RESUMO
Cone snails are carnivorous marine animals that prey on fish (piscivorous), worms (vermivorous), or other mollusks (molluscivorous). They produce a complex venom mostly made of disulfide-rich conotoxins and conopeptides in a compartmentalized venom gland. The pharmacology of cone snail venom has been increasingly investigated over more than half a century. The rising interest in cone snails was initiated by the surprising high human lethality rate caused by the defensive stings of some species. Although a vast amount of information has been uncovered on their venom composition, pharmacological targets, and mode of action of conotoxins, the venom-ecology relationships are still poorly understood for many lineages. This is especially important given the relatively recent discovery that some species can use different venoms to achieve rapid prey capture and efficient deterrence of aggressors. Indeed, via an unknown mechanism, only a selected subset of conotoxins is injected depending on the intended purpose. Some of these remarkable venom variations have been characterized, often using a combination of mass spectrometry and transcriptomic methods. In this review, we present the current knowledge on such specific predatory and defensive venoms gathered from sixteen different cone snail species that belong to eight subgenera: Pionoconus, Chelyconus, Gastridium, Cylinder, Conus, Stephanoconus, Rhizoconus, and Vituliconus. Further studies are needed to help close the gap in our understanding of the evolved ecological roles of many cone snail venom peptides.
Assuntos
Conotoxinas , Caramujo Conus , Humanos , Animais , Conotoxinas/toxicidade , Conotoxinas/química , Caramujo Conus/química , Venenos de Moluscos/química , Peptídeos , Peçonhas , CaramujosRESUMO
α-Conotoxins are well-known probes for the characterization of the various subtypes of nicotinic acetylcholine receptors (nAChRs). Identifying new α-conotoxins with different pharmacological profiles can provide further insights into the physiological or pathological roles of the numerous nAChR isoforms found at the neuromuscular junction, the central and peripheral nervous systems, and other cells such as immune cells. This study focuses on the synthesis and characterization of two novel α-conotoxins obtained from two species endemic to the Marquesas Islands, namely Conus gauguini and Conus adamsonii. Both species prey on fish, and their venom is considered a rich source of bioactive peptides that can target a wide range of pharmacological receptors in vertebrates. Here, we demonstrate the versatile use of a one-pot disulfide bond synthesis to achieve the α-conotoxin fold [Cys 1-3; 2-4] for GaIA and AdIA, using the 2-nitrobenzyl (NBzl) protecting group of cysteines for effective regioselective oxidation. The potency and selectivity of GaIA and AdIA against rat nicotinic acetylcholine receptors were investigated electrophysiologically and revealed potent inhibitory activities. GaIA was most active at the muscle nAChR (IC50 = 38 nM), whereas AdIA was most potent at the neuronal α6/3 ß2ß3 subtype (IC50 = 177 nM). Overall, this study contributes to a better understanding of the structure-activity relationships of α-conotoxins, which may help in the design of more selective tools.
Assuntos
Conotoxinas , Caramujo Conus , Receptores Nicotínicos , Animais , Ratos , Conotoxinas/farmacologia , Conotoxinas/química , Caramujo Conus/química , Caramujo Conus/fisiologia , Antagonistas Nicotínicos/farmacologia , Caramujos , PolinésiaRESUMO
Real-time quantification of reactive nitrogen and oxygen species (ROS) in cells is of paramount importance as they are essential for cellular functions. Their excessive formation contributes to the dysfunction of cells and organisms, ultimately leading to cell death. As ROS are mostly produced in the mitochondria, we have synthesized a fluorescent probe able to reach this organelle to detect and quantify, in real time, the variation of ROS by time-resolved microfluorimetry. The new probes are based on the long fluorescence lifetime of pyrene butyric acid (PBA). Two PBA isomers, attached at their 1- or 2-positions to a peptide vector to target mitochondria, were compared and were shown to allow the measurement of free radical species and oxygen, but not non-radical species such as H2 O2 .
