RESUMEN
A novel series of inhibitors for mitogen activated protein kinase-activated protein kinase 2 (MK-2) are reported. These squarate based inhibitors were identified via a high-throughput screen. An MK2 co-structure with the starting ligand was obtained and a structure based approach was followed to optimize potency and selectivity.
Asunto(s)
Ciclobutanos/química , Ciclobutanos/farmacología , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Sitios de Unión , Línea Celular Tumoral , Diseño de Fármacos , Humanos , Concentración 50 Inhibidora , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Modelos Moleculares , Monocitos/citología , Transporte de Proteínas , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Considerable effort exists within drug discovery to develop novel compounds to improve the underlying metabolic defects in type 2 diabetes. One approach is focused on inhibition of the tyrosine phosphatase, PTP1B, an important negative regulator of both insulin and leptin signaling. Historically, tyrosine phosphatase assays have used either small organic phosphates or, alternatively, phosphorylated peptides from the target proteins themselves. In characterizing inhibitors of PTP1B, measuring turnover of small organic phosphates is limited to evaluation of compounds that bind the active site itself. Peptide substrates allow identification of additional subsets of inhibitors (e.g., those that bind the second aryl-phosphate site), but assays of peptide turnover often involve detection steps that then limit full kinetic evaluation of inhibitors. Here we use a polyclonal antibody specific for the phosphorylated insulin receptor to allow much more sensitive detection of peptide phosphorylation. This kinetically robust enzyme-linked immunosorbent assay (ELISA) gives k(cat) and K(m) values for a phosphorylated insulin receptor peptide consistent with values determined by a continuous fluorescence-based assay. Furthermore, IC50 values determined for well-behaved active site inhibitors agree well with values determined for p-nitrophenyl phosphate cleavage. This assay permits full characterization of a larger subset of inhibitors as drug candidates for this promising target.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas Tirosina Fosfatasas/metabolismo , Receptor de Insulina/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Concentración 50 Inhibidora , Estructura Molecular , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
The following account describes our systematic effort to replace one of the carboxylate groups of our diacid thiophene PTP1B inhibitors. Active hits were validated using enzymatic assays before pursuing efforts to improve the potency. Only when the C2 carboxylic acid was replaced with another ionizable functional group was reversible and competitive inhibition retained. Use of a tetrazole ring or 1,2,5-thiadiazolidine-3-one-1,1-dioxide as a carboxylate mimetic led to the discovery of two unique starting series that showed improved permeability (PAMPA) and potency of the order of 300nM. The SAR from these efforts underscores some of the major challenges in developing small molecule inhibitors for PTP1B.