RESUMO
Through a phenotypic high-throughput screen using a serum response element luciferase promoter, we identified a novel 5-aryl-1,3,4-oxadiazol-2-ylthiopropionic acid lead inhibitor of Rho/myocardin-related transcription factor (MRTF)/serum response factor (SRF)-mediated gene transcription with good potency (IC50 = 180 nM). We were able to rapidly improve the cellular potency by 5 orders of magnitude guided by sharply defined and synergistic SAR. The remarkable potency and depth of the SAR, as well as the relatively low molecular weight of the series, suggests, but does not prove, that binding to the unknown molecular target may be occurring through a covalent mechanism. The series nevertheless has no observable cytotoxicity up to 100 µM. Ensuing pharmacokinetic optimization resulted in the development of two potent and orally bioavailable anti-fibrotic agents that were capable of dose-dependently reducing connective tissue growth factor gene expression in vitro as well as significantly reducing the development of bleomycin-induced dermal fibrosis in mice in vivo.
Assuntos
Ácidos Carboxílicos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Fibrose/tratamento farmacológico , Oxidiazóis/uso terapêutico , Fator de Resposta Sérica/antagonistas & inibidores , Transativadores/antagonistas & inibidores , Animais , Ácidos Carboxílicos/síntese química , Ácidos Carboxílicos/farmacocinética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Feminino , Fibrose/patologia , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/efeitos dos fármacos , Estrutura Molecular , Oxidiazóis/síntese química , Oxidiazóis/farmacocinética , Escleroderma Sistêmico/tratamento farmacológico , Escleroderma Sistêmico/patologia , Transdução de Sinais/efeitos dos fármacos , Pele/patologia , Relação Estrutura-Atividade , Transcrição Gênica/efeitos dos fármacos , Proteínas rho de Ligação ao GTP/antagonistas & inibidoresRESUMO
The Eph receptor tyrosine kinases mediate juxtacrine signals by interacting "in trans" with ligands anchored to the surface of neighboring cells via a GPI-anchor (ephrin-As) or a transmembrane segment (ephrin-Bs), which leads to receptor clustering and increased kinase activity. Additionally, soluble forms of the ephrin-A ligands released from the cell surface by matrix metalloproteases can also activate EphA receptor signaling. Besides these trans interactions, recent studies have revealed that Eph receptors and ephrins coexpressed in neurons can also engage in lateral "cis" associations that attenuate receptor activation by ephrins in trans with critical functional consequences. Despite the importance of the Eph/ephrin system in tumorigenesis, Eph receptor-ephrin cis interactions have not been previously investigated in cancer cells. Here we show that in cancer cells, coexpressed ephrin-A3 can inhibit the ability of EphA2 and EphA3 to bind ephrins in trans and become activated, while ephrin-B2 can inhibit not only EphB4 but also EphA3. The cis inhibition of EphA3 by ephrin-B2 implies that in some cases ephrins that cannot activate a particular Eph receptor in trans can nevertheless inhibit its signaling ability through cis association. We also found that an EphA3 mutation identified in lung cancer enhances cis interaction with ephrin-A3. These results suggest a novel mechanism that may contribute to cancer pathogenesis by attenuating the tumor suppressing effects of Eph receptor signaling pathways activated by ephrins in trans.