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1.
Nat Commun ; 11(1): 4303, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855401

RESUMO

The novel highly transmissible human coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Thus far, there is no approved therapeutic drug specifically targeting this emerging virus. Here we report the isolation and characterization of a panel of human neutralizing monoclonal antibodies targeting the SARS-CoV-2 receptor binding domain (RBD). These antibodies were selected from a phage display library constructed using peripheral circulatory lymphocytes collected from patients at the acute phase of the disease. These neutralizing antibodies are shown to recognize distinct epitopes on the viral spike RBD. A subset of the antibodies exert their inhibitory activity by abrogating binding of the RBD to the human ACE2 receptor. The human monoclonal antibodies described here represent a promising basis for the design of efficient combined post-exposure therapy for SARS-CoV-2 infection.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Betacoronavirus/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/metabolismo , Betacoronavirus/metabolismo , Chlorocebus aethiops , Mapeamento de Epitopos , Epitopos , Humanos , Biblioteca de Peptídeos , Peptidil Dipeptidase A/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero
2.
Nat Struct Mol Biol ; 27(9): 846-854, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32661423

RESUMO

The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (KD of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody-RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD-ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4-6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral , Receptores Virais/metabolismo , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/metabolismo , Anticorpos Neutralizantes/ultraestrutura , Anticorpos Antivirais/metabolismo , Anticorpos Antivirais/ultraestrutura , Afinidade de Anticorpos , Reações Antígeno-Anticorpo/imunologia , Betacoronavirus/metabolismo , Ligação Competitiva , Microscopia Crioeletrônica , Cristalografia por Raios X , Epitopos/imunologia , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Modelos Moleculares , Biblioteca de Peptídeos , Peptidil Dipeptidase A/ultraestrutura , Ligação Proteica , Conformação Proteica , Receptores Virais/ultraestrutura , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Anticorpos de Domínio Único/metabolismo , Anticorpos de Domínio Único/ultraestrutura , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/ultraestrutura
3.
Mol Immunol ; 125: 43-50, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32645549

RESUMO

The CD8 T cell response to the HLA-A2-restricted epitope LLWNGPMAV (LLW) of the non-structural protein 4b of Yellow Fever Virus (YFV) is remarkably immunodominant, highly prevalent and powerful in YFV-vaccinated humans. Here we used a combinatorial peptide library screening in the context of an A2/LLW-specific CD8 T cell clone to identify a superagonist that features a methionine to isoleucine substitution at position 7. Based on in silico modeling, the functional enhancement of this LLW-7I mutation was associated with alterations in the structural dynamics of the peptide in the major histocompatibility complex (pMHC) binding with the T cell receptor (TCR). While the TCR off-rate of LLW-7I pMHC is comparable to the wild type peptide, the rigidity of the 7I peptide seems to confer less entropy loss upon TCR binding. This LLW-7I superagonist is an example of improved functionality in human CD8 T cells associated with optimized ligand rigidity for TCR binding and not with changes in TCR:pMHC off-rate kinetics.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos Imunodominantes/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas não Estruturais Virais/imunologia , Vírus da Febre Amarela/imunologia , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Antígeno HLA-A2/química , Antígeno HLA-A2/imunologia , Humanos , Epitopos Imunodominantes/química , Modelos Moleculares , Mutação , Biblioteca de Peptídeos , Ligação Proteica/imunologia , Receptores de Antígenos de Linfócitos T/química
4.
MAbs ; 12(1): 1778435, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: covidwho-601168

