RESUMO
The osteopontin-derived peptide FOL-005 stimulates hair growth. Using ligand-receptor glyco-capture technology we identified neuropilin-1 (NRP-1), a known co-receptor for vascular endothelial growth factor (VEGF) receptors, as the most probable receptor for FOL-005 and the more stable analogue FOL-026. X-ray diffraction and microscale thermophoresis analysis revealed that FOL-026 shares binding site with VEGF in the NRP-1 b1-subdomain. Stimulation of human umbilical vein endothelial cells with FOL-026 resulted in phosphorylation of VEGFR-2, ERK1/2 and AKT, increased cell growth and migration, stimulation of endothelial tube formation and inhibition of apoptosis in vitro. FOL-026 also promoted angiogenesis in vivo as assessed by subcutaneous Matrigel plug and hind limb ischemia models. NRP-1 knock-down or treatment of NRP-1 antagonist EG00229 blocked the stimulatory effects of FOL-026 on endothelial cells. Exposure of human coronary artery smooth muscle cells to FOL-026 stimulated cell growth, migration, inhibited apoptosis, and induced VEGF gene expression and VEGFR-2/AKT phosphorylation by an NRP-1-dependent mechanism. RNA sequencing showed that FOL-026 activated pathways involved in tissue repair. These findings identify NRP-1 as the receptor for FOL-026 and show that its biological effects mimic that of growth factors binding to the VEGF receptor family. They also suggest that FOL-026 may have therapeutical potential in conditions that require vascular repair and/or enhanced angiogenesis.
Assuntos
Células Endoteliais da Veia Umbilical Humana , Neovascularização Fisiológica , Neuropilina-1 , Osteopontina , Neuropilina-1/metabolismo , Humanos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Animais , Neovascularização Fisiológica/efeitos dos fármacos , Osteopontina/metabolismo , Osteopontina/genética , Movimento Celular/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proliferação de Células/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Masculino , Peptídeos/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Apoptose/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Ligação Proteica , Isquemia/tratamento farmacológico , Isquemia/metabolismo , Camundongos , AngiogêneseRESUMO
There is a clinical need for conceptually new treatments that target the excessive activation of inflammatory pathways during systemic infection. Thrombin-derived C-terminal peptides (TCPs) are endogenous anti-infective immunomodulators interfering with CD14-mediated TLR-dependent immune responses. Here we describe the development of a peptide-based compound for systemic use, sHVF18, expressing the evolutionarily conserved innate structural fold of natural TCPs. Using a combination of structure- and in silico-based design, nuclear magnetic resonance spectroscopy, biophysics, mass spectrometry, cellular, and in vivo studies, we here elucidate the structure, CD14 interactions, protease stability, transcriptome profiling, and therapeutic efficacy of sHVF18. The designed peptide displays a conformationally stabilized, protease resistant active innate fold and targets the LPS-binding groove of CD14. In vivo, it shows therapeutic efficacy in experimental models of endotoxin shock in mice and pigs and increases survival in mouse models of systemic polymicrobial infection. The results provide a drug class based on Nature´s own anti-infective principles.
Assuntos
Lipopolissacarídeos , Receptores Toll-Like , Animais , Camundongos , Suínos , Lipopolissacarídeos/metabolismo , Receptores Toll-Like/metabolismo , Inflamação/patologia , Peptídeos/química , Peptídeo Hidrolases , Receptores de Lipopolissacarídeos/metabolismoRESUMO
Despite the recent clinical success of T cell checkpoint inhibition targeting the CTLA-4 and PD-1 pathways, many patients either fail to achieve objective responses or they develop resistance to therapy. In some cases, poor responses to checkpoint blockade have been linked to suboptimal CD28 costimulation and the inability to generate and maintain a productive adaptive anti-tumor immune response. To address this, here we utilize directed evolution to engineer a CD80 IgV domain with increased PD-L1 affinity and fuse this to an immunoglobulin Fc domain, creating a therapeutic (ALPN-202, davoceticept) capable of providing CD28 costimulation in a PD-L1-dependent fashion while also antagonizing PD-1 - PD-L1 and CTLA-4-CD80/CD86 interactions. We demonstrate that by combining CD28 costimulation and dual checkpoint inhibition, ALPN-202 enhances T cell activation and anti-tumor efficacy in cell-based assays and mouse tumor models more potently than checkpoint blockade alone and thus has the potential to generate potent, clinically meaningful anti-tumor immunity in humans.
