RESUMEN
Inhibitors of mitochondrial branched chain aminotransferase (BCATm), identified using fragment screening, are described. This was carried out using a combination of STD-NMR, thermal melt (Tm), and biochemical assays to identify compounds that bound to BCATm, which were subsequently progressed to X-ray crystallography, where a number of exemplars showed significant diversity in their binding modes. The hits identified were supplemented by searching and screening of additional analogues, which enabled the gathering of further X-ray data where the original hits had not produced liganded structures. The fragment hits were optimized using structure-based design, with some transfer of information between series, which enabled the identification of ligand efficient lead molecules with micromolar levels of inhibition, cellular activity, and good solubility.
Asunto(s)
Mitocondrias/enzimología , Transaminasas/antagonistas & inhibidores , Adipocitos/efectos de los fármacos , Adipocitos/enzimología , Cristalografía por Rayos X , Ensayos Analíticos de Alto Rendimiento , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Unión Proteica , Relación Estructura-ActividadRESUMEN
The hybridization of hits, identified by complementary fragment and high throughput screens, enabled the discovery of the first series of potent inhibitors of mitochondrial branched-chain aminotransferase (BCATm) based on a 2-benzylamino-pyrazolo[1,5-a]pyrimidinone-3-carbonitrile template. Structure-guided growth enabled rapid optimization of potency with maintenance of ligand efficiency, while the focus on physicochemical properties delivered compounds with excellent pharmacokinetic exposure that enabled a proof of concept experiment in mice. Oral administration of 2-((4-chloro-2,6-difluorobenzyl)amino)-7-oxo-5-propyl-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile 61 significantly raised the circulating levels of the branched-chain amino acids leucine, isoleucine, and valine in this acute study.
Asunto(s)
Proteínas Mitocondriales/antagonistas & inhibidores , Pirazoles/química , Pirimidinonas/química , Transaminasas/antagonistas & inhibidores , Adipocitos/efectos de los fármacos , Adipocitos/enzimología , Animales , Cristalografía por Rayos X , Humanos , Isoleucina/sangre , Leucina/sangre , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Modelos Moleculares , Pirazoles/síntesis química , Pirazoles/farmacología , Pirimidinonas/síntesis química , Pirimidinonas/farmacología , Relación Estructura-Actividad , Transaminasas/química , Valina/sangreRESUMEN
[reaction: see text] In this, the first of two Letters, we describe how a P3/P4 urea linking unit was used to greatly enhance the biochemical and replicon potency of inhibitors based upon the pyrrolidine-5,5-trans-lactam template. Compound 7b demonstrated a 100 nM IC(50) in the replicon cell-based surrogate HCV assay.
Asunto(s)
Antivirales/síntesis química , Hepacivirus/enzimología , Lactamas/química , Inhibidores de Proteasas/síntesis química , Pirrolidinas/química , Urea/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Antivirales/química , Antivirales/farmacología , Cristalografía por Rayos X , Hepacivirus/efectos de los fármacos , Hepacivirus/fisiología , Concentración 50 Inhibidora , Conformación Molecular , Estructura Molecular , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
[reaction: see text] In this, the second of two letters, we describe the elaboration of the pyrrolidine-5,5-trans-lactam template to delineate the requirements for optimal substitution of the pyrrolidine and lactam nitrogen atoms. Central to the strategy is the use of rapid iterative synthesis in conjunction with X-ray crystal structure determination of ligand-protein complexes.