RESUMO

Effective therapies are urgently needed for COVID-19. Here we describe the identification of a new stable human immunoglobulin G1 heavy-chain variable (VH) domain scaffold that was used for the construction of a large library, lCAT6, of engineered human VHs. This library was panned against the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) glycoprotein. Two VH domains (VH ab6 and VH m397) were selected and fused to Fc for increased half-life in circulation. The VH-Fc ab6 and m397 specifically neutralized SARS-CoV-2 with high potencies (50% neutralization at 0.35 µg/ml and 1.5 µg/ml, respectively) as measured by two independent replication-competent virus neutralization assays. Ab6 and m397 competed with ACE2 for binding to RBD, suggesting a competitive mechanism of virus neutralization. These VH domains may have potential applications for prophylaxis and therapy of COVID-19 alone or in combination, as well as for diagnosis and as tools for research.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus , Pandemias , Pneumonia Viral , Anticorpos de Domínio Único/imunologia , Anticorpos Monoclonais , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Biblioteca de Peptídeos
5.
Nat Commun ; 11(1): 3183, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576815

RESUMO

High-diversity genetically-encoded combinatorial libraries (108-1013 members) are a rich source of peptide-based binding molecules, identified by affinity selection. Synthetic libraries can access broader chemical space, but typically examine only ~ 106 compounds by screening. Here we show that in-solution affinity selection can be interfaced with nano-liquid chromatography-tandem mass spectrometry peptide sequencing to identify binders from fully randomized synthetic libraries of 108 members-a 100-fold gain in diversity over standard practice. To validate this approach, we show that binders to a monoclonal antibody are identified in proportion to library diversity, as diversity is increased from 106-108. These results are then applied to the discovery of p53-like binders to MDM2, and to a family of 3-19 nM-affinity, α/ß-peptide-based binders to 14-3-3. An X-ray structure of one of these binders in complex with 14-3-3σ is determined, illustrating the role of ß-amino acids in facilitating a key binding contact.


Assuntos
Biblioteca de Peptídeos , Peptídeos/química , Bibliotecas de Moléculas Pequenas/química , Sequência de Aminoácidos , Aminoácidos , Anticorpos Monoclonais/química , Afinidade de Anticorpos , Proteínas de Transporte/química , Cromatografia Líquida , Cristalografia por Raios X , Desenho de Fármacos , Descoberta de Drogas , Modelos Moleculares , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/metabolismo
6.
PLoS One ; 15(6): e0233961, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32479512

RESUMO

Hundreds of target specific peptides are routinely discovered by peptide display platforms. However, due to the high cost of peptide synthesis only a limited number of peptides are chemically made for further analysis. Here we describe an accurate and cost effective method to bin peptides on-phage based on binding region(s), without any requirement for peptide or protein synthesis. This approach, which integrates phage and yeast display platforms, requires display of target and its alanine variants on yeast. Flow cytometry was used to detect binding of peptides on-phage to the target on yeast. Once hits were identified, they were synthesized to confirm their binding region(s) by HDX (Hydrogen deuterium exchange) and crystallography. Moreover, we have successfully shown that this approach can be implemented as part of a panning process to deplete non-functional peptides. This technique can be applied to any target that can be successfully displayed on yeast; it narrows down the number of peptides requiring synthesis; and its utilization during selection results in enrichment of peptide population against defined binding regions on the target.


Assuntos
Técnicas de Visualização da Superfície Celular/métodos , Biblioteca de Peptídeos , Alanina/genética , Alanina/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Técnicas de Visualização da Superfície Celular/economia , Análise Custo-Benefício , Citometria de Fluxo/economia , Citometria de Fluxo/métodos , Subunidade p40 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/metabolismo , Mutação , Ligação Proteica/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
7.
Nat Rev Drug Discov ; 19(6): 389-413, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32494050

RESUMO

Dysregulation of peptide-activated pathways causes a range of diseases, fostering the discovery and clinical development of peptide drugs. Many endogenous peptides activate G protein-coupled receptors (GPCRs) - nearly 50 GPCR peptide drugs have been approved to date, most of them for metabolic disease or oncology, and more than 10 potentially first-in-class peptide therapeutics are in the pipeline. The majority of existing peptide therapeutics are agonists, which reflects the currently dominant strategy of modifying the endogenous peptide sequence of ligands for peptide-binding GPCRs. Increasingly, novel strategies are being employed to develop both agonists and antagonists, to both introduce chemical novelty and improve drug-like properties. Pharmacodynamic improvements are evolving to allow biasing ligands to activate specific downstream signalling pathways, in order to optimize efficacy and reduce side effects. In pharmacokinetics, modifications that increase plasma half-life have been revolutionary. Here, we discuss the current status of the peptide drugs targeting GPCRs, with a focus on evolving strategies to improve pharmacokinetic and pharmacodynamic properties.