Assuntos
Antígenos CD28 , Neoplasias , Animais , Antígeno B7-1/metabolismo , Antígenos CD28/metabolismo , Humanos , Ativação Linfocitária , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Linfócitos TRESUMO
We have obtained the X-ray crystal structure of the galectin-8 N-terminal domain (galectin-8N) with a previously reported quinoline-galactoside ligand at a resolution of 1.6 Å. Based on this X-ray structure, a collection of galactosides derivatised at O3 with triazole, benzimidazole, benzothiazole, and benzoxazole moieties were designed and synthesised. This led to the discovery of a 3-O-(N-methylbenzimidazolylmethyl)-galactoside with a Kd of 1.8 µM for galectin-8N, the most potent selective synthetic galectin-8N ligand to date. Molecular dynamics simulations showed that benzimidazole-galactoside derivatives bind the non-conserved amino acid Gln47, accounting for the higher selectivity for galectin-8N. Galectin-8 is a carbohydrate-binding protein that plays a key role in pathological lymphangiogenesis, modulation of the immune system, and autophagy. Thus, the benzimidazole-derivatised galactosides represent promising compounds for studies of the pathological implications of galectin-8, as well as a starting point for the development of anti-tumour and anti-inflammatory therapeutics targeting galectin-8.
Assuntos
Benzimidazóis/química , Desenho de Fármacos , Galactosídeos/química , Galectinas/química , Benzimidazóis/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Galactosídeos/metabolismo , Galectinas/genética , Galectinas/metabolismo , Humanos , Cinética , Ligantes , Simulação de Dinâmica Molecular , Ligação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Relação Estrutura-Atividade , TermodinâmicaRESUMO
The tenovins are a frequently studied class of compounds capable of inhibiting sirtuin activity, which is thought to result in increased acetylation and protection of the tumor suppressor p53 from degradation. However, as we and other laboratories have shown previously, certain tenovins are also capable of inhibiting autophagic flux, demonstrating the ability of these compounds to engage with more than one target. In this study, we present two additional mechanisms by which tenovins are able to activate p53 and kill tumor cells in culture. These mechanisms are the inhibition of a key enzyme of the de novo pyrimidine synthesis pathway, dihydroorotate dehydrogenase (DHODH), and the blockage of uridine transport into cells. These findings hold a 3-fold significance: first, we demonstrate that tenovins, and perhaps other compounds that activate p53, may activate p53 by more than one mechanism; second, that work previously conducted with certain tenovins as SirT1 inhibitors should additionally be viewed through the lens of DHODH inhibition as this is a major contributor to the mechanism of action of the most widely used tenovins; and finally, that small changes in the structure of a small molecule can lead to a dramatic change in the target profile of the molecule even when the phenotypic readout remains static.
