- Email: [email protected]
Selective inhibitors of neuronal nitric oxide synthase — is no NOS really good NOS for the nervous system?
R
E
E
I
V
W
Selective inhibitors of neuronal nitric oxide synthase - is no NOS really good NOS for the nervous system? Philip K. Moore and Rachel 1. C. Handy It is now ten years since NO was shown to account for the biological activity of endothelium-derived factor (EORF). It is also the tenth anniversary identification
of L-A/G monomethyl
relaxing of the
arginine (L-NMMA)
as the very first inhibitor of NO biosynthesis.
That EORF
and NO were one and the same sparked an explosion of interest in the biochemistry
and pharmacology
of NO
which has yet to subside. In contrast, the first ever nitric oxide synthase (NOS) inhibitor slipped seamiessly into the literature
virtually without comment at the time.
Over the following decade, L-NMMA inhibitors) have proved invaluable
(and like NOS
as tools for probing
the biological roles of NO in health and disease and, in particular,
have increased
our understanding
of the
function of NO in the nervous system. Further advances in this important area now require the development inhibitors selective
of
for the neuronal isoform of NOS
(nNOS). Here, Philip Moore and Rachel Handy provide an up-to-date biochemical
account of the literature and pharmacological
NOS inhibitors with particular with greater selectivity
P. K. Moore Reader II Pharmacology ant R. L. C. Handy Research Fellow Pharmacology Group Biomedical Science! Division, King’: College. University 0 London. Maws; Road, London UK SW36LX
204
regarding the
characterization
reference
of
to compounds
for the nNOS isoform.
The physiological and pathophysiological functions of NO and the molecular biology and biochemistry of NOS have both been extensively discussed and are beyond the scope of this review (for further information, see Refs 14). However, a brief description of the major biological roles of NO and of the nature of the individual NOS isoforms and the mechanism of NOS action does seem appropriate here if only to provide a rational basis for understanding the mechanism of action of NOS inhibitors. To date, the amino acid, mRNA and cDNA sequences of three distinct isoforms of NOS have been determined. These correspond to the neuronal (nNOS, type I), inducible (iNOS, type II) and endothelial (eNOS, type III) enzymes. The so-called constitutive isoforms of NOS (nNOS and eNOS) are located mainly (but not exclusively) in central and peripheral nerves and vascular
TiPS
-June
1997 (Vol. 18)
endothelial cells, respectively, and serve a wide range of predominantly physiological or ‘housekeeping’ functions. For example, NO derived from nNOS in the CNS has a number of important functions including the modification of pain perception, mediation of longterm potentiation (LTP) and memory, control of cerebral blood flow and in neurodegeneration following cerebral ischaemia. In addition, nNOS located in selected nonadrenergic, non-cholinergic (NANC) nerves in the periphery generates NO, which acts as a neurotransmitter to relax smooth muscle in the gastrointestinal and urogenital tracts and the airways. Furthermore, relaxation of vascular smooth muscle and inhibition of platelet adhesion and aggregation occur following activation of eNOS in vascular endothelial cells and most probably represents a fundamentally important mechanism for regulating blood pressure and tissue perfusion. In contrast, iNOS is synthesized de ~OUOwithin selected cell types a few hours after exposure to bacterial endotoxins or to a range of different cytokines. The iNOS isoform is believed to be of significance in pathophysiological situations playing a part, for example, in the body’s defensive response to disease and infections. Nitric oxide synthase catalyses the five-electron oxidation of L-arginine to NO and L-citrulline. A combination of mechanistic and molecular biological techniques has revealed that the active form of NOS is a dimer containing flavin (flavin adenine mononucleotide and flavin adenine dinucleotide, FMN and FAD), haem, NADPH (reduced nicotinamide adenine dinucleotide phosphate) and tetrahydrobiopterin as prosthetic groups. Nitric oxide formation commences with hydroxylation of L-arginine to form L-IF hydroxyarginine and the subsequent incorporation of a second atom of oxygen to yield NO and its co-product, citrulline. The part played by the various prosthetic groups in this reaction has been the subject of intense scrutiny and is of interest here since many inhibitors of this enzyme interfere with binding at these sites. Briefly, electron transfer from molecular oxygen proceeds across domains within the NOS enzyme in a linear sequence from NADPH to FAD/FMN and finally to the haem site. Calmodulin is activated by Ca2+ entering cells as a result of the opening of either receptor-operated [e.g. muscarinic receptors on endothelial cells or NMDA (Nmethyl-D-aspartate) receptors on neurones] or voltageoperated (e.g. NANC nerves) channels and promotes electron transfer between FAD/FMN and haem. Tetrahydrobiopterin may also act as a cofactor for NO formation but may have an even more fundamental role in linking the two monomers of NOS to yield the biochemically active NOS dimer.
The ‘A-Z’ of NOS inhibitors Drugs can, at least in theory, reduce or prevent the biological effects of NO in a number of ways (Fig. 1). These include inhibition of the uptake of L-arginine into the cell (A, Fig. l), reducing the cellular availability of necessary
0 1997, Elsevier Science Ltd
PII: SO165-6147(97)01064-X
REVIEW
cofactors by preventing their formation or promoting their breakdown (B, C), scavenging NO once formed (D) or inhibiting the cellular mechanisms leading to induction of the particular NOS isoform (E). Over recent years inhibition of the enzymatic activity of NOS (F-M) has been the most thoroughly investigated approach and has provided the majority of useful compounds. The multiplicity of binding sites on NOS for substrate (L-arginine), cofactor (NADPH, FAD, FMN, calmodulin), ‘stabilizer’ (possibly tetrahydrobiopterin) and the haem prosthetic moiety has provided researchers with ample scope for the identification of inhibitors acting at each of these sites. Indeed, in the ten years since the original report on the inhibitory effect of L-NMMA on NOS many more ‘novel’ inhibitors of NOS have been described. To date, there are well over a hundred NOS inhibitors in the literature with (almost) every letter of the alphabet represented (Box 1). The biochemical and pharmacological characterization of new NOS inhibitors is certainly a worthwhile research goal although the conduct of the experiments required and interpretation of the data obtained is a process that requires careful thought. Some of the problems associated with the identification of NOS inhibitors are described in Box 2. Many of the agents shown in Box 1 exhibit activity other than NOS inhibition and are thus unsuitable leads for the identification of selective nNOS inhibitors. For example, methylene blue is a potent inhibitor of soluble guanylate cyclase whilst iron-protoporphryn-IX inhibits haem oxygenase. Ebselen interacts with thiol groups on proteins and, as such, also inhibits NADPH oxidase and cytochrome B5 reductase. Finally, drugs that block NOS by binding at either the calmodulin (e.g. chlorpromazine) or NADPH (e.g. diphenyleneiodionium salts) sites or by disrupting cellular tetrahydrobiopterin formation (e.g. 2,4-diamino-6-hydroxypyrimidine) exhibit a wide range of alternative effects and are therefore of limited interest. Interestingly, of the compounds shown in Box 1 very few have been reported to exhibit any degree of selectivity for nNOS over the other isoforms.
Amino acid analogues as a source of nNOS-selective inhibitors? Many amino acid derivatives inhibit NOS enzyme activity both in vitro and in a range of isolated cells, biological tissues and intact animals6. In general, none of the compounds reported to date shows significant selectivity for nNOS either in vitro or in viva. Guanidino substituted analogues of L-arginine (e.g. L-NMMA (monomethylarginine), L-NGnitroarginine and its methyl ester, L-NOARG and L-NAME; see also compounds in Box 1) are potent NOS inhibitors both in vitro and in aivo. Whilst all analogues in this group inhibit NOS by competition with L-arginine for the substrate binding site the precise mechanism of action varies both with the analogue and with the NOS isoform studied. For example, L-NOARG produces a time-dependent, irreversible inhibition of constitutive NOS isoforms in
L-Arginine 74 A
NADPH CaM
FAD
NO + L-Citrulline
FMN
Ca2+
I NO
QLE
Fig.1.Inhibition of nitnc oxide synthase (NOS). Potential targets to intllbtt the synthesis or activity of NO in a stylized mammalian cell. NOS is shown in the centre of the cell equipped with seven binding sites for substrate or cofactors. Letters Indicate possible sites of drug action as follows. For drugs active at these various sites see Box 2. A. inhibition of uptake of Larginine; B. inhibition of tetrahydrobiopterin (BH,) biosynthesis; C, reduction of cellular concentration of cofactors, e.g. sequestration of Ca:+. D. induction of NDS, e.g. induction of inducible NOS (iNOS) by cytokines and endothelial NOS/neuronal NOS (eNDS/nNOS) by oestrogen; E, scavenging of extracellular NO; F. t-arglnine binding site; Ci, tetrahydrobiopterin binding site; H. haem site; J, NADPH (reduced nicotlnamide adenme dinucleotide phosphate) binding site; K. calmodulin site(CaM); L and M. binding sites for flavine adentne mononucleotide (FMN) and flavine adenine dinucleotide (FAD), respectively.
vitro and a long-lasting (>24h) inhibition of brain NOS following parenteral administration in the rat’. In contrast, L-NOARG causes competitive, reversible inhibition of iNOS. In general, guanidino-substituted analogues of L-arginine demonstrate little or no worthwhile selectivity for individual NOS isoforms in vitro. Furthermore, in isolated tissue preparations or in intact animals, administration of such analogues elicits a profile of pharmacological activity commensurate with inhibition of eNOS (e.g. reduction of endothelium vasodilatation in isolated blood vessels8, increase in arterial blood pressure in anaesthetized or conscious animal&lo), nNOS (e.g. antinociceptive activity”, inhibition of NANC relaxation of anococcygeus and other isolated muscle preparation+) and iNOS (e.g. increase in blood pressure in septic shockl3). In the majority of cases, the biological effect of the NOS inhibitor is either partially or totally reversed by I.-arginine. A range of other amino acid derivatives have also been assessed for their ability to inhibit NOS. Both L-N-iminoethylornithine (L-NIO)l” and L-iV6-iminoethyllysine (L-NIL)‘5 are relatively potent NOS inhibitors although neither shows selectivity for the nNOS isoform. Indeed, L-NIL is relatively selective for iNOS. Substituted citrulline compounds such as thiocitrulline and alkylthiocitrullines (e.g. S-methyl and S-ethylthiocitrulline, SMTC and SETC) also inhibit NOS. In this context, SMTC is of some interest in that it does indeed exhibit some degree
TilJS-Junr
lYY7
lVOl
18)
2 0 5
REVIEW Box 1. The ‘A-Z’ of nitric Compound and site of action’ Acetyl5-hydroxytryptamine (B)i Allylarginine (F)2 Aminoarginine (F)* 2-amino-5,6-dihydro-methylthiazine @MT)4 3-aminomethylbenzylacetamidine (14OOW)5 Aminoethylisothiourea (F/?H)6 Aminoguanidine (F/?H)7
2-amine-4-methylpyridine (F, ?H)n 3-amino-1,2,4triazole (B)i2 Arcaine Asymmetric NGNo-dimethylarginine (ADMA) (F)2 3-bromo 7nitroindazole (3Br 7-NI) (H/F/G)‘5 Calmidazolium (K)*6 Carbon monoxide (CO) (H)ir Canavanine Chlorpromazine (K)i9 Clotrimazole (H)zO Cylopropylarginine (F)zi DiaminoguanidineP Diamino-6-hydroxypyrimidine= Dicoumarol (B)i Dimethylguanidine23 Diphenyleneiodonium (J)24
Doxorubicin25 Ebselen26
Iminoethylornithinine (L-NIO)~~ Iminopiperidir+ S-isopropylisothioua (IPl7.J) (F/?H)6 KetoconazoIe (H)20 L-NC-substituted arginine analogues (F)
TOPS -June 1997(Vol.
