Abstract 1360: Design, synthesis, and target validation of T315, a putative ILK inhibitor
Authors: Lee Su-Lin , Hsu En-Chi , Chuang Hsiao-Ching , Kulp Samuel , Chen Ching-Shih 
Published: June 2012

Objectives of the study: Integrin-linked kinase (ILK) is an intracellular adaptor and kinase that links the cell adhesion receptors, integrins and growth factors to the actin cytoskeleton and is a component of multiple signaling pathways. Recent studies have demonstrated that ILK is highly expressed in various cancers, and that inhibition of ILK expression and activity may serve as an attractive strategy for cancer therapy. An important substrate of ILK is protein kinase B (PKB)/Akt, which ILK phosphorylates at the S473 site, thereby contributing to its activation. The objective of our work is to develop a novel potent small molecule inhibitor of ILK.

Methodology: Previously, we developed AR12 (formerly OSU-03012), a novel celecoxib-derived inhibitor of phosphoinositide-dependent protein kinase-1 (PDK1) that downregulates PKB/Akt phosphorylation at T308 (known as the PDK1 site) and induces apoptosis and growth inhibition in cancer cells by blocking the PI3K/Akt pathway. However, to become fully activated, PKB/Akt requires both phosphorylation at two sites_T308/S473. Using OSU-03012 as a lead, we synthesized a series of derivatives to generate a small kinase inhibitor library, which was screened for compounds that could specifically downregulate PKB/Akt phosphorylation at S473, which is known as the PDK2 target site. The MTT assay was used to examine cancer cell viability after drug treatment. A radiometric ILK kinase assay was used to assess the ILK kinase inhibitory activities of the compounds. Cellular protein levels and phosphorylation status of ILK downstream targets were determined in drug-treated cells by western blot analysis.

Results & Conclusions: The screening of our focused AR12-derived kinase inhibitor library identified T315 as an efficient inhibitor of PKB/Akt phosphorylation at S473 without altering phosphorylation at the T308 site. Moreover, T315 demonstrated strong cytotoxicity against prostate cancer cell lines (PC-3 and LNCaP cells; IC50, 2.0 and 1.8 μM, respectively), but not primary nonmalignant prostate epithelial cells. To date, the phosphorylation of PKB/Akt at its PDK2 site has been attributed to at least ten candidate kinases, including PKCα/βII, mTORC2, ILK, and ATM. After examination of several signaling pathways by western blotting, two downstream signaling effectors of ILK, GSK-3β and MLC2, were shown to be downregulated by T315 in a dose-dependent manner, while downstream targets of mTORC2, another PDK2 candidate, were unchanged by T315 treatment. Importantly, T315 also dose-dependently inhibited ILK kinase activity (IC50 &lt; 1 μM) as determined by radiometric kinase assay. Together, these findings indicate that T315 is a putative ILK inhibitor.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1360. doi:10.1158/1538-7445.AM2011-1360