Assuntos
Acetanilidas/farmacologia , Inibidores Enzimáticos/farmacologia , Neoplasias/tratamento farmacológico , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Polifarmacologia , Sirtuína 1/antagonistas & inibidores , Tioureia/análogos & derivados , Proteína Supressora de Tumor p53/metabolismo , Autofagia , Proliferação de Células , Di-Hidro-Orotato Desidrogenase , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Tioureia/farmacologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genéticaRESUMO
Ligand-based virtual screening of large compound collections, combined with fast bioactivity determination, facilitate the discovery of bioactive molecules with desired properties. Here, chemical similarity based machine learning and label-free differential scanning fluorimetry were used to rapidly identify new ligands of the anticancer target Pim-1 kinase. The three-dimensional crystal structure complex of human Pim-1 with ligand bound revealed an ATP-competitive binding mode. Generative de novo design with a recurrent neural network additionally suggested innovative molecular scaffolds. Results corroborate the validity of the chemical similarity principle for rapid ligand prototyping, suggesting the complementarity of similarity-based and generative computational approaches.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Inteligência Artificial , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-pim-1/química , Relação Quantitativa Estrutura-AtividadeRESUMO
Collagen VI is a ubiquitous extracellular matrix component that forms extensive microfibrillar networks in most connective tissues. In this study, we describe for the first time, to our knowledge, that the collagen VI von Willebrand factor type A-like domains exhibit a broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria in human skin infections in vivo. In silico sequence and structural analysis of VWA domains revealed that they contain cationic and amphipathic peptide sequence motifs, which might explain the antimicrobial nature of collagen VI. In vitro and in vivo studies show that these peptides exhibited significant antibacterial activity against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa through membrane disruption. Our findings shed new light on the role of collagen VI-derived peptides in innate host defense and provide templates for development of peptide-based antibacterial therapies.
Assuntos
Antibacterianos/imunologia , Colágeno Tipo VI/imunologia , Peptídeos/imunologia , Bactérias/imunologia , Infecções Bacterianas/imunologia , Humanos , Imunidade Inata/imunologia , Domínios Proteicos/imunologia , Pele/imunologia , Pele/microbiologia , Dermatopatias Bacterianas/imunologiaRESUMO
The original PDF version of this Article listed the authors as "Marcus J.G.W. Ladds," where it should have read "Marcus J. G. W. Ladds, Ingeborg M. M. van Leeuwen, Catherine J. Drummond et al.#".Also in the PDF version, it was incorrectly stated that "Correspondence and requests for materials should be addressed to S. Lín.", instead of the correct "Correspondence and requests for materials should be addressed to S. Laín."This has been corrected in the PDF version of the Article. The HTML version was correct from the time of publication.
RESUMO
The development of non-genotoxic therapies that activate wild-type p53 in tumors is of great interest since the discovery of p53 as a tumor suppressor. Here we report the identification of over 100 small-molecules activating p53 in cells. We elucidate the mechanism of action of a chiral tetrahydroindazole (HZ00), and through target deconvolution, we deduce that its active enantiomer (R)-HZ00, inhibits dihydroorotate dehydrogenase (DHODH). The chiral specificity of HZ05, a more potent analog, is revealed by the crystal structure of the (R)-HZ05/DHODH complex. Twelve other DHODH inhibitor chemotypes are detailed among the p53 activators, which identifies DHODH as a frequent target for structurally diverse compounds. We observe that HZ compounds accumulate cancer cells in S-phase, increase p53 synthesis, and synergize with an inhibitor of p53 degradation to reduce tumor growth in vivo. We, therefore, propose a strategy to promote cancer cell killing by p53 instead of its reversible cell cycle arresting effect.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Indazóis/farmacologia , Neoplasias/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Di-Hidro-Orotato Desidrogenase , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Proteólise/efeitos dos fármacos , Proteína Supressora de Tumor p53/genéticaRESUMO
Structural technologies are an essential component in the design of precision therapeutics. Precision medicine entails the development of therapeutics directed toward a designated target protein, with the goal to deliver the right drug to the right patient at the right time. In the field of oncology, protein structural variants are often associated with oncogenic potential. In a previous proteogenomic screen of patient-derived glioblastoma (GBM) tumor materials, we identified a sequence variant of human mitochondrial branched-chain amino acid aminotransferase 2 as a putative factor of resistance of GBM to standard-of-care-treatments. The enzyme generates glutamate, which is neurotoxic. To elucidate structural coordinates that may confer altered substrate binding or activity of the variant BCAT2 T186R, a ~45 kDa protein, we applied combined ETD and CID top-down mass spectrometry in a LC-FT-ICR MS at 21 T, and X-Ray crystallography in the study of both the variant and non-variant intact proteins. The combined ETD/CID fragmentation pattern allowed for not only extensive sequence coverage but also confident localization of the amino acid variant to its position in the sequence. The crystallographic experiments confirmed the hypothesis generated by in silico structural homology modeling, that the Lys59 side-chain of BCAT2 may repulse the Arg186 in the variant protein (PDB code: 5MPR), leading to destabilization of the protein dimer and altered enzyme kinetics. Taken together, the MS and novel 3D structural data give us reason to further pursue BCAT2 T186R as a precision drug target in GBM. Graphical Abstract á .
