RESUMO
Transient treatment with small molecule CDK inhibitors is toxic to cancer cells and leads to depletion of anti-apoptotic proteins and Chk1, coupled with DNA damage and induction of apoptosis. Here we have examined, which of these phenomena are necessary for CDK inhibitors to have an anti-proliferative effect. We find that 24 hours treatment with either a primarily CDK2-specific, or a primarily CDK7/9-specific, antagonist eliminates proliferative potential even if apoptosis is blocked and the tendency of CDK inhibition to result in DNA damage is overcome by expression of recombinant Chk1. Loss of proliferative potential is correlated with irreversible suppression of biomarkers of cell cycle progression. CDK inhibitors dramatically reduced levels of the anti-apoptotic proteins, Mcl-1 and XIAP, but siRNA-mediated suppression of Mcl-1 and XIAP did not induce cell death in the osteosarcoma cells used in this study. Finally, we found that many literature CDK inhibitors do not effectively suppress the CDK/cyclin complexes responsible for cell cycle progression at the minimum doses required to block proliferation: some are only effective after a substantial delay and may act via inhibition of CDK7.
Assuntos
Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Quinase 1 do Ponto de Checagem , Quinases Ciclina-Dependentes/metabolismo , Dano ao DNA , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Oxazóis/farmacologia , Proteínas Quinases/biossíntese , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , RNA Interferente Pequeno , Tiazóis/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
Virtual screening against a pCDK2/cyclin A crystal structure led to the identification of a potent and novel CDK2 inhibitor, which exhibited an unusual mode of interaction with the kinase binding motif. With the aid of X-ray crystallography and modelling, a medicinal chemistry strategy was implemented to probe the interactions seen in the crystal structure and to establish SAR. A fragment-based approach was also considered but a different, more conventional, binding mode was observed. Compound selectivity against GSK-3beta was improved using a rational design strategy, with crystallographic verification of the CDK2 binding mode.
Assuntos
Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Cristalografia por Raios X , Desenho de Fármacos , Inibidores de Proteínas Quinases/químicaRESUMO
Inhibition of the Chk1 kinase by small molecules binding to its active site is a strategy of great therapeutic interest for oncology. We report how computational modelling predicted the binding mode of ligands of special interest to the Chk1 ATP site, for representatives of an indazole series and debromohymenialdisine. These binding modes were subsequently confirmed by X-ray crystallography. The binding mode of a potent indazole derivative involves non-conventional C-H...O and N-H...pi-aromatic interactions with the protein. These interactions are formed in a buried pocket at the periphery of the ATP-binding site, the importance of which has previously been overlooked for ligand design against Chk1. It is demonstrated that filling this pocket can confer ligands with dramatically enhanced affinity for Chk1. Structural arguments in conjunction with assay data explain why targeting this pocket is also advantageous for selective binding to Chk1. Structural overlays of known inhibitors complexed with Chk1 show that only the indazole series utilizes the pocket of interest. Therefore, the analysis presented here should prove helpful in guiding future structure-based ligand design efforts against Chk1.
Assuntos
Azepinas/química , Simulação por Computador , Desenho de Fármacos , Inibidores Enzimáticos/química , Indazóis/química , Proteínas Quinases/química , Pirróis/química , Trifosfato de Adenosina/metabolismo , Azepinas/metabolismo , Sítios de Ligação , Quinase 1 do Ponto de Checagem , Cristalografia por Raios X , Humanos , Indazóis/metabolismo , Ligantes , Modelos Moleculares , Ligação Proteica/efeitos dos fármacos , Proteínas Quinases/efeitos dos fármacos , Pirróis/metabolismo , Relação Estrutura-AtividadeRESUMO
We report the discovery, synthesis, and crystallographic binding mode of novel furanopyrimidine and pyrrolopyrimidine inhibitors of the Chk1 kinase, an oncology target. These inhibitors are synthetically tractable and inhibit Chk1 by competing for its ATP site. A chronological account allows an objective comparison of modeled compound docking modes to the subsequently obtained crystal structures. The comparison provides insights regarding the interpretation of modeling results, in relationship to the multiple reasonable docking modes which may be obtained in a kinase-ATP site. The crystal structures were used to guide medicinal chemistry efforts. This led to a thorough characterization of a pair of ligand-protein complexes which differ by a single hydrogen bond. An analysis indicates that this hydrogen bond is expected to contribute a fraction of the 10-fold change in binding affinity, adding a valuable observation to the debate about the energetic role of hydrogen bonding in molecular recognition.