J . Med. Chem. 1992,35,1137-1144
1137
NG-Allyl-and NG-Cyclopropyl-L-arginine:Two Novel Inhibitors of Macrophage Nitric Oxide Synthase Norman M. Olken' and Michael A. Marletta*Jg* Interdepartmental Program in Medicinal Chemistry, College of Pharmacy and Department of Biological Chemistry, School of Medicine University of Michigan, Ann Arbor, Michigan 48109-1065. Received August 26,1991
p-Methyl-L-arginine has recently been shown to inactivate the inducible murine macrophage nitric oxide ('NO) synthase (Olken, N. M.; Rusche, K. M.; Richards, M. K.; Marletta, M. A. Biochem. Biophys. Res. Commun. 1991, 177,828-833). e-Allyl-L-arginine and p-cyclopropyl-L-argininewere synthesized as potential mechanism-based enzyme inhibitors to exploit the chemistry presumed to occur at the active site. p-Cyclopropyl-L-argininewas found to be a potent reversible inhibitor with a Ki = 7.7 pM. A@-Allyl-L-argininewas found to be both a potent reversible (ICi = 2.1 pM) and irreversible inhibitor of the enzyme. This irreversible inhibition demonstrated pseudo-fist-order inactivation kinetics with kh& = 0.026 min-' and KI= 3.4 pM. Stereospecific protection of the inactivation was afforded by L-arginine, and saturability of the inactivation rate was observed. Our studies indicate that both reversible and irreversible inhibition of the inducible 'NO synthase can be achieved with relatively simple modifications of the substrate L-arginine.
equilibrium with an electrophilic iminium species and the Introduction products of N-dealkylation, namely arginine and formThe manipulation of the recently discovered mammalian aldehyde. Indeed, cytochrome P450 has been shown to pathway of L-arginine oxidation to nitric oxide ('NO) has dealkylate methylguanidine to guanidine and formthe potential of broad therapeutic application.' *NO aldehyde.I4 To explore these mechanistic possibilities and produced in the vascular endothelium is critical to the homeostatic regulation of blood pressure and has an antiaggregatory effect on platelets.2 'NO liberated by immunostimulated macrophages has been shown to be crucial (1) Moncada, S.;Palmer, R. M. J.; Higgs, E. A. Nitric Oxide: to the cytotoxic and cytostatic function of these activated Physiology, Pathophysiology, and Pharmacology. Pharnaucol. Rev. 1991,43,109-142. cells? The 'NO synthases are a family of enzymes which (2) Mellion, B. T.; Ignarro, L. J.; Ohlstein, E. H.; Pontecorvo, E. synthesize 'NO by the oxidation of one of the two terminal G.; Hyman, A. L.; Kadowitz, P. J. Evidence for the Inhibitory guanido nitrogens of L-arginine (1) (Scheme I). Both Role of Cyclic GMP in ADP-Induced Human Platelet Aggreendothelial cells4and immunostimulated macrophage^^^^^^ gation in the Presence of Nitric Oxide and Related Vasodilahave been shown to generate [15N]'N0 following treatment tors. Blood 1981,77,946-955. Incubation of the mawith [15N-guanido-N2]-~-arginine. (3) Hibbs, J. B., Jr.; Taintor, R. R.; Vavrin, Z.; Rachlin, E. Nitric crophage enzyme with ['So]O2 results in l80incorporation Oxide: A Cytotoxic Macrophage Effector Molecule. Biochem. into the ureido group of citrulline, the other reaction Biophys. Res. Commun. 