Assuntos
Radicais Livres/análise , Microscopia de Fluorescência/métodos , Mitocôndrias/metabolismo , Peptídeos/química , Pirenos/química , Animais , Linhagem Celular , Citosol/química , Citosol/metabolismo , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Humanos , Mitocôndrias/química , Pirenos/síntese química , RatosRESUMO
Laxaphycins are a family of non-ribosomal lipopeptides that have been isolated from several cyanobacteria. Some of these compounds have presented cytotoxic activities, but their mechanism of action is poorly understood. In this work, the already described laxaphycins B and B3, and acyclolaxaphycins B and B3 were isolated from the marine cyanobacteria Anabaena torulosa. Moreover, two new acyclic compounds, [des-(Ala4-Hle5)] acyclolaxaphycins B and B3, were purified from the herviborous gastropod Stylocheilus striatus, with this being the first description of biotransformed laxaphycins. The structure of these new compounds was elucidated, together with the absolute configuration of acyclolaxaphycins B and B3. The bioactivities of the six peptides were determined in SH-SY5Y human neuroblastoma cells. Laxaphycins B and B3 were cytotoxic (IC50: 1.8 and 0.8 µM, respectively) through the induction of apoptosis. In comparison, acyclic laxaphycins did not show cytotoxicity but affected mitochondrial functioning, so their effect on autophagy-related protein expression was analyzed, finding that acyclic peptides affected this process by increasing AMPK phosphorylation and inhibiting mTOR. This work confirms the pro-apoptotic properties of cyclic laxaphycins B and is the first report indicating the effects on autophagy of their acyclic analogs. Moreover, gastropod-derived compounds presented ring opening and amino-acids deletion, a biotransformation that had not been previously described.
Assuntos
Antineoplásicos/farmacologia , Neuroblastoma/tratamento farmacológico , Peptídeos Cíclicos/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Concentração Inibidora 50 , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Peptídeos Cíclicos/química , Fosforilação , Conformação Proteica , Relação Estrutura-Atividade , Serina-Treonina Quinases TOR/metabolismoRESUMO
Five new laxaphycins were isolated and fully characterised from the bloom forming cyanobacteria Anabaena torulosa sampled from Moorea, French Polynesia: three acyclic laxaphycin A-type peptides, acyclolaxaphycin A (1), [des-Gly11]acyclolaxaphycin A (2) and [des-(Leu10-Gly11)]acyclolaxaphycin A (3), as well as two cyclic ones, [l-Val8]laxaphycin A (4) and [d-Val9]laxaphycin A (5). The absolute configuration of the amino acids, established using advanced Marfey's analysis for compounds 2-5, highlights a conserved stereochemistry at the Cα carbons of the peptide ring that is characteristic of this family. To the best of our knowledge, this is the first report of acyclic analogues within the laxaphycin A-type peptides. Whether these linear laxaphycins with the aliphatic ß-amino acid on the N-terminal are biosynthetic precursors or compounds obtained after enzymatic hydrolysis of the macrocycle is discussed. Biological evaluation of the new compounds together with the already known laxaphycin A shows that [l-Val8]laxaphycin A, [d-Val9]laxaphycin A and [des-Gly11]acyclolaxaphycin induce cellular toxicity whereas laxaphycin A and des-[(Leu10-Gly11)]acyclolaxaphycin A do not affect the cellular viability. An analysis of cellular death shows that the active peptides do not induce apoptosis or necrosis but instead, involve the autophagy pathway.
Assuntos
Peptídeos Cíclicos/química , Peptídeos/química , Anabaena/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Ressonância Magnética Nuclear Biomolecular , Peptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Conformação Proteica , Espécies Reativas de Oxigênio/metabolismoRESUMO
New chemiluminescence-based immunoassays for sensitive detection of 17-ß estradiol (E2) and ethinylestradiol (EE2) are described on the basis of the use of biotinylated estrogen derivatives. Estrogen derivatives bearing a carboxylic group (E2-COOH and EE2-COOH) on C-3 position were synthesized, covalently bound to aminated biotin and subsequently immobilized on avidin-coated microtiter plates. The assay principle was based on competition between free and immobilized estrogens for their binding to primary antibodies, with subsequent revelation using horseradish peroxidase (HRP)-labeled secondary antibodies. Under optimized conditions, the chemiluminescence immunoassays showed a highly sensitive response to E2 and EE2, with respective detection limits of 0.5 and 1.2 ng L-1. The LOD achieved using biotinylated E2 was in the same order of magnitude as those obtained using commercially available E2-bovine serum albumin conjugate (E2-BSA). The developed devices were successfully applied to analysis wastewater treatment plants effluents (WWTP) with negligible matrix effect.
