ABSTRACT:Null mutation of glutathione transferase (GST) M1 and GSTT1 was reported to correlate statistically with an abnormal increase in the plasma levels of alanine aminotransferase or aspartate aminotransferase caused by troglitazone in diabetic patients (Clin Pharmacol Ther, 73:435-455, 2003). This clinical evidence leads to the hypothesis that GSH conjugation catalyzed by GSTT1 and GSTM1 has a role in the elimination of reactive metabolites of troglitazone. However, the contribution of GST isoforms expressed in human liver to the detoxification of reactive metabolites of troglitazone has not yet been clarified. We investigated the involvement of human GST isoforms in the GSH conjugation of reactive metabolites of troglitazone using recombinant GST enzymes. Five reported GSH conjugates of reactive metabolites were produced from troglitazone after incubation with liver microsomes, NADPH, and GSH in a GSH concentration-dependent manner. Addition of human recombinant GSTA1, GSTA2, GSTM1, or GSTP1 protein to the incubation mixture further increased the GSH conjugates. However, the addition of GSTT1 did not show any catalytic effect. It is of interest that one of the reactive metabolites with a quinone structure was predominantly conjugated with GSH by GSTM1. Thus, we demonstrated that the GST isoforms contributed differently to the GSH conjugation of individual reactive metabolites of troglitazone, and GSTM1 is the most important GST isoform in the GSH conjugation of a specific reactive metabolite produced from the cytotoxic, quinone-form metabolite of troglitazone.

ABSTRACT:Clinical studies have suggested that a defect in both glutathione S-transferase (GST) M1 and GSTT1 increases the risk of drug-induced hepatotoxicity. The present study developed the method that enables genotyping of GSTM1 and GSTT1 directly using a small aliquot of blood samples based on an isothermal Smart amplification process version 2 (SmartAmp-2). SmartAmp-2 reaction could complete the genotyping of GSTM1 and GSTT1 within 40 min. The frequency of wild-type, GSTM1 null, GSTT1 null, and double null was 24, 21, 35, and 19%, respectively, consistent with previous reports in the Japanese population. The genotypes of 94 human genomic DNA samples determined by SmartAmp-2 were identical to those determined by the conventional polymerase chain reaction method. SmartAmp-2 was able to determine the genotypes of GSTM1 and GSTT1 even when human blood specimens were used. The SmartAmp-2 method is a rapid and accurate means of identifying the GSTM1 and GSTT1 genotypes, making it less time and more labor efficient in clinical practice than conventional methods requiring preparation of genomic DNA and electrophoresis. This will contribute to evaluate the susceptibility of disease and adverse reactions to drugs caused by deletion of GSTM1 and GSTT1.

Background: Wnt/β-catenin pathway controls many biological processes including cell proliferation and tissue development. In Wnt pathway, β-catenin is a key downstream factor which interacts with transcription factor T-cell factor (TCF) in the nucleus and induces expression of TCF responsive genes. β-catenin degradation is promoted by a cytoplasmic complex of APC, Axin and GSK3β. In many cancers, constitutive activation of Wnt/β-catenin pathway is observed due to mutations of the related genes and overexpression of Wnt ligands and receptors. For example, truncating mutation of the tumor suppressor APC are the most prevalent in colon cancer. APC deficiency leads to accumulation of nuclear β-catenin and promotes the transcription of downstream target genes. Hence, Wnt/β-catenin pathway inhibitor can be an attractive therapeutic agent for cancer patients. Results: To screen for Wnt/β-catenin pathway inhibitor, a reporter-based screen using a colon cancer cell line harboring APC mutation was performed. We identified a Wnt/β-catenin pathway inhibitor K-756 with an IC50 of 110 nmol/L, which was a distinct chemical structure from previously reported Wnt/β-catenin pathway inhibitors. From target identification study, it became clear that K-756 was a selective tankyrase inhibitor (AACR-NCI-EORTC 2013). Further development of the derivatives of K-756 using a structure-based drug design approach led to synthesis of K-476. K-476 inhibited Wnt/β-catenin pathway in APC mutant DLD-1 cell line with an IC50 of 0.3 nmol/L. K-476 stabilized Axin proteins and decreased active β-catenin. Wnt downstream target gene inhibition was also observed by K-476. PARP family profile assay was performed to evaluate the selectivity of K-476. K-476 inhibited tankyrase 1 and 2 and did not inhibit other PARP family isoforms even at 1000 nmol/L. Kinase profile assay was also performed towards 448 kinase but K-476 did not inhibit any of the kinases at 1000 nmol/L. Therefore K-476 is a highly selective tankyrase inhibitor. K-476 inhibited Wnt pathway signal and cell growth in colon cancer cell lines with an GI50 of less than 5 nmol/L. Oral dosing of K-476 resulted in inhibition of the reporter activity and Wnt downstream genes in a colon cancer xenograft mouse model. Taken together, K-476 represents a highly potent tankyrase inhibitor and is anticipated to be an important compound for further development of antitumor agent targeting the Wnt/β-catenin pathway regulation. Citation Format: Ryoko Okada, Yuichi Takahashi, Keiichi Motosawa, Yasuo Watanabe, Asae Igarashi, Ran Okada, Masahiro Ikkaku, Hikaru Miyagi, Yusuke Miura, Kazuki Asanome, Noriaki Uesaka, Jun-ichi Saito, Hiroshi Ishida, Ryuichiro Nakai. Discovery and characterization of a highly potent Wnt/ β -catenin pathway inhibitor targeting tankyrase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2715. doi:10.1158/1538-7445.AM2014-2715