Assuntos
Cristalografia por Raios X/métodos , Espectrometria de Massas/métodos , Antígenos de Histocompatibilidade Menor/química , Modelos Moleculares , Proteínas da Gravidez/química , Transaminases/química , Sequência de Aminoácidos , Humanos , Antígenos de Histocompatibilidade Menor/análise , Antígenos de Histocompatibilidade Menor/genética , Mutação , Medicina de Precisão , Proteínas da Gravidez/análise , Proteínas da Gravidez/genética , Transaminases/análise , Transaminases/genéticaRESUMO
Agents that trigger cell differentiation are highly efficacious in treating certain cancers, but such approaches are not generally effective in most malignancies. Compounds such as DMSO and hexamethylene bisacetamide (HMBA) have been used to induce differentiation in experimental systems, but their mechanisms of action and potential range of uses on that basis have not been developed. Here, we show that HMBA, a compound first tested in the oncology clinic over 25 years ago, acts as a selective bromodomain inhibitor. Biochemical and structural studies revealed an affinity of HMBA for the second bromodomain of BET proteins. Accordingly, both HMBA and the prototype BET inhibitor JQ1 induced differentiation of mouse erythroleukemia cells. As expected of a BET inhibitor, HMBA displaced BET proteins from chromatin, caused massive transcriptional changes, and triggered cell-cycle arrest and apoptosis in Myc-induced B-cell lymphoma cells. Furthermore, HMBA exerted anticancer effects in vivo in mouse models of Myc-driven B-cell lymphoma. This study illuminates the function of an early anticancer agent and suggests an intersection with ongoing clinical trials of BET inhibitor, with several implications for predicting patient selection and response rates to this therapy and starting points for generating BD2-selective BET inhibitors. Cancer Res; 76(8); 2376-83. ©2016 AACR.
Assuntos
Acetamidas/farmacologia , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Humanos , CamundongosRESUMO
Serine proteases of the S1 family have maintained a common structure over an evolutionary span of more than one billion years, and evolved a variety of substrate specificities and diverse biological roles, involving digestion and degradation, blood clotting, fibrinolysis and epithelial homeostasis. We here show that a wide range of C-terminal peptide sequences of serine proteases, particularly from the coagulation and kallikrein systems, share characteristics common with classical antimicrobial peptides of innate immunity. Under physiological conditions, these peptides exert antimicrobial effects as well as immunomodulatory functions by inhibiting macrophage responses to bacterial lipopolysaccharide. In mice, selected peptides are protective against lipopolysaccharide-induced shock. Moreover, these S1-derived host defense peptides exhibit helical structures upon binding to lipopolysaccharide and also permeabilize liposomes. The results uncover new and fundamental aspects on host defense functions of serine proteases present particularly in blood and epithelia, and provide tools for the identification of host defense molecules of therapeutic interest.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Macrófagos/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Serina Proteases/metabolismo , Choque Séptico/metabolismo , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Linhagem Celular , Modelos Animais de Doenças , Humanos , Imunidade Inata , Imunomodulação , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Filogenia , Relação Quantitativa Estrutura-Atividade , Serina Proteases/química , Serina Proteases/genética , Serina Proteases/imunologia , Choque Séptico/induzido quimicamente , Choque Séptico/imunologia , Homologia Estrutural de ProteínaRESUMO
Antibacterial peptides of the innate immune system combat pathogenic microbes, but often have additional roles in promoting inflammation and as growth factors during tissue repair. Midkine (MK) and pleiotrophin (PTN) are the only two members of a family of heparin-binding growth factors. They show restricted expression during embryogenesis and are up-regulated in neoplasia. In addition, MK shows constitutive and inflammation-dependent expression in some non-transformed tissues of the adult. In the present study, we show that both MK and PTN display strong antibacterial activity, present at physiological salt concentrations. Electron microscopy of bacteria and experiments using artificial lipid bilayers suggest that MK and PTN exert their antibacterial action via a membrane disruption mechanism. The predicted structure of PTN, employing the previously solved MK structure as a template, indicates that both molecules consist of two domains, each containing three antiparallel beta-sheets. The antibacterial activity was mapped to the unordered C-terminal tails of both molecules and the last beta-sheets of the N-terminals. Analysis of the highly conserved MK and PTN orthologues from the amphibian Xenopus laevis and the fish Danio rerio suggests that they also harbor antibacterial activity in the corresponding domains. In support of an evolutionary conserved function it was found that the more distant orthologue, insect Miple2 from Drosophila melanogaster, also displays strong antibacterial activity. Taken together, the findings suggest that MK and PTN, in addition to their earlier described activities, may have previously unrealized important roles as innate antibiotics.
Assuntos
Antibacterianos/química , Proteínas de Transporte/química , Citocinas/química , Evolução Molecular , Bicamadas Lipídicas/química , Fatores de Crescimento Neural/química , Animais , Antibacterianos/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Citocinas/genética , Citocinas/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Bicamadas Lipídicas/metabolismo , Midkina , Neoplasias/genética , Neoplasias/metabolismo , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Mapeamento de Peptídeos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Xenopus laevis , Peixe-ZebraRESUMO
BACKGROUND: The Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) blocks the Complement fragment C5a receptor (C5aR) and formylated peptide receptor (FPR) and is thereby a potent inhibitor of neutrophil chemotaxis and activation of inflammatory responses. The majority of the healthy human population has antibodies against CHIPS that have been shown to interfere with its function in vitro. The aim of this study was to define potential epitopes for human antibodies on the CHIPS surface. We also initiate the process to identify a mutated CHIPS molecule that is not efficiently recognized by preformed anti-CHIPS antibodies and retains anti-inflammatory activity. RESULTS: In this paper, we panned peptide displaying phage libraries against a pool of CHIPS specific affinity-purified polyclonal human IgG. The selected peptides could be divided into two groups of sequences. The first group was the most dominant with 36 of the 48 sequenced clones represented. Binding to human affinity-purified IgG was verified by ELISA for a selection of peptide sequences in phage format. For further analysis, one peptide was chemically synthesized and antibodies affinity-purified on this peptide were found to bind the CHIPS molecule as studied by ELISA and Surface Plasmon Resonance. Furthermore, seven potential conformational epitopes responsible for antibody recognition were identified by mapping phage selected peptide sequences on the CHIPS surface as defined in the NMR structure of the recombinant CHIPS31-121 protein. Mapped epitopes were verified by in vitro mutational analysis of the CHIPS molecule. Single mutations introduced in the proposed antibody epitopes were shown to decrease antibody binding to CHIPS. The biological function in terms of C5aR signaling was studied by flow cytometry. A few mutations were shown to affect this biological function as well as the antibody binding. CONCLUSION: Conformational epitopes recognized by human antibodies have been mapped on the CHIPS surface and amino acid residues involved in both antibody and C5aR interaction could be defined. This information has implications for the development of an effective anti-inflammatory agent based on a functional CHIPS molecule with low interaction with human IgG.