Irreversible inhibitor of nitric oxide synthase (NOS) Causes convulsions in dogs3 Potent and selective inducible NOS (iNOS) inhibitor Very potent and selective iNOS inhibitor Converted to mercaptoethylguanidi in vivo 26X more potent on iNOS than neuronal NOS (nNOS); iNOS inhibitor in vi&; nonselective, potent inhibitor of diamine oxidase, polyamine oxidase, riinucleotide reductaseYJ0 7X more potent on iNOS cf. endothelial NOS (eNOS) in vivo Inhibits cell tetrahydrobiopterin (BH4) formation which may inhibit synthesis of noradrenaline and 5-HT Endogenous inhibitor of NOS; increased plasma concentration in chronic kidney failure13 and in human schizophrenic@ 4~ more potent than 7nitroindazole (7-NI) on nNOS Does not inhibit iNOS since calmodulin is tightly bound in this isoform Potential role of CO in biological processes; physiological significance of interaction of CO and NOS is unclear Weak NOS inhibitor. Increases blood pressure in endotoxin-treated rats’* Also reduces induction of iNOS in lung from endotoxin-treated rats19 Antifungal Extremely potent inhibitor of all three isoforms; nonselective; interacts with NADPH (reduced nicotinamide adenine dinucleotide phosphate) site on other enzymes Seleno-organic compound, modifies thiol groups at or close to active site of NOS; nonselective, inhibits lipoxygenase, NADPH oxidase and protein kinase Cz7,reduces nuclear factor-lcB (NF-KB) activation28 and reacts with peroxynitrite29
S-ethylisothiourea (F/?H)6 Ethylthiopseudourea6,m Fluconazole (H)20 Guanethidir@ Guanidinoethyldisulphide (GED) (F/?H)32 Guinidinoglutaric acid (GGA) (F)B Haloperidol (K)H HF2035 (Ref. 35) Homothiocitrulline (H)x Imidazole (H)37 hninobiotin~ Iminoethyllysine (L-NIL)39
2 0 6
Comment
18)
Antifungal Sympatholytic
Anti-psychotic drug Inhibitor of nNOS/eNOS in vitro but less active against iNOS Very weak NOS inhibitor Equiactive on iNGS and nNOS in vitro 29X more potent on iNOS than nNOS; anti-inflammatory in a rat model of arthritis40 Irreversible inhibitor of iNOS in vitro -
Isoform nonselective isothiourea compound Antifungal D-isomers are biologically inactive; one report that D-NAME nitroarginine methyl ester inhibits endothelium-dependent relaxation of rat aorta”
REVIEW
oxide synthase inhibitors Compound and site of action’
Comment
LY83583 (Ref. 44) Mercaptoethylguanidine (MEG) (F)45
Also blocks soluble guanylate cyclase Also inhibits constitutive and inducible cyclooxygenase (COX-1 and COX-2)%; may be beneficial in the treatment of inflammatory conditions Anti-cancer agent Also inhibits soluble guanylate cyclase@ and xanthine oxida@
Methotrexate47 Methylene blue (?H)@ Methylguanidine23 S-methylisothiourea (SMT) (F/?H)6
Methylthiocitrulline (H)x Miconazole (H)20 Monomethylarginine (L-NMMA)
(F/?A)
Nitric oxide (H)x Nitroarginine (L-NOARG) (F)55 Nitroarginine methyl ester (L-NAME) (F)
‘I-nitroindazole (H/F/G) (7-NI)59
Oestrogen Paroxetinebl Phencyclidines
1,2- and 1,4-phenylimidazole (H)37 Pimozide (K) PIN (protein inhibitor of NOS)g
W/F/G)72
W-7 (K) Zinc (?H)73
Antifungal First reported ‘arginine-based’ NOS inhibito+; also inhibits arginine uptake into cell+; sometimes abbreviated to L-NMA Possible feedback inhibitor of NOS? Reported to inhibit arginase56; other abbreviations include L-NA and NOLA Demethylated in viva to yield active L-NOARG57; reported to antagonize muscarinic cholinoceptors58, not confirmed in other studies Relatively selective for nNOS in uivo; slightly more water soluble Na+ salt (7-NINA) available@; high concentrations relax smooth muscle by unknown mechanism61 High concentrations inhibit NOW; also induces eNOS and nNOS in intact rat63 Dissociative anaesthetic, psychotomimetic, drug of abuse; weak inhibitor of nNOS
Phenprocoumon (B)’ Phenylenebis(l,:! ethandiyl)bisisothiourea (PIBTtJ)m
Protoporphyrin-IX67 Putrescine (also D)69 Quercetin70 Retinoids Seminal plasma71 Spermidine (also D)69 Spermine (also D)@ Thiocitrulline (H)36 Trifluoperazine (K) Trifluoromethylphenylimidazole
Possibly more effective against iNOS but does increase blood pressure; reduces endotoxin-induced increase in plasma markers of liver injury in the rat51
‘Bis’ isothiourea compound; extremely potent iNOS inhibitor but limited access across cell membrane and highly toxic -
Anti-psychotic Endogenous inhibitor of nNOS with no reported effect on other NOS isoforms As zinc salt; nonselective, also inhibits haem oxygenasea Not found in spermatozoa; physiological relevance unclear
(TRIM)
Potent but isoform nonselective NOS inhibitor Does not inhibit iNOS Relatively selective nNOS inhibitor cf. eNOS in vitro and in vivo Binds at haem site; does not affect arginine binding to NOS
aLetters after named NOS inhibitors reflect site of action on enzyme as follows. Note that the absence of a letter indicates that site of action is not known. A, inhibition of uptake of L-arginine; B, inhibition of tetrahydrobiopterin (BH,) biosynthwis; C, reduction of cellular concentration of cofactors, e.g. sequestration of Ca *+; D, induction of NOS, e.g. induction of iNlOS by cytokines and eNOS/nNOS by oestrogen; E, scavenging of extracellular NO; F, L-arginine binding site; G, tetrahydrobiopterin binding site; H, haem site; J, NADPH binding site; K, calmodulin site; L and M, binding sites for FMN and FAD, respectively.
TIPS
June
1997
(Vu1
18)
2 0 7
REVIEW References for Box 1 1 Schoedon, G., BIau, N., Schneemann, M., Flury, G. and Schaffner, A. (1994)B&hem. Biophys.Res. Commun. 199,504-510 ‘2 Komori, Y.,Wallace, G. C. and Kukuto, J. M. (1994)Arch. Biockm. Biophys.315,213-218 3 Cobb, J. P. ef al. (1992)J. Exp. Med. 176,1175-1182 4 Nakane, M. et al. (1995)Mol. PkarmacoZ. 47,831-8&l 5 Garvey, E. P. et al. (1997)J. Biol.Chem.272,49594963 6 Southan, G. J., Szabo, C. and Thiemermann, C. (1994) Br. 1. Phanrracol. 114,510-516 7 Corbett, A., Tilton, R. G. and Chang, K. (1992) Diabetes 41, 552-556 8 Stefanovicracic, M. ef al. (1995)J. Rheumutol.22,1922-1928 9 Biegamki,T. et al. (1983)&o&m. Biophys.AC~Q756,196-203 10 01, I? and Wolff, S. P. (1993)B&&m. Pharmacol. 46,1139-1144 11 Faraci,W. S. et al. (1996)Br. J Pharmacol. 119,1101-1108 12 BuchmuIler-RouiIIer, Y., Schneider, P., Betz-Corradin, S., Smith, J. and Mauel, C. (1992)Biochem. Biophys. Res. Commun. 183, 150-155 13 Valiance, P., Leone, A., Calver, A., Collier, J. and Moncada, S. (1992)Lancet393,575-577 14 Das, I., Khan, N. S., Puri, B. K. and Hirsch, S. R. (1996)Neurosci. Left. 215,209-211 15 Bland-Ward, P. A. and Moore, P. K. (1995)Lif Sci. 57,131-135 16 Schini, V. B. and Vanhoutte, P. M. (1992)J. Pharmacol.Exp. Ther. 261,553-559 17 White, K. A. and Marletta, M. A. (1992) Biochemisfry 31, 6627-6631 18 Teale, D. M. and Atkinson, A. M. (1994)Eur. 1. Pharmacol.271, 87-92 19 Palacios, M. et al. (1993) Biochem. Biophys. Res. Commun. 196, 280-286 20 Wolff, D. J., Datto, G. A. and Samatowicz, R. A. (1993)J. Biol. them. 268,9430-9436 21 Lambert, L. E. et al. (1992)Eur. J. Phnrmacol. 216,131-134 22 Hasan, K. ef al. (1993)Eur. J. Pharmacol. 249,101-106 23 Gross, S. S. and Levi, R. (1992)J. BioI.Chem.267,3886-3888 24 Stuehr, D. J. et al. (1991)FASEBJ. 5,98-103 25 Luo, D. S. and Vincent, S. R. (1994) Biochem. Pharmacol. 47, 2111-2112 26 Zembowicz, A., Hatchett, R. J., Radziszewski, W. and Gryglewski, R. J. (1993)J. Pharmacol. Exp. T’her.267,1112-1118 27 Schewe, T. (1995)Gen. Pharmacol.26,1153-1169 28 Eizirik, D. L., Flodstrom, M. and DemeIlo, M. A. R. (1996) Diabetolagia 39,387
Psychol. Bull. 32,447 65 Osawa, Y. and Davila, J. C. (1993)Biochem. Biophys. Res. Commun.
