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1.
Nature ; 593(7859): 424-428, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33767445

RESUMO

Neutralizing antibodies that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein are among the most promising approaches against COVID-191,2. A bispecific IgG1-like molecule (CoV-X2) has been developed on the basis of C121 and C135, two antibodies derived from donors who had recovered from COVID-193. Here we show that CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable spike binding to the cellular receptor of the virus, angiotensin-converting enzyme 2 (ACE2). Furthermore, CoV-X2 neutralizes wild-type SARS-CoV-2 and its variants of concern, as well as escape mutants generated by the parental monoclonal antibodies. We also found that in a mouse model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, the simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, and combines the advantages of antibody cocktails with those of single-molecule approaches.


Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Neutralizantes/imunologia , COVID-19/imunologia , COVID-19/virologia , Imunoglobulina G/imunologia , SARS-CoV-2/imunologia , Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/uso terapêutico , Peso Corporal , COVID-19/prevenção & controle , Dependovirus/genética , Modelos Animais de Doenças , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Feminino , Humanos , Evasão da Resposta Imune/genética , Camundongos , Camundongos Endogâmicos C57BL , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Tratamento Farmacológico da COVID-19
3.
Nat Methods ; 15(11): 901-904, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30377366

RESUMO

We present a 'hit-and-return' (HARE) method for time-resolved serial synchrotron crystallography with time resolution from milliseconds to seconds or longer. Timing delays are set mechanically, using the regular pattern in fixed-target crystallography chips and a translation stage system. Optical pump-probe experiments to capture intermediate structures of fluoroacetate dehalogenase binding to its ligand demonstrated that data can be collected at short (30 ms), medium (752 ms) and long (2,052 ms) intervals.


Assuntos
Cristalografia por Raios X , Hidrolases/química , Conformação Proteica , Rodopseudomonas/enzimologia , Síncrotrons/instrumentação , Desenho de Equipamento , Modelos Moleculares , Fatores de Tempo
4.
Chemistry ; 25(49): 11416-11421, 2019 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-31407832

RESUMO

Trypanothione reductase (TR) plays a key role in the unique redox metabolism of trypanosomatids, the causative agents of human African trypanosomiasis (HAT), Chagas' disease, and leishmaniases. Introduction of a new, lean propargylic vector to a known class of TR inhibitors resulted in the strongest reported competitive inhibitor of Trypanosoma (T.) brucei TR, with an inhibition constant Ki of 73 nm, which is fully selective against human glutathione reductase (hGR). The best ligands exhibited in vitro IC50 values (half-maximal inhibitory concentration) against the HAT pathogen, T. brucei rhodesiense, in the mid-nanomolar range, reaching down to 50 nm. X-Ray co-crystal structures confirmed the binding mode of the ligands and revealed the presence of a HEPES buffer molecule in the large active site. Extension of the propargylic vector, guided by structure-based design, to replace the HEPES buffer molecule should give inhibitors with low nanomolar Ki and IC50 values for in vivo studies.


Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/química , NADH NADPH Oxirredutases/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Trypanosoma brucei brucei/enzimologia , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Concentração Inibidora 50 , Ligantes , Simulação de Dinâmica Molecular , NADH NADPH Oxirredutases/metabolismo , Proteínas de Protozoários/metabolismo , Relação Estrutura-Atividade
5.
Glob Chall ; 7(10): 2300088, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37829677

RESUMO

Neutralizing monoclonal antibodies have achieved great efficacy and safety for the treatment of numerous infectious diseases. However, their neutralization potency is often rapidly lost when the target antigen mutates. Instead of isolating new antibodies each time a pathogen variant arises, it can be attractive to adapt existing antibodies, making them active against the new variant. Potential benefits of this approach include reduced development time, cost, and regulatory burden. Here a methodology is described to rapidly evolve neutralizing antibodies of proven activity, improving their function against new pathogen variants without losing efficacy against previous ones. The reported procedure is based on structure-guided affinity maturation using combinatorial mutagenesis and phage display technology. Its use against the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is demonstrated, but it is suitable for any other pathogen. As proof of concept, the method is applied to CoV-X2, a human bispecific antibody that binds with high affinity to the early SARS-CoV-2 variants but lost neutralization potency against Delta. Antibodies emerging from the affinity maturation selection exhibit significantly improved neutralization potency against Delta and no loss of efficacy against the other viral sequences tested. These results illustrate the potential application of structure-guided affinity maturation in facilitating the rapid adaptation of neutralizing antibodies to pathogen variants.

6.
Sci Immunol ; 8(81): eade0958, 2023 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-36701425

RESUMO

Emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike glycoprotein that are both functionally relevant and averse to change, we identified human neutralizing antibodies to highly conserved viral epitopes. Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four genera, including the nine human coronaviruses, through recognition of a conserved motif that includes the S2' site of proteolytic cleavage. Antibody hr2.016 targets the stem helix and neutralizes SARS-CoV-2 variants. Antibody sd1.040 binds to subdomain 1, synergizes with antibody rbd.042 for neutralization, and, similar to fp.006 and hr2.016, protects mice expressing human angiotensin-converting enzyme 2 against infection when present as a bispecific antibody. Thus, coldspot-guided antibody discovery reveals donor-derived neutralizing antibodies that are cross-reactive with Orthocoronavirinae, including SARS-CoV-2 variants.


