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1.
Front Immunol ; 14: 1209059, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37483599

RESUMO

Long-distance migratory animals such as birds and bats have evolved to withstand selection imposed by pathogens across the globe, and pathogen richness is known to be particularly high in tropical regions. Immune genes, so-called Major Histocompatibility Complex (MHC) genes, are highly duplicated in songbirds compared to other vertebrates, and this high MHC diversity has been hypothesised to result in a unique adaptive immunity. To understand the rationale behind the evolution of the high MHC genetic diversity in songbirds, we determined the structural properties of an MHC class I protein, Acar3, from a long-distance migratory songbird, the great reed warbler Acrocephalus arundinaceus (in short: Acar). The structure of Acar3 was studied in complex with pathogen-derived antigens and shows an overall antigen presentation similar to human MHC class I. However, the peptides bound to Acar3 display an unusual conformation: Whereas the N-terminal ends of the peptides display enhanced flexibility, the conformation of their C-terminal halves is rather static. This uncommon peptide-binding mode in Acar3 is facilitated by a central Arg residue within the peptide-binding groove that fixes the backbone of the peptide at its central position, and potentially permits successful interactions between MHC class I and innate immune receptors. Our study highlights the importance of investigating the immune system of wild animals, such as birds and bats, to uncover unique immune mechanisms which may neither exist in humans nor in model organisms.


Assuntos
Quirópteros , Aves Canoras , Animais , Humanos , Aves Canoras/genética , Aves Canoras/metabolismo , Antígenos de Histocompatibilidade Classe I , Peptídeos/metabolismo , Apresentação de Antígeno , Antígenos HLA
2.
ACS Infect Dis ; 7(4): 746-758, 2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33710875

RESUMO

The enoyl-acyl carrier protein (ACP) reductase (ENR) is a key enzyme within the bacterial fatty-acid synthesis pathway. It has been demonstrated that small-molecule inhibitors carrying the diphenylether (DPE) scaffold bear a great potential for the development of highly specific and effective drugs against this enzyme class. Interestingly, different substitution patterns of the DPE scaffold have been shown to lead to varying effects on the kinetic and thermodynamic behavior toward ENRs from different organisms. Here, we investigated the effect of a 4'-pyridone substituent in the context of the slow tight-binding inhibitor SKTS1 on the inhibition of the Staphylococcus aureus enoyl-ACP-reductase saFabI and the closely related isoenzyme from Mycobacterium tuberculosis, InhA, and explored a new interaction site of DPE inhibitors within the substrate-binding pocket. Using high-resolution crystal structures of both complexes in combination with molecular dynamics (MD) simulations, kinetic measurements, and quantum mechanical (QM) calculations, we provide evidence that the 4'-pyridone substituent adopts different tautomeric forms when bound to the two ENRs. We furthermore elucidate the structural determinants leading to significant differences in the residence time of SKTS1 on both enzymes.


Assuntos
Inibidores Enzimáticos/farmacologia , Isoenzimas , Oxirredutases/antagonistas & inibidores , Isomerismo , Mycobacterium tuberculosis/enzimologia , Staphylococcus aureus/enzimologia
3.
Structure ; 28(2): 215-222.e3, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31831212

RESUMO

The aquaglyceroporin 7 (AQP7) facilitates permeation of glycerol through cell membranes and is crucial for lipid metabolism in humans. Glycerol efflux in human adipocytes is controlled by translocation of AQP7 to the plasma membrane upon hormone stimulation. Here we present two X-ray structures of human AQP7 at 1.9 and 2.2 Å resolution. The structures combined with molecular dynamics simulations suggest that AQP7 is a channel selective for glycerol and that glycerol may hamper water permeation through the channel. Moreover, the high resolution of the structures facilitated a detailed analysis of the orientation of glycerol in the pore, disclosing unusual positions of the hydroxyl groups. The data suggest that glycerol is conducted by a partly rotating movement through the channel. These observations provide a framework for understanding the basis of glycerol efflux and selectivity in aquaglyceroporins and pave the way for future design of AQP7 inhibitors.


Assuntos
Aquaporinas/química , Aquaporinas/metabolismo , Glicerol/metabolismo , Transporte Biológico , Cristalografia por Raios X , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína , Água/metabolismo
4.
J Am Chem Soc ; 139(9): 3417-3429, 2017 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-28151657

RESUMO

A critical goal of lead compound selection and optimization is to maximize target engagement while minimizing off-target binding. Since target engagement is a function of both the thermodynamics and kinetics of drug-target interactions, it follows that the structures of both the ground states and transition states on the binding reaction coordinate are needed to rationally modulate the lifetime of the drug-target complex. Previously, we predicted the structure of the rate-limiting transition state that controlled the time-dependent inhibition of the enoyl-ACP reductase InhA. This led to the discovery of a triazole-containing diphenyl ether with an increased residence time on InhA due to transition-state destabilization rather than ground-state stabilization. In the present work, we evaluate the inhibition of InhA by 14 triazole-based diphenyl ethers and use a combination of enzyme kinetics and X-ray crystallography to generate a structure-kinetic relationship for time-dependent binding. We show that the triazole motif slows the rate of formation for the final drug-target complex by up to 3 orders of magnitude. In addition, we identify a novel inhibitor with a residence time on InhA of 220 min, which is 3.5-fold longer than that of the INH-NAD adduct formed by the tuberculosis drug, isoniazid. This study provides a clear example in which the lifetime of the drug-target complex is controlled by interactions in the transition state for inhibitor binding rather than the ground state of the enzyme-inhibitor complex, and demonstrates the important role that on-rates can play in drug-target residence time.