Assuntos
Técnicas de Química Analítica/métodos , Monitoramento Ambiental/métodos , Estradiol/análise , Etinilestradiol/análise , Imunoensaio , Medições Luminescentes , Animais , Biotinilação , Bovinos , Limite de Detecção , Soroalbumina Bovina/análise , Soroalbumina Bovina/química , Poluentes Químicos da Água/análiseRESUMO
Angiogenesis is tightly regulated through the binding of vascular endothelial growth factors (VEGFs) to their receptors (VEGFRs). In this context, we showed that human VEGFR1 domain 2 crystallizes in the presence of Zn2+, Co2+ or Cu2+ as a dimer that forms via metal-ion interactions and interlocked hydrophobic surfaces. SAXS, NMR and size exclusion chromatography analyses confirm the formation of this dimer in solution in the presence of Co2+, Cd2+ or Cu2+. Since the metal-induced dimerization masks the VEGFs binding surface, we investigated the ability of metal ions to displace the VEGF-A binding to hVEGFR1: using a competition assay, we evidenced that the metals displaced the VEGF-A binding to hVEGFR1 extracellular domain binding at micromolar level.
Assuntos
Cátions Bivalentes/farmacologia , Simulação de Acoplamento Molecular , Multimerização Proteica , Fator A de Crescimento do Endotélio Vascular/química , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/química , Sítios de Ligação , Humanos , Ligação Proteica/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
A concise synthetic pathway yielding to hydrophobic α,α-disubstituted disilylated amino acids based on a hydrosilylation reaction is described. As a first example of utilization in solid-phase peptide synthesis, TESDpg was introduced in replacement of Aib in an alamethicin sequence, leading to analogues with modified physicochemical properties and conserved helical structures. This study highlights the potential of these new amino acids as tools for peptide modulation.
Assuntos
Aminoácidos/síntese química , Peptídeos/síntese química , Silanos/síntese química , Técnicas de Síntese em Fase Sólida , Alameticina/química , Aminoácidos/química , Estrutura Molecular , Peptídeos/química , Silanos/química , EstereoisomerismoRESUMO
A diisopropylcarbodiimide/Oxyma (ethyl 2-cyano-2-(hydroxyimino)acetate) coupling cocktail was successfully incorporated into the automated microwave-assisted synthesis of two peptaibols and one analog, whose previously reported syntheses were complicated by steric hindrance. This method utilizes commercially available reagents and affords alamethicin F50/5 and bergofungin D in high yields and purities along with an appreciable reduction of synthesis time and cost when compared to previously reported methods.
Assuntos
Micro-Ondas , Peptaibols/síntese química , Acetatos/química , Alameticina/síntese química , Alameticina/química , Peptídeos Catiônicos Antimicrobianos , Carbodi-Imidas/química , Indicadores e Reagentes , Estrutura Molecular , Oximas/química , Peptaibols/química , Peptídeos/síntese química , Peptídeos/químicaRESUMO
OBJECTIVES: We studied whether plasma levels of angiogenic factors VEGF and placental growth factor (PlGF) in coronary artery disease patients or undergoing cardiac surgery are modified, and whether those factors modulate endothelial progenitor's angiogenic potential. METHODS AND RESULTS: A total of 143 patients' plasmas from two different studies were analyzed (30 coronary artery disease patients, 30 patients with stable angina, coupled with 30 age and sex-matched controls; 53 patients underwent cardiac surgery). Among factors screened, only PlGF was found significantly increased in these pathological populations. PlGF-1 and PlGF-2 were then tested on human endothelial-colony-forming cells (ECFCs). We found that PlGF-1 and PlGF-2 induce VEGFR1 phosphorylation and potentiate ECFCs tubulogenesis in vitro. ECFCs VEGFR1 was further inhibited using a specific small interfering RNA (siRNA) and the chemical compound 4321. We then observed that the VEGFR1-siRNA and the compound 4321 decrease ECFCs tubulogenesis potential in vitro. Finally, we tested the compound 4321 in the preclinical Matrigel(®)-plug model with C57Bl/6J mice as well as in the murine hindlimb ischemia model. We found that 4321 inhibited the plug vascularization, attested by the hemoglobin content and the VE-Cadherin expression level and that 4321 inhibited the post-ischemic revascularization. CONCLUSION: PlGF plasma levels were found increased in cardiovascular patients. Disrupting PlGF/VEGFR1 pathway could modulate ECFC-induced tubulogenesis, the cell type responsible for newly formed vessels in vivo.