Assuntos
Anticorpos Neutralizantes , COVID-19 , Humanos , Animais , Camundongos , SARS-CoV-2 , Epitopos , Glicoproteína da Espícula de Coronavírus , Anticorpos Antivirais , Testes de Neutralização
7.
bioRxiv ; 2022 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-36482967

RESUMO

Emergence of SARS-CoV-2 variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike that are both functionally relevant and averse to change, we identified human neutralizing antibodies to highly conserved viral epitopes. Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four genera , including the nine human coronaviruses, through recognition of a conserved motif that includes the S2' site of proteolytic cleavage. Antibody hr2.016 targets the stem helix and neutralizes SARS-CoV-2 variants. Antibody sd1.040 binds to subdomain 1, synergizes with antibody rbd.042 for neutralization and, like fp.006 and hr2.016, protects mice when present as bispecific antibody. Thus, coldspot-guided antibody discovery reveals donor-derived neutralizing antibodies that are cross-reactive with Orthocoronavirinae , including SARS-CoV-2 variants. One sentence summary: Broadly cross-reactive antibodies that protect from SARS-CoV-2 variants are revealed by virus coldspot-driven discovery.

8.
Nat Commun ; 12(1): 3532, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112780

RESUMO

In systemic light chain amyloidosis (AL), pathogenic monoclonal immunoglobulin light chains (LC) form toxic aggregates and amyloid fibrils in target organs. Prompt diagnosis is crucial to avoid permanent organ damage, but delayed diagnosis is common because symptoms usually appear only after strong organ involvement. Here we present LICTOR, a machine learning approach predicting LC toxicity in AL, based on the distribution of somatic mutations acquired during clonal selection. LICTOR achieves a specificity and a sensitivity of 0.82 and 0.76, respectively, with an area under the receiver operating characteristic curve (AUC) of 0.87. Tested on an independent set of 12 LCs sequences with known clinical phenotypes, LICTOR achieves a prediction accuracy of 83%. Furthermore, we are able to abolish the toxic phenotype of an LC by in silico reverting two germline-specific somatic mutations identified by LICTOR, and by experimentally assessing the loss of in vivo toxicity in a Caenorhabditis elegans model. Therefore, LICTOR represents a promising strategy for AL diagnosis and reducing high mortality rates in AL.


Assuntos
Caenorhabditis elegans/metabolismo , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/toxicidade , Amiloidose de Cadeia Leve de Imunoglobulina/diagnóstico , Amiloidose de Cadeia Leve de Imunoglobulina/genética , Aprendizado de Máquina , Algoritmos , Sequência de Aminoácidos , Animais , Anticorpos/genética , Caenorhabditis elegans/genética , Bases de Dados Genéticas , Expressão Gênica , Humanos , Cadeias Leves de Imunoglobulina/química , Modelos Moleculares , Mutação , Proteínas Recombinantes
9.
bioRxiv ; 2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33501434

RESUMO

Neutralizing antibodies targeting the receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) are among the most promising approaches against coronavirus disease 2019 (COVID-19) 1,2 . We developed a bispecific, IgG1-like molecule (CoV-X2) based on two antibodies derived from COVID-19 convalescent donors, C121 and C135 3 . CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable S binding to Angiotensin-Converting Enzyme 2 (ACE2), the virus cellular receptor. Furthermore, CoV-X2 neutralizes SARS-CoV-2 and its variants of concern, as well as the escape mutants generated by the parental monoclonals. In a novel animal model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, combining into a single molecule the advantages of antibody cocktails.

10.
ChemMedChem ; 13(9): 957-967, 2018 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-29624890

RESUMO

The tropical diseases human African trypanosomiasis, Chagas disease, and the various forms of leishmaniasis are caused by parasites of the family of trypanosomatids. These protozoa possess a unique redox metabolism based on trypanothione and trypanothione reductase (TR), making TR a promising drug target. We report the optimization of properties and potency of cyclohexylpyrrolidine inhibitors of TR by structure-based design. The best inhibitors were freely soluble and showed competitive inhibition constants (Ki ) against Trypanosoma (T.) brucei TR and T. cruzi TR and in vitro activities (half-maximal inhibitory concentration, IC50 ) against these parasites in the low micromolar range, with high selectivity against human glutathione reductase. X-ray co-crystal structures confirmed the binding of the ligands to the hydrophobic wall of the "mepacrine binding site" with the new, solubility-providing vectors oriented toward the surface of the large active site.


Assuntos
Inibidores Enzimáticos/farmacologia , NADH NADPH Oxirredutases/antagonistas & inibidores , Pirrolidinas/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma/efeitos dos fármacos , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Ligantes , Modelos Moleculares , Estrutura Molecular , NADH NADPH Oxirredutases/metabolismo , Oxirredução , Testes de Sensibilidade Parasitária , Pirrolidinas/síntese química , Pirrolidinas/química , Solubilidade , Tripanossomicidas/síntese química , Tripanossomicidas/química , Trypanosoma/metabolismo
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