RESUMO
The main protease (3CLp) of the SARS-CoV-2, the causative agent for the COVID-19 pandemic, is one of the main targets for drug development. To be active, 3CLp relies on a complex interplay between dimerization, active site flexibility, and allosteric regulation. The deciphering of these mechanisms is a crucial step to enable the search for inhibitors. In this context, using NMR spectroscopy, we studied the conformation of dimeric 3CLp from the SARS-CoV-2 and monitored ligand binding, based on NMR signal assignments. We performed a fragment-based screening that led to the identification of 38 fragment hits. Their binding sites showed three hotspots on 3CLp, two in the substrate binding pocket and one at the dimer interface. F01 is a non-covalent inhibitor of the 3CLp and has antiviral activity in SARS-CoV-2 infected cells. This study sheds light on the complex structure-function relationships of 3CLp and constitutes a strong basis to assist in developing potent 3CLp inhibitors.
Assuntos
Antivirais/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , SARS-CoV-2/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Antivirais/química , Sítios de Ligação , Chlorocebus aethiops , Proteases 3C de Coronavírus/química , Inibidores de Cisteína Proteinase/química , Avaliação Pré-Clínica de Medicamentos , Testes de Sensibilidade Microbiana , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Multimerização Proteica , SARS-CoV-2/química , Bibliotecas de Moléculas Pequenas/química , Células VeroRESUMO
Insulin-degrading enzyme, IDE, is a metalloprotease implicated in the metabolism of key peptides such as insulin, glucagon, ß-amyloid peptide. Recent studies have pointed out its broader role in the cell physiology. In order to identify new drug-like inhibitors of IDE with optimal pharmacokinetic properties to probe its multiple roles, we ran a high-throughput drug repurposing screening. Ebselen, cefmetazole and rabeprazole were identified as reversible inhibitors of IDE. Ebselen is the most potent inhibitor (IC50(insulin)â¯=â¯14â¯nM). The molecular mode of action of ebselen was investigated by biophysical methods. We show that ebselen induces the disorder of the IDE catalytic cleft, which significantly differs from the previously reported IDE inhibitors. IDE inhibition by ebselen can explain some of its reported activities in metabolism as well as in neuroprotection.
Assuntos
Azóis/farmacologia , Reposicionamento de Medicamentos , Inibidores Enzimáticos/farmacologia , Insulisina/antagonistas & inibidores , Compostos Organosselênicos/farmacologia , Azóis/química , Biocatálise , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Ensaios de Triagem em Larga Escala , Humanos , Insulisina/metabolismo , Isoindóis , Estrutura Molecular , Compostos Organosselênicos/química , Relação Estrutura-AtividadeRESUMO
Insulin-degrading enzyme (IDE) is a protease that cleaves insulin and other bioactive peptides such as amyloid-ß. Knockout and genetic studies have linked IDE to Alzheimer's disease and type-2 diabetes. As the major insulin-degrading protease, IDE is a candidate drug target in diabetes. Here we have used kinetic target-guided synthesis to design the first catalytic site inhibitor of IDE suitable for in vivo studies (BDM44768). Crystallographic and small angle X-ray scattering analyses show that it locks IDE in a closed conformation. Among a panel of metalloproteases, BDM44768 selectively inhibits IDE. Acute treatment of mice with BDM44768 increases insulin signalling and surprisingly impairs glucose tolerance in an IDE-dependent manner. These results confirm that IDE is involved in pathways that modulate short-term glucose homeostasis, but casts doubt on the general usefulness of the inhibition of IDE catalytic activity to treat diabetes.
Assuntos
Ácidos Hidroxâmicos/síntese química , Insulisina/antagonistas & inibidores , Triazóis/síntese química , Animais , Células CACO-2 , Domínio Catalítico , Diabetes Mellitus/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Teste de Tolerância a Glucose , Humanos , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microssomos Hepáticos , Terapia de Alvo Molecular , Distribuição Aleatória , Relação Estrutura-Atividade , Triazóis/farmacologia , Triazóis/uso terapêuticoRESUMO
In this Letter we describe the design, synthesis, screening, and optimization of a new family of ADAMTS-5 inhibitors. These inhibitors display an original 1,2,4-triazole-3-thiol scaffold as a putative zinc binding-group. In vitro results are rationalized by in silico docking of the compounds in ADAMTS-5's crystal structure.
Assuntos
Proteínas ADAM/antagonistas & inibidores , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacologia , Triazóis/síntese química , Triazóis/farmacologia , Proteína ADAMTS5 , Sítios de Ligação , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos , Humanos , Indicadores e Reagentes , Modelos Moleculares , Osteoartrite/tratamento farmacológico , Inibidores de Proteases/química , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Triazóis/química , Difração de Raios XRESUMO
Prolyl endopeptidases (PEPs) have been found in numerous species. Inhibitors of human enzyme could correct cognitive deficits in Alzheimer patients while inhibition of Trypanosoma cruzi PEP could prevent invasion phase in Chagas disease. A structure-activity relationship study carried out in a tetrahydroisoquinoline series allowed to obtain potent competitive inhibitors superior to SUAM-1221. Besides, inhibitors expected to act according to an irreversible mechanism revealed to be superior to JPT-4819, for applications linked to human enzyme inhibition.