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Epitopos , Imunoglobulina G/metabolismo , Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação de Anticorpos/genética , Sítios de Ligação de Anticorpos/imunologia , Quimiotaxia , Mapeamento de Epitopos , Imunoglobulina G/química , Imunoglobulina G/imunologia , Conformação Molecular , Mutagênese Sítio-Dirigida , Mutação , Neutrófilos/metabolismo , Neutrófilos/patologia , Biblioteca de Peptídeos , Peptídeos/síntese química , Peptídeos/imunologia , Peptídeos/metabolismo , Ligação Proteica , Receptor da Anafilatoxina C5a , Receptores de Complemento/imunologia , Receptores de Complemento/metabolismo , Receptores de Formil Peptídeo/imunologia , Receptores de Formil Peptídeo/metabolismo , Transdução de Sinais/imunologia , Staphylococcus aureus , Ressonância de Plasmônio de SuperfícieRESUMO
Protein-protein interactions are widely found in biological systems controlling diverse cellular events. Because these interactions are implicated in many diseases such as autoimmunity and cancer, regulation of protein-protein interactions provides ideal targets for drug intervention. The CD80-CD28 costimulatory pathway plays a critical role in regulation of the immune response and thus constitutes an attractive target for therapeutic manipulation of autoimmune diseases. The objective of this study is to identify small compounds disrupting these pivotal protein-protein interactions. Compounds that specifically blocked binding of CD80 to CD28 were identified using a strategy involving a cell-based scintillation proximity assay as the initial step. Secondary screening (e.g., by analyzing the direct binding of these compounds to the target immobilized on a biosensor surface) revealed that these compounds are highly selective CD80 binders. Screening of structurally related derivatives led to the identification of the chemical features required for inhibition of the CD80-CD28 interaction. In addition, the optimization process led to a 10-fold increase in binding affinity of the CD80 inhibitors. Using this approach, the authors identify low-molecular-weight compounds that specifically and with high potency inhibit the interaction between CD80 and CD28. These compounds serve as promising starting points for further development of CD80 inhibitors as potential immunomodulatory drugs.
Assuntos
Antígeno B7-1/metabolismo , Desenho de Fármacos , Animais , Ligação Competitiva , Células CHO , Cricetinae , Cricetulus , Cabras , Humanos , Camundongos , Ligação Proteica , Mapeamento de Interação de Proteínas , CoelhosRESUMO
Growth factors, comprising diverse protein and peptide families, are involved in a multitude of developmental processes, including embryogenesis, angiogenesis, and wound healing. Here we show that peptides derived from HB-EGF, amphiregulin, hepatocyte growth factor, PDGF-A and PDGF-B, as well as various FGFs are antimicrobial, demonstrating a previously unknown activity of growth factor-derived peptides. The peptides killed the Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa, and the Gram-positive Bacillus subtilis, as well as the fungus Candida albicans. Several peptides were also active against the Gram-positive S. aureus. Electron microscopy analysis of peptide-treated bacteria, paired with analysis of peptide effects on liposomes, showed that the peptides exerted membrane-breaking effects similar to those seen after treatment with the "classical" human antimicrobial peptide LL-37. Furthermore, HB-EGF was antibacterial per se, and its epitope GKRKKKGKGLGKKRDPCLRKYK retained its activity in presence of physiological salt and plasma. No discernible hemolysis was noted for the growth factor-derived peptides. Besides providing novel templates for design of peptide-based antimicrobials, our findings demonstrate a previously undisclosed link between the family of growth factors and antimicrobial peptides, both of which are induced during tissue remodelling and repair.
