RESUMO
A HTS campaign identified compound 1, an excellent hit-like molecule to initiate medicinal chemistry efforts to optimize a dual ROCK1 and ROCK2 inhibitor. Substitution (2-Cl, 2-NH2, 2-F, 3-F) of the pyridine hinge binding motif or replacement with pyrimidine afforded compounds with a clean CYP inhibition profile. Cocrystal structures of an early lead compound were obtained in PKA, ROCK1, and ROCK2. This provided critical structural information for medicinal chemistry to drive compound design. The structural data indicated the preferred configuration at the central benzylic carbon would be ( R), and application of this information to compound design resulted in compound 16. This compound was shown to be a potent and selective dual ROCK inhibitor in both enzyme and cell assays and efficacious in the retinal nerve fiber layer model after oral dosing. This tool compound has been made available through the AbbVie Compound Toolbox. Finally, the cocrystal structures also identified that aspartic acid residues 176 and 218 in ROCK2, which are glutamic acids in PKA, could be targeted as residues to drive both potency and kinome selectivity. Introduction of a piperidin-3-ylmethanamine group to the compound series resulted in compound 58, a potent and selective dual ROCK inhibitor with excellent predicted drug-like properties.
Assuntos
Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Administração Oral , Animais , Disponibilidade Biológica , Cristalografia por Raios X , Inibidores do Citocromo P-450 CYP2C9/química , Inibidores do Citocromo P-450 CYP2C9/farmacologia , Inibidores do Citocromo P-450 CYP3A/química , Inibidores do Citocromo P-450 CYP3A/farmacologia , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Meia-Vida , Humanos , Camundongos Endogâmicos C57BL , Traumatismos do Nervo Óptico/tratamento farmacológico , Traumatismos do Nervo Óptico/patologia , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Quinases Associadas a rho/químicaRESUMO
A new class of checkpoint kinase 1 (CHK-1) inhibitors bearing a 1,4-dihydroindeno[1,2-c]pyrazole core was developed after initial hits from high throughput screening. The efficient hit-to-lead process was facilitated by X-ray crystallography and led to potent inhibitors (<10nM) against CHK-1. X-ray co-crystal structures of bound inhibitors demonstrated that two sub-series of this class of compounds, exemplified by 21 and 41, exhibit distinctive hydrogen bonding patterns in the specificity pocket of the active site. Two compounds, 41 and 43, were capable of potentiating doxorubicin and camptothecin, both DNA-damaging agents, in cell proliferation assays (MTS and soft agar assays) and abrogating G2/M checkpoint in a mechanism-based FACS assay.
Assuntos
Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Pirazóis/síntese química , Pirazóis/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Camptotecina/farmacologia , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quinase 1 do Ponto de Checagem , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Células HeLa , Humanos , Ligação de Hidrogênio , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Especificidade por SubstratoRESUMO
Kinase enzymes are involved in a vast array of biological processes associated with human disease; therefore, selective kinase inhibition by small molecules and therapeutic antibodies is an area of intense study. The authors show that drug candidates with immediate value for biological preclinical evaluation can be identified directly through ultra-efficient affinity screening of kinase enzymes and random compound mixtures. The screening process comprises sampling and trapping equilibrium binding between candidate ligands and protein in solution, followed by removal of unbound ligands via 3 rounds of ultrafiltration and direct identification of bound ligands by mass spectrometry. Evaluation of significant peaks is facilitated by automated integration and collation of the mass spectral data and import into custom software for analysis. One Chk1-selective ligand found by using this process is presented in detail. The compound is potent in both enzymatic and Chk1-dependent cellular assays, and specific contacts in the Chk1 active site are shown by X-ray crystallography.