29 Masumoto, H. and Sies, H. (1996)Chem.Res. Toxicol.9,1057-1062 GaTvey,E. P. ef al. (1994)J. Biol. Chem. 269,26669-26675
66 Jaffrey, S. R. and Snyder, S. H. (1996)Science274,774-777
Yokoi, I. et al. (1996)Neurcchem.Res. 21,1187-1192 Szabo, C. et al. (1996)Br. 1. Phnrmucol.118,1659-1668 Yokoi, I. et al. (1994)J. Neurochem. 63,1565-1567 Iwahashi, K. ef al. (1996) Neuropsychobioogy33,76-79 Win, N. H. H. ei al. (1996)Eur. J Pharmacol.299,119-126 36 Frey, C. ef al. (1994)J. Biol.Chem.269,26083-26091 37 WoIff, D. J., Datto, G. A., Samatowicz, R. A. and Tempsick, R. A.
67 Wolff, D. J, Naddelman, R. A., Lube&e, A. and Saks, D. A. (1996)Arch. BiochemBiophys.333,27-34 68 Chernick, R. J., Mark.& R. D., Levere, R., Margreiter, R. and Abraham, N. G. (1989)Hepatology 10,365-369 69 Hu, J. G., Mahmoud, M. I. and El-Fakahany, E. E. (1994)Neurosci. Lett. 175,41-45 70 Chiesi, M. and Schwaller, R. (1995) Biochem. Pharmacol. 49,
(1993) J Bioi. Chem. 268,9425-9429 38 Sup, S. J., Green, B. G. and Grant, S. K. (1994)Biockm. Biophyi. Res. Commwn. 204,962~968 39 Moore, W. M. et al. (1994)J. Med. Chem. 37,3886-3888 40 Connor, J. R. et al. (1995)Eur. J. Pharmacol. 273,15-24
495-501 7l Schaad, N. C. et al. (1996)Hum. Reprod. 11,561~565 72 Handy, R. L. C. et al. (1996)Br. 1. Pharmacol. 119,423-431 73 Perschini, A., M&i&m, K. and Masters, 8. S. S. (1995) Biochemistry 34,15091-15095
30 31 32 33 34 35
ratios on the two isoforms ranging from 10 (Ref. 16) to 40 (Ref. 17). However, SMTC is also a very potent vasopressor agent in the anaesthetized rat’8 and an inhibitor of endothelium-dependent relaxation responses of the isolated rat aorta with a potency similar to that of L-NMMA (Ref. 19). From these data it seems likely that SMTC causes significant eNOS inhibition in viva and, consequently, should not be used to study the function of nNOS-derived NO in intact animals.
2 0 8
41 McCall, T. H., Fee&h, M., Palmer, R. M. J. and Moncada, S. (1991)Br. 1. Pharmacol. 102,234-238 42 Southan G. J., Szabo, C., O’Connor, M. P., Salzman, A. L. and ThiemermaM, C. (1995)Eur. I. Pharmacol. 291,311-318 43 Wang, Y-X.,Poon, C. I. and Pang, C. C. Y. (1993)J. Pharmucol.EXQ. Ther.26!?,112-119 44 Luo, D., Das, S. and Vincent, S. R. (1995)Eur. 1. Pharmacol. 290, 247-251 45 Southan, G. J., Zingarelli, B., O’Connor, M., Salkman, A. L. and Szabo, C. (1996)Br. J. Pharmacol. 117,61%32 46 Zingarelli, B. et al. (1996)Br. J. Pharmacol.120,357-366 47 Cattell, R. J., Hamon, C. G. B. and Blair, J. A. (1988)Biol.Chem. Hoppe-Styler369,545 48 Mayer, B., Brunner, F. and Schmidt, K. (1993)Biochem. Pharmacol. 45‘367-374 49 Miki, N., Kawabe, Y. and Kuriyama, K. (1977)Biochem. Biophys. Res. Commun.75851-856 50 Salaris, S. C., Babbs, C. F. and Voorhees, W. D., III (1991)B&hem. Phat7nacol.42,499-506 51 Szabo, C., Southan, G. J. and Thiemermann, C. (1994)Proc. Nafl. Acad. Sci. U. S. A. 91,12472-12476 52 Hibbs, J. B., Jr, Traintor, R. R. and Zavrin, Z. (1987)Science235, 473-476 53 Bogle, R. G., Moncada, S., Pearson, J. D. and Mann, G. E. (1992) Br. 1. Pharmacol. 105,768-770 54 Griscavage, J. M., Fukuto, J. M., Komori, Y. and Ignarro, L. J. (1994)J. Biol.Chem.269‘21644-21649 55 Moore, P. K. et al. (1990)Br. J Pharmacol. 99,408-412 56 Robertson, C. A. et a!. (1993)Biochem.Biophys. Res. Commun. 197, 523-528 57 Pfeiffer, S., Leopold, E., Schmidt, K., Bnmner, F. and Mayer, B. (1996)Br. 1. Pharmacol. 118,1433-1440 58 Buxton, H. 0. et al. (1993)Circ. Res. 72,387-395 59 Moore, P. K., Wallace, P., Gaffen, Z., Hart, S. L. and Babbedge, R. C. (1993)Br. 1. Pharmacol.110,219-224 60 Silva, M. T. et al. (1995)Br. J Pharmacol.114,257-258 61 AIlawi, H. S. et al. (1994)Br. 1. Pharmacol. 113,282-286 62 Hayashi, T. et al. (1994) Biochem. Biophys. Res. Commun. 203, 1013-1019 63 Weiner, C. P. et al. (1994) Proc. NatI. Acad. Sci. U. S. A. 91, 5212-5216 64 Finkel, M. S., Laghrissithode, F., Pollock, 8. G. and Rong, J. (1996)
TiPS -June
1997 (Vol. 18)
194,1435-1439
A range of heterocyclic compounds have been shown to inhibit NOS. These include the nitroindazole group of fused heterocycles [e.g. 7-nitroindazole, 7-NI (Ref. 20); 3-bromo-7-nitroindazole and 2,7-dinitroindazolezl] as well as imidazole and its various substituted derivatives [e.g. l-, 2- and 4-phenylimidazole22; econazole, miconazole and fluconazole23; I-(2-trifluoromethylphenyl) imidazole, trimethylphenylfluoroimidazole (TRIM)*41.
REVIEW
Box 2. Identification of nitric oxide synthase (NO!3 inhibitors - some pitfalls for the unwary? Biochemical assays Many NO!3 inhibitors are identified solely by the ability to reduce enzyme activity in vitro. Even a cursory perusal of the literature reveals large variations in the incubation conditions employed between (and sometimes even within) different laboratories. These include: (1) the source of NOS (crude tissue homogenate, semi-purified or genetically engineered enzyme); (2) presence or absence and absolute concentration of cofactors; (3) presence or absence of protease inhibitors; (4) time of exposure of inhibitor to enzyme; (5) reaction time; (6) composition and pH of buffer containing enzyme. Such methodological variations render comparison of data between laboratories difficult if not meaningless and cause excessive duplication by which newly described inhibitors are ‘checked out’ in each laboratory. Some attempt to standardize screens for NOS inhibitors is worthwhile. Minimally, purified or cloned enzymes should be employed and putative inhibitors tested over a range of substrate concentrations and exposure periods in the presence of saturating concentrations of cofactors. However, experiments that utilize crude tissue homogenates should not be
Both nitroindazoles (nitro group showing electron withdrawing activity) and imidazole-based compounds (possessing a lone pair of electrons at Nl) carry a net electronegative charge and, as a consequence, are believed to bind to the haem prosthetic group of NOS, thereby disrupting electron flow through the enzyme and preventing NO formation. Interestingly, by binding at the haem site, both 7-NI and TRIM interfere with the binding of both L-arginine and tetrahydrobiopterin, thus explaining the apparent competitive inhibitory nature of 7-NI and TRIM at these siteWs. The isoform selectivity of 7-NI and TRIM has also been studied. Whilst both 7-NI [K, 2.8 pM; 5.6 FM and 7.0 pM (Refs 26,27 and 28, respectively)] and TRIM (K, 47 PM) inhibit brain nNOS in vitro, only TRIM shows relative selectivity towards nNOS in vitro, being -37 times more potent as an inhibitor of nNOS than eNOS. 7-Nitroindazole is equipotent as an inhibitor of both nNOS and eNOS in aitro. The lack of effect of TRIM on eNOS in vitro is reflected in its inability to influence acetylcholineinduced endothelium-dependent relaxation of the isolated rabbit aorta preparation and failure to increase arterial blood pressure in the anaesthetized mouse and rat24. Perhaps more surprisingly, despite a potent eNOS inhibitory activity i~ vitro, 7-NI does not inhibit acetylcholine-induced endothelium-dependent relaxation of the isolated rabbit aorta preparation and is without effect on arterial blood pressure in a range of species including
ignored as they may provide useful data concerning the stability of the inhibitor in biological conditions. The need for complete biochemical and pharmacological profile NOS inhibitors seem to differ greatly (in terms of potency and isoform selectivity) in vitro and in vivo. Inability to penetrate biological membranes will considerably reduce the biological effects of NOS inhibitors in vivo whilst some compounds may exhibit unexpected isoform selectivity in vivo (which may not have been obvious in vitro) as a result of selective accumulation or catabolism of the compound in different target cells. A detailed profile of information (i.e. mechanism of action, efficacy in whole-cell preparations and biological activity in intact animals) is required. How selective is selective? Precisely how great the potency gap must be before a compound can be considered ‘isoform selective’ is a subjective decision. Certainly, researchers have been known to claim isoform ‘selectivity’ with a potency ratio of 5:l. A potency ratio of 1OO:l or more would seem worthwhile provided that a similar degree of selectivity could also be achieved in vivo. Interestingly, by this criterion there is no known selective neuronal NOS (nNOS) inhibitor.
