RESUMEN
The IAPs are key regulators of the apoptotic pathways and are commonly overexpressed in many cancer cells. IAPs contain one to three BIR domains that are crucial for their inhibitory function. The pro-survival properties of XIAP come from binding of the BIR domains to the pro-apoptotic caspases. The BIR3 domain of XIAP binds and inhibits caspase 9, while the BIR2 domain binds and inhibits the terminal caspases 3 and 7. While XIAP BIR3 inhibitors have previously been reported, they also inhibit cIAP1/2 and promote the release of TNFα, potentially limiting their therapeutic utility. This paper will focus on the optimization of selective XIAP BIR2 inhibitors leading to the discovery of highly potent benzodiazepinone 36 (IC50 = 45 nM), which has high levels of selectivity over XIAP BIR3 and cIAP1 BIR2/3 and shows efficacy in a xenograft pharmacodynamic model monitoring caspase activity while not promoting the release of TNFα in vitro.
Asunto(s)
Compuestos Heterocíclicos/síntesis química , Compuestos Heterocíclicos/farmacología , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Proteína Inhibidora de la Apoptosis Ligada a X/antagonistas & inhibidores , Alanina/análogos & derivados , Alanina/síntesis química , Alanina/farmacocinética , Alanina/farmacología , Animales , Apoptosis/efectos de los fármacos , Benzodiazepinonas/síntesis química , Benzodiazepinonas/farmacocinética , Benzodiazepinonas/farmacología , Western Blotting , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Femenino , Compuestos Heterocíclicos/farmacocinética , Humanos , Proteínas Inhibidoras de la Apoptosis/química , Proteínas Inhibidoras de la Apoptosis/metabolismo , Ratones , Ratones Desnudos , Modelos Químicos , Modelos Moleculares , Estructura Molecular , Estructura Terciaria de Proteína , Ubiquitina-Proteína Ligasas , Proteína Inhibidora de la Apoptosis Ligada a X/química , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
MDM2 negatively regulates p53 stability and many human tumors overproduce MDM2 as a mechanism to restrict p53 function. Thus, inhibitors of p53-MDM2 binding that can reactivate p53 in cancer cells may offer an effective approach for cancer therapy. RG7112 is a potent and selective member of the nutlin family of MDM2 antagonists currently in phase I clinical studies. RG7112 binds MDM2 with high affinity (K(D) ~ 11 nmol/L), blocking its interactions with p53 in vitro. A crystal structure of the RG7112-MDM2 complex revealed that the small molecule binds in the p53 pocket of MDM2, mimicking the interactions of critical p53 amino acid residues. Treatment of cancer cells expressing wild-type p53 with RG7112 activated the p53 pathway, leading to cell-cycle arrest and apoptosis. RG7112 showed potent antitumor activity against a panel of solid tumor cell lines. However, its apoptotic activity varied widely with the best response observed in osteosarcoma cells with MDM2 gene amplification. Interestingly, inhibition of caspase activity did not change the kinetics of p53-induced cell death. Oral administration of RG7112 to human xenograft-bearing mice at nontoxic concentrations caused dose-dependent changes in proliferation/apoptosis biomarkers as well as tumor inhibition and regression. Notably, RG7112 was highly synergistic with androgen deprivation in LNCaP xenograft tumors. Our findings offer a preclinical proof-of-concept that RG7112 is effective in treatment of solid tumors expressing wild-type p53.
Asunto(s)
Antineoplásicos/farmacología , Imidazolinas/farmacología , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Animales , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Humanos , Imidazolinas/química , Ratones , Simulación del Acoplamiento Molecular , Neoplasias/patología , Unión Proteica/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-mdm2/química , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Carga Tumoral/efectos de los fármacos , Proteína p53 Supresora de Tumor/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cross-feedback activation of MAPK and AKT pathways is implicated as a resistance mechanism for cancer therapeutic agents targeting either RAF/MEK or PI3K/AKT/mTOR. It is thus important to have a better understanding of the molecular resistance mechanisms to improve patient survival benefit from these agents. Here we show that BRAFV600E is a negative regulator of the AKT pathway. Expression of BRAFV600E in NIH3T3 cells significantly suppresses MEK inhibitor (RG7167) or mTORC1 inhibitor (rapamycin) induced AKT phosphorylation (pAKT) and downstream signal activation. Treatment-induced pAKT elevation is found in BRAF wild type melanoma cells but not in a subset of melanoma cell lines harboring BRAFV600E. Knock-down of BRAFV600E in these melanoma cells elevates basal pAKT and downstream signals, whereas knock-down of CRAF, MEK1/2 or ERK1/2 or treatment with a BRAF inhibitor have no impact on pAKT. Mechanistically, we show that BRAFV600E interacts with rictor complex (mTORC2) and regulates pAKT through mTORC2. BRAFV600E is identified in mTORC2 after immunoprecipitation of rictor. Knock-down of rictor abrogates BRAFV600E depletion induced pAKT. Knock-down of BRAFV600E enhances cellular enzyme activity of mTORC2. Aberrant activation of AKT pathway by PTEN loss appears to override the negative impact of BRAFV600E on pAKT. Taken together, our findings suggest that in a subset of BRAFV600E melanoma cells, BRAFV600E negatively regulates AKT pathway in a rictor-dependent, MEK/ERK and BRAF kinase-independent manner. Our study reveals a novel molecular mechanism underlying the regulation of feedback loops between the MAPK and AKT pathways.