In the past three decades, nitric oxide has been well established as an important bioactive molecule implicated in regulation of cardiovascular, nervous, and immune systems. Therefore, it is not surprising that much effort has been made to find specific inhibitors of nitric oxide synthases (NOS), the enzymes responsible for production of nitric oxide. Among the many NOS inhibitors developed to date, inhibitors based on derivatives and analogues of arginine are of special interest, as this category includes a relatively high number of compounds with good potential for experimental as well as clinical application. Though this group of inhibitors covers early nonspecific compounds, modern drug design strategies such as biochemical screening and computer-aided drug design have provided NOS-isoform-specific inhibitors. With an emphasis on major advances in this field, a comprehensive list of inhibitors based on their structural characteristics is discussed in this paper. We provide a summary of their biochemical properties as well as their observed effects both in vitro and in vivo. Furthermore, we focus in particular on their pharmacology and use in recent clinical studies. The potential of newly designed specific NOS inhibitors developed by means of modern drug development strategies is highlighted.

International Clinical Research Center Center of Biomolecular and Cell Engineering St Anne's University Hospital Brno 656 91 Brno Czech Republic

Zobrazit více v PubMed

Alderton WK, Cooper CE, Knowles RG. Nitric oxide synthases: structure, function and inhibition. Biochemical Journal. 2001;357(3):593–615. PubMed PMC

Daff S. NO synthase: structures and mechanisms. Nitric Oxide. 2010;23(1):1–11. PubMed

Ji H, Tan S, Igarashi J, et al. Selective neuronal nitric oxide synthase inhibitors and the prevention of cerebral palsy. Annals of Neurology. 2009;65(2):209–217. PubMed PMC

Loscalzo J, Welch G. Nitric oxide and its role in the cardiovascular system. Progress in Cardiovascular Diseases. 1995;38(2):87–104. PubMed

Jaffrey SR, Snyder SH. Nitric oxide: a neural messenger. Annual Review of Cell and Developmental Biology. 1995;11:417–440. PubMed

Bogdan C. Nitric oxide and the immune response. Nature Immunology. 2001;2(10):907–916. PubMed

Hobbs AJ, Higgs A, Moncada S. Inhibition of nitric oxide synthase as a potential therapeutic target. Annual Review of Pharmacology and Toxicology. 1999;39:191–220. PubMed

Tinker AC, Wallace AV. Selective inhibitors of inducible nitric oxide synthase: potential agents for the treatment of inflammatory diseases? Current Topics in Medicinal Chemistry. 2006;6(2):77–92. PubMed

Silverman RB. Design of selective neuronal nitric oxide synthase inhibitors for the prevention and treatment of neurodegenerative diseases. Accounts of Chemical Research. 2009;42(3):439–451. PubMed PMC

Madaford S, Annedi SC, Ramnauth J, Rakhit S. Advancements in the development of nitric oxide synthase inhibitors. In: Macor JE, editor. Annual Reports in Medicinal Chemistry. Elsevier; 2009. pp. 27–50.

Garcin ED, Arvai AS, Rosenfeld RJ, et al. Anchored plasticity opens doors for selective inhibitor design in nitric oxide synthase. Nature Chemical Biology. 2008;4(11):700–707. PubMed PMC

Ji H, Li H, Martásek P, Roman LJ, Poulos TL, Silverman RB. Discovery of highly potent and selective inhibitors of neuronal nitric oxide synthase by fragment hopping. Journal of Medicinal Chemistry. 2009;52(3):779–797. PubMed PMC

Bedford MT, Clarke SG. Protein arginine methylation in mammals: who, what, and why. Molecular Cell. 2009;33(1):1–13. PubMed PMC

Palmer RMJ, Rees DD, Ashton DS, Moncada S. L-arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation. Biochemical and Biophysical Research Communications. 1988;153(3):1251–1256. PubMed

Sakuma I, Stuehr DJ, Gross SS, Nathan C, Levi R. Identification of arginine as a precursor of endothelium-derived relaxing factor. Proceedings of the National Academy of Sciences of the United States of America. 1988;85(22):8664–8667. PubMed PMC

Aisaka K, Gross SS, Griffith OW, Levi R. N(G)-methylarginine, an inhibitor of endothelium-derived nitric oxide synthesis, is a potent pressor agent in the guinea pig: does nitric oxide regulate blood pressure in vivo? Biochemical and Biophysical Research Communications. 1989;160(2):881–886. PubMed

Lowenstein CJ, Dinerman JL, Snyder SH. Nitric oxide: a physiologic messenger. Annals of Internal Medicine. 1994;120(3):227–237. PubMed

Calver A, Collier J, Vallance P. Nitric oxide and cardiovascular control. Experimental Physiology. 1993;78(3):303–326. PubMed

Zhang J, Snyder SH. Nitric-oxide in the nervous-system. Annual Review of Pharmacology and Toxicology. 1995;35:213–233. PubMed

Reif DW, McCreedy SA. N-Nitro-L-arginine and N-monomethyl-L-arginine exhibit a different pattern of inactivation toward the three nitric oxide synthases. Archives of Biochemistry and Biophysics. 1995;320(1):170–176. PubMed

Olken NM, Osawa Y, Marletta MA. Characterization of the inactivation of nitric oxide synthase by N(G)- methyl-L-arginine: evidence for heme loss. Biochemistry. 1994;33(49):14784–14791. PubMed

Griffith OW, Kilbourn RG. Nitric oxide synthase inhibitors: amino acids. In: Kartsatos AG, editor. Nitric Oxide, Part A: Sources and Detection of No; No Synthase. Vol. 268. San Diego: Academic Press; 1996. pp. 375–392. (Methods in Enzymology). PubMed