Assuntos
Diferenciação Celular , Células Endoteliais/metabolismo , Células-Tronco/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Procedimentos Cirúrgicos Cardíacos , Diferenciação Celular/efeitos dos fármacos , Ensaios de Migração Celular , Proliferação de Células/efeitos dos fármacos , Colágeno/metabolismo , Ensaio de Unidades Formadoras de Colônias , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/patologia , Combinação de Medicamentos , Células Endoteliais/efeitos dos fármacos , Membro Posterior/irrigação sanguínea , Membro Posterior/patologia , Humanos , Isquemia/patologia , Laminina/metabolismo , Proteínas de Membrana/sangue , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteoglicanas/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/farmacologia , Células-Tronco/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/sangueRESUMO
The design, synthesis, conformational studies and binding affinity for VEGF receptors of a collection of linear and cyclic peptide analogues of the N-terminal α-helix fragments 13-25 of VEGF and 1-13 of Vammin are described. Linear 13(14)-mer peptides were designed with the help of an AGADIR algorithm and prepared following peptide solid-phase synthetic protocols. Cyclic peptide derivatives were prepared on-resin from linear precursors with conveniently located Glu and Lys residues, by the formation of amide linkages. Conformational analysis, CD and NMR, showed that most synthesized peptides have a clear tendency to be structured as α-helices in solution. Some of the peptides were able to bind a VEGFR-1 receptor with moderate affinity. In addition to the described key residues (Phe17, Tyr21 and Tyr25), Val14 and Val20 seem to be relevant for affinity.
Assuntos
Peptídeos/química , Receptores de Fatores de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/química , Venenos de Víboras/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Peptídeos/síntese química , Peptídeos/metabolismo , Conformação Proteica , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Novel electrochemical immunosensors for sensitive detection of 17-ß estradiol (E2) and ethinylestradiol (EE2) are described on the basis of the use of magnetic beads (MBs) as solid support and screen-printed electrodes as sensing platforms. Four synthetic estrogen derivatives containing either a carboxylic group or an amine group at the C-3 position were synthesized and covalently bound to MBs functionalized with amine or carboxyl groups, respectively. The assay was based on competition between the free and immobilized estrogen for the binding sites of the primary antibody, with subsequent revelation using alkaline phosphatase-labeled secondary antibody. Preliminary colorimetric tests were performed in order to validate the applicability of the synthetic estrogens to immuno-recognition and to optimize different experimental parameters. In a second step, electrochemical detection was carried out by square wave voltammetry (SWV). Under the optimized working conditions, the electrochemical immunosensors showed a highly sensitive response to E2 and EE2, with respective detection limits of 1 and 10 ng/L. Cross-reactivity evaluated against other hormones demonstrated an excellent selectivity. The developed devices were successfully applied to analysis of spiked and natural water samples. These new immunosensors offer the advantages of being highly sensitive, easy, and rapid to prepare, with a short assay time.
Assuntos
Técnicas Eletroquímicas , Estradiol/análise , Etinilestradiol/análise , Imunoensaio , Águas Residuárias/química , Poluentes Químicos da Água/análise , Anticorpos/imunologia , Sítios de Ligação , Técnicas Biossensoriais , Colorimetria , Estradiol/imunologia , Estradiol/isolamento & purificação , Etinilestradiol/imunologia , Etinilestradiol/isolamento & purificação , Separação Imunomagnética , Poluentes Químicos da Água/imunologia , Poluentes Químicos da Água/isolamento & purificaçãoRESUMO
The present study was conducted to assess the structures of the main unknown oxygenated metabolites of EAPB0203. The first step was to assign all the (1)H and (13)C NMR of both EAPB0203 and its demethylated metabolite (EAPB0202) to the corresponding atoms in their molecular structures and to elucidate the fragmentation pathways for the [M + H](+) ions of these compounds using high-resolution mass spectrometry (MS). MS/MS spectra showed that both protonated molecules possessing an even number of electrons were unexpectedly losing radicals such as H(â¢), CH(3)(â¢), or even C(7)H(7)(â¢) giving stable radical cations. In vitro metabolism studies were investigated in rat and dog liver microsomes and in the filamentous fungus Cunninghamella elegans. Structural elucidation of six oxygenated metabolites was performed based on the following: (i) their fragmentation pathways in liquid chromatography-MS/MS (LC-MS/MS) analyses; (ii) comparison of their changes in their molecular masses and fragment ions with those of the parent drugs; and (iii) the results of online H/D exchange experiments that provided additional evidence in differentiating hydoxylated metabolites from N-oxides. Structures of the metabolites were elucidated by LC-MS/MS and comparison with synthetic standards; structures of these standards were confirmed using one- and two-dimensional (1)H NMR spectroscopies.
