RESUMO
Although Hsp90 inhibitors can inhibit multiple tumorigenic pathways in cancer cells, their anticancer activity has been disappointingly modest. However, by forcing Hsp90 inhibitors into the mitochondria with mitochondrial delivery vehicles, they were converted into potent drugs targeting the mitochondrial Hsp90 paralog TRAP1. Here, to improve mitochondrial drug accumulation without using the mitochondrial delivery vehicle, we increased freely available drug concentrations in the cytoplasm by reducing the binding of the drugs to the abundant cytoplasmic Hsp90. After analyzing X-ray cocrystal structures, the purine ring of the Hsp90 inhibitor 2 (BIIB021) was modified to pyrazolopyrimidine scaffolds. One pyrazolopyrimidine, 12b (DN401), bound better to TRAP1 than to Hsp90, inactivated the mitochondrial TRAP1 in vivo, and it exhibited potent anticancer activity. Therefore, the rationale and feasible guidelines for developing 12b can potentially be exploited to design a potent TRAP1 inhibitor.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Pirazóis/química , Pirazóis/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cristalografia por Raios X , Proteínas de Choque Térmico HSP90/metabolismo , Células HeLa , Humanos , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Simulação de Acoplamento Molecular , Neoplasias/metabolismo , Neoplasias/patologia , Pirazóis/farmacocinética , Pirazóis/uso terapêutico , Pirimidinas/farmacocinética , Pirimidinas/uso terapêuticoRESUMO
The mitochondrial pool of Hsp90 and its mitochondrial paralogue, TRAP1, suppresses cell death and reprograms energy metabolism in cancer cells; therefore, Hsp90 and TRAP1 have been suggested as target proteins for anticancer drug development. Here, we report that the actual target protein in cancer cell mitochondria is TRAP1, and current Hsp90 inhibitors cannot effectively inactivate TRAP1 because of their insufficient accumulation in the mitochondria. To develop mitochondrial TRAP1 inhibitors, we determined the crystal structures of human TRAP1 complexed with Hsp90 inhibitors. The isopropyl amine of the Hsp90 inhibitor PU-H71 was replaced with the mitochondria-targeting moiety triphenylphosphonium to produce SMTIN-P01. SMTIN-P01 showed a different mode of action from the nontargeted PU-H71, as well as much improved cytotoxicity to cancer cells. In addition, we determined the structure of a TRAP1-adenylyl-imidodiphosphate (AMP-PNP) complex. On the basis of comparative analysis of TRAP1 structures, we propose a molecular mechanism of ATP hydrolysis that is crucial for chaperone function.
Assuntos
Benzodioxóis/química , Benzodioxóis/farmacologia , Desenho de Fármacos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/química , Mitocôndrias/efeitos dos fármacos , Purinas/química , Purinas/farmacologia , Aminas/química , Linhagem Celular Tumoral , Cristalografia por Raios X , Humanos , Mitocôndrias/metabolismo , Modelos Moleculares , Compostos Organofosforados/química , Multimerização Proteica , Estabilidade Proteica , Estrutura Quaternária de ProteínaRESUMO
Hsp90 is a molecular chaperone responsible for the assembly and regulation of many cellular client proteins. In particular, Trap1, a mitochondrial Hsp90 homologue, plays a pivotal role in maintaining mitochondrial integrity, protecting against apoptosis in cancer cells. The N (N-terminal)-M (middle) domain of human Trap1 was crystallized in complex with Hsp90 inhibitors (PU-H71 and BIIB-021) by the hanging-drop vapour-diffusion method at pH 6.5 and 293â K using 15% PEG 8K as a precipitant. Diffraction data were collected from crystals of the Trap1-PU-H71 (2.7â Å) and Trap1-BIIB-021 (3.1â Å) complexes to high resolution at a synchrotron-radiation source. Preliminary X-ray diffraction analysis revealed that both crystals belonged to space group P41212 or P43212, with unit-cell parameters a = b = 69.2, c = 252.5â Å, and contained one molecule per asymmetric unit according to Matthews coefficient calculations.