Assuntos
Inibinas/antagonistas & inibidores , Termodinâmica , Triazóis/farmacologia , Cristalografia por Raios X , Humanos , Inibinas/metabolismo , Cinética , Modelos Moleculares , Estrutura Molecular , Fatores de Tempo , Triazóis/química
5.
Biochemistry ; 56(13): 1865-1878, 2017 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-28225601

RESUMO

There is growing awareness of the link between drug-target residence time and in vivo drug activity, and there are increasing efforts to determine the molecular factors that control the lifetime of a drug-target complex. Rational alterations in the drug-target residence time require knowledge of both the ground and transition states on the inhibition reaction coordinate, and we have determined the structure-kinetic relationship for 22 ethyl- or hexyl-substituted diphenyl ethers that are slow-binding inhibitors of bpFabI1, the enoyl-ACP reductase FabI1 from Burkholderia pseudomallei. Analysis of enzyme inhibition using a two-dimensional kinetic map demonstrates that the ethyl and hexyl diphenyl ethers fall into two distinct clusters. Modifications to the ethyl diphenyl ether B ring result in changes to both on and off rates, where residence times of up to ∼700 min (∼11 h) are achieved by either ground state stabilization (PT444) or transition state destabilization (slower on rate) (PT404). By contrast, modifications to the hexyl diphenyl ether B ring result in residence times of 300 min (∼5 h) through changes in only ground state stabilization (PT119). Structural analysis of nine enzyme:inhibitor complexes reveals that the variation in structure-kinetic relationships can be rationalized by structural rearrangements of bpFabI1 and subtle changes to the orientation of the inhibitor in the binding pocket. Finally, we demonstrate that three compounds with residence times on bpFabI1 from 118 min (∼2 h) to 670 min (∼11 h) have in vivo efficacy in an acute B. pseudomallei murine infection model using the virulent B. pseudomallei strain Bp400.


Assuntos
Antibacterianos/química , Proteínas de Bactérias/antagonistas & inibidores , Burkholderia pseudomallei/efeitos dos fármacos , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/antagonistas & inibidores , Inibidores Enzimáticos/química , Melioidose/dietoterapia , Éteres Fenílicos/química , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Burkholderia pseudomallei/enzimologia , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/crescimento & desenvolvimento , Contagem de Colônia Microbiana , Cristalografia por Raios X , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/genética , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Expressão Gênica , Cinética , Pulmão/efeitos dos fármacos , Pulmão/microbiologia , Melioidose/tratamento farmacológico , Melioidose/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Simulação de Dinâmica Molecular , Éteres Fenílicos/farmacologia , Ligação Proteica , Estrutura Secundária de Proteína , Baço/efeitos dos fármacos , Baço/microbiologia , Relação Estrutura-Atividade
6.
J Biol Chem ; 289(23): 15987-6005, 2014 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-24739388

RESUMO

Determining the molecular basis for target selectivity is of particular importance in drug discovery. The ideal antibiotic should be active against a broad spectrum of pathogenic organisms with a minimal effect on human targets. CG400549, a Staphylococcus-specific 2-pyridone compound that inhibits the enoyl-acyl carrier protein reductase (FabI), has recently been shown to possess human efficacy for the treatment of methicillin-resistant Staphylococcus aureus infections, which constitute a serious threat to human health. In this study, we solved the structures of three different FabI homologues in complex with several pyridone inhibitors, including CG400549. Based on these structures, we rationalize the 65-fold reduced affinity of CG400549 toward Escherichia coli versus S. aureus FabI and implement concepts to improve the spectrum of antibacterial activity. The identification of different conformational states along the reaction coordinate of the enzymatic hydride transfer provides an elegant visual depiction of the relationship between catalysis and inhibition, which facilitates rational inhibitor design. Ultimately, we developed the novel 4-pyridone-based FabI inhibitor PT166 that retained favorable pharmacokinetics and efficacy in a mouse model of S. aureus infection with extended activity against Gram-negative and mycobacterial organisms.


Assuntos
Antibacterianos/farmacologia , Desenho de Fármacos , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Piridonas/farmacologia , Animais , Antibacterianos/química , Antibacterianos/farmacocinética , Sequência de Bases , Cristalografia por Raios X , Primers do DNA , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Feminino , Camundongos , Camundongos Endogâmicos ICR , Testes de Sensibilidade Microbiana , Estrutura Molecular , Reação em Cadeia da Polimerase , Piridonas/química , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento
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