Antitumor activity of ZSTK474, a new phosphatidylinositol 3-kinase inhibitor.

BACKGROUND We previously synthesized a novel s-triazine derivative, ZSTK474 [2-(2-difluoromethylbenzimidazol-1-yl)-4,6-dimorpholino-1,3,5-triazine], that strongly inhibited the growth of tumor cells. We identified its molecular target, investigated its effects on cellular signaling pathways, and examined its antitumor efficacy and toxicity in vivo. METHODS We used COMPARE analysis of chemosensitivity measurements from 39 human cancer cell lines and identified phosphatidylinositol 3-kinase (PI3K) as a molecular target for ZSTK474. PI3K was immunoprecipitated from A549 cell lysates, and its activity was measured by assessing the incorporation of 32P into phosphatidylinositol. We used the crystal structure of the PI3K-LY294002 complex to model the binding of ZSTK474 to PI3K (where LY294002 is a known PI3K inhibitor). PI3K downstream activity was analyzed by immunoblotting. Antitumor activity of ZSTK474 was examined against A549, PC-3, and WiDr xenografts in nude mice. Phosphorylation of Akt, a serine/threonine protein kinase and a major signaling component downstream of PI3K, was assessed in vivo by immunohistochemistry. RESULTS PI3K was identified as a molecular target for ZSTK474 by COMPARE analysis. We confirmed that ZSTK474 directly inhibited PI3K activity more efficiently than the PI3K inhibitor LY294002. At concentrations of 1 microM, ZSTK474 and LY2194002 reduced PI3K activity to 4.7% (95% confidence interval [CI] = 3.2% to 6.1%) and 44.6% (95% CI = 38.9% to 50.3%), respectively, of the untreated control level. Molecular modeling of the PI3K-ZSTK474 complex indicated that ZSTK474 could bind to the ATP-binding pocket of PI3K. ZSTK474 inhibited phosphorylation of signaling components downstream from PI3K, such as Akt and glycogen synthase kinase 3beta, and mediated a decrease in cyclin D1 levels. ZSTK474 administered orally to mice had strong antitumor activity against human cancer xenografts without toxic effects in critical organs. Akt phosphorylation was reduced in xenograft tumors after oral administration of ZSTK474. CONCLUSION ZSTK474 is a new PI3K inhibitor with strong antitumor activity against human cancer xenografts without toxic effects in critical organs. ZSTK474 merits further investigation as an anticancer drug.