RESUMEN
The vitamin D receptor (VDR) is a nuclear hormone receptor that regulates cell proliferation, cell differentiation, and calcium homeostasis. The receptor is activated by vitamin D analogues that induce the disruption of VDR-corepressor binding and promote VDR-coactivator interactions. The interactions between VDR and coregulators are essential for VDR-mediated transcription. Small molecule inhibition of VDR-coregulator binding represents an alternative method to the traditional ligand-based approach in order to modulate the expression of VDR target genes. A high throughput fluorescence polarization screen that quantifies the inhibition of binding between VDR and a fluorescently labeled steroid receptor coactivator 2 peptide was applied to discover the new small molecule VDR-coactivator inhibitors, 3-indolylmethanamines. Structure-activity relationship studies with 3-indolylmethanamine analogues were used to determine their mode of VDR-binding and to produce the first VDR-selective and irreversible VDR-coactivator inhibitors with the ability to regulate the transcription of the human VDR target gene TRPV6.
Asunto(s)
Indoles/síntesis química , Metilaminas/síntesis química , Coactivadores de Receptor Nuclear/antagonistas & inhibidores , Receptores de Calcitriol/antagonistas & inhibidores , Línea Celular , Ensayos Analíticos de Alto Rendimiento , Humanos , Indoles/química , Indoles/farmacología , Membranas Artificiales , Metilaminas/química , Metilaminas/farmacología , Coactivador 2 del Receptor Nuclear/antagonistas & inhibidores , Coactivador 2 del Receptor Nuclear/metabolismo , Coactivador 3 de Receptor Nuclear/antagonistas & inhibidores , Coactivador 3 de Receptor Nuclear/metabolismo , Coactivadores de Receptor Nuclear/metabolismo , Permeabilidad , Unión Proteica , Receptores de Calcitriol/metabolismo , Solubilidad , Relación Estructura-Actividad , Canales Catiónicos TRPV/genética , Transcripción Genética/efectos de los fármacosRESUMEN
OBJECTIVE: In human trials calcitriol and its analogs displayed unacceptable systemic toxicities including hypercalcemia. This study was designed to evaluate a novel non-hypercalcemic vitamin-D derivative (MT19c) and its anticancer effects in cultured ovarian cancer cell model. METHODS: We modified the Ergocalciferol structure to generate MT19c, a heterocyclic vitamin-D derivative. Hypercalcemic liabilities of MT19c were assessed by estimating the blood calcium levels in drug treated animals. VDR agonistic or antagonistic properties of MT19c were determined via a VDR-coactivator binding assay. The anticancer effects of MT19c were evaluated by (i) cytotoxicity studies in cancer cell lines and the National Cancer Institute (NCI(60)) cell lines, (ii) identification of apoptosis markers by microscopy and western blots, (iii) cell cycle analysis, and (iv) by studying the insulin receptor substrate-1/2 (IRS1/2) signaling in ovarian cancer cells (SKOV-3) by western blotting. RESULTS: MT19c treatment did not cause hypercalcemia in mice and showed minor VDR antagonistic activity. In a NCI(60) screen MT19c revealed cell-type specific growth inhibition. MT19c displayed superior cytotoxicity to cisplatin, calcitriol, EB1089 and Iressa in SKOV-3 cell-lines and was comparable to Taxol in our in vitro assays. In SKOV-3 cells MT19c showed caspase dependent apoptosis, DNA fragmentation and cell cycle arrest. MT19c did not alter VDR but downregulated the IGFR/IRS-1/2-MEK-ras-ERK1/2-pathway via activated TNFα-receptor/SAPK/JNK component. CONCLUSION: Our results demonstrate how structural optimization of the vitamin-D scaffold leads to identification of a non-hypercalcemic compound MT19c which exerts cytotoxicity in vitro based on a VDR-independent signaling pathway and displays potent anti-cancer activity in ovarian cancer cell models.