Assuntos
Desenho de Fármacos , Peptídeos , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Ensaios Clínicos como Assunto , Avaliação Pré-Clínica de Medicamentos , Humanos , Ligantes , Terapia de Alvo Molecular , Biblioteca de Peptídeos , Peptídeos/farmacocinética , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Ligação Proteica , Transdução de Sinais
8.
MAbs ; 12(1): 1778435, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-32544372

RESUMO

Effective therapies are urgently needed for COVID-19. Here we describe the identification of a new stable human immunoglobulin G1 heavy-chain variable (VH) domain scaffold that was used for the construction of a large library, lCAT6, of engineered human VHs. This library was panned against the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) glycoprotein. Two VH domains (VH ab6 and VH m397) were selected and fused to Fc for increased half-life in circulation. The VH-Fc ab6 and m397 specifically neutralized SARS-CoV-2 with high potencies (50% neutralization at 0.35 µg/ml and 1.5 µg/ml, respectively) as measured by two independent replication-competent virus neutralization assays. Ab6 and m397 competed with ACE2 for binding to RBD, suggesting a competitive mechanism of virus neutralization. These VH domains may have potential applications for prophylaxis and therapy of COVID-19 alone or in combination, as well as for diagnosis and as tools for research.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus , Pandemias , Pneumonia Viral , Anticorpos de Domínio Único/imunologia , Anticorpos Monoclonais , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Biblioteca de Peptídeos
9.
Cell Host Microbe ; 27(6): 891-898.e5, 2020 06 10.
Artigo em Inglês | MEDLINE | ID: covidwho-260276

RESUMO

The worldwide spread of COVID-19 highlights the need for an efficient approach to rapidly develop therapeutics and prophylactics against SARS-CoV-2. The SARS-CoV-2 spike protein, containing the receptor-binding domain (RBD) and S1 subunit involved in receptor engagement, is a potential therapeutic target. We describe the development of a phage-displayed single-domain antibody library by grafting naive complementarity-determining regions (CDRs) into framework regions of a human germline immunoglobulin heavy chain variable region (IGHV) allele. Panning this library against SARS-CoV-2 RBD and S1 subunit identified fully human single-domain antibodies targeting five distinct epitopes on SARS-CoV-2 RBD with subnanomolar to low nanomolar affinities. Some of these antibodies neutralize SARS-CoV-2 by targeting a cryptic epitope located in the spike trimeric interface. Collectively, this work presents a versatile platform for rapid antibody isolation and identifies promising therapeutic anti-SARS-CoV-2 antibodies as well as the diverse immogneic profile of the spike protein.


Assuntos
Biblioteca de Peptídeos , Anticorpos de Domínio Único/isolamento & purificação , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Epitopos/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas , Região Variável de Imunoglobulina , Modelos Moleculares , Domínios Proteicos , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/química
10.
Nat Biomed Eng ; 4(5): 560-571, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32393891

RESUMO

The oral administration of peptide drugs is hampered by their metabolic instability and limited intestinal uptake. Here, we describe a method for the generation of small target-specific peptides (less than 1,600 Da in size) that resist gastrointestinal proteases. By using phage display to screen large libraries of genetically encoded double-bridged peptides on protease-resistant fd bacteriophages, we generated a peptide inhibitor of the coagulation Factor XIa with nanomolar affinity that resisted gastrointestinal proteases in all regions of the gastrointestinal tract of mice after oral administration, enabling more than 30% of the peptide to remain intact, and small quantities of it to reach the blood circulation. We also developed a gastrointestinal-protease-resistant peptide antagonist for the interleukin-23 receptor, which has a role in the pathogenesis of Crohn's disease and ulcerative colitis. The de novo generation of targeted peptides that resist proteolytic degradation in the gastrointestinal tract should help the development of effective peptides for oral delivery.


