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Xinhua Ji1, Ajai Pal2, Ravi Kalathur1, Xun Hu2, Yijun Gu1,2, Joseph E Saavedra3, Gregory S Buzard3, Aloka Srinivasan4, Larry K Keefer4, Shivendra V Singh21Macromolecular Crystallography Laboratory, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA; 2Department of Pharmacology and University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA; 3Basic Research Program, SAIC-Frederick Inc., Frederick, MD 21702, USA; 4Laboratory of Comparative Carcinogenesis, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USAAbstract: Glutathione S-transferase (GST) is a superfamily of detoxification enzymes, represented by GSTα, GSTµ, GSTπ, etc. GSTα is the predominant isoform of GST in human liver, playing important roles for our well being. GSTπ is overexpressed in many forms of cancer, thus presenting an opportunity for selective targeting of cancer cells. Our structure-based design of prodrugs intended to release cytotoxic levels of nitric oxide in GSTπ-overexpressing cancer cells yielded PABA/NO, which exhibited anticancer activity both in vitro and in vivo with a potency similar to that of cisplatin. Here, we present the details on structural modification, molecular modeling, and enzymatic characterization for the design of PABA/NO. The design was efficient because it was on the basis of the reaction mechanism and the structures of related GST isozymes at both the ground state and the transition state. The ground-state structures outlined the shape and property of the substrate-binding site in different isozymes, and the structural information at the transition-state indicated distinct conformations of the Meisenheimer complex of prodrugs in the active site of different isozymes, providing guidance for the modifications of the molecular structure of the prodrug molecules. Two key alterations of a GSTα-selective compound led to the GSTπ-selective PABA/NO.Keywords: structure-based, drug design, anticancer, prodrug, PABA/NO

Endogenously generated nitric oxide (NO) mediates a host of important physiological functions, playing roles in the vascular, immunological, and neurological systems. As a result, exogenous agents that release NO have become important therapeutic interventions and research tools. O 2 -Vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO) is a prodrug designed with the hypothesis that it might release nitric oxide via epoxidation of the vinyl group by cytochrome P450, followed by enzymatic and/or spontaneous epoxide hydration to release the ultimate NO-donating moiety, 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (PYRRO/NO) ion. In this study, we investigated this hypothetical activation mechanism quantitatively for V-PYRRO/NO using cDNA-expressed human cytochrome P450 (CYP)2E1. Incubation with CYP2E1 and an NADPH-regenerating system resulted in a time-dependent decomposition of V-PYRRO/NO, with a turnover rate of 2.0 nmol/min/pmol CYP2E1. Nitrate and nitrite were detected in high yield as metabolites of NO. The predicted organic metabolites pyrrolidine and glycolaldehyde were also detected in near-quantitative yields. The enzymatic decomposition of V-PYRRO/NO was also catalyzed, albeit at lower rates, by CYP2A6 and CYP2B6. We conclude that the initial step in the metabolism of V-PYRRO/NO to NO in the liver is catalyzed efficiently but not exclusively by the alcohol-inducible form of cytochrome P450 (CYP2E1). The results confirm the proposed activation mechanism involving enzymatic oxidation of the vinyl group in V-PYRRO/NO followed by epoxide hydration and hydrolytic decomposition of the resulting PYRRO/NO ion to generate nitric oxide.

The involvement of nitric oxide in numerous biological functions has led to the intense study of nitric oxide (NO) generation by the nitric oxide synthases (NOS) responsible. In addition to NO, nitric oxide synthases produceNG-hydroxy- l -arginine, superoxide anion and, indirectly, NOx species such as peroxynitrite and, possibly, nitrous oxide (N2O). Consequently, the interactions ofNG-hydroxy- l -arginine with NO and other oxides of nitrogen (NOx) are of considerable interest.NG-Hydroxy- l -arginine and other monosubstituted hydroxyguanidines react with aqueous aerobic NO, peroxynitrite, and various NOx and nitrosating agents to form compounds that subsequently release NO and N2O. Spectrometric data indicate that the nitrosation product ofNG-hydroxy- l -arginine is of the sameN-nitroso-N-hydroxy/diazeniumdiolate (formerly “NONOate”) structure as previously found for the nitrosation products of other model hydroxyguanidines. These decompose in aqueous solution in a pH-dependent manner to yield mainly NO and ureas at low pH, N2O and cyanamides at basic pH, and what appear to be primary nitrosamines/nitrosoimines. Studies on purified iNOS using a mass spectrometer with a gas-permeable membrane inlet identified both NO and N2O (or15NO and15N15NO with15N-labeled l -arginine as substrate) as products of NOS activity. These experiments suggest that much more NO than N2O is produced under the conditions studied and that N2O formation can be rationalized via the reaction of NOx species withNG-hydroxy- l -arginine.