mouse20, rat29 and pigeon.30 The mechanism whereby 7-NI inhibits eNOS activity in vitro but, apparently, not in viva is still unclear and warrants further study. Early studies of the pharmacological actions of TRIM and 7-NI in terms of CNS effects revealed that both corn.pounds elicit an L-arginine-reversible antinociception determined either as inhibition of formalin-induced hindpaw licking behaviour or acetic acid-induced abdominal constrictions in the mouse2OJ.1.Since this time, the profile of activity of 7-NI in particular has been extensively examined. To date, 7-NI has been reported to inhibit spinal cord ‘wind-up’ (increased firing of sensory neurones in the spinal cord) associated with peripheral noxious stimuli in the anaesthetized rat”‘, reduce cerebral blood flow implying an important role for nNOSderived NO in the regulation of blood flow within the brain32, inhibit the behavioural consequences of opioid withdrawal33 and prevent convulsions following administration of cocaine31 or NMDA (Ref. 35) to the mouse. Interestingly, 7-NI administration also reduces neuronal death following middle cerebral artery occlusion in the rat, which is widely used as an animal model of stroke3”. Numerous researchers have also utilized 7-NI to probe the role of NO in neurodegenerative disorders. Thus, parenteral administration of 7-NI reduces striatal dopamine and tyrosine hydroxylase loss, which occurs five days after methamphetamine injection in the mouse37, protects against hippocampal neurone loss
7-IPS-
lunf
lYY7 (Vol. 18)
2
0
9
REVIEW after bilateral carotid occlusion in the gerbil38and striatal neurone loss, after 1-methyl4phenyl-1,2,3,6+etrahydropyridine (MPTP) injection in the mouse39 and baboon@. Intriguingly, 7-NI pretreatment also ameliorates the neurological defect associated with mild head trauma in the mouse41.Because 7-NI has been demonstrated to promote the release of both noradrenalinehzand dopamine43 from hippocampal and striatal neurones, respectively, it is conceivable that some of these various effects of the inhibitor on central function may be secondary to changes in the release of conventional neurotransmitters. Peripheral nNOS activity is also associated with NANC nerves innervating a wide range of smooth muscle in the gastrointestinal, airways and urogenital systems”. Nitro arginine methyl ester and many other NOS inhibitors inhibit the smooth muscle relaxation elicited by electrical stimulation in such preparations. The effects of 7-NI and TRIM on such NANC responses have yet to be reported in the literature. This seems to be an interesting omission: whether 7-NI and TRIM affect NANC transmission to smooth muscle in vivo is likely to have a major bearing on the side-effect profile of nNOS inhibitors and thus their potential for clinical use. Endogenous inhibitors of NOS The possibility that the body may produce an ‘endogenous inhibitor’ of NOS has been a matter for debate over several years. For example, asymmetric dimethyl NGNCL-arginine (L-ADMA) is a potent NOS inhibitor that is found in many sites in the body including plasma, brain and kidney45. In addition, a high molecular weight (>lOOOO)inhibitor of NOS was demonstrated in crude homogenates of rat and rabbit brain&. Recently, a naturally occurring protein inhibitor (PIN) of NOS was extracted from rat brain47. PIN contains 82 amino acids and is a potent and highly selective inhibitor of nNOS. The discovery of PIN raises a number of important issues. Does PIN affect nNOS activity in the central and peripheral nervous systems in vivo? If so, does PIN play a part in the physiological and/or pathophysiological roles of NO in the nervous system? Does the concentration of PIN in the brain alter in different physiological or disease states? Importantly, the precise mechanism by which PIN inhibits nNOS at the molecular level is still to be determined. Information of this type is clearly required before the full biological significance (if any) of PIN can be appreciated. Whether the discovery of PIN will provide useful leads to the development of additional nNOS inhibitors remains to be seen. Potential therapeutic applications of nNOS inhibitors - is no NOS good NOS? Identification of potent and selective inhibitors of nNOS is clearly a very worthwhile goal in terms of providing tools with which to investigate the many and varied biological functions of NO in the nervous system. Whether selective nNOS inhibitors also have a future in the clinic is less clear cut. This uncertainty goes some
2 1 0
TIPS -June
1997 (Vol. 18)
way to explaining the relative dearth of such compounds in the literature compared with, for example, the plethora of iNOS inhibitors for which therapeutic targets are more clearly defined. Two problems that may hinder the future clinical exploitation of nNOS inhibitors can be identified. The first relates to our present incomplete understanding of the precise role of nNOS-derived NO in CNS function. At least part of the reason for this must be the use of isoform nonselective NOS inhibitors which, in addition to inhibition of brain nNOS, markedly increase blood pressure by reducing endothelial NO formation. It is, for example, conceivable that this ‘dual’ effect of L-NAME on nNOS and eNOS underlies reports in the literature of increased, decreased or unaltered neuronal damage following focal ischaemic damage in experimental animals pretreated with L-NAME. Indeed, similar confusion exists with respect to the effect of L-NAME (and like NOS inhibitors) on pain perception, convulsive behaviour and memory. It is hoped that the more widespread use of selective nNOS inhibitors by researchers will help to clarify the part played by NO in these important processes. The second problem is the possibility of a range of unwanted side-effects. Based on animal studies, using for the most part isoform nonselective NOS inhibitors, ‘expected’ side-effects of nNOS inhibitors include impairment of memory acquisition/retrieval and blockade of peripheral NANC nerves leading, amongst other things, to disordered stomach motility and impotence. Such side-effects may indeed present problems in the clinic, particularly if chronic treatment is envisaged. However, loss of memory and failure to gain an erection would not necessarily rule out the use of nNOS inhibitors in, for example, stroke or when administered prior to surgery to treat postoperative pain. In addition, further experiments are also required to determine the ability of the presently available nNOS inhibitors (e.g. 7-NI, TRIM) to influence NANC transmission. Given the current interest of the pharmaceutical industry in the development of NOS inhibitors it seems likely that additional, even more potent and more selective, nNOS inhibitors than are currently known will become available in the next few years. It must be hoped that the arrival of such compounds for pharmacological evaluation will provide the fundamental knowledge on which matters of clinical usage can be based. Selected references 1 Marletta. M. (1994) Cell 78.927-930 2 Forstermann; U. and KIeinert, H. (1995) Nuunyn-SchmiudeberX’s Arch. Pharmacol. 352,351-364 3 Anggard, E. (1994) Laticet 343,1199-1206 4 Dalkara, T. and Moskowitz, M. A. (1994) Brain Pathol. 4,49-57 5 Lyons, C. R. (1995) Adv. htm~umol. 60,32kL371 6 Griffiths. 0. W. and Kilboum. R. G. (1996) Methods Enzwmol. ~ _I 268, 375-392 7 Dwyer, M. A., Bredt, D. S. and Snyder, S. H. (1991) Biocllem. Biophys. Res. Commun. 176,11361141 8 Rees, D. D., PaImer, R. M. J., Schulz, R., Hodson, H. F. and Moncada, S. (1990) Br. 1. Pharmacol. 101,74&752
R 9 Rees, D. D., Palmer, R. M. J. and Moncada, S. (1990) Proc. Natl. Acad. Sci. U. S. A. 86,3375-3378 10 Gardiner, S. M., Compton, A. M., Bennett, T., Palmer, R. M. J. and Moncada, S. (1990) Eur. J. Pharmacol. 213,449-451 11 Moore, P. K., Oluyomi, A. O., Babbedge, R. C., Wallace, P. and Hart, S. L. (1991) Br. I. Pharmucol. 102,198-202 12 Gibson, A., Mirzazadeh, S., Hobbs, A. J. and Moore, l’. K. (1990) Br. J. Pharmacol. 99,602-606 13 Nava, E., Palmer, R. M. J. and Moncada, S. (1991) Lancet 338, 1555-1557 14 McCall, T. H., Feelisch, M., Palmer, R. M. J. and Moncada, S. (1991) Br. 1. Pharmacol. 102,234-238 1.5 Moore, W. M. et al. (1994) J. Med. Chem. 37,388&3888 16 Furfine, E. S. ct al. (1994) 1. Biof. Chem. 269,26677-26683 17 Nagafuji, T. et RI. (1995) NeuroReport 6,1541-1545 18 Narayanan, K. et al. (1995) 1. Biol. Chem. 270,11103-11110 19 Joly, G. A., Narayanan, K., Griffith, 0. W. and Kilboum, R. G. (1995) Br. J. Pharmacol. 115,491-497 20 Moore, P. K., Wallace, P., Gaffen, Z., Hart, S. L. and Babbedge, R. C. (1993) Br. 1. Pharmacol. 110,219-224 21 Bland-Ward, I’. A. and Moore, P. K. (1995) L$e Sci. 57,131-135 22 Wolff, D. J., Datto, G. A., Samatovicz, R. A. and Tempsick, R. A. (1993) Proc. Nntl. Acad. Sci. U. S. A. 268,9425-9429 23 Wolff, D. J., Datto, G. A. and Samatovicz, R. A. (1993) 1, Biol. Chem. 268,9430-9436 24 Handy, R. L. C. et al. (1996) Br. 1. Pharmacol. 119,423431 25 Klatt, P. et RI (1994) 1. Bio2.Chenr. 269,13861-13866 26 Mayer, B., Klatt, I’., Werner, R. and Schmidt, K. (1994) Neurophnrmncology 33,1253-1259 27 Babbedge, R. C., Bland-Ward, P. A., Hart, S. L. and Moore, P. K. (1993) Br. 1. Pharmncol. 110,225226 28 Wolff, D. J. and Gribin, B. J. (1994) Arch. Biochem. Biophys. 311, 300-306 29 Beierwaltes, W. H. (1995) Am. J. Physiol. 38, F134-F139 30 Zagvazdin, Y. S., Fitzgerald, M. E. C., Sancesario, G. and Reiner, A. (1996) Inrwf. O$zfhdmol. Vis. Sri. 37,666-672 31 Stanfa, L. C., Misra, C. and Dickenson, A. H. (1996) Brain Res. 737, 92-98 32 Kelly, I’. A. T., Ritchie, 1. M. and Arbuthnott, G. W. (1995) J. Cereb. Blood Flozc M&b. 15, 766-773
Gene targeting - homing in on qadrenoceptorsubtype function Ewen
MacDonald,
Brian K. Kobilka and Mika Scheinin
The qadrenoceptorwas a2A-1 a2El-
subdivided into three subtypes:
and cl,,-adrenoceptors
Since then, the search develop subtype-selective
agonists and antagonists,
as yet no major breakthrough
and but
has been made. In the past
year, several strains of genetically become available,
almost ten years ago.
has been on to discover
engineered
either overexpressing,
mice have
totally lacking
or expressing heavily modified c*,-adrenoceptor
subtypes.
