Abstract B229: Novel ezatiostat analogues disrupt binding of GSTP1 to all three major MAP kinases (JNK, ERK and p38) and exhibit context-dependent antitumor activity.

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Abstract Background: The glutathione S-transferase P1 (GSTP1) inhibitor, ezatiostat (TLK199; Telintra®), a diethyl ester prodrug hydrolyzed intracellularly to the active diacid metabolite, has demonstrated significant clinical efficacy in patients with myelodysplastic syndrome (Blood 2009, 113[26]:6533–6540) and preclinical anticancer activity in multiple cancer cell lines. The effects of TLK199 on normal cell proliferation and in causing cancer cell death have been suggested to involve disruption of the binding of GSTP1 to Jun N-terminal kinase, JNK, resulting in increased intracellular JNK activity and ROS levels. In previous efforts, we synthesized several TLK199 analogues for determining the critical structural pre-requisites for enhanced GSTP1 selectivity and biological potency. The present study seeks to gain insight into the mechanism of action of these compounds that could facilitate their further development. Depending upon cellular context and stimulus, all MAP kinases can regulate cell proliferation and/or cell death. We, therefore, examined the ability of the analogues to interfere with the physical and functional interactions of GSTP1 with all three major classes of human MAP kinases, namely, JNK, ERK and p38, and for their antitumor activity in vitro. Methods: GSTP1, with/without prior phosphorylation by EGFR, was incubated with each of analogues, and the MAPK added; tumor cells were treated with selected analogues. The reaction mixtures and cell extracts were immunoprecipitated with a MAPK specific antibody, western blotted for total and phosphorylated protein, and the GSTP1-MAPK complex quantitated. Antitumor activity was measured by real time cell survival sensing and by determining the level of apoptotic induction, following treatment with 0–50 uM of analogue. Results: We showed that 1) GSTP1 binds to all three MAP kinases; prior EGFR-dependent tyrosine phosphorylation of GSTP1 increased the GSTP1-MAPK binding, significantly. 2) The analogues differ considerably in their inhibition of GSTP1-MAPK binding; 9 of 28 analogues inhibited GSTP1 binding to, at least, one MAPK. Analogue diesters showed little to no activity in the cell-free system. 3) 14 compounds showed significant anti-tumor activity (> 70% kill at 50 uM) in human cell lines of glioblastoma, inflammatory breast cancer and malignant melanoma; analogues with an S-substituted biphenyl group were among the most active. Conclusions: The results indicate that: 1) regulation of all three major MAPKs (JNK, ERK and p38) may be involved in the mechanisms of antitumor action of ezatiostat and its active analogues; 2) the specific GSTP1-MAPK interaction affected and the anti-tumor activity are dependent upon the chemical structure of the analogue, the cell line/tumor type and the MAPK/GSTP1 status of the tumor cells; 3) S-biphenyl substitution in ezatiostat enhances MAPK binding and antitumor activity. These findings should be useful in the further design and development of more potent and more tumor-specific next generation ezatiostat GSTP1 inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B229.