Assuntos
Peptídeos/administração & dosagem , Peptídeos/uso terapêutico , Proteólise , Administração Oral , Sequência de Aminoácidos , Animais , Técnicas de Visualização da Superfície Celular , Cristalografia por Raios X , Feminino , Trato Gastrointestinal/metabolismo , Humanos , Isomerismo , Camundongos Endogâmicos BALB C , Modelos Moleculares , Peptídeo Hidrolases/metabolismo , Biblioteca de Peptídeos , Peptídeos/química , Estabilidade Proteica , Estrutura Secundária de Proteína , Receptores de Interleucina/antagonistas & inibidores , Receptores de Interleucina/metabolismo
11.
Mol Immunol ; 123: 7-17, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32387766

RESUMO

The identification of T cell epitopes derived from tumour specific antigens remains a significant challenge for the development of peptide-based vaccines and immunotherapies. The use of mass spectrometry-based approaches (immunopeptidomics) can provide powerful new avenues for the identification of such epitopes. In this study we report the use of complementary peptide antigen enrichment methods and a comprehensive mass spectrometric acquisition strategy to provide in-depth immunopeptidome data for the THP-1 cell line, a cell line used widely as a model of human leukaemia. To accomplish this, we combined robust experimental workflows that incorporated ultrafiltration or off-line reversed phase chromatography to enrich peptide ligand as well as a multifaceted data acquisition strategy using an Orbitrap Fusion LC-MS instrument. Using the combined datasets from the two ligand enrichment methods we gained significant depth in immunopeptidome coverage by identifying a total of 41,816 HLA class I peptides from THP-1 cells, including a significant number of peptides derived from different oncogenes or over expressed proteins associated with cancer. The physicochemical properties of the HLA-bound peptides dictated their recovery using the two ligand enrichment approaches and their distribution across the different precursor charge states considered in the data acquisition strategy. The data highlight the complementarity of the two enrichment procedures, and in cases where sample is not limiting, suggest that the combination of both approaches will yield the most comprehensive immunopeptidome information.


Assuntos
Antígenos de Neoplasias/análise , Mineração de Dados/métodos , Epitopos de Linfócito T/imunologia , Leucemia Mieloide Aguda/metabolismo , Peptídeos/análise , Proteoma/análise , Antígenos de Neoplasias/química , Antígenos de Neoplasias/imunologia , Células Cultivadas , Cromatografia Líquida/métodos , Bases de Dados de Proteínas , Humanos , Imunoterapia/métodos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/terapia , Ligantes , Espectrometria de Massas/métodos , Biblioteca de Peptídeos , Peptídeos/química , Proteoma/química , Proteômica/métodos , Células THP-1
12.
Artigo em Inglês | MEDLINE | ID: mdl-32413276

RESUMO

The worldwide spread of COVID-19 highlights the need for an efficient approach to rapidly develop therapeutics and prophylactics against SARS-CoV-2. The SARS-CoV-2 spike protein, containing the receptor-binding domain (RBD) and S1 subunit involved in receptor engagement, is a potential therapeutic target. We describe the development of a phage-displayed single-domain antibody library by grafting naive complementarity-determining regions (CDRs) into framework regions of a human germline immunoglobulin heavy chain variable region (IGHV) allele. Panning this library against SARS-CoV-2 RBD and S1 subunit identified fully human single-domain antibodies targeting five distinct epitopes on SARS-CoV-2 RBD with subnanomolar to low nanomolar affinities. Some of these antibodies neutralize SARS-CoV-2 by targeting a cryptic epitope located in the spike trimeric interface. Collectively, this work presents a versatile platform for rapid antibody isolation and identifies promising therapeutic anti-SARS-CoV-2 antibodies as well as the diverse immogneic profile of the spike protein.


