Abstract 5505: Potent redox-based inhibition of human MGMT for improving the efficacy of anticancer alkylating agents

There is an urgent need for new strategies for inhibiting the DNA repair protein MGMT (O 6 -methylguanine-DNA methyltransferase) and improving the efficacy of alkylating agents. MGMT is highly expressed in human cancers and prevents the formation of cytotoxic lesions in alkylated DNA. Current clinical trials involving MGMT depletion by O 6 -benzylguanine (BG), although promising, are beset with severe toxicity to the bone marrow, which has necessitated the transduction of BG-resistant MGMT gene into hematopoietic stem cells. Here, we exploited the highly reactive nature of Cysteine 145 of MGMT which accepts the alkyl groups for pharmacological intervention. Cys145 has a pKa of 4.8 due to its microenvironment and is susceptible for glutathionylation (Niture & Srivenugopal, Proc of AACR 47, abst. 789, 2006) and nitrosylation (Liu et al. Cancer Res. 62, 3037, 2002). S-Thiolation and S-nitrosylation are reversible posttranslational mechanisms that gauge the intracellular redox and transduce them into functional responses. This study investigated the effect of two nontoxic small molecules which readily react with reactive cysteines, namely, the NCX-4016 (nitro-aspirin capable of S-nitrosylation) and disulfiram (capable of thiol-conjugation). NCX-4016, also called a fatty aspirin, is degraded by plasma and tissue esterases to release NO in a sustained manner. In three MGMT-proficient human cancer cell lines (HT29, T47D, and HCT116), nitro-aspirin at very low concentrations (5-10 µM) caused a 90% inhibition of MGMT activity within 1 h of exposure. Interestingly, the MGMT protein also disappeared with similar kinetics after NCX-4016 treatment; approx. 80-90% of MGMT was degraded after 5 µM NO-aspirin treatment for 2 h. These data are highly comparable or better than those reported for BG. Further, purified MGMT or tumor cell extracts exposed to NCX-4016 failed to bind the biotin-labeled BG, indicating Cys145 to be the site of nitrosylation. >60% of MGMT protein was regenerated at 24 h when NO-aspirin treated HT29 cells were post-incubated in drug-free medium, indicating a transient inhibition and rapid repletion. Disulfiram (DS), the alcohol deterrent drug, also curtailed MGMT activity in HT29 and HCT116 cells with a 20 h 400 µM treatment causing a 95% inhibition. DS at 200 µM induced about 70% degradation of MGMT at 12 h. Other redox-sensitive proteins such as the wild-type and mutant p53, NF-κB, and ubiquitin E1 were all degraded by DS in a dose- dependent manner. Studies to evaluate MGMT inhibition by NO-aspirin and DS in animal tissues are in progress. Because NO-aspirin, is non-toxic (IC 50 >500 µM for cell lines), yields a chemopreventive by-product, unlikely to elicit tumor resistance, and is lipophilic enough to cross the BBB, we believe it can be exploited for glioma therapy. Our studies show that the redox-regulated proteins are ‘druggable’ and highlight options for redox- driven therapeutic strategies. 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 5505. doi:10.1158/1538-7445.AM2011-5505