Ewen MacDonald,
Scheinin
Brian
Kobilka
and Mika
describe how these mice may be utilized to elucidate the physiological
functions of the receptor subtypes and the
properties of future subtype-selective
CD1947, Elsevier Science Ltd
drugs.
E
V
E
I
W-
33 Vaupel, D. B., Kines, A. S. and London, E. D. (1995) Ps!/chopharmncology 18,361-368 34 Itzhak, Y. and Ali, S. F. (1996) J. Neurochem. 67,1770-1773 35 Przegalinski, E., Baran, L. and Siwanowicz, J. (1996) Nnrrosci. Left. 217,145-148 36 Dalkara, T., Yoshida, T., Irikura, K. and Moskowitz, M. A. (1994) Neuropharmacology 33,1447-1452 37 Dimonte, D. A., Royland, J. E., Jakowec, M. W. and Langston, J. W (1996) J. Neurochem. 67,244s2450 38 O’Neill, M. J., Hicks, C. and Ward, M. (1996) Eur. 1. Phnrmacol. 310. 115-122 39 Schulz, J. B., Matthews, R. T., Muqit, M. M. K., Browne, S. E. and Beal, M. F. (1995) J. krrochem. 64,936-939 40 Hantraye, P. et a/. (1996) Nat. Med. 2, 1017-1021 41 Mesenge, C., Verrecchia, C., Allix, M., Boulu, R. R. and Plotkine, M. (1996) J. Neurotrauma 13,209-214 42 Kiss, J. P., Sershen, H., Lajtha, A. and Vizi, E. S. (1996) Nrurosci. Lrff. 215,115-118 43 Silva, M. T. et al. (1995) Br. 1. Pharmncof. 114,257-258 44 Rand, M. J. and Li, C. J. (1995) AH~I~. RCP Physiol 57, 659-682 45 Valiance, I’., Leone, A., Claver, A., Collier, J. and Moncada, S. (1992) J. Cardiovusc. Pharmacol. 20,560-562 46 Moore, P. K., al-Swayeh, 0. A. and Evans, R. (1990) Br. 1. Pharmacoi’. 101,865+368 47 Jaffrey, S. R. and Snyder, S. H. (1996) Sciencr 274,771777
r Chemical names HF2035: 2-[2-aminoethyl]-N-[2,4,5-trichlorobenzenesulphonyl] amino-N-[4-chlorocinnamyl]N-methylbenzylamine LY83583: 6-anilino-5,Squinolinedione HF2035: 2-[2-aminoethyl]-N-[2,4,Strichlorobenzenesulphonyllamino-N-[4-chlorocinnamyl]N-methylbenzylamine
One of the greatest contributions that molecular biology has made to pharmacology is the clarification of receptor classification, particularly the functional contributions of varying receptor subtypes. Before molecular cloning as a means for receptor identification, classification of a new receptor subtype relied on pharmacological methods (e.g. order of agonist potency) that were often confounded in interpretation due to variable receptor reserve in different tissues or the exploitation of differing effecters in different settings by receptor subpopulations. Currently, receptor subtypes are often cloned before specific ligands binding to them are available, i.e. so-called orphan receptors posing a new set of challenges for pharmacologists and the pharmaceutical industry. In many ways, the a,-adrenoceptors exemplify the above scenario. There are now three well characterized receptor subtypes; aZA, aZB and (tic (Ref. 1; Table 1). However, available ligands have only marginal subtype selectivity. This review will describe strategies other than the evaluation of subtype-selective ligands to allocate physiological effects to distinct g-adrenoceptor subtypes and subsequently predict the pharmacological and therapeutic properties of subtype-selective drugs under development.
1’11:5016%6147(97)01063-8
TIPS-June
1997 (VOI 18)
E. MacDonald.
Lecturer, Departmentof Pharmacologyand Toxicology, Urwers~ty of Ku&o. PL 1627, FIN-7021 1 Kuoplo, Finland, B. K. Kobilka. Associate Professor, Department of Molecular and Cellular Physiology. AssIstant Investigator, Howard Hughes MedIcal Institute, Stanford Unwerslty, Stanford, CA 94305, USA. and M. Scheinin. Professor of Clwcal Pharmacology, Department of Pharmacology and Climcal Pharmacology, Unwerslty of Turku, FIN-20520 Turku. Finland
'11
E
E
I
V
W
Selective inhibitors of neuronal nitric oxide synthase - is no NOS really good NOS for the nervous system? Philip K. Moore and Rachel 1. C. Handy It is now ten years since NO was shown to account for the biological activity of endothelium-derived factor (EORF). It is also the tenth anniversary identification
of L-A/G monomethyl
relaxing of the
arginine (L-NMMA)
as the very first inhibitor of NO biosynthesis.
That EORF
and NO were one and the same sparked an explosion of interest in the biochemistry
and pharmacology
of NO
which has yet to subside. In contrast, the first ever nitric oxide synthase (NOS) inhibitor slipped seamiessly into the literature
virtually without comment at the time.
Over the following decade, L-NMMA inhibitors) have proved invaluable
(and like NOS
as tools for probing
the biological roles of NO in health and disease and, in particular,
have increased
our understanding
of the
function of NO in the nervous system. Further advances in this important area now require the development inhibitors selective
of
for the neuronal isoform of NOS
(nNOS). Here, Philip Moore and Rachel Handy provide an up-to-date biochemical
account of the literature and pharmacological
NOS inhibitors with particular with greater selectivity
P. K. Moore Reader II Pharmacology ant R. L. C. Handy Research Fellow Pharmacology Group Biomedical Science! Division, King’: College. University 0 London. Maws; Road, London UK SW36LX
204
regarding the
characterization
reference
of
to compounds
for the nNOS isoform.
The physiological and pathophysiological functions of NO and the molecular biology and biochemistry of NOS have both been extensively discussed and are beyond the scope of this review (for further information, see Refs 14). However, a brief description of the major biological roles of NO and of the nature of the individual NOS isoforms and the mechanism of NOS action does seem appropriate here if only to provide a rational basis for understanding the mechanism of action of NOS inhibitors. To date, the amino acid, mRNA and cDNA sequences of three distinct isoforms of NOS have been determined. These correspond to the neuronal (nNOS, type I), inducible (iNOS, type II) and endothelial (eNOS, type III) enzymes. The so-called constitutive isoforms of NOS (nNOS and eNOS) are located mainly (but not exclusively) in central and peripheral nerves and vascular
TiPS
-June
1997 (Vol. 18)
endothelial cells, respectively, and serve a wide range of predominantly physiological or ‘housekeeping’ functions. For example, NO derived from nNOS in the CNS has a number of important functions including the modification of pain perception, mediation of longterm potentiation (LTP) and memory, control of cerebral blood flow and in neurodegeneration following cerebral ischaemia. In addition, nNOS located in selected nonadrenergic, non-cholinergic (NANC) nerves in the periphery generates NO, which acts as a neurotransmitter to relax smooth muscle in the gastrointestinal and urogenital tracts and the airways. Furthermore, relaxation of vascular smooth muscle and inhibition of platelet adhesion and aggregation occur following activation of eNOS in vascular endothelial cells and most probably represents a fundamentally important mechanism for regulating blood pressure and tissue perfusion. In contrast, iNOS is synthesized de ~OUOwithin selected cell types a few hours after exposure to bacterial endotoxins or to a range of different cytokines. The iNOS isoform is believed to be of significance in pathophysiological situations playing a part, for example, in the body’s defensive response to disease and infections. Nitric oxide synthase catalyses the five-electron oxidation of L-arginine to NO and L-citrulline. A combination of mechanistic and molecular biological techniques has revealed that the active form of NOS is a dimer containing flavin (flavin adenine mononucleotide and flavin adenine dinucleotide, FMN and FAD), haem, NADPH (reduced nicotinamide adenine dinucleotide phosphate) and tetrahydrobiopterin as prosthetic groups. Nitric oxide formation commences with hydroxylation of L-arginine to form L-IF hydroxyarginine and the subsequent incorporation of a second atom of oxygen to yield NO and its co-product, citrulline. The part played by the various prosthetic groups in this reaction has been the subject of intense scrutiny and is of interest here since many inhibitors of this enzyme interfere with binding at these sites. Briefly, electron transfer from molecular oxygen proceeds across domains within the NOS enzyme in a linear sequence from NADPH to FAD/FMN and finally to the haem site. Calmodulin is activated by Ca2+ entering cells as a result of the opening of either receptor-operated [e.g. muscarinic receptors on endothelial cells or NMDA (Nmethyl-D-aspartate) receptors on neurones] or voltageoperated (e.g. NANC nerves) channels and promotes electron transfer between FAD/FMN and haem. Tetrahydrobiopterin may also act as a cofactor for NO formation but may have an even more fundamental role in linking the two monomers of NOS to yield the biochemically active NOS dimer.