Assuntos
Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Substâncias de Crescimento/química , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Bactérias/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/genética , Escherichia coli/efeitos dos fármacos , Substâncias de Crescimento/genética , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intercelular , Testes de Sensibilidade Microbiana , Microscopia Eletrônica , Modelos Moleculares , Dados de Sequência Molecular , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacosRESUMO
The human anaphylatoxin peptide C3a, generated during complement activation, exerts antimicrobial effects. Phylogenetic analysis, sequence analyses, and structural modeling studies paired with antimicrobial assays of peptides from known C3a sequences showed that, in particular in vertebrate C3a, crucial structural determinants governing antimicrobial activity have been conserved during the evolution of C3a. Thus, regions of the ancient C3a from Carcinoscorpius rotundicauda as well as corresponding parts of human C3a exhibited helical structures upon binding to bacterial lipopolysaccharide permeabilized liposomes and were antimicrobial against gram-negative and gram-positive bacteria. Human C3a and C4a (but not C5a) were antimicrobial, in concert with the separate evolutionary development of the chemotactic C5a. Thus, the results demonstrate that, notwithstanding a significant sequence variation, functional and structural constraints imposed on C3a during evolution have preserved critical properties governing antimicrobial activity.
Assuntos
Complemento C3a , Sequência de Aminoácidos , Anafilatoxinas/química , Anafilatoxinas/genética , Anafilatoxinas/metabolismo , Animais , Anti-Infecciosos/química , Anti-Infecciosos/metabolismo , Complemento C3a/química , Complemento C3a/genética , Complemento C3a/metabolismo , Complemento C4a/química , Complemento C4a/genética , Complemento C4a/metabolismo , Complemento C5a/química , Complemento C5a/genética , Complemento C5a/metabolismo , Evolução Molecular , Caranguejos Ferradura , Humanos , Invertebrados , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Alinhamento de SequênciaRESUMO
The B7 ligands CD80 and CD86 on APCs deliver either costimulatory or inhibitory signals to the T cell when interacting with their counter-receptors CD28 and CD152 (CTLA-4) on the T cell surface. Although crucial for lymphocyte regulation, the structural basis of these interactions is still not completely understood. Using multivalent presentation and conditions mimicking clustering, believed to be essential for signaling through these receptors, and by applying a combined differential mass spectrometry and structural mapping approach to these conditions, we were able to identify a putative contact area involving hydrophilic regions on both CD28 and CD80 as well as a putative CD28 oligomerization interface induced by B7 ligation. Analysis of the CD80-CD28 interaction site reveals a well-defined interface structurally distinct from that of CD80 and CD152 and thus provides valuable information for therapeutic intervention targeted at this pathway, suggesting a general approach for other receptors.
Assuntos
Antígeno B7-1/química , Antígenos CD28/química , Animais , Antígenos CD , Antígenos de Diferenciação/química , Antígeno B7-1/fisiologia , Antígenos CD28/fisiologia , Células CHO , Antígeno CTLA-4 , Cricetinae , Dimerização , Humanos , Transdução de SinaisRESUMO
Monoclonal antibodies have a potential for cancer therapy that may be further improved by linking them to effector molecules such as superantigens. Tumor targeting of a superantigen leads to a powerful T cell attack against the tumour tissue. Encouraging results have been observed preclinically and in patients using the superantigen staphylococcal enterotoxin A, SEA. To further improve the concept, we have reduced the reactivity to antibodies against superantigens, which is found in all individuals. Using epitope mapping, antibody binding sites in SEA and SEE were found around their MHC class II binding sites. These epitopes were removed genetically and a large number of synthetic superantigens were produced in an iterative engineering procedure. Properties such as decreased binding to anti-SEA as well as higher selectivity to induce killing of tumour cells compared to MHC class II expressing cells, were sequentially improved. The lysine residues 79, 81, 83 and 84 are all part of major antigenic epitopes, Gln204, Lys74, Asp75 and Asn78 are important for optimal killing of tumour cells while Asp45 affects binding to MHC class II. The production properties were optimised by further engineering and a novel synthetic superantigen, SEA/E-120, was designed. It is recognised by approximately 15% of human anti-SEA antibodies and have more potent tumour cell killing properties than SEA. SEA/E-120 is likely to have a low toxicity due to its reduced capacity to mediate killing of MHC class II expressing cells. It is produced as a Fab fusion protein at approximately 35 mg/l in Escherichia coli.