Abstract 3246: Synthesis and biochemical characterization of novel analogues of ezatiostat hydrochloride (TLK199, Telintra®)
Authors: Cai Danying , Cheng Mingshan , Keck James G. , Laborde Edgardo , Macsata Robert W. 
Published: June 2012

Ezatiostat hydrochloride (TLK199, Telintra®) is the first glutathione S-transferase P1-1 (GST P1-1) inhibitor prodrug to demonstrate a clinically significant improvement in the cytopenias affecting patients with myelodysplastic syndrome (MDS), thereby validating this enzyme as a candidate target for pharmacologic intervention (Blood 2009; 113(26):6533-6540).

Structurally, ezatiostat is a tripeptide glutathione analog diethyl ester that is metabolized in vivo to the diacid, which is a potent and selective inhibitor of GST P1-1, an enzyme overexpressed in many human hematologic cancers. This inhibition induces a dissociation of GST P1-1 from its complex with c-jun N-terminal kinase (JNK)/c-Jun and activates signaling pathways that lead to cell proliferation and differentiation of normal hematopoietic cells and apoptosis of malignant cells.

To improve the inhibitory potency and selectivity for the target enzyme GST P1-1, a series of diacid analogs of ezatiostat bearing different substituents on the cysteinyl sulfur were synthesized and evaluated in several biological assays. Replacement of the benzyl group by a 2- or 4-biphenylmethyl or a 1-naphthylethyl substituent resulted in a 30- to 130-fold improvement in IC50 against GST P1-1 and 3- to 10-fold higher selectivity relative to the GST A1-1 and GST M1-1 isoforms.

The most active analogs were esterified and tested in cell-based and in vivo assays. The esters induced growth arrest and cellular apoptosis in human leukemia cells (HL-60) with lower or similar CC50 values as ezatiostat (6-17 μM range). In an HL-60 differentiation assay measuring induction of cell surface CD11b expression, the compounds increased CD11b expression by approximately 2-fold. Similarly to ezatiostat, the compounds also increased the production of reactive oxygen species in HL-60 and 293T cells by 2- to 3-fold after a 2 hour exposure, as measured by the generation of a fluorescent signal from a redox-sensitive dye.

Select ezatiostat analogs were evaluated in rodents for toxicity as well as for acceleration of recovery of neutrophil levels in a standard model of 5-FU chemotherapy-induced neutropenia. The compounds were well tolerated when administered at doses up to 200 mg/kg and, as ezatiostat, led to a significant &gt;1 day accelerated recovery in the 5-FU-induced neutropenia model in rats compared to growth factor treatment.

Substitution at the cysteinyl sulfur was established as the most critical position in the structure-activity relationship analysis and is consistent with the unique mechanism of action of these tripeptide inhibitors. Further investigation of the most potent and selective analogs in biochemical and cellular assays is underway. The availability of more selective and potent GST P1-1 inhibitors may help elucidate the role of GST P1-1 in the development and progression of MDS.

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 3246. doi:10.1158/1538-7445.AM2011-3246