The ‘A-Z’ of NOS inhibitors Drugs can, at least in theory, reduce or prevent the biological effects of NO in a number of ways (Fig. 1). These include inhibition of the uptake of L-arginine into the cell (A, Fig. l), reducing the cellular availability of necessary
0 1997, Elsevier Science Ltd
PII: SO165-6147(97)01064-X
REVIEW
cofactors by preventing their formation or promoting their breakdown (B, C), scavenging NO once formed (D) or inhibiting the cellular mechanisms leading to induction of the particular NOS isoform (E). Over recent years inhibition of the enzymatic activity of NOS (F-M) has been the most thoroughly investigated approach and has provided the majority of useful compounds. The multiplicity of binding sites on NOS for substrate (L-arginine), cofactor (NADPH, FAD, FMN, calmodulin), ‘stabilizer’ (possibly tetrahydrobiopterin) and the haem prosthetic moiety has provided researchers with ample scope for the identification of inhibitors acting at each of these sites. Indeed, in the ten years since the original report on the inhibitory effect of L-NMMA on NOS many more ‘novel’ inhibitors of NOS have been described. To date, there are well over a hundred NOS inhibitors in the literature with (almost) every letter of the alphabet represented (Box 1). The biochemical and pharmacological characterization of new NOS inhibitors is certainly a worthwhile research goal although the conduct of the experiments required and interpretation of the data obtained is a process that requires careful thought. Some of the problems associated with the identification of NOS inhibitors are described in Box 2. Many of the agents shown in Box 1 exhibit activity other than NOS inhibition and are thus unsuitable leads for the identification of selective nNOS inhibitors. For example, methylene blue is a potent inhibitor of soluble guanylate cyclase whilst iron-protoporphryn-IX inhibits haem oxygenase. Ebselen interacts with thiol groups on proteins and, as such, also inhibits NADPH oxidase and cytochrome B5 reductase. Finally, drugs that block NOS by binding at either the calmodulin (e.g. chlorpromazine) or NADPH (e.g. diphenyleneiodionium salts) sites or by disrupting cellular tetrahydrobiopterin formation (e.g. 2,4-diamino-6-hydroxypyrimidine) exhibit a wide range of alternative effects and are therefore of limited interest. Interestingly, of the compounds shown in Box 1 very few have been reported to exhibit any degree of selectivity for nNOS over the other isoforms.
Amino acid analogues as a source of nNOS-selective inhibitors? Many amino acid derivatives inhibit NOS enzyme activity both in vitro and in a range of isolated cells, biological tissues and intact animals6. In general, none of the compounds reported to date shows significant selectivity for nNOS either in vitro or in viva. Guanidino substituted analogues of L-arginine (e.g. L-NMMA (monomethylarginine), L-NGnitroarginine and its methyl ester, L-NOARG and L-NAME; see also compounds in Box 1) are potent NOS inhibitors both in vitro and in aivo. Whilst all analogues in this group inhibit NOS by competition with L-arginine for the substrate binding site the precise mechanism of action varies both with the analogue and with the NOS isoform studied. For example, L-NOARG produces a time-dependent, irreversible inhibition of constitutive NOS isoforms in
L-Arginine 74 A
NADPH CaM
FAD
NO + L-Citrulline
FMN
Ca2+
I NO
QLE
Fig.1.Inhibition of nitnc oxide synthase (NOS). Potential targets to intllbtt the synthesis or activity of NO in a stylized mammalian cell. NOS is shown in the centre of the cell equipped with seven binding sites for substrate or cofactors. Letters Indicate possible sites of drug action as follows. For drugs active at these various sites see Box 2. A. inhibition of uptake of Larginine; B. inhibition of tetrahydrobiopterin (BH,) biosynthesis; C, reduction of cellular concentration of cofactors, e.g. sequestration of Ca:+. D. induction of NDS, e.g. induction of inducible NOS (iNOS) by cytokines and endothelial NOS/neuronal NOS (eNDS/nNOS) by oestrogen; E, scavenging of extracellular NO; F. t-arglnine binding site; Ci, tetrahydrobiopterin binding site; H. haem site; J, NADPH (reduced nicotlnamide adenme dinucleotide phosphate) binding site; K. calmodulin site(CaM); L and M. binding sites for flavine adentne mononucleotide (FMN) and flavine adenine dinucleotide (FAD), respectively.
vitro and a long-lasting (>24h) inhibition of brain NOS following parenteral administration in the rat’. In contrast, L-NOARG causes competitive, reversible inhibition of iNOS. In general, guanidino-substituted analogues of L-arginine demonstrate little or no worthwhile selectivity for individual NOS isoforms in vitro. Furthermore, in isolated tissue preparations or in intact animals, administration of such analogues elicits a profile of pharmacological activity commensurate with inhibition of eNOS (e.g. reduction of endothelium vasodilatation in isolated blood vessels8, increase in arterial blood pressure in anaesthetized or conscious animal&lo), nNOS (e.g. antinociceptive activity”, inhibition of NANC relaxation of anococcygeus and other isolated muscle preparation+) and iNOS (e.g. increase in blood pressure in septic shockl3). In the majority of cases, the biological effect of the NOS inhibitor is either partially or totally reversed by I.-arginine. A range of other amino acid derivatives have also been assessed for their ability to inhibit NOS. Both L-N-iminoethylornithine (L-NIO)l” and L-iV6-iminoethyllysine (L-NIL)‘5 are relatively potent NOS inhibitors although neither shows selectivity for the nNOS isoform. Indeed, L-NIL is relatively selective for iNOS. Substituted citrulline compounds such as thiocitrulline and alkylthiocitrullines (e.g. S-methyl and S-ethylthiocitrulline, SMTC and SETC) also inhibit NOS. In this context, SMTC is of some interest in that it does indeed exhibit some degree
TilJS-Junr
lYY7
lVOl
18)
2 0 5
REVIEW Box 1. The ‘A-Z’ of nitric Compound and site of action’ Acetyl5-hydroxytryptamine (B)i Allylarginine (F)2 Aminoarginine (F)* 2-amino-5,6-dihydro-methylthiazine @MT)4 3-aminomethylbenzylacetamidine (14OOW)5 Aminoethylisothiourea (F/?H)6 Aminoguanidine (F/?H)7
2-amine-4-methylpyridine (F, ?H)n 3-amino-1,2,4triazole (B)i2 Arcaine Asymmetric NGNo-dimethylarginine (ADMA) (F)2 3-bromo 7nitroindazole (3Br 7-NI) (H/F/G)‘5 Calmidazolium (K)*6 Carbon monoxide (CO) (H)ir Canavanine Chlorpromazine (K)i9 Clotrimazole (H)zO Cylopropylarginine (F)zi DiaminoguanidineP Diamino-6-hydroxypyrimidine= Dicoumarol (B)i Dimethylguanidine23 Diphenyleneiodonium (J)24
Doxorubicin25 Ebselen26
Iminoethylornithinine (L-NIO)~~ Iminopiperidir+ S-isopropylisothioua (IPl7.J) (F/?H)6 KetoconazoIe (H)20 L-NC-substituted arginine analogues (F)
TOPS -June 1997(Vol.
Irreversible inhibitor of nitric oxide synthase (NOS) Causes convulsions in dogs3 Potent and selective inducible NOS (iNOS) inhibitor Very potent and selective iNOS inhibitor Converted to mercaptoethylguanidi in vivo 26X more potent on iNOS than neuronal NOS (nNOS); iNOS inhibitor in vi&; nonselective, potent inhibitor of diamine oxidase, polyamine oxidase, riinucleotide reductaseYJ0 7X more potent on iNOS cf. endothelial NOS (eNOS) in vivo Inhibits cell tetrahydrobiopterin (BH4) formation which may inhibit synthesis of noradrenaline and 5-HT Endogenous inhibitor of NOS; increased plasma concentration in chronic kidney failure13 and in human schizophrenic@ 4~ more potent than 7nitroindazole (7-NI) on nNOS Does not inhibit iNOS since calmodulin is tightly bound in this isoform Potential role of CO in biological processes; physiological significance of interaction of CO and NOS is unclear Weak NOS inhibitor. Increases blood pressure in endotoxin-treated rats’* Also reduces induction of iNOS in lung from endotoxin-treated rats19 Antifungal Extremely potent inhibitor of all three isoforms; nonselective; interacts with NADPH (reduced nicotinamide adenine dinucleotide phosphate) site on other enzymes Seleno-organic compound, modifies thiol groups at or close to active site of NOS; nonselective, inhibits lipoxygenase, NADPH oxidase and protein kinase Cz7,reduces nuclear factor-lcB (NF-KB) activation28 and reacts with peroxynitrite29
S-ethylisothiourea (F/?H)6 Ethylthiopseudourea6,m Fluconazole (H)20 Guanethidir@ Guanidinoethyldisulphide (GED) (F/?H)32 Guinidinoglutaric acid (GGA) (F)B Haloperidol (K)H HF2035 (Ref. 35) Homothiocitrulline (H)x Imidazole (H)37 hninobiotin~ Iminoethyllysine (L-NIL)39
2 0 6
Comment
18)
Antifungal Sympatholytic
Anti-psychotic drug Inhibitor of nNOS/eNOS in vitro but less active against iNOS Very weak NOS inhibitor Equiactive on iNGS and nNOS in vitro 29X more potent on iNOS than nNOS; anti-inflammatory in a rat model of arthritis40 Irreversible inhibitor of iNOS in vitro -
Isoform nonselective isothiourea compound Antifungal D-isomers are biologically inactive; one report that D-NAME nitroarginine methyl ester inhibits endothelium-dependent relaxation of rat aorta”
REVIEW
oxide synthase inhibitors Compound and site of action’
Comment
LY83583 (Ref. 44) Mercaptoethylguanidine (MEG) (F)45
Also blocks soluble guanylate cyclase Also inhibits constitutive and inducible cyclooxygenase (COX-1 and COX-2)%; may be beneficial in the treatment of inflammatory conditions Anti-cancer agent Also inhibits soluble guanylate cyclase@ and xanthine oxida@
Methotrexate47 Methylene blue (?H)@ Methylguanidine23 S-methylisothiourea (SMT) (F/?H)6
Methylthiocitrulline (H)x Miconazole (H)20 Monomethylarginine (L-NMMA)
(F/?A)
Nitric oxide (H)x Nitroarginine (L-NOARG) (F)55 Nitroarginine methyl ester (L-NAME) (F)
‘I-nitroindazole (H/F/G) (7-NI)59
Oestrogen Paroxetinebl Phencyclidines
1,2- and 1,4-phenylimidazole (H)37 Pimozide (K) PIN (protein inhibitor of NOS)g
W/F/G)72
W-7 (K) Zinc (?H)73
Antifungal First reported ‘arginine-based’ NOS inhibito+; also inhibits arginine uptake into cell+; sometimes abbreviated to L-NMA Possible feedback inhibitor of NOS? Reported to inhibit arginase56; other abbreviations include L-NA and NOLA Demethylated in viva to yield active L-NOARG57; reported to antagonize muscarinic cholinoceptors58, not confirmed in other studies Relatively selective for nNOS in uivo; slightly more water soluble Na+ salt (7-NINA) available@; high concentrations relax smooth muscle by unknown mechanism61 High concentrations inhibit NOW; also induces eNOS and nNOS in intact rat63 Dissociative anaesthetic, psychotomimetic, drug of abuse; weak inhibitor of nNOS
Phenprocoumon (B)’ Phenylenebis(l,:! ethandiyl)bisisothiourea (PIBTtJ)m
Protoporphyrin-IX67 Putrescine (also D)69 Quercetin70 Retinoids Seminal plasma71 Spermidine (also D)69 Spermine (also D)@ Thiocitrulline (H)36 Trifluoperazine (K) Trifluoromethylphenylimidazole
Possibly more effective against iNOS but does increase blood pressure; reduces endotoxin-induced increase in plasma markers of liver injury in the rat51
‘Bis’ isothiourea compound; extremely potent iNOS inhibitor but limited access across cell membrane and highly toxic -
Anti-psychotic Endogenous inhibitor of nNOS with no reported effect on other NOS isoforms As zinc salt; nonselective, also inhibits haem oxygenasea Not found in spermatozoa; physiological relevance unclear
(TRIM)
Potent but isoform nonselective NOS inhibitor Does not inhibit iNOS Relatively selective nNOS inhibitor cf. eNOS in vitro and in vivo Binds at haem site; does not affect arginine binding to NOS
aLetters after named NOS inhibitors reflect site of action on enzyme as follows. Note that the absence of a letter indicates that site of action is not known. A, inhibition of uptake of L-arginine; B, inhibition of tetrahydrobiopterin (BH,) biosynthwis; C, reduction of cellular concentration of cofactors, e.g. sequestration of Ca *+; D, induction of NOS, e.g. induction of iNlOS by cytokines and eNOS/nNOS by oestrogen; E, scavenging of extracellular NO; F, L-arginine binding site; G, tetrahydrobiopterin binding site; H, haem site; J, NADPH binding site; K, calmodulin site; L and M, binding sites for FMN and FAD, respectively.