Assuntos
Biblioteca de Peptídeos , Anticorpos de Domínio Único/isolamento & purificação , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Epitopos/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas , Região Variável de Imunoglobulina , Modelos Moleculares , Domínios Proteicos , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/química
13.
Exp Parasitol ; 212: 107885, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32234306

RESUMO

A phage-display library was generated using a Bus thalamus scorpion toxin (BTK-2) as a peptide scaffold. BTK-2 belongs to the disulfide-rich family of proteins with pronounced structural stability due to the presence of three disulfide bridges that connects antiparallel beta-sheets and one alpha helix. Using BTK-2 as a phage display scaffold, we introduced mutations in five residues located in the alpha-helix and two residues located in the smaller loop, keeping intact the disulfide bridges to create a peptide phage-displayed library with disulfide-rich family properties. The library was subjected to in vivo and in vitro phage display selections against Trypanosoma evansi, the etiological agent of "Surra", a disease that affects a wide range of mammals. The development of T. evansi specific biomarkers is essential to improve diagnostic methods and epidemiological studies leading to a more accurate clinical decision for the treatment of this disease of economic impact for commercial livestock production. In this study, we identified two disulfide-rich peptides targeting T. evansi parasites. Further specificity studies are necessary to investigate the potential of selected peptides as new biomarkers to aid diagnostic and treatment procedures of T. evansi infections.


Assuntos
Dissulfetos , Peptídeos , Trypanosoma/química , Tripanossomíase/diagnóstico , Tripanossomíase/terapia , Sequência de Aminoácidos , Animais , Biomarcadores , Clonagem Molecular , Dissulfetos/química , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese , Oligonucleotídeos/química , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/genética , Venenos de Escorpião/química , Venenos de Escorpião/genética
14.
Food Chem ; 321: 126685, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32240918

RESUMO

In this study, we demonstrated the feasibility of isolating recombinant phage-antibodies against gluten from a non-immunized library of human single-domain antibodies (dAbs). Phage display technology enabled the selection of affinity probes by successive rounds of biopanning against a biotinylated synthetic peptide comprising repetitive immunogenic gluten motifs. The analysis of a wide representation of heterologous plant species corroborated that two of the isolated clones were specific to wheat, barley and rye proteins. The phage antibody selected as the most appropriate clone for the detection of gluten in foods (dAb8E-phage) was further applied in an indirect ELISA to the analysis of 50 commercial food samples. Although the limit of detection achieved did not improve those of current immunoassays, the proposed methodology could provide promising new pathways for the generation of recombinant antibodies that allow a comprehensive determination of gluten in foods, whilst replacing the need for animal immunization.


Assuntos
Alérgenos/imunologia , Análise de Alimentos , Glutens/imunologia , Hordeum/imunologia , Proteínas de Plantas/imunologia , Secale/imunologia , Triticum/imunologia , Ensaio de Imunoadsorção Enzimática , Alimentos , Glutens/análise , Hordeum/química , Humanos , Fragmentos de Imunoglobulinas/imunologia , Biblioteca de Peptídeos , Proteínas de Plantas/química , Secale/química , Triticum/química
15.
Proc Natl Acad Sci U S A ; 117(16): 8870-8875, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32245816

RESUMO

The ability to precisely design large proteins with diverse shapes would enable applications ranging from the design of protein binders that wrap around their target to the positioning of multiple functional sites in specified orientations. We describe a protein backbone design method for generating a wide range of rigid fusions between helix-containing proteins and use it to design 75,000 structurally unique junctions between monomeric and homo-oligomeric de novo designed and ankyrin repeat proteins (RPs). Of the junction designs that were experimentally characterized, 82% have circular dichroism and solution small-angle X-ray scattering profiles consistent with the design models and are stable at 95 °C. Crystal structures of four designed junctions were in close agreement with the design models with rmsds ranging from 0.9 to 1.6 Å. Electron microscopic images of extended tetrameric structures and ∼10-nm-diameter "L" and "V" shapes generated using the junctions are close to the design models, demonstrating the control the rigid junctions provide for protein shape sculpting over multiple nanometer length scales.


