dose-finding Phase 2 study with 15-70 mg of CIGB300 concomitant to chemoradiotherapy in cervical cancer, the combining
setting exhibited higher antitumor response than chemoradiotherapy alone.
These findings provide perspectives for CIGB-300 as potential adjuvant
therapy to chemoradiotherapy in cervical cancer and useful clues for the
next Phase 3 clinical trial. Collectively, our data outline important new
clinical and non-clinical insights that reinforce CIGB-300 a novel peptidebased candidate with perspectives to treat solid and blood cancer.
Synthetic Peptides Varadero 2015
Thursday, April 9st
One-bead-one-compound (oboc) combinatorial peptides libraries:
design, synthesis and application in the biomedicine.
Authors: Yordanka Masforrol1, Jeovanis Gil2, Luis J. González2, Yasset
Pérez-Riverol2, Jorge Fernández-de-Cossío3, Aniel Sánchez2, Lázaro
Betancourt2, Hilda E. Garay1, Ania Cabrales1, Fernando Albericio5,
Hongquian Yang6, Roman Zubarev6, Vladimir Besada2, Yasser Perera4,
Evelin Caballero4, Silvio Perea4 and Osvaldo Reyes1.
Affiliations: Division of 1Physical-Chemistry, 2Biology of Systems,
3
Informatics, 4Pharmaceutic, Center for Genetic Engineering and
Biotechnology. PO Box 6162. Havana. CUBA. 5University of Yachay,
Ecuador. 6Division of Molecular Biometry, Department of Medical
Biochemistry and Biophysics, Karolinska Institutet, Stockholm, SWEDEN.
[email protected]
Abstract:
Combinatorial chemistry holds the promise of minimizing what constitutes
the longest stage during the development of a new drug: its initial discovery.
There are several advantages to the use of peptides in preparing
combinatorial libraries for drug discovery, including their exquisite
selectivity when used as drugs, and the fact that solid-phase peptide
synthesis is a robust, well-known technology. At CIGB we designed,
obtained and used a combinatorial synthetic hexapeptide library “one-beadone-compound” (Masforrol Y. et al.; ACS Comb Sci., 2012; 14(3):145-9).
The library containing sixteen aminoacids (different sequences) was
synthesized on a Tentagel resin previously modified with a dipeptide linker
(Asp-Pro). This peptide bond is highly susceptible to cleavage under mild
acidic conditions in a salt-free solution prepared with H216O/H218O (60/40
%v/v). In the hydrolysis hexapeptides are released with an additional Asp
residue partially labeled with 18O at the C-terminus. The hydrolysis
conditions release enough peptide for characterization by MS and
complementary analyses such as stepwise Edman degradation. Several
different projects at CIGB have used the OBOC library to screen for
potentially therapeutic peptides. Currently, three peptides that tested
positive in biological assays on the NCI-H125 cell line have already
advanced to the stage of sequence optimization.
Synthetic Peptides Varadero 2015
Thursday, April 9st
Identification and optimization of an Interleukin-15 antagonist
peptide
Author: Yunier Rodríguez
[email protected]
CIGB55 is a 10 aminoacids synthetic peptide with IL-15 antagonist activity.
This peptide binds to IL-15Ra and blocks IL-15 binding in ELISA assays in
a dose dependent manner. Likewise, we have observed that CIGB55 inhibits
IL-15-induced proliferation of CTLL-2 and KiT225 cell lines, thus
demonstrating its antagonist capacity. Ala scanning of the peptide was used
to study contribution of each amino acid to the antagonist effect. Here, we
found that Phe and Cys are important for peptide binding to IL-15R. We also
investigated other single site mutations and dimer formation of this peptide.
The resulting peptide [K6T]CIGB55 exhibited a higher activity than
CIGB55 peptide and dimer formed by S:S bridge is active and more stable
than monomeric form. CIGB55 inhibited IL-6 induced by IL-15Ralpha-IL15 membrane binding and showed inhibitory activity in both a skin allograft
and in Rheumatoid arthritis models.