McDonald KK, Rouhani R, Handlogten ME, et al. Inhibition of endothelial cell amino acid transport System y+ by arginine analogs that inhibit nitric oxide synthase. Biochimica et Biophysica Acta. Biomembranes. 1997;1324(1):133–141. PubMed

Schwartz S, Clare R, Devereux K, Fook Sheung C. Pharmacokinetics, disposition and metabolism of 546C88 (L-N(G)- methylarginine hydrochloride) in rat and dog. Xenobiotica. 1997;27(12):1259–1271. PubMed

Maurer TS, Mishra Y, Fung HL. Nonlinear pharmacokinetics of L-N-G-methyl-arginine in rats: characterization by an improved HPLC assay. Biopharmaceutics & Drug Disposition. 1999;20(8):397–400. PubMed

Ueno SI, Sano A, Kotani K, Kondoh K, Kakimoto Y. Distribution of free methylarginines in rat tissues and in the bovine brain. Journal of Neurochemistry. 1992;59(6):2012–2016. PubMed

Hussein Z, Beerahee M, Grover R, et al. Pharmacokinetics of the nitric oxide synthase inhibitor L-N(G)- methylarginine hydrochloride in patients with septic shock. Clinical Pharmacology and Therapeutics. 1999;65(1):1–9. PubMed

Umans JG, Levi R. Nitric oxide in the regulation of blood flow and arterial pressure. Annual Review of Physiology. 1995;57:771–790. PubMed

Kilbourn RG, Jubran A, Gross SS, et al. Reversal of endotoxin-mediated shock by N(G)-methyl-L-arginine, an inhibitor of nitric oxide synthesis. Biochemical and Biophysical Research Communications. 1990;172(3):1132–1138. PubMed

Kilbourn RG, Gross SS, Jubran A, et al. N(G)-methyl-L-arginine inhibits tumor necrosis factor-induced hypotension: implications for the involvement of nitric oxide. Proceedings of the National Academy of Sciences of the United States of America. 1990;87(9):3629–3632. PubMed PMC

Kilbourn RG, Szabó C, Traber DL. Beneficial versus detrimental effects of nitric oxide synthase inhibitors in circulatory shock: lessons learned from experimental and clinical studies. Shock. 1997;7(4):235–246. PubMed

Haynes WG, Noon JP, Walker BR, Webb DJ. Inhibition of nitric oxide synthesis increases blood pressure in healthy humans. Journal of Hypertension. 1993;11(12):1375–1380. PubMed

Stamler JS, Loh E, Roddy MA, Currie KE, Creager MA. Nitric oxide regulates basal systemic and pulmonary vascular resistance in healthy humans. Circulation. 1994;89(5):2035–2040. PubMed

Nyberg M, Mortensen SP, Saltin B, Hellsten Y, Bangsbo J. Low blood flow at onset of moderate-intensity exercise does not limit muscle oxygen uptake. American Journal of Physiology - Regulatory Integrative and Comparative Physiology. 2010;298(3):R843–R848. PubMed

Petros A, Bennett D, Vallance P. Effect of nitric oxide synthase inhibitors on hypotension in patients with septic shock. Lancet. 1991;338(8782-8783):1557–1558. PubMed

Petros A, Lamb G, Leone A, Moncada S, Bennett D, Vallance P. Effects of a nitric oxide synthase inhibitor in human with septic shock. Cardiovascular Research. 1994;28(1):34–39. PubMed

Grover R, Zaccardelli D, Colice G, Guntupalli K, Watson D, Vincent JL. An open-label dose escalation study of the nitric oxide synthase inhibitor, N(G)-methyl-L-arginine hydrochloride (546C88), in patients with septic shock. Critical Care Medicine. 1999;27(5):913–922. PubMed

Bakker J, Grover R, McLuckie A, et al. Administration of the nitric oxide synthase inhibitor NG-methyl- L-arginine hydrochloride (546C88) by intravenous infusion for up to 72 hours can promote the resolution of shock in patients with severe sepsis: results of a randomized, double-blind, placebo-controlled multicenter study (study no. 144-002) Critical Care Medicine. 2004;32(1):1–12. PubMed

Watson D, Grover R, Anzueto A, et al. Cardiovascular effects of the nitric oxide synthase inhibitor N G-methyl-L-arginine hydrochloride (546C88) in patients with septic shock: results of a randomized, double-blind, placebo-controlled multicenter study (study no. 144-002) Critical Care Medicine. 2004;32(1):13–20. PubMed

López A, Lorente JA, Steingrub J, et al. Multiple-center, randomized, placebo-controlled, double-blind study of the nitric oxide synthase inhibitor 546C88: effect on survival in patients with septic shock. Critical Care Medicine. 2004;32(1):21–30. PubMed

Lassen LH, Ashina M, Christiansen I, et al. Nitric oxide synthase inhibition: a new principle in the treatment of migraine attacks. Cephalalgia. 1998;18(1):27–32. PubMed

Ashina M, Lassen LH, Bendtsen L, Jensen R, Olesen J. Effect of inhibition of nitric oxide synthase on chronic tension-type headache: a randomised crossover trial. Lancet. 1999;353(9149):287–289. PubMed

Lassen LH, Christiansen I, Iversen HK, Jansen-Olesen I, Olesen J. The effect of nitric oxide synthase inhibition on histamine induced headache and arterial dilatation in migraineurs. Cephalalgia. 2003;23(9):877–886. PubMed

Hjorth Lassen L, Klingenberg Iversen H, Olesen J. A dose-response study of nitric oxide synthase inhibition in different vascular beds in man. European Journal of Clinical Pharmacology. 2003;59(7):499–505. PubMed

