RESUMO
Heat shock protein 90 (Hsp90) is a molecular chaperone protein implicated in stabilizing the conformation and maintaining the function of many cell-signaling proteins. Many oncogenic proteins are more dependent on Hsp90 in maintaining their conformation, stability, and maturation than their normal counterparts. Furthermore, recent data show that Hsp90 exists in an activated form in malignant cells but in a latent inactive form in normal tissues, suggesting that inhibitors selective for the activated form could provide a high therapeutic index. Hence, Hsp90 is emerging as an exciting new target for the treatment of cancer. We now report on a novel series of 2-amino-6-halopurine Hsp90 inhibitors exemplified by 2-amino-6-chloro-9-(4-iodo-3,5-dimethylpyridin-2-ylmethyl)purine (30). These highly potent inhibitors (IC50 of 30 = 0.009 microM in a HER-2 degradation assay) also display excellent antiproliferative activity against various tumor cell lines (IC50 of 30 = 0.03 microM in MCF7 cells). Moreover, this class of inhibitors shows higher affinity for the activated form of Hsp90 compared to our earlier 8-sulfanylpurine Hsp90 inhibitor series. When administered orally to mice, these compounds exhibited potent tumor growth inhibition (>80%) in an N87 xenograft model, similar to that observed with 17-allylamino-17-desmethoxygeldanamycin (17-AAG), which is a compound currently in phase I/II clinical trials.
Assuntos
Adenina/análogos & derivados , Antineoplásicos/síntese química , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Purinas/síntese química , Piridinas/síntese química , Adenina/síntese química , Adenina/química , Adenina/farmacologia , Administração Oral , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Disponibilidade Biológica , Linhagem Celular Tumoral , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Purinas/química , Purinas/farmacologia , Piridinas/química , Piridinas/farmacologia , Receptor ErbB-2/metabolismo , Relação Estrutura-Atividade , Transplante HeterólogoRESUMO
Orally active Hsp90 inhibitors are of interest as potential chemotherapeutic agents. Recently, fully synthetic 8-benzyladenines and 8-sulfanyladenines such as 4 were disclosed as Hsp90 inhibitors, but these compounds are not water soluble and consequently have unacceptably low oral bioavailabilities. We now report that water-solubility can be achieved by inserting an amino functionality in the N(9) side chain. This results in compounds that are potent, soluble in aqueous media, and orally bioavailable. In an HER-2 degradation assay, the highest potency was achieved with the neopentylamine 42 (HER-2 IC(50) = 90 nM). In a murine tumor xenograft model (using the gastric cancer cell line N87), the H(3)PO(4) salts of the amines 38, 39, and 42 induced tumor growth inhibition when administered orally at 200 mg/kg/day. The amines 38, 39, and 42 are the first Hsp90 inhibitors shown to inhibit tumor growth upon oral dosage.
Assuntos
Adenina/análogos & derivados , Antineoplásicos/síntese química , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Purinas/síntese química , Adenina/síntese química , Adenina/química , Adenina/farmacologia , Administração Oral , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Disponibilidade Biológica , Feminino , Camundongos , Camundongos Nus , Purinas/química , Purinas/farmacologia , Solubilidade , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
NB1011, a phosphoramidate derivative of (E)-5-(2-bromovinyl)-2'-deoxyuridine, is a novel small molecule anticancer agent. NB1011 is selectively active against tumor cells expressing high levels of thymidylate synthase (TS), a critical enzyme in DNA biosynthesis. NB1011 is different from the current TS-targeted drugs, which require inhibition of TS to be effective, because NB1011 cytotoxicity depends upon activation by TS. Here we report a dose-dependent, antitumor activity of NB1011 against established Tomudex-resistant breast cancer (MCF7TDX) xenografts in athymic mice. Against 5-fluorouracil-resistant colon carcinoma (H630R10) xenografts, NB1011 was as efficacious as irinotecan, a drug recently approved for the treatment of 5-fluorouracil-resistant colon cancer. To gain insight into the mechanisms NB1011 uses to suppress cellular growth, we analyzed the downstream molecular events in the high TS-expressing MCF7TDX and RKOTDX cell lines upon NB1011 treatment. NB1011 treatment increased the mRNA levels of p21, Bax, and GADD45. Furthermore, NB1011 induced p53, p21, and Bax proteins specifically in high TS-expressing tumor cells, whereas no induction was observed in low TS-expressing tumor cells (MCF7) or normal cells (WI38). Cell cycle analysis demonstrated that NB1011 treatment of MCF7TDX and RKOTDX cells resulted in an accumulation of cells in the G2-M phase of the cell cycle. Altogether, our data indicate that the induction of the p53 target genes p21, bax, and GADD45, with a concomitant deregulation of the cell cycle, may represent one of the mechanisms by which NB1011 exerts its growth-suppressive effects.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Bromodesoxiuridina/farmacologia , Neoplasias do Colo/patologia , Proteína Oncogênica p21(ras)/biossíntese , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas/biossíntese , Timidilato Sintase/metabolismo , Proteína Supressora de Tumor p53/biossíntese , Animais , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Neoplasias da Mama/enzimologia , Bromodesoxiuridina/análogos & derivados , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Neoplasias do Colo/enzimologia , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , Ciclinas/genética , Células Epiteliais/enzimologia , Feminino , Fibroblastos/enzimologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Nus , Proteína Oncogênica p21(ras)/genética , Proteínas/genética , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/biossíntese , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína X Associada a bcl-2 , Proteínas GADD45RESUMO
The geldanamycin derivative 17-allyamino-17-demethoxygeldanamycin (17-AAG) is a clinical stage ATP-competitive HSP90 inhibitor that induces degradation of HSP90 client proteins. 17-AAG contains 1 ansamycin moiety and is highly potent in conventional cell killing assays. Since active Hsp90 exists as a dimer, we hypothesized that dimeric compounds containing 2 ansamycin pharmacophores might inhibit Hsp90 function more efficiently than 17-AAG. Here, we show that monomeric and dimeric ansamycins exert their activity in distinct ways. Under conditions of continuous exposure, 17-AAG induced client degradation and cell growth inhibition more readily than the dimeric drugs CF237 and CF483. By contrast, 24 hr treatment of various tumor cells with 17-AAG followed by drug washout caused temporary client degradation and cell cycle arrest but minimal cell death, whereas both dimers induced massive apoptosis. CF237 remained bound to Hsp90 for days after drug withdrawal and, while both monomeric and dimeric compounds caused accumulation of the inactive intermediate Hsp90 complex, this effect disappeared following washout of 17-AAG but not CF237. The dimer was also retained for longer in tumor xenografts and displayed superior antitumor activity in vivo. These results indicate that monomeric and dimeric Hsp90 inhibitors have distinct biological profiles and work differentially toward target inhibition.