Assuntos
Antineoplásicos/química , Antineoplásicos/metabolismo , Espectrometria de Massas , Quinoxalinas/química , Quinoxalinas/metabolismo , Animais , Cromatografia Líquida , Medição da Troca de Deutério , Cães , Espectroscopia de Ressonância Magnética , Microssomos Hepáticos/metabolismo , Oxigênio/química , RatosRESUMO
Protein-protein interactions play a central role in medicine, and their modulation with small organic compounds remains an enormous challenge. Because it has been noted that the macromolecular complexes modulated to date have a relatively pronounced binding cavity at the interface, we decided to perform screening experiments over the vascular endothelial growth factor receptor (VEGFR), a validated target for antiangiogenic treatments with a very flat interface. We focused the study on the VEGFR-1 D2 domain, and 20 active compounds were identified. These small compounds contained a (3-carboxy-2-ureido)thiophen unit and had IC(50) values in the low micromolar range. The most potent compound inhibited the VEGF-induced VEGFR-1 transduction pathways. Our findings suggest that our best hit may be a promising scaffold to probe this macromolecular complex and for the development of treatments of VEGFR-1-dependent diseases.
Assuntos
Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Inibidores da Angiogênese/farmacologia , Sítios de Ligação , Células Cultivadas , Desenho de Fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Tiofenos/química , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
The design, synthesis, conformational studies and binding affinity for VEGFR-1 receptors of a collection of linear and cyclic peptide analogues of the ß-hairpin fragment VEGF(81-91) are described. Cyclic 11-mer peptide derivatives were prepared from linear precursors with conveniently located Cys, Asp or Dap residues, by the formation of disulfide and amide bridges, using solid-phase synthesis. Molecular modelling studies indicated a tendency to be structured around the central ß-turn of the VEGF(81-91) ß-hairpin in most synthesized cyclic compounds. This structural behavior was confirmed by NMR conformational analysis. The NHCO cyclic derivative 7 showed significant affinity for VEGFR-1, slightly higher than the native linear fragment, thus supporting the design of mimics of this fragment as a valid approach to disrupt the VEGF/VEGFR-1 interaction.
Assuntos
Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Fator A de Crescimento do Endotélio Vascular/química , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Amidas/química , Sequência de Aminoácidos , Inibidores da Angiogênese/síntese química , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Dissulfetos/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Peptídeos Cíclicos/síntese química , Ligação Proteica , Conformação Proteica , Técnicas de Síntese em Fase SólidaRESUMO
BACKGROUND: The application (WO2010075475) is involved in the diagnosis of angiogenesis-dependent diseases related with the soluble FMS-like tyrosine kinase-1 (sFlt-1) biomarker. OBJECTIVE: It aims to identify, characterize and produce antibodies raised against sFlt-1 for the diagnosis of preeclampsia as well as other cardiovascular diseases. METHODS: The diagnostic kit is based on a double mAb sandwich assay, comprising of a capture anti-sFlt-1 mAb, grafted onto paramagnetic particles that are able to bind sFlt-1 both in bound and free forms. An acridinium conjugated anti-sFlt-1 antibody, which binds to s-Flt1 on another epitope, serves as a chemioluminescent label. RESULTS: Using this assay, the total amount of sFlt-1 could be estimated in various biological samples. CONCLUSION: Antibodies against the free and bound forms of sFlt-1 offer new opportunities in the diagnostics of preeclampsia and other angiogenesis-dependent disorders. Furthermore, as demonstrated in this patent, the immunoassay could be automated and is fast and reliable.