Lassen LH, Sperling B, Andersen AR, Olesen J. The effect of i.v. L-NG methylarginine hydrochloride (L-NMMA: 546C88) on basal and acetazolamide (Diamox) induced changes of blood velocity in cerebral arteries and regional cerebral blood flow in man. Cephalalgia. 2005;25(5):344–352. PubMed

Vallance P, Leone A, Calver A, Collier J, Moncada S. Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure. Lancet. 1992;339(8793):572–575. PubMed

Vallance P, Leone A, Calver A, Collier J, Moncada S. Endogenous dimethylarginine as an inhibitor of nitric oxide synthesis. Journal of Cardiovascular Pharmacology. 1992;20(12):S60–S62. PubMed

Böger RH. Asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase, explains the "L-arginine paradox" and acts as a novel cardiovascular risk factor. Journal of Nutrition. 2004;134(10):S2842–S2847. PubMed

Komori Y, Wallace GC, Fukuto JM. Inhibition of purified nitric oxide synthase from rat cerebellum and macrophage by L-arginine analogs. Archives of Biochemistry and Biophysics. 1994;315(2):213–218. PubMed

Cardounel AJ, Zweier JL. Endogenous methylarginines regulate neuronal nitric-oxide synthase and prevent excitotoxic injury. Journal of Biological Chemistry. 2002;277(37):33995–34002. PubMed

Kielstein JT, Zoccali C. Asymmetric dimethylarginine: a cardiovascular risk factor and a uremic toxin coming of age? American Journal of Kidney Diseases. 2005;46(2):186–202. PubMed

Böger RH. The emerging role of asymmetric dimethylarginine as a novel cardiovascular risk factor. Cardiovascular Research. 2003;59(4):824–833. PubMed

Teerlink T, Luo Z, Palm F, Wilcox CS. Cellular ADMA: regulation and action. Pharmacological Research. 2009;60(6):448–460. PubMed PMC

Vallance P, Leiper J. Cardiovascular biology of the asymmetric dimethylarginine:dimethylarginine dimethylaminohydrolase pathway. Arteriosclerosis, Thrombosis, and Vascular Biology. 2004;24(6):1023–1030. PubMed

Wolf SS. The protein arginine methyltransferase family: an update about function, new perspectives and the physiological role in humans. Cellular and Molecular Life Sciences. 2009;66(13):2109–2121. PubMed PMC

Moore PK, Al-Swayeh OA, Chong NWS, Evans RA, Gibson A. L-N(G)-nitro arginine (L-NOARG), a novel, L-arginine-reversible inhibitor of endothelium-dependent vasodilatation in vitro. British Journal of Pharmacology. 1990;99(2):408–412. PubMed PMC

Rees DD, Palmer RMJ, Schulz R, Hodson HF, Moncada S. Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo. British Journal of Pharmacology. 1990;101(3):746–752. PubMed PMC

Furfine ES. Selective inhibition of constitutive nitric oxide synthase by L-NG-nitroarginine. Biochemistry. 1993;32(33):8512–8517. PubMed

Moore WM, Webber RK, Fok KF, et al. Inhibitors of human nitric oxide synthase isoforms with the carbamidine moiety as a common structural element. Bioorganic and Medicinal Chemistry. 1996;4(9):1559–1564. PubMed

Furfine ES, Harmon MF, Paith JE, et al. Potent and selective inhibition of human nitric oxide synthases. Selective inhibition of neuronal nitric oxide synthase by S-methyl-L-thiocitrulline and S-ethyl-L-thiocitrulline. Journal of Biological Chemistry. 1994;269(43):26677–26683. PubMed

Klatt P, Schmidt K, Brunner F, Mayer B. Inhibitors of brain nitric oxide synthase. Binding kinetics, metabolism, and enzyme inactivation. Journal of Biological Chemistry. 1994;269(3):1674–1680. PubMed

Schmidt K, Klatt P, Mayer B. Characterization of endothelial cell amino acid transport systems involved in the actions of nitric oxide synthase inhibitors. Molecular Pharmacology. 1993;44(3):615–621. PubMed

Kubes P, Suzuki M, Granger DN. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proceedings of the National Academy of Sciences of the United States of America. 1991;88(11):4651–4655. PubMed PMC

Dawson VL, Dawson TM, Bartley DA, Uhl GR, Snyder SH. Mechanisms of nitric oxide-mediated neurotoxicity in primary brain cultures. Journal of Neuroscience. 1993;13(6):2651–2661. PubMed PMC

Boje KM, Arora PK. Microglial-produced nitric oxide and reactive nitrogen oxides mediate neuronal cell death. Brain Research. 1992;587(2):250–256. PubMed

Papapetropoulos A, García-Cardeña G, Madri JA, Sessa WC. Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells. Journal of Clinical Investigation. 1997;100(12):3131–3139. PubMed PMC

Ziche M, Morbidelli L, Masini E, et al. Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P. Journal of Clinical Investigation. 1994;94(5):2036–2044. PubMed PMC

Balligand JL, Kelly RA, Marsden PA, Smith TW, Michel T. Control of cardiac muscle cell function by an endogenous nitric oxide signaling system. Proceedings of the National Academy of Sciences of the United States of America. 1993;90(1):347–351. PubMed PMC

Tabrizi-Fard MA, Fung HL. Pharmacokinetics and steady-state tissue distribution of L- and D-isomers of nitroarginine in rats. Drug Metabolism and Disposition. 1996;24(11):1241–1246. PubMed

Kidd EJ, Michel AD, Humphrey PPA. Autoradiographic distribution of [3H]L-N(G)-Nitro-arginine binding in rat brain. Neuropharmacology. 1995;34(1):63–73. PubMed

