RESUMO
Dihydroorotate dehydrogenase (DHODH) enzymatic activity impacts many aspects critical to cell proliferation and survival. Recently, DHODH has been identified as a target for acute myeloid differentiation therapy. In preclinical models of AML, the DHODH inhibitor Brequinar (BRQ) demonstrated potent anti-leukemic activity. Herein we describe a carboxylic acid isostere study of Brequinar which revealed a more potent non-carboxylic acid derivative with improved cellular potency and good pharmacokinetic properties.
Assuntos
Antineoplásicos/farmacologia , Compostos de Bifenilo/farmacologia , Ácidos Carboxílicos/farmacologia , Inibidores Enzimáticos/farmacologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Animais , Antineoplásicos/química , Compostos de Bifenilo/química , Ácidos Carboxílicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Di-Hidro-Orotato Desidrogenase , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Humanos , Camundongos , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Relação Estrutura-AtividadeRESUMO
We report the results of a series of chemical, EPR, ENDOR, and HYSCORE spectroscopic investigations of the mechanism of action (and inhibition) of GcpE, E-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate (HMBPP) synthase, also known as IspG, an Fe(4)S(4) cluster-containing protein. We find that the epoxide of HMBPP when reduced by GcpE generates the same transient EPR species as observed on addition of the substrate, 2-C-methyl-D-erythritol-2, 4-cyclo-diphosphate. ENDOR and HYSCORE spectra of these transient species (using (2)H, (13)C and (17)O labeled samples) indicate formation of an Fe-C-H containing organometallic intermediate, most likely a ferraoxetane. This is then rapidly reduced to a ferracyclopropane in which the HMBPP product forms an eta(2)-alkenyl pi- (or pi/sigma) complex with the 4th Fe in the Fe(4)S(4) cluster, and a similar "metallacycle" also forms between isopentenyl diphosphate (IPP) and GcpE. Based on this metallacycle concept, we show that an alkyne (propargyl) diphosphate is a good (K(i) approximately 300 nM) GcpE inhibitor, and supported again by EPR and ENDOR results (a (13)C hyperfine coupling of approximately 7 MHz), as well as literature precedent, we propose that the alkyne forms another pi/sigma metallacycle, an eta(2)-alkynyl, or ferracyclopropene. Overall, the results are of broad general interest because they provide new mechanistic insights into GcpE catalysis and inhibition, with organometallic bond formation playing, in both cases, a key role.