Synthetic Peptides Varadero 2015
Friday, April 10th
Quantitation of therapeutic peptide in human plasma by using mass
spectrometry: application to pharmacokinetic studies in clinical trials
Author: Ania Cabrales-Rico
[email protected]
One of the major driving forces in the development of mass spectrometry is
its usefulness in the discovery and development of drugs in the
pharmaceutical industry. The increasing demand for ADME-Tox studies at
earlier stages in the drug discovery process has been fulfilled with the stateof-the-art mass spectrometers due to their impressive capabilities in terms of
sensitivity, resolving power, mass accuracy and different scan-modes
versatility. However, measurement of therapeutic peptides presents
particular problems for quantitative mass spectrometry from selectivity and
sensitivity perspectives. The availability of internal standards, strategies for
their selection/synthesis and a proper method of detection are also key
elements for a successful outcome. Peptides are complex molecules with a
wide diversity of physicochemical properties, so the development of
''generic'' methodologies will not be as implemented as easy as it has been
the case of small organic molecules. In consequence, all strategies involving
the peptide quantitation in biological fluids are still a challenge and need to
be tailored. We present here the development and validation of two
customized methods applied to pharmacokinetic studies included in phase I
clinical trials, for the quantitation in plasma of two therapeutics peptides.
CIGB-500 (GHRP-6) is a peptide with a potent cytoprotective effect and
CIGB-300 is an antitumor cell penetrating peptide that impairs CK2mediated phosphorylation and induces apoptosis in tumor cells. For the
quantitation of both peptides, alternatives of AQUA® methodology were
used, but the design of internal standards, as well as the sample processing
and mass spectrometry techniques were optimized.
Synthetic Peptides Varadero 2015
Friday, April 10th
CIGB-814, a peptide as immunomodulator for the treatment of
arthritis reumatoide
Authors: Lorenzo N1, Ancizar JA1, Torres AM2, Hernandez MV2,
Hernandez I2, Gil R2, Altruda F3, Silengo L3, Padrón GR1 and Domínguez
MC1.
1
Centro de Ingeniería Genética y Biotecnología, La Habana, Cuba. 2
Servicio Nacional de Reumatología, La Habana, Cuba. 3 Universidad de
Turín, Italia.
[email protected]
Induction of immune tolerance as therapeutic approach for autoimmune
diseases constitutes a current research focal point. In this sense, we aimed to
evaluate an Altered Peptides Ligand (APL) for induction of peripheral
tolerance in patients with Rheumatoid Arthritis (RA). A novel T cell epitope
from human heat-shock protein 60 (Hsp60), an autoantigen involved in the
pathogenesis of RA, was identified by bioinformatics tools and an APL was
design from this epitope (CIGB-814). Firstly, we investigated the ability of
this peptide for inducing regulatory T cells (Treg cells) in mice. CIGB-814
induced an increase of the proportions of Treg cells in the draining lymph
nodes of the injected site in mice. On other hand, this peptide increases the
proportions of the CD4+CD25highFoxP3+ Treg cells in ex vivo assays using
PBMC isolated from RA patients. In addition, we evaluated the therapeutic
effect of this APL in two animal models: adjuvant induced arthritis (AA) in
Lewis rat and collagen induced arthritis (CIA) in DBA/1 mice. Our
approach was compared to metotrexate (MTX), the gold standard treatment
for RA, in CIA model. Clinical score, Treg, TNFα levels and histopathology
were monitored. CIGB-814 efficiently inhibited the course of AA and CIA,
with significant reduction of the clinical and histopathogy score. The
therapeutic effect induced by CIGB-814 is mediated by an increase of the
proportions of Treg cells and a decrease of TNFα levels. These results
indicate a therapeutic potentiality of this peptide and support further
investigation of this candidate drugs for treatment of RA. Interference of the
pathogenic T cell function in a specific manner using an APL derived from
an autoantigen that can induce tolerance mediated by activation of Tregs as
shows here, represents an attractive therapeutic approach for autoimmune
diseases.
Synthetic Peptides Varadero 2015
Friday, April 10th
CIGB-210: a novel approach to treat hiv infection
Authors: Celia Fernandez-Ortega1, Anna Ramirez1, Dionne Casillas1,
Taimi Paneque1, Marta Dubed2, Leonor Navea2, Eladio Silva2, Lila
Castellanos1, Carlos Duarte1, Viviana Falcon1, Ramos Yasser1, Alejandro
Fuentes1, Osvaldo Reyes1, Hilda Garay1, Raimundo Ubieta1
1
Center for Genetic Engineering and Biotechnology, Ave 31 /158 y 190,
Cubanacan, Playa, PO Box: 6162, Havana 10600, CUBA
2
Laboratory for AIDS Research, Carretera de Tapaste y Autopista Nacional,
San José de las Lajas, CP 32700, Mayabeque, CUBA
[email protected]
Objective: New biological approaches have been proposed in order to battle
HIV infection. One of them is the use of cellular proteins as therapeutic
target. In this work we expose the validation of a cytoskeleton protein as
therapeutic target against HIV infection and a new peptide targeting that
protein with notable anti-HIV activity.