Rao VLR, Butterworth RF. Kinetics, pharmacology, and autoradiographic distribution of L-[3H]nitroarginine binding sites in rat cerebellum. Journal of Neurochemistry. 1996;66(2):701–709. PubMed

Pfeiffer S, Leopold E, Schmidt K, Brunner F, Mayer B. Inhibition of nitric oxide synthesis by N(G)-nitro-L-arginine methyl ester (L-NAME): requirement for bioactivation to the free acid, N(G)-nitro-L-arginine. British Journal of Pharmacology. 1996;118(6):1433–1440. PubMed PMC

Frandsen U, Bangsbo J, Sander M, et al. Exercise-induced hyperaemia and leg oxygen uptake are not altered during effective inhibition of nitric oxide synthase with NG-nitro-L-arginine methyl ester in humans. Journal of Physiology. 2001;531(1):257–264. PubMed PMC

Avontuur JAM, Buijk SLCE, Bruining HA. Distribution and metabolism of N(G)-nitro-L-arginine methyl ester in patients with septic shock. European Journal of Clinical Pharmacology. 1998;54(8):627–631. PubMed

Zhang HQ, Fast W, Marletta MA, Martasek P, Silverman RB. Potent and selective inhibition of neuronal nitric oxide synthase by N(ω)-propyl-L-arginine. Journal of Medicinal Chemistry. 1997;40(24):3869–3870. PubMed

Zhang HQ, Dixon RP, Marletta MA, Nikolic D, Van Breemen R, Silverman RB. Mechanism of inactivation of neuronal nitric oxide synthase by N(ω)-allyl-L-arginine. Journal of the American Chemical Society. 1997;119(45):10888–10902.

Olken NM, Marietta MA. NG-allyl- and NG-cyclopropyl-L-arginine: two novel inhibitors of macrophage nitric oxide synthase. Journal of Medicinal Chemistry. 1992;35(6):1137–1144. PubMed

Wolff DJ, Lubeskie A. Inactivation of nitric oxide synthase isoforms by diaminoguanidine and NG-amino-L-arginine. Archives of Biochemistry and Biophysics. 1996;325(2):227–234. PubMed

Huang H, Martasek P, Roman LJ, Masters BSS, Silverman RB. N(ω)-nitroarginine-containing dipeptide amides. Potent and highly selective inhibitors of neuronal nitric oxide synthase. Journal of Medicinal Chemistry. 1999;42(16):3147–3153. PubMed

Silverman RB, Huang H, Marletta MA, Martasek P. Selective inhibition of neuronal nitric oxide synthase by N(ω)- nitroarginine- and phenylalanine-containing dipeptides and dipeptide esters. Journal of Medicinal Chemistry. 1997;40(18):2813–2817. PubMed

Ji H, Gómez-Vidal JA, Martásek P, Roman LJ, Silverman RB. Conformationally restricted dipeptide amides as potent and selective neuronal nitric oxide synthase inhibitors. Journal of Medicinal Chemistry. 2006;49(21):6254–6263. PubMed PMC

Hah JM, Roman LJ, Martásek P, Silverman RB. Reduced amide bond peptidomimetics. (4S)-N-(4-amino-5-[aminoalkyl]aminopentyl)-N′-nitroguanidines, potent and highly selective inhibitors of neuronal nitric oxide synthase. Journal of Medicinal Chemistry. 2001;44(16):2667–2670. PubMed

Hah JM, Martásek P, Roman LJ, Silverman RB. Aromatic reduced amide bond peptidomimetics as selective inhibitors of neuronal nitric oxide synthase. Journal of Medicinal Chemistry. 2003;46(9):1661–1669. PubMed

Park JM, Higuchi T, Kikuchi K, et al. Selective inhibition of human inducible nitric oxide synthase by S-alkyl-L-isothiocitrulline-containing dipeptides. British Journal of Pharmacology. 2001;132(8):1876–1882. PubMed PMC

Babu BR, Griffith OW. N5-(1-imino-3-butenyl)-L-ornithine. A neuronal isoform selective mechanism-based inactivator of nitric oxide synthase. Journal of Biological Chemistry. 1998;273(15):8882–8889. PubMed

Litzinger EA, Martásek P, Roman LJ, Silverman RB. Design, synthesis, and biological testing of potential heme-coordinating nitric oxide synthase inhibitors. Bioorganic and Medicinal Chemistry. 2006;14(9):3185–3198. PubMed

Young RJ, Beams RM, Carter K, et al. Inhibition of inducible nitric oxide synthase by acetamidine derivatives of hetero-substituted lysine and homolysine. Bioorganic and Medicinal Chemistry Letters. 2000;10(6):597–600. PubMed

Alderton WK, Angell ADR, Craig C, et al. GW274150 and GW273629 are potent and highly selective inhibitors of inducible nitric oxide synthase in vitro and in vivo. British Journal of Pharmacology. 2005;145(3):301–312. PubMed PMC

Ijuin R, Umezawa N, Higuchi T. Design, synthesis, and evaluation of new type of l-amino acids containing pyridine moiety as nitric oxide synthase inhibitor. Bioorganic and Medicinal Chemistry. 2006;14(10):3563–3570. PubMed

Ulhaq S, Chinje EC, Naylor MA, Jaffar M, Stratford IJ, Threadgill MD. S-2-Amino-5-azolylpentanoic acids related to L-ornithine as inhibitors of the isoforms of nitric oxide synthase (NOS) Bioorganic and Medicinal Chemistry. 1998;6(11):2139–2149. PubMed