1988,157,87-94. product.' Marletta and colleagues originally proposed5p6 (4) Palmer, R. M. J.; Ashton, D. S.; Moncada, S. Vascular Enthat hydroxylation of L-arginine to P-hydroxy-L-arginine dothelial Cells Synthesize Nitric Oxide from L-Arginine. Nature 1987,333,664-666. (2) was the first enzymatic step in this reaction. In support (5) Iyengar, R.; Stuehr, D. J.; Marletta, M. A. Macrophage Synof this hypothesis,incubation of macrophage 'NO synthase thesis of Nitrite, Nitrate, and N-Nitrosamines: Precursors and with chemically synthesized [ 15N-OH]fl-hydroxy-~-arthe Role of the Respiratory Burst. h o c . Natl. Acad. Sci. ginine was found to produce [15N]'N0 e x c l ~ s i v e l y . ~ ~ ~ U.S.A. 1987,84,6369-6373. Furthermore, P-hydroxy-L-arginine has been shown to (6) Marletta, M. A.; Yoon, P. S.; Iyengar, R.; Leaf,C. D.; Wishnok, have vasoactive properties indistinguishable from those J. S.Macrophage Oxidation of L-Arginine to Nitrite and Niof L-arginine.'O Analogous to oxidations performed by the trate Nitric Oxide Is an Intermediate. Biochemistry 1988,27, cytochromes P4501' and the pterin-dependent amino acid 8707-8711. hydroxylases,12the mechanism of this hydroxylation may (7) Kwon, N. S.;Nathan, C. F.; Gilker, C.; Griffith, 0. W.; Matinvolve single electron oxidation of a terminal guanido thews, D. E.; Stuehr, D. J. L-Citrulline Production from LAginine by Macrophage Nitric Oxide Synthase. The Ureido nitrogen followed by radical recombination with a metOxygen Derives from Dioxygen. J. Biol. Chem. 1990,265, al-oxygen complex at the active site. 13442-13445. We have recently reported that P-methyl-L-arginine (8) Stuehr, D. J.; Kwon, N. S.; Nathan, C. F.; Griffith, 0. W.; (L-NMA, 3) is a mechanism-based enzyme inhibitor of Feldman, D. L.; Wiseman, J. Nu-Hydroxy-L-Arginine Is an murine macrophage *NO synthase.13 Typical pseudoIntermediate in the Biosynthesis of Nitric Oxide from LAfirst-order inactivation kinetics were found, with kinact= ginine. J. Biol. Chem. 1991,266,6259-6263. 0.050min-' and KI = 4.2 pM at 37 "C. We speculated that (9) Pufahl, R. A.; Nanjappan, P.; Woodard, R. W.; Marletta, M. either single electron oxidation or the NG-methylatedniA. Unpublished results. trogen followed by deprotonation or direct oxidation of the (10) Wallace, G. C.; Fukuto, J. M. Synthesis and Bioactivity of methyl group could produce a carbon-centeredfree radical N-Hydroxyarginine: A Possible Intermediate in the Biosynthesis of Nitric Oxide from Arginine. J. Med. Chem. 1991,34, capable of enzyme alkylation (Scheme 11, R = H). Sub1746-1748. sequent hydroxylation of the methyl group would result (11) Guengerich, F. P.; Macdonald, T. L. Mechanisms of Cytoin the formation of a carbinolamine at the active site of chrome P-450Catalysis. FASEB J. 1991,4,2453-2459. the *NO synthase. This carbinolamine would then be in ~
*Address correspondence to this author a t the College of Phannacy, 428 Church Street, University of Michigan, Ann Arbor, MI 48109-1065. +Collegeof Pharmacy. Department of Biological Chemistry.
(12) Dix, T.A,; Benkovic, S.J. Mechanism of Oxygen Activation by Pteridine-Dependent Monooxygenases. Acc. Chem. Res. 1988,21,101-107. (13) Olken, N. M.; Rusche, K. M.; Richards, M. K.; Marletta, M. A. Inactivation of Macrophage Nitric Oxide Synthase Activity By NO-Methyl-L-Arginine. Biochem. Biophys. Res. Commun. 1991,177,828-833.
0022-2623/92/l835-1137$03.00/0 0 1992 American Chemical Society
Olken and Marletta
1138 Journal of Medicinal Chemistry, 1992, Vol. 35, No. 6
Scheme I. Reaction Catalyzed by *NOSynthasea
L-arginine ( 1 )
NG-hydroxy-L-arginine ( 2 )
citrulline
nitric oxide
OThe oxidation of L-arginine (1) to citrulline and nitric oxide catalyzed by 'NO synthase, showing the reaction intermediate hydroxy-L-arginine (2).
fl-
Scheme 11. General Rationale for the Inactivation of the 'NO Synthase by fl-Alkyl-substituted-L-argininesa
+
+
'+
.
+
?H
"7N- CH2R +
+
\ I 3 u H i L o -
+
NH-~HR
R
ComDound Compound
HflN*7NH -CHR
\
L
L - NMA (3)
- CH-CH2
L - ALA (4)
YN +
+ H$J
carbinolamine
carbon-centered
-
H2NY N= +
+
COO-
+
?H
H20
COO-
imine
ftwrdical
R
8,
