Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 3 de 3
Filtrar
Mais filtros

Base de dados
Tipo de documento
Intervalo de ano de publicação
1.
J Chem Inf Model ; 64(10): 3977-3991, 2024 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-38727192

RESUMO

The worldwide spread of the metallo-ß-lactamases (MBL), especially New Delhi metallo-ß-lactamase-1 (NDM-1), is threatening the efficacy of ß-lactams, which are the most potent and prescribed class of antibiotics in the clinic. Currently, FDA-approved MBL inhibitors are lacking in the clinic even though many strategies have been used in inhibitor development, including quantitative high-throughput screening (qHTS), fragment-based drug discovery (FBDD), and molecular docking. Herein, a machine learning-based prediction tool is described, which was generated using results from HTS of a large chemical library and previously published inhibition data. The prediction tool was then used for virtual screening of the NIH Genesis library, which was subsequently screened using qHTS. A novel MBL inhibitor was identified and shown to lower minimum inhibitory concentrations (MICs) of Meropenem for a panel of E. coli and K. pneumoniae clinical isolates expressing NDM-1. The mechanism of inhibition of this novel scaffold was probed utilizing equilibrium dialyses with metal analyses, native state electrospray ionization mass spectrometry, UV-vis spectrophotometry, and molecular docking. The uncovered inhibitor, compound 72922413, was shown to be 9-hydroxy-3-[(5-hydroxy-1-oxa-9-azaspiro[5.5]undec-9-yl)carbonyl]-4H-pyrido[1,2-a]pyrimidin-4-one.


Assuntos
Aprendizado de Máquina , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Inibidores de beta-Lactamases , beta-Lactamases , beta-Lactamases/metabolismo , beta-Lactamases/química , Inibidores de beta-Lactamases/farmacologia , Inibidores de beta-Lactamases/química , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/enzimologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Ensaios de Triagem em Larga Escala
2.
J Am Chem Soc ; 143(22): 8314-8323, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34038127

RESUMO

New Delhi metallo-ß-lactamase (NDM) grants resistance to a broad spectrum of ß-lactam antibiotics, including last-resort carbapenems, and is emerging as a global antibiotic resistance threat. Limited zinc availability adversely impacts the ability of NDM-1 to provide resistance, but a number of clinical variants have emerged that are more resistant to zinc scarcity (e.g., NDM-15). To provide a novel tool to better study metal ion sequestration in host-pathogen interactions, we describe the development of a fluorescent probe that reports on the dynamic metalation state of NDM within Escherichia coli. The thiol-containing probe selectively coordinates the dizinc metal cluster of NDM and results in a 17-fold increase in fluorescence intensity. Reversible binding enables competition and time-dependent studies that reveal fluorescence changes used to detect enzyme localization, substrate and inhibitor engagement, and changes to metalation state through the imaging of live E. coli using confocal microscopy. NDM-1 is shown to be susceptible to demetalation by intracellular and extracellular metal chelators in a live-cell model of zinc dyshomeostasis, whereas the NDM-15 metalation state is shown to be more resistant to zinc flux. The development of this reversible turn-on fluorescent probe for the metalation state of NDM provides a new tool for monitoring the impact of metal ion sequestration by host defense mechanisms and for detecting inhibitor-target engagement during the development of therapeutics to counter this resistance determinant.


Assuntos
Quelantes/farmacologia , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/farmacologia , Compostos de Sulfidrila/farmacologia , Zinco/farmacologia , beta-Lactamases/metabolismo , Quelantes/química , Inibidores Enzimáticos/química , Escherichia coli/enzimologia , Corantes Fluorescentes/química , Estrutura Molecular , Compostos de Sulfidrila/química , Zinco/química
3.
Chem Sci ; 11(33): 8999-9010, 2020 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34123154

RESUMO

We use mass spectrometry (MS), under denaturing and non-denaturing solution conditions, along with ultraviolet photodissociation (UVPD) to characterize structural variations in New Delhi metallo-ß-lactamase (NDM) upon perturbation by ligands or mutation. Mapping changes in the abundances and distributions of fragment ions enables sensitive detection of structural alterations throughout the protein. Binding of three covalent inhibitors was characterized: a pentafluorphenyl ester, an O-aryloxycarbonyl hydroxamate, and ebselen. The first two inhibitors modify Lys211 and maintain dizinc binding, although the pentafluorophenyl ester is not selective (Lys214 and Lys216 are also modified). Ebselen reacts with the sole Cys (Cys208) and ejects Zn2 from the active site. For each inhibitor, native UVPD-MS enabled simultaneous detection of the closing of a substrate-binding beta-hairpin loop, identification of covalently-modified residue(s), reporting of the metalation state of the enzyme, and in the case of ebselen, observation of the induction of partial disorder in the C-terminus of the protein. Owing to the ability of native UVPD-MS to track structural changes and metalation state with high sensitivity, we further used this method to evaluate the impact of mutations found in NDM clinical variants. Changes introduced by NDM-4 (M154L) and NDM-6 (A233V) are revealed to propagate through separate networks of interactions to direct zinc ligands, and the combination of these two mutations in NDM-15 (M154L, A233V) results in additive as well as additional structural changes. Insight from UVPD-MS helps to elucidate how distant mutations impact zinc affinity in the evolution of this antibiotic resistance determinant. UVPD-MS is a powerful tool capable of simultaneous reporting of ligand binding, conformational changes and metalation state of NDM, revealing structural aspects of ligand recognition and clinical variants that have proven difficult to probe.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA