RESUMEN
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.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Furanos/síntesis química , Proteínas de Choque Térmico/antagonistas & inhibidores , Purinas/síntesis química , Adenosina Trifosfatasas/química , Adenosina Trifosfato/química , Adenilil Imidodifosfato/química , Sitios de Unión , Calorimetría , Cristalografía por Rayos X , Chaperón BiP del Retículo Endoplásmico , Furanos/química , Proteínas del Choque Térmico HSC70/química , Proteínas HSP70 de Choque Térmico/química , Proteínas de Choque Térmico/química , Ligandos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/química , Purinas/química , Estereoisomerismo , Relación Estructura-Actividad , Resonancia por Plasmón de Superficie , TermodinámicaRESUMEN
PURPOSE: The anti-apoptotic function of the 70 kDa family of heat shock proteins and their role in cancer is well documented. Dual targeting of Hsc70 and Hsp70 with siRNA induces proteasome-dependent degradation of Hsp90 client proteins and extensive tumor specific apoptosis as well as the potentiation of tumor cell apoptosis following pharmacological Hsp90 inhibition. METHODS: We have previously described the discovery and synthesis of novel adenosine-derived inhibitors of the 70 kDa family of heat shock proteins; the first inhibitors described to target the ATPase binding domain. The in vitro activity of VER-155008 was evaluated in HCT116, HT29, BT474 and MDA-MB-468 carcinoma cell lines. Cell proliferation, cell apoptosis and caspase 3/7 activity was determined for VER-155008 in the absence or presence of small molecule Hsp90 inhibitors. RESULTS: VER-155008 inhibited the proliferation of human breast and colon cancer cell lines with GI(50)s in the range 5.3-14.4 microM, and induced Hsp90 client protein degradation in both HCT116 and BT474 cells. As a single agent, VER-155008 induced caspase-3/7 dependent apoptosis in BT474 cells and non-caspase dependent cell death in HCT116 cells. VER-155008 potentiated the apoptotic potential of a small molecule Hsp90 inhibitor in HCT116 but not HT29 or MDA-MB-468 cells. In vivo, VER-155008 demonstrated rapid metabolism and clearance, along with tumor levels below the predicted pharmacologically active level. CONCLUSION: These data suggest that small molecule inhibitors of Hsc70/Hsp70 phenotypically mimic the cellular mode of action of a small molecule Hsp90 inhibitor and can potentiate the apoptotic potential of a small molecule Hsp90 inhibitor in certain cell lines. The factors determining whether or not cells apoptose in response to Hsp90 inhibition or the combination of Hsp90 plus Hsc70/Hsp70 inhibition remain to be determined.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas del Choque Térmico HSC70/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Nucleósidos de Purina/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Sistemas de Liberación de Medicamentos , Sinergismo Farmacológico , Femenino , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Nucleósidos de Purina/farmacocinéticaRESUMEN
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.
Asunto(s)
Adenosina/farmacología , Diseño de Fármacos , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Adenosina/química , Línea Celular Tumoral , Cristalografía por Rayos X , Inmunoensayo de Polarización Fluorescente , Humanos , Estructura MolecularRESUMEN
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.
Asunto(s)
Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Diseño de Fármacos , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Triazoles/química , Línea Celular Tumoral , Quinasa 2 Dependiente de la Ciclina/metabolismo , Humanos , Concentración 50 Inhibidora , Estructura Molecular , Inhibidores de Proteínas Quinasas/química , Pirimidinas/síntesis química , Relación Estructura-ActividadRESUMEN
The protein structure guided design of a series of pyrazolo[1,5-a]pyrimidines with high potency for human cyclin-dependent kinase 2 (CDK2) is described. Some examples were shown to inhibit the growth of human colon tumour cells, were equipotent for CDK1 and were selective against GSK-3beta and other kinases.