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1.
Molecules ; 26(17)2021 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-34500777

RESUMO

Human neutrophil elastase (HNE) is a uniquely destructive serine protease with the ability to unleash a wave of proteolytic activity by destroying the inhibitors of other proteases. Although this phenomenon forms an important part of the innate immune response to invading pathogens, it is responsible for the collateral host tissue damage observed in chronic conditions such as chronic obstructive pulmonary disease (COPD), and in more acute disorders such as the lung injuries associated with COVID-19 infection. Previously, a combinatorially selected activity-based probe revealed an unexpected substrate preference for oxidised methionine, which suggests a link to oxidative pathogen clearance by neutrophils. Here we use oxidised model substrates and inhibitors to confirm this observation and to show that neutrophil elastase is specifically selective for the di-oxygenated methionine sulfone rather than the mono-oxygenated methionine sulfoxide. We also posit a critical role for ordered solvent in the mechanism of HNE discrimination between the two oxidised forms methionine residue. Preference for the sulfone form of oxidised methionine is especially significant. While both host and pathogens have the ability to reduce methionine sulfoxide back to methionine, a biological pathway to reduce methionine sulfone is not known. Taken together, these data suggest that the oxidative activity of neutrophils may create rapidly cleaved elastase "super substrates" that directly damage tissue, while initiating a cycle of neutrophil oxidation that increases elastase tissue damage and further neutrophil recruitment.


Assuntos
Imunidade Inata , Elastase de Leucócito/metabolismo , Metionina/análogos & derivados , Neutrófilos/imunologia , Biocatálise , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Domínio Catalítico/genética , Ensaios Enzimáticos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Elastase de Leucócito/antagonistas & inibidores , Elastase de Leucócito/genética , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Metionina/metabolismo , Simulação de Dinâmica Molecular , Infiltração de Neutrófilos , Neutrófilos/enzimologia , Oxirredução/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/patologia , SARS-CoV-2/imunologia , Especificidade por Substrato/imunologia
2.
Mar Drugs ; 17(12)2019 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-31842369

RESUMO

Serine proteases play pivotal roles in normal physiology and a spectrum of patho-physiological processes. Accordingly, there is considerable interest in the discovery and design of potent serine protease inhibitors for therapeutic applications. This led to concerted efforts to discover versatile and robust molecular scaffolds for inhibitor design. This investigation is a bioprospecting study that aims to isolate and identify protease inhibitors from the cnidarian Actinia tenebrosa. The study isolated two Kunitz-type protease inhibitors with very similar sequences but quite divergent inhibitory potencies when assayed against bovine trypsin, chymostrypsin, and a selection of human sequence-related peptidases. Homology modeling and molecular dynamics simulations of these inhibitors in complex with their targets were carried out and, collectively, these methodologies enabled the definition of a versatile scaffold for inhibitor design. Thermal denaturation studies showed that the inhibitors were remarkably robust. To gain a fine-grained map of the residues responsible for this stability, we conducted in silico alanine scanning and quantified individual residue contributions to the inhibitor's stability. Sequences of these inhibitors were then used to search for Kunitz homologs in an A. tenebrosa transcriptome library, resulting in the discovery of a further 14 related sequences. Consensus analysis of these variants identified a rich molecular diversity of Kunitz domains and expanded the palette of potential residue substitutions for rational inhibitor design using this domain.


Assuntos
Cnidários/classificação , Serina Proteases/efeitos dos fármacos , Inibidores de Serino Proteinase/farmacologia , Animais , Bovinos , Quimotripsina/antagonistas & inibidores , Quimotripsina/metabolismo , Simulação por Computador , Humanos , Simulação de Dinâmica Molecular , Serina Proteases/metabolismo , Inibidores de Serino Proteinase/isolamento & purificação , Tripsina/efeitos dos fármacos , Tripsina/metabolismo , Inibidores da Tripsina/isolamento & purificação , Inibidores da Tripsina/farmacologia
3.
PLoS One ; 14(1): e0210842, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30668585

RESUMO

Engagement of an extended ß-sheet is a common substrate/inhibitor interaction at the active site of serine proteases and is an important feature of Laskowski mechanism inhibitors that present a substrate-like loop to a target protease. This loop is cleaved but subsequently relegated forming a stable inhibitor/protease complex. Laskowski inhibitors are ubiquitous in nature and are used extensively in serine protease inhibitor design. However, most studies concentrate on introducing new sidechain interactions rather than the direct contributions of the substrate-like ß-sheet to enzyme inhibition. Here we report the crystal structure of an simplified ß-sheet inhibitory motif within the Sunflower Trypsin Inhibitor (SFTI) in complex with trypsin. We show that the intramolecular hydrogen bond network of this SFTI variant (SFTI-TCTR) engages the inhibitor sidechains that would normally interact with a target protease, giving mainchain interactions a more prominent role in complex formation. Despite having reduced sidechain interactions, this SFTI variant is remarkably potent and inhibits a diverse range of serine proteases. Crystal structural analysis and molecular modelling of SFTI-TCTR complexes again indicates an interface dominated by ß-sheet interactions, highlighting the importance of this motif and the adaptability of SFTI as a scaffold for inhibitor design.


Assuntos
Inibidores de Serino Proteinase/química , Inibidores de Serino Proteinase/farmacologia , Tripsina/química , Motivos de Aminoácidos , Animais , Bovinos , Cristalografia por Raios X , Helianthus/química , Ligação de Hidrogênio , Modelos Moleculares , Simulação de Dinâmica Molecular , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Proteínas de Plantas/química , Proteínas de Plantas/farmacologia , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Eletricidade Estática , Inibidores da Tripsina/química , Inibidores da Tripsina/farmacologia
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