Assuntos
Anticorpos Monoclonais , Doenças Cardiovasculares/diagnóstico , Pré-Eclâmpsia/diagnóstico , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/imunologia , Animais , Feminino , Humanos , Patentes como Assunto , Gravidez , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/análiseRESUMO
The design, synthesis and binding affinity for VEGFR-1 receptors of a small library of linear and cyclic analogues of the VEGF(81-91) fragment are described. Cyclic 11- and 10-mer peptide derivatives were prepared using parallel solid-phase protocols. The formation of hydrocarbon alkene-bridged cyclic peptides was achieved through optimized ring-closing metathesis reactions from linear derivatives with conveniently located allylGly residues. Alkane-bridged analogues were successfully obtained by ulterior on-resin hydrogenation. Binding assays showed that some of these compounds were able to compete with labeled VEGF for interaction with the VEGFR-1 receptor. Several peptide derivatives, 2, 7 and 8, showed modest but significant binding affinity, indicating that the designed peptide could mimic the VEGF(81-91) fragment and therefore disrupt the VEGF/VEGFR-1 interaction. This fact opens the way for using these peptides as the starting point for biological/pharmacological tools to deeply investigate this protein-protein system.
Assuntos
Hidrocarbonetos/química , Bibliotecas de Moléculas Pequenas/síntese química , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fatores de Crescimento do Endotélio Vascular/química , Sequência de Aminoácidos , Hidrogenação , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologia , Ligação Proteica , Bibliotecas de Moléculas Pequenas/química , Fatores de Crescimento do Endotélio Vascular/síntese químicaRESUMO
Cyclic peptide antagonist c[YYDEGLEE]-NH(2), which disrupts the interaction between vascular endothelial growth factor (VEGF) and its receptors (VEGFRs), represents a promising tool in the fight against cancer and age-related macular degeneration. Furthermore, coupled to a cyclen derivative, this ligand could be used as a medicinal imaging agent. Nevertheless, before generating such molecular probes, some preliminary studies need to be undertaken in order to define the more suitable positions for introduction of the cyclen macrocycle. Through an Ala-scan study on this peptide, we identified its binding motif, and an NMR study highlights its binding sites on the VEGFR-1D2 Ig-like domain. Guided by the structural relationship results deduced from the effect of the peptides on endothelial cells, new peptides were synthesized and grafted on beads. Used in a pull-down assay, these new peptides trap the VEGFRs, thus confirming that the identified amino acid positions are suitable for further derivatization.
Assuntos
Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ciclamos , Compostos Heterocíclicos/química , Humanos , Medições Luminescentes , Lisina , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Imagem Molecular , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Sondas Moleculares/farmacologia , Peptídeos Cíclicos/farmacologia , Fosforilação/efeitos dos fármacos , Ligação Proteica , Conformação Proteica , Receptores de Fatores de Crescimento do Endotélio Vascular/química , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Especificidade por Substrato , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
The interaction of the vascular endothelial growth factor (VEGF) with its cellular receptors exerts a central role in the regulation of angiogenesis. Among these receptors, the VEGF receptor 1 may be implicated in pathological angiogenesis. Here, we report the first total chemical synthesis of the VEGF-binding domain of the VEGF receptor 1. Aggregation issues were overcome by the use of a low-substituted resin and the stepwise introduction of pseudoproline dipeptides and Dmb-glycines. The folding of the protein was achieved by air oxidation and its biological activity was verified on ELISA-based assays.
Assuntos
Peptídeos/química , Peptídeos/síntese química , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/química , Humanos , Prolina/análogos & derivados , Prolina/química , Dobramento de Proteína , Estrutura Terciária de Proteína , Tiazóis/químicaRESUMO
Dysregulated angiogenesis is implicated in several pathologies, including cancer and age-related macular degeneration. A potential antiangiogenic strategy consists in developing VEGF receptor ligands capable of preventing VEGF binding and the subsequent activation of these receptors. Herein, we describe the structure-based design of a VEGF-mimicking peptide, VG3F. This 25-mer peptide was doubly cyclized, on-resin, by formation of both a disulfide bridge and an intramolecular amide bond to constrain it to adopt a bioactive conformation. Tested on in vitro assays, VG3F was able to prevent VEGF binding to VEGF receptor 1 and inhibit both VEGF-induced signal transduction and cell migration.