Abstract 4685: Discovery and characterization of nitroaspirin (NCX-4016) as a powerful and clinically relevant inhibitor of human MGMT for increasing the efficacy of alkylating agents
Authors: Paranjpe Ameya , Srivenugopal Kalkunte S. 
Published: August 2012

Abstract
The DNA repair protein MGMT (O6-methylguanine-DNA methyltransferase) is a major determinant of resistance to many alkylating agents. Currently, clinical trials with O6-benzylguanine (BG) to inhibit MGMT and induce its degradation are ongoing; however, this is beset with severe alkylation damage to bone marrow and the need for hematopoietic rescue using BG-resistant MGMT gene therapy. Therefore, an urgent need exists for non-toxic regimens that deplete MGMT and increase tumor antitumor efficacy. Here, we have exploited the highly reactive nature of Cysteine 145 which accepts the alkyl groups for novel drug design. Cys145 has a pKa of 4.8 and is susceptible for glutathionylation and nitrosylation, both of which inactivate MGMT. This study characterized the effect of nitro-aspirin or NCX-4016, which is a potent nitrosylator of reactive cysteines and tyrosines. NCX-4016 is a well characterized chemopreventive agent without the GI adverse effects of aspirin and 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), nitroaspirin (NA) at pharmacologically achievable concentrations (5-10 µM) caused 90% inhibition of MGMT activity within 1 h of exposure. Interestingly, the MGMT protein disappeared very rapidly with similar kinetics after NA treatment; approx. 80-90% of MGMT was degraded after 10 µM NA treatment for 2 h. These data are highly comparable and/or superior to those reported for BG. Further, purified MGMT or tumor cell extracts exposed to NCX-4016 failed to bind the biotin-labeled BG, indicating Cys145 as the nitrosylation site. Tyr114, a residue critical for MGMT catalysis was another target for nitrosylation. The involvement of the ub-proteasome pathway in the degradation of nitrosylated MGMT both in vitro and in cells was also demonstrated. In cultured cells, MGMT suppression by BG was more prolonged than nitroaspirin, however, the 24-36 h curtailment by NA was adequate for increased production of cytotoxic lesions. Pre-exposure of tumor cells to NA followed by BCNU resulted in (i) a significantly greater Induction and persistence of DNA interstrand crosslinks, and (ii) at least a 2-fold increase in the G2/M cell cycle blockade. Experiments in mice have shown that a single injection (100 mg/kg) of NCX-4016 causes 50-60% inhibition of MGMT activity in mice brain and liver. Because NO-aspirin, is non-toxic (IC50&gt;500 µM for cell lines), can be administered at high levels, yields a chemopreventive by-product, unlikely to elicit tumor resistance, is lipophilic enough to cross the blood-brain barrier, and its pleiotropic actions are actually beneficial for chemotherapy, we believe NA's ability to potently inhibit MGMT holds great promise for efficient brain tumor therapy (supported by RO3 CA125872 and Assoc. for Res of Childhood Cancer).
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4685. doi:1538-7445.AM2012-4685