RESUMO
Inhibitors of the Hsp90 molecular chaperone are showing considerable promise as potential chemotherapeutic agents for cancer. Here, we describe the structure-based design, synthesis, structure-activity relationships and pharmacokinetics of potent small-molecule inhibitors of Hsp90 based on the 4,5-diarylisoxazole scaffold. Analogues from this series have high affinity for Hsp90, as measured in a fluorescence polarization (FP) competitive binding assay, and are active in cancer cell lines where they inhibit proliferation and exhibit a characteristic profile of depletion of oncogenic proteins and concomitant elevation of Hsp72. Compound 40f (VER-52296/NVP-AUY922) is potent in the Hsp90 FP binding assay (IC50 = 21 nM) and inhibits proliferation of various human cancer cell lines in vitro, with GI50 averaging 9 nM. Compound 40f is retained in tumors in vivo when administered i.p., as evaluated by cassette dosing in tumor-bearing mice. In a human colon cancer xenograft model, 40f inhibits tumor growth by approximately 50%.
Assuntos
Antineoplásicos/síntese química , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Isoxazóis/síntese química , Resorcinóis/síntese química , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Ligação Competitiva , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Polarização de Fluorescência , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Isoxazóis/farmacocinética , Isoxazóis/farmacologia , Camundongos , Camundongos Nus , Modelos Moleculares , Transplante de Neoplasias , Resorcinóis/farmacocinética , Resorcinóis/farmacologia , Relação Estrutura-Atividade , Transplante HeterólogoRESUMO
78 kDa glucose-regulated protein (Grp78) is a heat shock protein (HSP) involved in protein folding that plays a role in cancer cell proliferation. Binding of adenosine-derived inhibitors to Grp78 was characterized by surface plasmon resonance and isothermal titration calorimetry. The most potent compounds were 13 (VER-155008) with K(D) = 80 nM and 14 with K(D) = 60 nM. X-ray crystal structures of Grp78 bound to ATP, ADPnP, and adenosine derivative 10 revealed differences in the binding site between Grp78 and homologous proteins.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Furanos/síntese química , Proteínas de Choque Térmico/antagonistas & inibidores , Purinas/síntese química , Adenosina Trifosfatases/química , Trifosfato de Adenosina/química , Adenilil Imidodifosfato/química , Sítios de Ligação , Calorimetria , Cristalografia por Raios X , Chaperona BiP do Retículo Endoplasmático , Furanos/química , Proteínas de Choque Térmico HSC70/química , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico/química , Ligantes , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Purinas/química , Estereoisomerismo , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , TermodinâmicaRESUMO
BACKGROUND: Checkpoint kinase 1 and 2 (Chk1/Chk2), and the Aurora kinases play a critical role in the activation of the DNA damage response and mitotic spindle checkpoints. We have identified a novel inhibitor of these kinases and utilized this molecule to probe the functional interplay between these two checkpoints. PRINCIPAL FINDINGS: Fragment screening, structure guided design, and kinase cross screening resulted in the identification of a novel, potent small molecule kinase inhibitor (VER-150548) of Chk1 and Chk2 kinases with IC(50)s of 35 and 34 nM as well as the Aurora A and Aurora B kinases with IC(50)s of 101 and 38 nM. The structural rationale for this kinase specificity could be clearly elucidated through the X-ray crystal structure. In human carcinoma cells, VER-150548 induced reduplication and the accumulation of cells with >4N DNA content, inhibited histone H3 phosphorylation and ultimately gave way to cell death after 120 hour exposure; a phenotype consistent with cellular Aurora inhibition. In the presence of DNA damage induced by cytotoxic chemotherapeutic drugs, VER-150548 abrogated DNA damage induced cell cycle checkpoints. Abrogation of these checkpoints correlated with increased DNA damage and rapid cell death in p53 defective HT29 cells. In the presence of DNA damage, reduplication could not be observed. These observations are consistent with the Chk1 and Chk2 inhibitory activity of this molecule. CONCLUSIONS: In the presence of DNA damage, we suggest that VER-150548 abrogates the DNA damage induced checkpoints forcing cells to undergo a lethal mitosis. The timing of this premature cell death induced by Chk1 inhibition negates Aurora inhibition thereby preventing re-entry into the cell cycle and subsequent DNA reduplication. This novel kinase inhibitor therefore serves as a useful chemical probe to further understand the temporal relationship between cell cycle checkpoint pathways, chemotherapeutic agent induced DNA damage and cell death.
Assuntos
Ciclo Celular , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Aurora Quinase B , Aurora Quinases , Morte Celular/efeitos dos fármacos , Quinase 1 do Ponto de Checagem , Quinase do Ponto de Checagem 2 , Cristalografia por Raios X , Dano ao DNA , Citometria de Fluxo , Células HT29 , Humanos , Modelos Moleculares , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
The design and synthesis of novel adenosine-derived inhibitors of HSP70, guided by modeling and X-ray crystallographic structures of these compounds in complex with HSC70/BAG-1, is described. Examples exhibited submicromolar affinity for HSP70, were highly selective over HSP90, and some displayed potency against HCT116 cells. Exposure of compound 12 to HCT116 cells caused significant reduction in cellular levels of Raf-1 and Her2 at concentrations similar to that which caused cell growth arrest.
Assuntos
Adenosina/farmacologia , Desenho de Fármacos , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Adenosina/química , Linhagem Celular Tumoral , Cristalografia por Raios X , Imunoensaio de Fluorescência por Polarização , Humanos , Estrutura MolecularRESUMO
Inhibitors of the Hsp90 molecular chaperone are showing considerable promise as potential molecular therapeutic agents for the treatment of cancer. Here we describe novel 2-aminothieno[2,3-d]pyrimidine ATP competitive Hsp90 inhibitors, which were designed by combining structural elements of distinct low affinity hits generated from fragment-based and in silico screening exercises in concert with structural information from X-ray protein crystallography. Examples from this series have high affinity (IC50 = 50-100 nM) for Hsp90 as measured in a fluorescence polarization (FP) competitive binding assay and are active in human cancer cell lines where they inhibit cell proliferation and exhibit a characteristic profile of depletion of oncogenic proteins and concomitant elevation of Hsp72. Several examples (34a, 34d and 34i) caused tumor growth regression at well tolerated doses when administered orally in a human BT474 human breast cancer xenograft model.
Assuntos
Antineoplásicos/síntese química , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Pirimidinas/síntese química , Administração Oral , Animais , Antineoplásicos/farmacologia , Ligação Competitiva , Cristalografia por Raios X , Feminino , Polarização de Fluorescência , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pirimidinas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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
Crystallographic and modelling data, in conjunction with a medicinal chemistry template-hopping approach, led to the identification of a series of novel and potent inhibitors of human cyclin-dependent kinase 2 (CDK2), with selectivity over glycogen synthase kinase-3beta (GSK-3beta). One example had a CDK2 IC(50) of 120 nM and showed selectivity over GSK-3beta of 167-fold.