Ulhaq S, Chinje EC, Naylor MA, Jaffar M, Stratford IJ, Threadgill MD. Heterocyclic analogues of L-citrulline as inhibitors of the isoforms of nitric oxide synthase (NOS) and identification of N(δ)-(4,5-dihydrothiazol-2-yl)ornithine as a potent inhibitor. Bioorganic and Medicinal Chemistry. 1999;7(9):1787–1796. PubMed

Scheer M, Grote A, Chang A, et al. BRENDA, the enzyme information system in 2011. Nucleic Acids Research. 2011;39(1):D670–D676. PubMed PMC

Babu BR, Griffith OW. Design of isoform-selective inhibitors of nitric oxide synthase. Current Opinion in Chemical Biology. 1998;2(4):491–500. PubMed

Garvey EP, Oplinger JA, Tanoury GJ, et al. Potent and selective inhibition of human nitric oxide synthases. Inhibition by non-amino acid isothioureas. Journal of Biological Chemistry. 1994;269(43):26669–26676. PubMed

Hagen TJ, Bergmanis AA, Kramer SW, et al. 2-Iminopyrrolidines as potent and selective inhibitors of human inducible nitric oxide synthase. Journal of Medicinal Chemistry. 1998;41(19):3675–3683. PubMed

Collins JL, Shearer BG, Oplinger JA, et al. N-phenylamidines as selective inhibitors of human neuronal nitric oxide synthase: structure-activity studies and demonstration of in vivo activity. Journal of Medicinal Chemistry. 1998;41(15):2858–2871. PubMed

Seo J, Martásek P, Roman LJ, Silverman RB. Selective l-nitroargininylaminopyrrolidine and l-nitroargininylaminopiperidine neuronal nitric oxide synthase inhibitors. Bioorganic and Medicinal Chemistry. 2007;15(5):1928–1938. PubMed PMC

Sonntag M, Deussen A, Schrader J. Role of nitric oxide in local blood flow control in the anaesthetized dog. Pflugers Archiv European Journal of Physiology. 1992;420(2):194–199. PubMed

Van Buren GA, Yang D, Clark KE. Estrogen-induced uterine vasodilatation is antagonized by L-nitroarginine methyl ester, an inhibitor of nitric oxide synthesis. American Journal of Obstetrics and Gynecology. 1992;167(3):828–833. PubMed

Vials A, Burnstock G. Effects of nitric oxide synthase inhibitors, L-N(G)-nitroarginine and L-N(G)-nitroarginine methyl ester, on responses to vasodilators of the guinea-pig coronary vasculature. British Journal of Pharmacology. 1992;107(2):604–609. PubMed PMC

Lorente JA, Landin L, De Pablo R, Renes E, Liste D. L-arginine pathway in the sepsis syndrome. Critical Care Medicine. 1993;21(9):1287–1295. PubMed

Avontuur JAM, Tutein Nolthenius RP, Van Bodegom JW, Bruining HA. Prolonged inhibition of nitric oxide synthesis in severe septic shock: a clinical study. Critical Care Medicine. 1998;26(4):660–667. PubMed

Yates DH, Kharitonov SA, Thomas PS, Barnes PJ. Endogenous nitric oxide is decreased in asthmatic patients by an inhibitor of inducible nitric oxide synthase. American Journal of Respiratory and Critical Care Medicine. 1996;154(1):247–250. PubMed

Fast W, Levsky ME, Marletta MA, Silverman RB. N(ω)-Propargyl-L-arginine and N(ω)-hydroxy-N(ω)-propargyl-L-arginine are inhibitors, but not activators, of neuronal and macrophage nitric oxide synthases. Bioorganic and Medicinal Chemistry. 1997;5(8):1601–1608. PubMed

Raoul C, Estévez AG, Nishimune H, et al. Motoneuron death triggered by a specific pathway downstream of fas: potentiation by ALS-linked SOD1 mutations. Neuron. 2002;35(6):1067–1083. PubMed

Caron MH, Alling C. Role of nitric oxide in ethanol-induced up-regulation of muscarinic acetylcholine receptors in SH-SY5Y cells. Alcoholism: Clinical and Experimental Research. 2001;25(8):1107–1113. PubMed

Cooper GR, Mialkowski K, Wolff DJ. Cellular and enzymatic studies of N(ω)-propyl-L-arginine and S-ethyl-N- [4-(trifluoromethyl)phenyl]isothiourea as reversible, slowly dissociating inhibitors selective for the neuronal nitric oxide synthase isoform. Archives of Biochemistry and Biophysics. 2000;375(1):183–194. PubMed

Oceandy D, Cartwright EJ, Emerson M, et al. Neuronal nitric oxide synthase signaling in the heart is regulated by the sarcolemmal calcium pump 4b. Circulation. 2007;115(4):483–492. PubMed

Isaak A, Ellrich J. Neuronal nitric oxide synthase is involved in the induction of nerve growth factor-induced neck muscle nociception. Headache. 2011;51(5):734–743. PubMed

Klamer D, Engel JA, Svensson L. The neuronal selective nitric oxide synthase inhibitor, N ω-propyl-l-arginine, blocks the effects of phencyclidine on prepulse inhibition and locomotor activity in mice. European Journal of Pharmacology. 2004;503(1–3):103–107. PubMed

Kellogg DL, Zhao JL, Wu Y. Roles of nitric oxide synthase isoforms in cutaneous vasodilation induced by local warming of the skin and whole body heat stress in humans. Journal of Applied Physiology. 2009;107(5):1438–1444. PubMed PMC

Fukuto JM, Wood KS, Byrns RE, Ignarro LJ. N(G)-Amino-L-arginine: a new potent antagonist of L-arginine-mediated endothelium-dependent relaxation. Biochemical and Biophysical Research Communications. 1990;168(2):458–465. PubMed

Gross SS, Stuehr DJ, Aisaka K, Jaffe EA, Levi R, Griffith OW. Macrophage and endothelial cell nitric oxide synthesis: cell-type selective inhibition by N(G)-aminoarginine, N(G)-nitroarginine and N(G)-methylarginine. Biochemical and Biophysical Research Communications. 1990;170(1):96–103. PubMed

Hecker M, Walsh DT, Vane JR. On the substrate specificity of nitric oxide synthase. FEBS Letters. 1991;294(3):221–224. PubMed

Silverman RB, Huang H, Zhang HQ. Inhibition of nitric oxide synthase by amino acids and dipeptides. USA patent US6274557B1, Northwestern University, Evanston, Ill, USA, 2001.