Assuntos
Modelos Moleculares , Engenharia de Proteínas/métodos , Proteínas/ultraestrutura , Sequências Repetitivas de Aminoácidos/genética , Dicroísmo Circular , Microscopia Eletrônica , Biblioteca de Peptídeos , Conformação Proteica em alfa-Hélice/genética , Dobramento de Proteína , Proteínas/química , Proteínas/genética , Espalhamento a Baixo Ângulo , Difração de Raios X
16.
Adv Exp Med Biol ; 1248: 531-546, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32185724

RESUMO

Peptides, as a large group of molecules, are composed of amino acid residues and can be divided into linear or cyclic peptides according to the structure. Over 13,000 molecules of natural peptides have been found and many of them have been well studied. In artificial peptide libraries, the number of peptide diversity could be up to 1 × 1013. Peptides have more complex structures and higher affinity to target proteins comparing with small molecular compounds. Recently, the development of targeting cancer immune checkpoint (CIP) inhibitors is having a very important role in tumor therapy. Peptides targeting ligands or receptors in CIP have been designed based on three-dimensional structures of target proteins or directly selected by random peptide libraries in biological display systems. Most of these targeting peptides work as inhibitors of protein-protein interaction and improve CD8+ cytotoxic T-lymphocyte (CTL) activation in the tumor microenvironment, for example, PKHB1, Ar5Y4 and TPP1. Peptides could be designed to regulate CIP protein degradation in vivo, such as PD-LYSO and PD-PALM. Besides its use in developing therapeutic drugs for targeting CIP, targeting peptides could be used in drug's targeted delivery and diagnosis in tumor immune therapy.


Assuntos
Sistemas de Liberação de Medicamentos , Terapia de Alvo Molecular , Peptídeos/metabolismo , Peptídeos/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/imunologia , Humanos , Ligantes , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , Biblioteca de Peptídeos , Peptídeos/química
17.
PLoS One ; 15(3): e0229080, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32196507

RESUMO

Therapeutic monoclonal antibodies have the potential to work as biological therapeutics. OKT3, Herceptin, Keytruda and others have positively impacted healthcare. Antibodies evolved naturally to provide high specificity and high affinity once mature. These characteristics can make them useful as therapeutics. However, we may be missing characteristics that are not obvious. We present a means of measuring antibodies in an unbiased manner that may highlight therapeutic activity. We propose using a microarray of random peptides to assess antibody properties. We tested twenty-four different commercial antibodies to gain some perspective about how much information can be derived from binding antibodies to random peptide libraries. Some monoclonals preferred to bind shorter peptides, some longer, some preferred motifs closer to the C-term, some nearer the N-term. We tested some antibodies with clinical activity but whose function was blinded to us at the time. We were provided with twenty-one different monoclonal antibodies, thirteen mouse and eight human IgM. These antibodies produced a variety of binding patterns on the random peptide arrays. When unblinded, the antibodies with polyspecific binding were the ones with the greatest therapeutic activity. The protein target to these therapeutic monoclonals is still unknown but using common sequence motifs from the peptides we predicted several human and mouse proteins. The same five highest proteins appeared in both mouse and human lists.


Assuntos
Anticorpos Monoclonais/análise , Mapeamento de Epitopos/métodos , Biblioteca de Peptídeos , Análise Serial de Proteínas , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Ensaios de Triagem em Larga Escala/métodos , Humanos , Imunoglobulina M/análise , Imunoglobulina M/metabolismo , Camundongos , Peptídeos/química , Peptídeos/imunologia , Peptídeos/metabolismo , Análise Serial de Proteínas/métodos , Ligação Proteica , Proteoma/análise
18.
J Chromatogr A ; 1620: 460986, 2020 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-32173023

