RESUMO
A lead generation and optimization program delivered the highly selective and potent CatC inhibitor 10 as an in vivo tool compound and potential development candidate. Structural studies were undertaken to generate SAR understanding.
Assuntos
Catepsina C/antagonistas & inibidores , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacologia , Humanos , Indicadores e Reagentes , Modelos Moleculares , Conformação Molecular , Proteínas Recombinantes/química , Espectrometria de Fluorescência , Relação Estrutura-Atividade , Especificidade por Substrato , Difração de Raios XRESUMO
Dipeptidyl peptidase 1 (DPP1) (EC 3.4.14.1; also known as cathepsin C, cathepsin J, dipeptidyl aminopeptidase, and dipeptidyl aminotransferase) is a lysosomal cysteinyl protease of the papain family involved in the intracellular degradation of proteins. Isolated enzyme assays for DPP1 activity using a variety of synthetic substrates such as dipeptide or peptide linked to amino-methyl-coumarin (AMC) or other fluorophores are well established. There is, however, no report of a simple whole-cell-based assay for measuring lysosomal DPP1 activity other than the use of flow cytometry (fluorescence-activated cell sorting) or the use of invasive activity-based probes or the production of physiological products such as neutrophil elastase. The authors investigated a number of DPP1 fluorogenic substrates that have the potential to access the lysosome and enable the measurement of DPP1 enzyme activity in situ. They describe the development and evaluation of a simple noninvasive fluorescence assay for measuring DPP1 activity in fresh or cryopreserved human THP-1 cells using the substrate H-Gly-Phe-AFC (amino-fluoro-coumarin). This cell-based fluorescence assay can be performed in a 96-well plate format and is ideally suited for determining the cell potency of potential DPP1 enzyme inhibitors.