Silverman RB. Selective neuronal nitric oxide synthase inhibitors. USA patent WO03000198(A2), Northwestern University, 2003.

Kobayashi N, Higuchi T, Hirobe M, Nagano T. Selective inhibitor for nitrogen monooxide synthase. Dainippon Pharmaceutical, Japan patent JP10237097(A), 1998.

Kobayashi N, Higuchi T, Urano Y, Kikuchi K, Hirobe M, Nagano T. Dipeptides containing L-arginine analogs: new isozyme-selective inhibitors of nitric oxide synthase. Biological and Pharmaceutical Bulletin. 1999;22(9):936–940. PubMed

Rogers NE, Ignarro LJ. Constitutive nitric oxide synthase from cerebellum is reversibly inhibited by nitric oxide formed from L-arginine. Biochemical and Biophysical Research Communications. 1992;189(1):242–249. PubMed

Narayanan K, Griffith OW. Synthesis of L-thiocitrulline, L-homothiocitrulline, and S-methyl-L-thiocitrulline: a new class of potent nitric oxide synthase inhibitors. Journal of Medicinal Chemistry. 1994;37(7):885–887. PubMed

Frey C, Narayanan K, McMillan K, et al. L-thiocitrulline: a stereospecific, heme-binding inhibitor of nitric- oxide synthases. Journal of Biological Chemistry. 1994;269(42):26083–26091. PubMed

Narayanan K, Spack L, McMillan K, et al. S-alkyl-L-thiocitrullines. Potent stereoselective inhibitors of nitric oxide synthase with strong pressor activity in vivo. Journal of Biological Chemistry. 1995;270(19):11103–11110. PubMed

Ijuin R, Umezawa N, Nagai SI, Higuchi T. Evaluation of 3-substituted arginine analogs as selective inhibitors of human nitric oxide synthase isozymes. Bioorganic and Medicinal Chemistry Letters. 2005;15(11):2881–2885. PubMed

Zhang J, Xu M, Dence CS, Sherman ELC, McCarthy TJ, Welch MJ. Synthesis, in vivo evaluation and PET study of a carbon-11 -labeled neuronal nitric oxide synthase (nNOS) inhibitor S-Methyl-L-thiocitrulline. Journal of Nuclear Medicine. 1997;38(8):1273–1278. PubMed

Zanzinger J, Czachurski J, Seller H. Neuronal nitric oxide reduces sympathetic excitability by modulation of central glutamate effects in pigs. Circulation Research. 1997;80(4):565–571. PubMed

Kelley JB, Anderson KL, Altmann SL, Itzhak Y. Long-term memory of visually cued fear conditioning: roles of the neuronal nitric oxide synthase gene and cyclic AMP response element-binding protein. Neuroscience. 2011;174:91–103. PubMed PMC

Ichihara A, Inscho EW, Imig JD, Navar LG. Neuronal nitric oxide synthase modulates rat renal microvascular function. American Journal of Physiology, Renal Physiology. 1998;274(3):F516–F524. PubMed

Komers R, Oyama TT, Chapman JG, Allison KM, Anderson S. Effects of systemic inhibition of neuronal nitric oxide synthase in diabetic rats. Hypertension. 2000;35(2):655–661. PubMed

Komers R, Lindsley JN, Oyama TT, Allison KM, Anderson S. Role of neuronal nitric oxide synthase (NOS1) in the pathogenesis of renal hemodynamic changes in diabetes. American Journal of Physiology, Renal Physiology. 2000;279(3):F573–F583. PubMed

McCall TB, Feelisch M, Palmer RMJ, Moncada S. Identification of N-iminoethyl-L-ornithine as an irreversible inhibitor of nitric oxide synthase in phagocytic cells. British Journal of Pharmacology. 1991;102(1):234–238. PubMed PMC

Moore WM, Webber RK, Jerome GM, Tjoeng FS, Misko TP, Currie MG. L-N6-(1-Iminoethyl)lysine: a selective inhibitor of inducible nitric oxide synthase. Journal of Medicinal Chemistry. 1994;37(23):3886–3888. PubMed

Hallinan EA, Tsymbalov S, Dorn CR, et al. Synthesis and biological characterization of L-N6-(1-iminoethyl)lysine 5-tetrazole-amide, a prodrug of a selective iNOS inhibitor. Journal of Medicinal Chemistry. 2002;45(8):1686–1689. PubMed

Assreuy J, Cunha FQ, Epperlein M, et al. Production of nitric oxide and superoxide by activated macrophages and killing of Leishmania major. European Journal of Immunology. 1994;24(3):672–676. PubMed

De Graaf JC, Banga JD, Moncada S, Palmer RMJ, De Groot PG, Sixma JJ. Nitric oxide functions as an inhibitor of platelet adhesion under flow conditions. Circulation. 1992;85(6):2284–2290. PubMed

