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1.
Biochim Biophys Acta ; 1798(2): 87-93, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19835839

RESUMO

Neisseria meningitidis is a major cause of meningitis. Although protective vaccination is available against some pathogenic serogroups, serogroup B meningococci have been a challenge for vaccinologists. A family of outer membrane lipoproteins, LP2086 (or factor H binding proteins, fHbp), has been shown to elicit bactericidal antibodies and is currently part of a cocktail vaccine candidate. The NMR structure of the variant LP2086-B01 in micellar solution provided insights on the topology of this family of proteins on the biological membrane. Based on flow cytometry experiments on whole meningococcal cells, binding experiments with monoclonal antibodies, and the NMR structure in micellar solution, we previously proposed that LP2086-B01 anchors the outer bacterial membrane through its lipidated N-terminal cysteine, while a flexible 20 residue linker positions the protein above the layer of lipo-oligosaccharides that surrounds the bacteria. This topology was suggested to increase the antigen exposure to the immune system. In the present work, using micellar solution as a membrane mimicking system, we characterized the backbone dynamics of the variant LP2086-B01 in both its lipidated and unlipidated forms. In addition, binding experiments with a Fab fragment derived from the monoclonal MN86-1042-2 were also performed. Our data suggests that due to the length and flexibility of the N-terminal linker, the antigen is not in contact with the micelle, thus making both N- and C-domains highly available to the host immune system. This dynamic model, combined with the binding data obtained with MN86-1042-2, supports our previously proposed arrangement that LP2086-B01 exposes one face to the extracellular space. Binding of MN86-1042-2 antibody shows that the N-domain is the primary target of this monoclonal, providing further indication that this domain is immunologically important for this family of proteins.


Assuntos
Anticorpos Antibacterianos/química , Anticorpos Monoclonais/química , Antígenos de Bactérias/química , Proteínas de Bactérias/química , Lipopolissacarídeos/química , Modelos Moleculares , Neisseria meningitidis/química , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Humanos , Lipopolissacarídeos/imunologia , Camundongos , Micelas , Neisseria meningitidis/imunologia , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína/fisiologia
2.
Antimicrob Agents Chemother ; 55(10): 4552-9, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21807982

RESUMO

The integrase inhibitor (INI) dolutegravir (DTG; S/GSK1349572) has significant activity against HIV-1 isolates with raltegravir (RAL)- and elvitegravir (ELV)-associated resistance mutations. As an initial step in characterizing the different resistance profiles of DTG, RAL, and ELV, we determined the dissociation rates of these INIs with integrase (IN)-DNA complexes containing a broad panel of IN proteins, including IN substitutions corresponding to signature RAL and ELV resistance mutations. DTG dissociates slowly from a wild-type IN-DNA complex at 37°C with an off-rate of 2.7 × 10(-6) s(-1) and a dissociative half-life (t(1/2)) of 71 h, significantly longer than the half-lives for RAL (8.8 h) and ELV (2.7 h). Prolonged binding (t(1/2), at least 5 h) was observed for DTG with IN-DNA complexes containing E92, Y143, Q148, and N155 substitutions. The addition of a second substitution to either Q148 or N155 typically resulted in an increase in the off-rate compared to that with the single substitution. For all of the IN substitutions tested, the off-rate of DTG from IN-DNA complexes was significantly slower (from 5 to 40 times slower) than the off-rate of RAL or ELV. These data are consistent with the potential for DTG to have a higher genetic barrier to resistance, provide evidence that the INI off-rate may be an important component of the mechanism of INI resistance, and suggest that the slow dissociation of DTG may contribute to its distinctive resistance profile.


Assuntos
DNA Viral/metabolismo , Inibidores de Integrase de HIV/metabolismo , Integrase de HIV/metabolismo , HIV-1/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/metabolismo , Pirrolidinonas/metabolismo , Quinolonas/metabolismo , Substituição de Aminoácidos , DNA Complementar , Farmacorresistência Viral , Genótipo , Integrase de HIV/genética , Inibidores de Integrase de HIV/química , Inibidores de Integrase de HIV/farmacologia , HIV-1/genética , Compostos Heterocíclicos com 3 Anéis/farmacologia , Mutação , Oxazinas , Piperazinas , Piridonas , Pirrolidinonas/farmacologia , Quinolonas/farmacologia , Raltegravir Potássico
3.
J Med Chem ; 55(16): 7193-207, 2012 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-22827572

RESUMO

Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is activated in response to a variety of endoplasmic reticulum stresses implicated in numerous disease states. Evidence that PERK is implicated in tumorigenesis and cancer cell survival stimulated our search for small molecule inhibitors. Through screening and lead optimization using the human PERK crystal structure, we discovered compound 38 (GSK2606414), an orally available, potent, and selective PERK inhibitor. Compound 38 inhibits PERK activation in cells and inhibits the growth of a human tumor xenograft in mice.


Assuntos
Adenina/análogos & derivados , Antineoplásicos/síntese química , Indóis/síntese química , Pirimidinas/síntese química , Pirróis/síntese química , eIF-2 Quinase/antagonistas & inibidores , Adenina/síntese química , Adenina/química , Adenina/farmacologia , Administração Oral , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Disponibilidade Biológica , Linhagem Celular Tumoral , Cristalografia por Raios X , Cães , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Indóis/química , Indóis/farmacologia , Masculino , Camundongos , Camundongos Nus , Modelos Moleculares , Transplante de Neoplasias , Fosforilação , Conformação Proteica , Pirimidinas/química , Pirimidinas/farmacologia , Pirróis/química , Pirróis/farmacologia , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Transplante Heterólogo
4.
J Biol Chem ; 284(13): 8738-46, 2009 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-19103601

RESUMO

LP2086 is a family of outer membrane lipoproteins from Neisseria meningitidis, which elicits bactericidal antibodies and are currently undergoing human clinical trials in a bivalent formulation where each antigen represents one of the two known LP2086 subfamilies. Here we report the NMR structure of the recombinant LP2086 variant B01, a representative of the LP2086 subfamily B. The structure reveals a novel fold composed of two domains: a "taco-shaped" N-terminal beta-sheet and a C-terminal beta-barrel connected by a linker. The structure in micellar solution is consistent with a model of LP2086 anchored to the outer membrane bilayer through its lipidated N terminus. A long flexible chain connects the folded part of the protein to the lipid anchor and acts as spacer, making both domains accessible to the host immune system. Antibodies broadly reactive against members from both subfamilies have been mapped to the N terminus. A surface of subfamily-defining residues was identified on one face of the protein, offering an explanation for the induction of subfamily-specific bactericidal antibodies.


Assuntos
Anticorpos Antibacterianos/química , Antígenos de Bactérias/química , Proteínas de Bactérias/química , Bicamadas Lipídicas/química , Vacinas Meningocócicas/química , Micelas , Neisseria meningitidis/química , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Sequência de Bases , Humanos , Bicamadas Lipídicas/imunologia , Vacinas Meningocócicas/genética , Vacinas Meningocócicas/imunologia , Camundongos , Dados de Sequência Molecular , Neisseria meningitidis/genética , Neisseria meningitidis/imunologia , Ressonância Magnética Nuclear Biomolecular/métodos , Mapeamento de Peptídeos/métodos , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia
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