Assuntos
Antineoplásicos/uso terapêutico , Benzoquinonas/uso terapêutico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Lactamas Macrocíclicas/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Animais , Western Blotting , Proliferação de Células/efeitos dos fármacos , Dimerização , Citometria de Fluxo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Receptor ErbB-2/metabolismo , Receptor IGF Tipo 1/metabolismo , Rifabutina/análogos & derivados , Rifabutina/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
NB1011, a phosphoramidate derivative of (E)-5-(2-bromovinyl)-2'-deoxyuridine, is a novel anti-cancer agent that selectively targets tumor cells expressing high levels of thymidylate synthase (TS), an enzyme required for DNA biosynthesis. NB1011 treatment of high-TS-expressing breast carcinoma cells (MCF7TDX) results in the induction of p53 and p21 protein levels, whereas no p53 or p21 induction is observed in the low-TS-expressing MCF7 tumor cells. Furthermore, MCF7TDX cells accumulate in the G(2)/M phase of the cell cycle in response to NB1011. In this study, the effect of NB1011 on the phosphorylation status of p53 was analyzed. We demonstrate that NB1011 treatment of various tumor cell lines expressing high TS results in the phosphorylation of p53 on Ser15, whereas this p53 phosphorylation is not observed in low-TS-expressing tumor cells. Also, we examined the role of several key cell cycle regulators in the growth inhibition observed in response to NB1011. Our results show that the mRNA and protein levels of the G(2)/M regulators cdc2, cyclin B1 and cdc25C are down-regulated in MCF7TDX cells, while unaffected in MCF7 cells. The mRNA and protein levels of 14-3-3sigma, also a direct transcriptional target of p53, are up-regulated in MCF7TDX cells following NB1011 treatment, while unchanged in MCF7 cells. Taken together, our data indicate that the growth inhibition caused by NB1011 in MCF7TDX cells is mediated through phosphorylation of p53 and activation of the G(2)/M checkpoint.
Assuntos
Adenocarcinoma/tratamento farmacológico , Neoplasias da Mama/tratamento farmacológico , Bromodesoxiuridina/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Timidilato Sintase/efeitos dos fármacos , Proteína Supressora de Tumor p53/efeitos dos fármacos , Bromodesoxiuridina/análogos & derivados , Bromodesoxiuridina/farmacologia , Humanos , Fosforilação/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
NB1011, a novel anticancer agent, targets tumor cells expressing high levels of thymidylate synthase (TS). NB1011 is converted intracellularly to bromovinyldeoxyuridine monophosphate (BVdUMP) which competes with the natural substrate, deoxyuridine monophosphate, for binding to TS. Unlike inhibitors, NB1011 becomes a reversible substrate for TS catalysis. Thus, TS retains activity and converts BVdUMP into cytotoxic product(s). In vitro cytotoxicity studies demonstrate NB1011's preferential activity against tumor cells expressing elevated TS protein levels. Additionally, NB1011 has antitumor activity in vivo. To identify drugs which interact synergistically with NB1011, we screened 13 combinations of chemotherapeutic agents with NB1011 in human tumor and normal cells. Dipyridamole and p-nitrobenzylthioinosine (NBMPR), potent inhibitors of equilibrative nucleoside transport, synergized with NB1011 selectively against 5-fluorouracil (5-FU)-resistant H630R10 colon carcinoma cells [combination index (CI)=0.75 and 0.35] and Tomudex-resistant MCF7TDX breast carcinoma cells (CI=0.51 and 0.57), both TS overexpressing cell lines. These agents produced no synergy with NB1011 in Det551 and CCD18co normal cells (CI > 1.1) lacking TS overexpression. Dipyridamole potentiated NB1011's cytotoxicity in medium lacking nucleosides and bases, suggesting a non-salvage-dependent mechanism. We demonstrate that nucleoside transport inhibitors, dipyridamole and NBMPR, show promise for clinically efficacious combination with NB1011.