Jenkins DC, Charles IG, Thomsen LL, et al. Roles of nitric oxide in tumor growth. Proceedings of the National Academy of Sciences of the United States of America. 1995;92(10):4392–4396. PubMed PMC

Duffield JS, Erwig LP, Wei XQ, Liew FY, Rees AJ, Savill JS. Activated macrophages direct apoptosis and suppress mitosis of mesangial cells. Journal of Immunology. 2000;164(4):2110–2119. PubMed

Brahmachari S, Fung YK, Pahan K. Induction of glial fibrillary acidic protein expression in astrocytes by nitric oxide. Journal of Neuroscience. 2006;26(18):4930–4939. PubMed PMC

Zhang JY, Wang Y, Milton MN, Kraus L, Breau AP, Paulson SK. Disposition and Pharmacokinetics of L-N6-(1-Iminoethyl)Lysine-5-Tetrazole-Amide, a Selective iNOS Inhibitor, in Rats. Journal of Pharmaceutical Sciences. 2004;93(5):1229–1240. PubMed

Zhang JY, Wang YF, Milton MN, et al. Pharmacokinetics, metabolism and excretion of an inhibitor of inducible nitric oxide synthase, L-NIL-TA, in dog. Xenobiotica. 2003;33(11):1159–1171. PubMed

László F, Whittle BJR. Actions of isoform-selective and non-selective nitric oxide synthase inhibitors on endotoxin-induced vascular leakage in rat colon. European Journal of Pharmacology. 1997;334(1):99–102. PubMed

Connor JR, Manning PT, Settle SL, et al. Suppression of adjuvant-induced arthritis by selective inhibition of inducible nitric oxide synthase. European Journal of Pharmacology. 1995;273(1-2):15–24. PubMed

Macmicking JD, North RJ, Lacourse R, Mudgett JS, Shah SK, Nathan CF. Identification of nitric oxide synthase as a protective locus against tuberculosis. Proceedings of the National Academy of Sciences of the United States of America. 1997;94(10):5243–5248. PubMed PMC

Salvemini D, Wang ZQ, Wyatt PS, et al. Nitric oxide: a key mediator in the early and late phase of carrageenan-induced rat paw inflammation. British Journal of Pharmacology. 1996;118(4):829–838. PubMed PMC

Pelletier JP, Jovanovic D, Fernandes JC, et al. Reduced progression of experimental osteoarthritis in vivo by selective inhibition of inducible nitric oxide synthase. Arthritis & Rheumatism. 1998;41(7):1275–1286. PubMed

Dumont M, Wille E, Calingasan NY, Nathan C, Beal MF, Lin MT. N-iminoethyl-l-lysine improves memory and reduces amyloid pathology in a transgenic mouse model of amyloid deposition. Neurochemistry International. 2010;56(2):345–351. PubMed

Crowell JA, Steele VE, Sigman CC, Fay JR. Is inducible nitric oxide synthase a target for chemoprevention? Molecular Cancer Therapeutics. 2003;2(8):815–823. PubMed

Eynott PR, Groneberg DA, Caramori G, et al. Role of nitric oxide in allergic inflammation and bronchial hyperresponsiveness. European Journal of Pharmacology. 2002;452(1):123–133. PubMed

Neufeld AH, Kawai SI, Das S, et al. Loss of retinal ganglion cells following retinal ischemia: the role of inducible nitric oxide synthase. Experimental Eye Research. 2002;75(5):521–528. PubMed

Hansel TT, Kharitonov SA, Donnelly LE, et al. A selective inhibitor of inducible nitric oxide synthase inhibits exhaled breath nitric oxide in healthy volunteers and asthmatics. The FASEB Journal. 2003;17(10):1298–1300. PubMed

Hodson HF, Palmer RMJ, Sawyer DA, et al. Enzyme inhibitors. US patent WO9534534(A1), 1995.

Baydoun AR, Bertran J, Thakur S, Dawson J, Palacín M, Knowles RG. y+ LAT-1 mediates transport of the potent and selective iNOS inhibitor, GW274150, in control J774 macrophages. Amino Acids. 2006;31(2):101–109. PubMed

O’Brien AJ, Wilson AJ, Sibbald R, Singer M, Clapp LH. Temporal variation in endotoxin-induced vascular hyporeactivity in a rat mesenteric artery organ culture model. British Journal of Pharmacology. 2001;133(3):351–360. PubMed PMC

Mattiussi S, Lazzari C, Truffa S, et al. Homeodomain interacting protein kinase 2 activation compromises endothelial cell response to laminar flow: protective role of p21waf1,cip1,sdi1. PLoS One. 2009;4(8)e6603 PubMed PMC

Chatterjee PK, Kvale EO, Patel NSA, Thiemermann C. GW274150 inhibits nitric oxide production by primary cultures of rat proximal tubular cells. Medical Science Monitor. 2003;9(10):BR357–BR362. PubMed

Arnhold S, Faßbender A, Klinz FJ, et al. NOS-II is involved in early differentiation of murine cortical, retinal and ES cell-derived neurons - An immunocytochemical and functional approach. International Journal of Developmental Neuroscience. 2002;20(2):83–92. PubMed

De Alba J, Clayton NM, Collins SD, Colthup P, Chessell I, Knowles RG. GW274150, a novel and highly selective inhibitor of the inducible isoform of nitric oxide synthase (iNOS), shows analgesic effects in rat models of inflammatory and neuropathic pain. Pain. 2006;120(1-2):170–181. PubMed

McDonald MC, Izumi M, Cuzzocrea S, Thiemermann C. A novel, potent and selective inhibitor of the activity of inducible nitric oxide synthase (GW274150) reduces the organ injury in hemorrhagic shock. Journal of Physiology and Pharmacology. 2002;53(4 I):555–569. PubMed

