RESUMO
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.
Assuntos
Mitocôndrias/enzimologia , Transaminases/antagonistas & inibidores , Adipócitos/efeitos dos fármacos , Adipócitos/enzimologia , Cristalografia por Raios X , Ensaios de Triagem em Larga Escala , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Proteínas Mitocondriais/antagonistas & inibidores , Pirazóis/química , Pirimidinonas/química , Transaminases/antagonistas & inibidores , Adipócitos/efeitos dos fármacos , Adipócitos/enzimologia , Animais , Cristalografia por Raios X , Humanos , Isoleucina/sangue , Leucina/sangue , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Moleculares , Pirazóis/síntese química , Pirazóis/farmacologia , Pirimidinonas/síntese química , Pirimidinonas/farmacologia , Relação Estrutura-Atividade , Transaminases/química , Valina/sangueRESUMO
[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.
Assuntos
Antivirais/síntese química , Hepacivirus/enzimologia , Lactamas/química , Inibidores de Proteases/síntese química , Pirrolidinas/química , Ureia/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/química , Antivirais/farmacologia , Cristalografia por Raios X , Hepacivirus/efeitos dos fármacos , Hepacivirus/fisiologia , Concentração Inibidora 50 , Conformação Molecular , Estrutura Molecular , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
[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.