TIPS
June
1997
(Vu1
18)
2 0 7
REVIEW References for Box 1 1 Schoedon, G., BIau, N., Schneemann, M., Flury, G. and Schaffner, A. (1994)B&hem. Biophys.Res. Commun. 199,504-510 ‘2 Komori, Y.,Wallace, G. C. and Kukuto, J. M. (1994)Arch. Biockm. Biophys.315,213-218 3 Cobb, J. P. ef al. (1992)J. Exp. Med. 176,1175-1182 4 Nakane, M. et al. (1995)Mol. PkarmacoZ. 47,831-8&l 5 Garvey, E. P. et al. (1997)J. Biol.Chem.272,49594963 6 Southan, G. J., Szabo, C. and Thiemermann, C. (1994) Br. 1. Phanrracol. 114,510-516 7 Corbett, A., Tilton, R. G. and Chang, K. (1992) Diabetes 41, 552-556 8 Stefanovicracic, M. ef al. (1995)J. Rheumutol.22,1922-1928 9 Biegamki,T. et al. (1983)&o&m. Biophys.AC~Q756,196-203 10 01, I? and Wolff, S. P. (1993)B&&m. Pharmacol. 46,1139-1144 11 Faraci,W. S. et al. (1996)Br. J Pharmacol. 119,1101-1108 12 BuchmuIler-RouiIIer, Y., Schneider, P., Betz-Corradin, S., Smith, J. and Mauel, C. (1992)Biochem. Biophys. Res. Commun. 183, 150-155 13 Valiance, P., Leone, A., Calver, A., Collier, J. and Moncada, S. (1992)Lancet393,575-577 14 Das, I., Khan, N. S., Puri, B. K. and Hirsch, S. R. (1996)Neurosci. Left. 215,209-211 15 Bland-Ward, P. A. and Moore, P. K. (1995)Lif Sci. 57,131-135 16 Schini, V. B. and Vanhoutte, P. M. (1992)J. Pharmacol.Exp. Ther. 261,553-559 17 White, K. A. and Marletta, M. A. (1992) Biochemisfry 31, 6627-6631 18 Teale, D. M. and Atkinson, A. M. (1994)Eur. 1. Pharmacol.271, 87-92 19 Palacios, M. et al. (1993) Biochem. Biophys. Res. Commun. 196, 280-286 20 Wolff, D. J., Datto, G. A. and Samatowicz, R. A. (1993)J. Biol. them. 268,9430-9436 21 Lambert, L. E. et al. (1992)Eur. J. Phnrmacol. 216,131-134 22 Hasan, K. ef al. (1993)Eur. J. Pharmacol. 249,101-106 23 Gross, S. S. and Levi, R. (1992)J. BioI.Chem.267,3886-3888 24 Stuehr, D. J. et al. (1991)FASEBJ. 5,98-103 25 Luo, D. S. and Vincent, S. R. (1994) Biochem. Pharmacol. 47, 2111-2112 26 Zembowicz, A., Hatchett, R. J., Radziszewski, W. and Gryglewski, R. J. (1993)J. Pharmacol. Exp. T’her.267,1112-1118 27 Schewe, T. (1995)Gen. Pharmacol.26,1153-1169 28 Eizirik, D. L., Flodstrom, M. and DemeIlo, M. A. R. (1996) Diabetolagia 39,387
Psychol. Bull. 32,447 65 Osawa, Y. and Davila, J. C. (1993)Biochem. Biophys. Res. Commun.
29 Masumoto, H. and Sies, H. (1996)Chem.Res. Toxicol.9,1057-1062 GaTvey,E. P. ef al. (1994)J. Biol. Chem. 269,26669-26675
66 Jaffrey, S. R. and Snyder, S. H. (1996)Science274,774-777
Yokoi, I. et al. (1996)Neurcchem.Res. 21,1187-1192 Szabo, C. et al. (1996)Br. 1. Phnrmucol.118,1659-1668 Yokoi, I. et al. (1994)J. Neurochem. 63,1565-1567 Iwahashi, K. ef al. (1996) Neuropsychobioogy33,76-79 Win, N. H. H. ei al. (1996)Eur. J Pharmacol.299,119-126 36 Frey, C. ef al. (1994)J. Biol.Chem.269,26083-26091 37 WoIff, D. J., Datto, G. A., Samatowicz, R. A. and Tempsick, R. A.
67 Wolff, D. J, Naddelman, R. A., Lube&e, A. and Saks, D. A. (1996)Arch. BiochemBiophys.333,27-34 68 Chernick, R. J., Mark.& R. D., Levere, R., Margreiter, R. and Abraham, N. G. (1989)Hepatology 10,365-369 69 Hu, J. G., Mahmoud, M. I. and El-Fakahany, E. E. (1994)Neurosci. Lett. 175,41-45 70 Chiesi, M. and Schwaller, R. (1995) Biochem. Pharmacol. 49,
(1993) J Bioi. Chem. 268,9425-9429 38 Sup, S. J., Green, B. G. and Grant, S. K. (1994)Biockm. Biophyi. Res. Commwn. 204,962~968 39 Moore, W. M. et al. (1994)J. Med. Chem. 37,3886-3888 40 Connor, J. R. et al. (1995)Eur. J. Pharmacol. 273,15-24
495-501 7l Schaad, N. C. et al. (1996)Hum. Reprod. 11,561~565 72 Handy, R. L. C. et al. (1996)Br. 1. Pharmacol. 119,423-431 73 Perschini, A., M&i&m, K. and Masters, 8. S. S. (1995) Biochemistry 34,15091-15095
30 31 32 33 34 35
ratios on the two isoforms ranging from 10 (Ref. 16) to 40 (Ref. 17). However, SMTC is also a very potent vasopressor agent in the anaesthetized rat’8 and an inhibitor of endothelium-dependent relaxation responses of the isolated rat aorta with a potency similar to that of L-NMMA (Ref. 19). From these data it seems likely that SMTC causes significant eNOS inhibition in viva and, consequently, should not be used to study the function of nNOS-derived NO in intact animals.
2 0 8
41 McCall, T. H., Fee&h, M., Palmer, R. M. J. and Moncada, S. (1991)Br. 1. Pharmacol. 102,234-238 42 Southan G. J., Szabo, C., O’Connor, M. P., Salzman, A. L. and ThiemermaM, C. (1995)Eur. I. Pharmacol. 291,311-318 43 Wang, Y-X.,Poon, C. I. and Pang, C. C. Y. (1993)J. Pharmucol.EXQ. Ther.26!?,112-119 44 Luo, D., Das, S. and Vincent, S. R. (1995)Eur. 1. Pharmacol. 290, 247-251 45 Southan, G. J., Zingarelli, B., O’Connor, M., Salkman, A. L. and Szabo, C. (1996)Br. J. Pharmacol. 117,61%32 46 Zingarelli, B. et al. (1996)Br. J. Pharmacol.120,357-366 47 Cattell, R. J., Hamon, C. G. B. and Blair, J. A. (1988)Biol.Chem. Hoppe-Styler369,545 48 Mayer, B., Brunner, F. and Schmidt, K. (1993)Biochem. Pharmacol. 45‘367-374 49 Miki, N., Kawabe, Y. and Kuriyama, K. (1977)Biochem. Biophys. Res. Commun.75851-856 50 Salaris, S. C., Babbs, C. F. and Voorhees, W. D., III (1991)B&hem. Phat7nacol.42,499-506 51 Szabo, C., Southan, G. J. and Thiemermann, C. (1994)Proc. Nafl. Acad. Sci. U. S. A. 91,12472-12476 52 Hibbs, J. B., Jr, Traintor, R. R. and Zavrin, Z. (1987)Science235, 473-476 53 Bogle, R. G., Moncada, S., Pearson, J. D. and Mann, G. E. (1992) Br. 1. Pharmacol. 105,768-770 54 Griscavage, J. M., Fukuto, J. M., Komori, Y. and Ignarro, L. J. (1994)J. Biol.Chem.269‘21644-21649 55 Moore, P. K. et al. (1990)Br. J Pharmacol. 99,408-412 56 Robertson, C. A. et a!. (1993)Biochem.Biophys. Res. Commun. 197, 523-528 57 Pfeiffer, S., Leopold, E., Schmidt, K., Bnmner, F. and Mayer, B. (1996)Br. 1. Pharmacol. 118,1433-1440 58 Buxton, H. 0. et al. (1993)Circ. Res. 72,387-395 59 Moore, P. K., Wallace, P., Gaffen, Z., Hart, S. L. and Babbedge, R. C. (1993)Br. 1. Pharmacol.110,219-224 60 Silva, M. T. et al. (1995)Br. J Pharmacol.114,257-258 61 AIlawi, H. S. et al. (1994)Br. 1. Pharmacol. 113,282-286 62 Hayashi, T. et al. (1994) Biochem. Biophys. Res. Commun. 203, 1013-1019 63 Weiner, C. P. et al. (1994) Proc. NatI. Acad. Sci. U. S. A. 91, 5212-5216 64 Finkel, M. S., Laghrissithode, F., Pollock, 8. G. and Rong, J. (1996)
TiPS -June
1997 (Vol. 18)
194,1435-1439
A range of heterocyclic compounds have been shown to inhibit NOS. These include the nitroindazole group of fused heterocycles [e.g. 7-nitroindazole, 7-NI (Ref. 20); 3-bromo-7-nitroindazole and 2,7-dinitroindazolezl] as well as imidazole and its various substituted derivatives [e.g. l-, 2- and 4-phenylimidazole22; econazole, miconazole and fluconazole23; I-(2-trifluoromethylphenyl) imidazole, trimethylphenylfluoroimidazole (TRIM)*41.