Cuzzocrea S, Chatterjee PK, Mazzon E, et al. Beneficial effects of GW274150, a novel, potent and selective inhibitor of iNOS activity, in a rodent model of collagen-induced arthritis. European Journal of Pharmacology. 2002;453(1):119–129. PubMed

Chatterjee PK, Patel NSA, Sivarajah A, et al. GW274150, a potent and highly selective inhibitor of iNOS reduces experimental renal ischemia/reperfusion injury. Kidney International. 2003;63(3):853–865. PubMed

Dugo L, Marzocco S, Mazzon E, et al. Effects of GW274150, a novel and selective inhibitor of iNOS activity, in acute lung inflammation. British Journal of Pharmacology. 2004;141(6):979–987. PubMed PMC

Di Paola R, Mazzon E, Patel NSA, et al. Beneficial effects of GW274150 treatment on the development of experimental colitis induced by dinitrobenzene sulfonic acid. European Journal of Pharmacology. 2005;507(1–3):281–289. PubMed

Broom L, Marinova-Mutafchieva L, Sadeghian M, Davis JB, Medhurst AD, Dexter DT. Neuroprotection by the selective iNOS inhibitor GW274150 in a model of Parkinson disease. Free Radical Biology and Medicine. 2011;50(5):633–640. PubMed

Singh D, Richards D, Knowles RG, et al. Selective inducible nitric oxide synthase inhibition has no effect on allergen challenge in asthma. American Journal of Respiratory and Critical Care Medicine. 2007;176(10):988–993. PubMed

Palmer JE, Guillard FL, Laurijssens BE, Wentz AL, Dixon RM, Williams PM. A randomised, single-blind, placebo-controlled, adaptive clinical trial of GW274150, a selective iNOS inhibitor, in the treatment of acute migraine. Cephalalgia. 2009;29(1):p. 124.

Høivik HO, Laurijssens BE, Harnisch LO, et al. Lack of efficacy of the selective iNOS inhibitor GW274150 in prophylaxis of migraine headache. Cephalalgia. 2010;30(12):1458–1467. PubMed

Rogerson TD, Wilkinson CF, Hetarski K. Steric factors in the inhibitory interaction of imidazoles with microsomal enzymes. Biochemical Pharmacology. 1977;26(11):1039–1042. PubMed

Rees DD, Palmer RMJ, Moncada S. Role of endothelium-derived nitric oxide in the regulation of blood pressure. Proceedings of the National Academy of Sciences of the United States of America. 1989;86(9):3375–3378. PubMed PMC

Cheshire DR, Åberg A, Andersson GMK, et al. The discovery of novel, potent and highly selective inhibitors of inducible nitric oxide synthase (iNOS) Bioorganic and Medicinal Chemistry Letters. 2011;21(8):2468–2471. PubMed

Fullmer T. Rethinking mechanisms of drug discovery. SciBX. 2011;4(27)

Huang H, Martásek P, Roman LJ, Silverman RB. Synthesis and evaluation of peptidomimetics as selective inhibitors and active site probes of nitric oxide synthases. Journal of Medicinal Chemistry. 2000;43(15):2938–2945. PubMed

Ekins S, Mestres J, Testa B. In silico pharmacology for drug discovery: methods for virtual ligand screening and profiling. British Journal of Pharmacology. 2007;152(1):9–20. PubMed PMC

Guido RVC, Oliva G, Andricopulo AD. Virtual screening and its integration with modern drug design technologies. Current Medicinal Chemistry. 2008;15(1):37–46. PubMed

Hoffer L, Renaud JP, Horvath D. Fragment-based drug design: computational and experimental state of the art. Combinatorial Chemistry and High Throughput Screening. 2011;14(6):500–520. PubMed

Tinker AC, Beaton HG, Boughton-Smith N, et al. 1,2-Dihydro-4-quinazolinamines: potent, highly selective inhibitors of inducible nitric oxide synthase which show antiinflammatory activity in vivo. Journal of Medicinal Chemistry. 2003;46(6):913–916. PubMed

Ji H, Stanton BZ, Igarashi J, et al. Minimal pharmacophoric elements and fragment hopping, an approach directed at molecular diversity and isozyme selectivity. Design of selective neuronal nitric oxide synthase inhibitors. Journal of the American Chemical Society. 2008;130(12):3900–3914. PubMed PMC

Ekins S, Mestres J, Testa B. In silico pharmacology for drug discovery: applications to targets and beyond. British Journal of Pharmacology. 2007;152(1):21–37. PubMed PMC

Ji H, Li H, Flinspach M, Poulos TL, Silverman RB. Computer modeling of selective regions in the active site of nitric oxide synthases: implication for the design of isoform-selective inhibitors. Journal of Medicinal Chemistry. 2003;46(26):5700–5711. PubMed

Fröhlich LG, Kotsonis P, Traub H, et al. Inhibition of neuronal nitric oxide synthase by 4-amino pteridine derivatives: structure-activity relationship of antagonists of (6R)-5,6,7,8- tetrahydrobiopterin cofactor. Journal of Medicinal Chemistry. 1999;42(20):4108–4121. PubMed

Matter H, Kotsonis P, Klingler O, et al. Structural requirements for inhibition of the neuronal nitric oxide synthase (NOS-I): 3D-QSAR analysis of 4-oxo- and 4-amino-pteridine-based inhibitors. Journal of Medicinal Chemistry. 2002;45(14):2923–2941. PubMed

Age-dependent redox status in the brain stem of NO-deficient hypertensive rats