RESUMEN
The p53 tumor suppressor protein is regulated by its interaction with HDM2, which serves as a ubiquitin ligase (E3) to target p53 for degradation. We have identified a family of small molecules (HLI98) that inhibits HDM2's E3 activity. These compounds show some specificity for HDM2 in vitro, although at higher concentrations effects on unrelated RING and HECT domain E3s are detectable, which could be due, at least in part, to effects on E2-ubiquitin thiol-ester levels. In cells, the compounds allow the stabilization of p53 and HDM2 and activation of p53-dependent transcription and apoptosis, although other p53-independent toxicity was also observed.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Línea Celular Tumoral , Quinasa 4 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte , Inhibidores Enzimáticos/química , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Flavinas/química , Expresión Génica/efectos de los fármacos , Humanos , Ratones , Estructura Molecular , Ubiquitina-Proteína Ligasas Nedd4 , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Unión Proteica/efectos de los fármacos , Proteínas/antagonistas & inhibidores , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Transfección , Proteína p53 Supresora de Tumor/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The conjugation of proteins with ubiquitin plays numerous regulatory roles through both proteasomal-dependent and nonproteasomal-dependent functions. Alterations in ubiquitylation are observed in a wide range of pathologic conditions, including numerous malignancies. For this reason, there is great interest in targeting the ubiquitin-proteasome system in cancer. Several classes of proteasome inhibitors, which block degradation of ubiquitylated proteins, are widely used in research, and one, Bortezomib, is now in clinical use. Despite the well-defined and central role of the ubiquitin-activating enzyme (E1), no cell permeable inhibitors of E1 have been identified. Such inhibitors should, in principle, block all functions of ubiquitylation. We now report 4[4-(5-nitro-furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester (PYR-41) as the first such inhibitor. Unexpectedly, in addition to blocking ubiquitylation, PYR-41 increased total sumoylation in cells. The molecular basis for this is unknown; however, increased sumoylation was also observed in cells harboring temperature-sensitive E1. Functionally, PYR-41 attenuates cytokine-mediated nuclear factor-kappaB activation. This correlates with inhibition of nonproteasomal (Lys-63) ubiquitylation of TRAF6, which is essential to IkappaB kinase activation. PYR-41 also prevents the downstream ubiquitylation and proteasomal degradation of IkappaBalpha. Furthermore, PYR-41 inhibits degradation of p53 and activates the transcriptional activity of this tumor suppressor. Consistent with this, it differentially kills transformed p53-expressing cells. Thus, PYR-41 and related pyrazones provide proof of principle for the capacity to differentially kill transformed cells, suggesting the potential for E1 inhibitors as therapeutics in cancer. These inhibitors can also be valuable tools for studying ubiquitylation.