Methods: Comparative proteomic of a leukocyte extract was used to
identify proteins potentially involved in HIV infection in MT4 cell line.
Gene silencing using RNA interference was assayed to elucidate the effect
of the protein on the HIV replicative cycle. Infection assays with replication
competent HIV-1 or pseudotyped virus were performed. Fluorescence and
transmission electron microscopy were used too.
Results: A cytoskeleton protein was identified as a potential therapeutic
target for HIV infection. Stably silenced cells were challenged with a
lentiviral vector based on HIV-1 gene and carrying eGFP, or with a
competent replication HIV-1. Strong reduction in eGFP expression and in
CAp24 HIV-1 viral protein was observed in knockdown cells. A synthetic
peptide designated as CIGB-210 showed a remarkable anti-HIV activity
showing a half maximal inhibitory dose in nanomolar range and a low
cytotoxicity, resulting in a selectivity index greater than 150000. Peptide
acts on supramolecular structure of the protein as was shown by
transmission electron microscopy.
Conclusions: A cytoskeleton protein can be a suitable target to inhibit
HIV-1. CIGB-210 exhibited low cytotoxicity and high inhibitory activity
on HIV-1 replication in a dose-dependent manner indicating that CIGB210 may be a proper drug candidate against HIV-1 infection.
Synthetic Peptides Varadero 2015
Friday, April 10th
Peptide formulation strategy for the development of therapeutics
Authors: Héctor Santana1, Yasser Zárate1, Yaima González1, Yairet García2,
Maribel Guerra3.
1
Department of Pharmaceutical Technology, 2Department of ChemicalPhysical and 3Department of Pharmaceutics. Center for Genetic
Engineering and Biotechnology, Havana, Cuba.
[email protected]
Peptides have increasingly gained wider attention in drug development as
judged by over 80 ongoing clinical trials and significant approvals. Peptides
are powerful drugs, but specific formulation issues should be addressed,
such as solubility, stability and compatibility. This presentation will
showcase the points to consider for formulation development for peptide
drug products. Advances made in the pharmaceutical development of
peptides will be presented along with a discussion on the applicability of
ICHQ8R2 guideline. Real-life case study of investigational peptide drug
product will be presented. Advances in the formulations including
liposomal, polymer and nanoparticle-based delivery vehicles have largely
contributed to recent promises and successes.
Synthetic Peptides Varadero 2015
Friday, April 10th
QbD: ¿Reality or Utopia?
Author: David Diago Abreu
[email protected]
Actually an aspect that has key role in the Development of
Biopharmaceutical and Pharmaceutical process and product is the
implementation of Quality by Design (QbD), it´s a challenge and
requirement to access to the market and be successful in a business.
This conference shortly describes some ideas and principles
according to obtain short development timelines as well as develop
robust processes and product.
Synthetic Peptides Varadero 2015
Next Meetings
The CIGB Biomedical Research Direction is pleased to announce as part of the series of
events on OMICs and Bioinformatics the International Meeting OMICs Varadero 2015 which
will be held on October 27th-30th in Varadero, Cuba.
The Meeting will cover topics such as bioinformatics and statistical methods for genomic and
proteomic research, technologies for OMICS data generation, cancer genomics, mass spectra
data processing and computational proteomics, next Generation Sequencing: RNASeq,
ChipSeq, DNA methylation, data management and analysis, metagenomics; personalized
medicine: selection of drug-response markers, translational biomarkers, epigenetics,
integrative OMICS data analysis, genomic technology and methodology development and
system biology based bioinformatics software development.
The CIGB's Direction of Biomedical Research invites you to attend Basic Wound Healing
Varadero 2016, an International Meeting on basic aspects of physiological and pathological
cutaneous healing which is going to be held on Oct 25-28, 2016.
The Meeting will gather some of the world's top researchers and specialists in these hot
topics and is intended for clinicians and scientists interested on the cellular and molecular
mechanisms of cutaneous tissue repair.
Major clinical conditions to be addressed: Basic science of burn injury, diabetic foot ulcers
and pressure ulcers.
IEpithelial response to injury.
IIDermal response to injury.
IIIImmuno-inflammatory cells response in wound healing.
IVGrowth factors and stem cells in wound healing.
We cordially invite you to be part of these exciting meetings. For more information please
visit http://biomed.cigb.edu.cu
LEGEND
Synthetic Peptides Varadero 2015
Synthetic Peptides Varadero 2015
Synthetic Peptides Varadero 2015