RESUMO

Human plasminogen Kringle 5 is known to pose a more potent anti-angiogenesis effect by inducing endothelial cell apoptosis. Our previous studies have identified the peptide IGNSNTL as a binding sequence of Kringle 5 using Ph.D.-7 phage display peptide library and enzyme-linked immunosorbent assay. Here, eleven proteins were screened and summarized by BLAST, laminin α3 chain G1 domain (LG1) was considered as the most potential receptor based on E value and domain function. The specific interaction of them was directly revealed through ligand blot and a strong concentration-dependent manner occurred between them (Ka 4.30 × 105 L mol-1) in frontal chromatography observation. Moreover, R10A/P83R substitution Kringle 5 decreased the affinity capacity to LG1. Furthermore, a remarkable conformational change from random coil3 to α helix and α1 helix to random coil were observed to the structural compactness and stability for LG1. Surface loops and coils also showed fluctuations up to some extent, giving the binding surface greater flexibility and correspondingly allowing for induced-fit binding, which was -23.87 kcal mol-1 of the free energy with electrostatic force as a main driver. Taken together, not only effective theoretical prediction and experiment validated that LG1 is receptor of Kringle 5, but also give an new perspective of the binding mechanism of Kringle 5 and its specific receptor and could facilitate the development of novel agent targeted toward pathologic angiogenesis.


Assuntos
Células Endoteliais/metabolismo , Laminina/química , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/metabolismo , Plasminogênio/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Cinética , Ligantes , Proteínas Mutantes/química , Biblioteca de Peptídeos , Ligação Proteica , Domínios Proteicos , Termodinâmica
19.
Acta Crystallogr D Struct Biol ; 76(Pt 3): 193-208, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32133985

RESUMO

The analysis of large structural databases reveals general features and relationships among proteins, providing useful insight. A different approach is required to characterize ubiquitous secondary-structure elements, where flexibility is essential in order to capture small local differences. The ALEPH software is optimized for the analysis and the extraction of small protein folds by relying on their geometry rather than on their sequence. The annotation of the structural variability of a given fold provides valuable information for fragment-based molecular-replacement methods, in which testing alternative model hypotheses can succeed in solving difficult structures when no homology models are available or are successful. ARCIMBOLDO_BORGES combines the use of composite secondary-structure elements as a search model with density modification and tracing to reveal the rest of the structure when both steps are successful. This phasing method relies on general fold libraries describing variations around a given pattern of ß-sheets and helices extracted using ALEPH. The program introduces characteristic vectors defined from the main-chain atoms as a way to describe the geometrical properties of the structure. ALEPH encodes structural properties in a graph network, the exploration of which allows secondary-structure annotation, decomposition of a structure into small compact folds, generation of libraries of models representing a variation of a given fold and finally superposition of these folds onto a target structure. These functions are available through a graphical interface designed to interactively show the results of structure manipulation, annotation, fold decomposition, clustering and library generation. ALEPH can produce pictures of the graphs, structures and folds for publication purposes.


Assuntos
Biblioteca de Peptídeos , Conformação Proteica , Software , Modelos Moleculares , Anotação de Sequência Molecular , Fragmentos de Peptídeos , Dobramento de Proteína , Estrutura Secundária de Proteína , Interface Usuário-Computador
20.
Nat Commun ; 11(1): 1548, 2020 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-32214105

RESUMO

Data-independent acquisition approaches typically rely on experiment-specific spectrum libraries, requiring offline fractionation and tens to hundreds of injections. We demonstrate a library generation workflow that leverages fragmentation and retention time prediction to build libraries containing every peptide in a proteome, and then refines those libraries with empirical data. Our method specifically enables rapid, experiment-specific library generation for non-model organisms, which we demonstrate using the malaria parasite Plasmodium falciparum, and non-canonical databases, which we show by detecting missense variants in HeLa.


Assuntos
Cromatografia Líquida/métodos , Peptídeos/análise , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Algoritmos , Bases de Dados de Proteínas , Células HeLa , Humanos , Biblioteca de Peptídeos , Peptídeos/química , Proteoma/análise , Proteoma/química , Reprodutibilidade dos Testes , Fluxo de Trabalho
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