Examination of N-hydroxylation as a Prerequisite Mechanism of Nitric Oxide Synthase Inactivation Tristan S. Maurer, y Jianping Pan, { Brian P. Booth, x Thomas I. Kalman and Ho-Leung Fung* Department of Pharmaceutics and Department of Medicinal Chemistry, School of Pharmacy, University at Bualo, State University of New York, Bualo, NY 14260-1200, USA Received 26 January 2000; accepted 10 March 2000 AbstractÐl-N 5 -(1-Hydroxyiminoethyl)-ornithine (l-NHIO) and l-N 6 -(1-hydroxyiminoethyl)-lysine (l-NHIL) were synthesized and tested as potential intermediates in the mechanism-based inactivation of nitric oxide synthase (NOS) by l-N 5 -iminoethyl- ornithine (l-NIO) and l-N 6 -iminoethyllysine (l-NIL). Although these compounds were determined to be competitive inhibitors, mechanism-based inactivation was not observed. # 2000 Elsevier Science Ltd. All rights reserved. Amino acid-based inhibitors of nitric oxide synthase (NOS) have shown promise in the treatment of many in¯ammatory diseases. 1 4 However, insucient potency and selectivity for the inducible isoform (NOS II) limits the therapeutic potential of many NOS inhibitors. 5 Although binding selectivity to the NOS II isoform has been improved, 6 8 overall inhibitory selectivity is frequently confounded by mechanism-based inactivation. 9 12 Cur- rently, very little is known about the fundamental mechanism for NOS II inactivation. The natural sub- strate, l-arginine, and the mechanism-based inactivator, l-N G -methylarginine (l-NMMA), are similarly N- hydroxylated by NOS II to the intermediates l-N G - hydroxyarginine and l-N G -hydroxy-N G -methylarginine, respectively. 13 Both l-N G -hydroxyarginine and l-N G - hydroxy-N G -methylarginine display lower anities for NOS II, but are metabolized more rapidly than their non-hydroxylated parent compounds. 13 Unlike l-N G - hydroxyarginine, further metabolism of l-N G -hydroxy- N G -methylarginine results in mechanism-based inacti- vation. 13 This inactivation is associated with both cova- lent modi®cation of the NOS protein and loss of NOS associated heme. 11,14 These eects on the NOS protein have been postulated to arise from the release of formal- dehyde or nitrosomethane radical cation during the metabolism of l-N G -hydroxy-N G - methylarginine. 9,12 14 Unlike l-NMMA, inactivation by l-N 5 iminoethyl-orni- thine (l-NIO) and l-N 6 -iminoethyl-lysine (l-NIL) is not associated with covalent modi®cation of the NOS pro- tein, but instead, is associated only with heme loss. 11,12 In addition, the release of formaldehyde or nitro- somethane radical cation that follows N-hydroxylation is not likely to occur in the case of l-NIO and l-NIL, because it would require a C±C instead of a C±N bond cleavage. Herein, we examined whether N-hydroxyla- tion is a prerequisite for the inactivation of NOS by l- NIO and l-NIL. Results and Discussion Using eq 1 and PCNONLIN 4.2, we were able to describe the observed inhibition kinetics of l-NIO and l-NIL (Fig. 2A and B). The concentration dependence and saturability of enzyme inactivation, typical of mechanism-based inactivation, is apparent. Inactivation of NOS by l-NIO and l-NIL was dependent upon the presence of NADPH (data not shown). Based upon this analysis, the K I =k inact parameters of l-NIO and l-NIL were estimated to be 1.42  0.34 mM/0.16  0.01 min 1 and 2.16  0.27 mM/0.35  0.01 min 1 , respectively. These parameters are in close agreement with those previously reported for these compounds. 12 Unlike l-N G -hydroxy- N G -methylarginine, the presumed metabolic intermediates of NOS inactivation by l-NIO and l-NIL, l-NHIO 0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0960-894X(00)00171-2 Bioorganic & Medicinal Chemistry Letters 10 (2000) 1077±1080 *Corresponding author. Tel.: +1-716-645-2842, ext. 222; fax: +1-716- 645-3693; e-mail: hlfung@acsu.bualo.edu y Present address: P®zer Central Research, Department of Drug Meta- bolism. Groton, CT 06340, USA. { Present address: Schering-Plough Research Institute, Chemical Research, Kenilworth, NJ 07033, USA. x Present address: Food and Drug Administration, Center for Drug Evaluation and Research. Rockville, MD 20857, USA.