REVIEW
Box 2. Identification of nitric oxide synthase (NO!3 inhibitors - some pitfalls for the unwary? Biochemical assays Many NO!3 inhibitors are identified solely by the ability to reduce enzyme activity in vitro. Even a cursory perusal of the literature reveals large variations in the incubation conditions employed between (and sometimes even within) different laboratories. These include: (1) the source of NOS (crude tissue homogenate, semi-purified or genetically engineered enzyme); (2) presence or absence and absolute concentration of cofactors; (3) presence or absence of protease inhibitors; (4) time of exposure of inhibitor to enzyme; (5) reaction time; (6) composition and pH of buffer containing enzyme. Such methodological variations render comparison of data between laboratories difficult if not meaningless and cause excessive duplication by which newly described inhibitors are ‘checked out’ in each laboratory. Some attempt to standardize screens for NOS inhibitors is worthwhile. Minimally, purified or cloned enzymes should be employed and putative inhibitors tested over a range of substrate concentrations and exposure periods in the presence of saturating concentrations of cofactors. However, experiments that utilize crude tissue homogenates should not be
Both nitroindazoles (nitro group showing electron withdrawing activity) and imidazole-based compounds (possessing a lone pair of electrons at Nl) carry a net electronegative charge and, as a consequence, are believed to bind to the haem prosthetic group of NOS, thereby disrupting electron flow through the enzyme and preventing NO formation. Interestingly, by binding at the haem site, both 7-NI and TRIM interfere with the binding of both L-arginine and tetrahydrobiopterin, thus explaining the apparent competitive inhibitory nature of 7-NI and TRIM at these siteWs. The isoform selectivity of 7-NI and TRIM has also been studied. Whilst both 7-NI [K, 2.8 pM; 5.6 FM and 7.0 pM (Refs 26,27 and 28, respectively)] and TRIM (K, 47 PM) inhibit brain nNOS in vitro, only TRIM shows relative selectivity towards nNOS in vitro, being -37 times more potent as an inhibitor of nNOS than eNOS. 7-Nitroindazole is equipotent as an inhibitor of both nNOS and eNOS in aitro. The lack of effect of TRIM on eNOS in vitro is reflected in its inability to influence acetylcholineinduced endothelium-dependent relaxation of the isolated rabbit aorta preparation and failure to increase arterial blood pressure in the anaesthetized mouse and rat24. Perhaps more surprisingly, despite a potent eNOS inhibitory activity i~ vitro, 7-NI does not inhibit acetylcholine-induced endothelium-dependent relaxation of the isolated rabbit aorta preparation and is without effect on arterial blood pressure in a range of species including
ignored as they may provide useful data concerning the stability of the inhibitor in biological conditions. The need for complete biochemical and pharmacological profile NOS inhibitors seem to differ greatly (in terms of potency and isoform selectivity) in vitro and in vivo. Inability to penetrate biological membranes will considerably reduce the biological effects of NOS inhibitors in vivo whilst some compounds may exhibit unexpected isoform selectivity in vivo (which may not have been obvious in vitro) as a result of selective accumulation or catabolism of the compound in different target cells. A detailed profile of information (i.e. mechanism of action, efficacy in whole-cell preparations and biological activity in intact animals) is required. How selective is selective? Precisely how great the potency gap must be before a compound can be considered ‘isoform selective’ is a subjective decision. Certainly, researchers have been known to claim isoform ‘selectivity’ with a potency ratio of 5:l. A potency ratio of 1OO:l or more would seem worthwhile provided that a similar degree of selectivity could also be achieved in vivo. Interestingly, by this criterion there is no known selective neuronal NOS (nNOS) inhibitor.
mouse20, rat29 and pigeon.30 The mechanism whereby 7-NI inhibits eNOS activity in vitro but, apparently, not in viva is still unclear and warrants further study. Early studies of the pharmacological actions of TRIM and 7-NI in terms of CNS effects revealed that both corn.pounds elicit an L-arginine-reversible antinociception determined either as inhibition of formalin-induced hindpaw licking behaviour or acetic acid-induced abdominal constrictions in the mouse2OJ.1.Since this time, the profile of activity of 7-NI in particular has been extensively examined. To date, 7-NI has been reported to inhibit spinal cord ‘wind-up’ (increased firing of sensory neurones in the spinal cord) associated with peripheral noxious stimuli in the anaesthetized rat”‘, reduce cerebral blood flow implying an important role for nNOSderived NO in the regulation of blood flow within the brain32, inhibit the behavioural consequences of opioid withdrawal33 and prevent convulsions following administration of cocaine31 or NMDA (Ref. 35) to the mouse. Interestingly, 7-NI administration also reduces neuronal death following middle cerebral artery occlusion in the rat, which is widely used as an animal model of stroke3”. Numerous researchers have also utilized 7-NI to probe the role of NO in neurodegenerative disorders. Thus, parenteral administration of 7-NI reduces striatal dopamine and tyrosine hydroxylase loss, which occurs five days after methamphetamine injection in the mouse37, protects against hippocampal neurone loss
7-IPS-
lunf
lYY7 (Vol. 18)
2
0
9
REVIEW after bilateral carotid occlusion in the gerbil38and striatal neurone loss, after 1-methyl4phenyl-1,2,3,6+etrahydropyridine (MPTP) injection in the mouse39 and baboon@. Intriguingly, 7-NI pretreatment also ameliorates the neurological defect associated with mild head trauma in the mouse41.Because 7-NI has been demonstrated to promote the release of both noradrenalinehzand dopamine43 from hippocampal and striatal neurones, respectively, it is conceivable that some of these various effects of the inhibitor on central function may be secondary to changes in the release of conventional neurotransmitters. Peripheral nNOS activity is also associated with NANC nerves innervating a wide range of smooth muscle in the gastrointestinal, airways and urogenital systems”. Nitro arginine methyl ester and many other NOS inhibitors inhibit the smooth muscle relaxation elicited by electrical stimulation in such preparations. The effects of 7-NI and TRIM on such NANC responses have yet to be reported in the literature. This seems to be an interesting omission: whether 7-NI and TRIM affect NANC transmission to smooth muscle in vivo is likely to have a major bearing on the side-effect profile of nNOS inhibitors and thus their potential for clinical use. Endogenous inhibitors of NOS The possibility that the body may produce an ‘endogenous inhibitor’ of NOS has been a matter for debate over several years. For example, asymmetric dimethyl NGNCL-arginine (L-ADMA) is a potent NOS inhibitor that is found in many sites in the body including plasma, brain and kidney45. In addition, a high molecular weight (>lOOOO)inhibitor of NOS was demonstrated in crude homogenates of rat and rabbit brain&. Recently, a naturally occurring protein inhibitor (PIN) of NOS was extracted from rat brain47. PIN contains 82 amino acids and is a potent and highly selective inhibitor of nNOS. The discovery of PIN raises a number of important issues. Does PIN affect nNOS activity in the central and peripheral nervous systems in vivo? If so, does PIN play a part in the physiological and/or pathophysiological roles of NO in the nervous system? Does the concentration of PIN in the brain alter in different physiological or disease states? Importantly, the precise mechanism by which PIN inhibits nNOS at the molecular level is still to be determined. Information of this type is clearly required before the full biological significance (if any) of PIN can be appreciated. Whether the discovery of PIN will provide useful leads to the development of additional nNOS inhibitors remains to be seen. Potential therapeutic applications of nNOS inhibitors - is no NOS good NOS? Identification of potent and selective inhibitors of nNOS is clearly a very worthwhile goal in terms of providing tools with which to investigate the many and varied biological functions of NO in the nervous system. Whether selective nNOS inhibitors also have a future in the clinic is less clear cut. This uncertainty goes some
2 1 0
TIPS -June
1997 (Vol. 18)
way to explaining the relative dearth of such compounds in the literature compared with, for example, the plethora of iNOS inhibitors for which therapeutic targets are more clearly defined. Two problems that may hinder the future clinical exploitation of nNOS inhibitors can be identified. The first relates to our present incomplete understanding of the precise role of nNOS-derived NO in CNS function. At least part of the reason for this must be the use of isoform nonselective NOS inhibitors which, in addition to inhibition of brain nNOS, markedly increase blood pressure by reducing endothelial NO formation. It is, for example, conceivable that this ‘dual’ effect of L-NAME on nNOS and eNOS underlies reports in the literature of increased, decreased or unaltered neuronal damage following focal ischaemic damage in experimental animals pretreated with L-NAME. Indeed, similar confusion exists with respect to the effect of L-NAME (and like NOS inhibitors) on pain perception, convulsive behaviour and memory. It is hoped that the more widespread use of selective nNOS inhibitors by researchers will help to clarify the part played by NO in these important processes. The second problem is the possibility of a range of unwanted side-effects. Based on animal studies, using for the most part isoform nonselective NOS inhibitors, ‘expected’ side-effects of nNOS inhibitors include impairment of memory acquisition/retrieval and blockade of peripheral NANC nerves leading, amongst other things, to disordered stomach motility and impotence. Such side-effects may indeed present problems in the clinic, particularly if chronic treatment is envisaged. However, loss of memory and failure to gain an erection would not necessarily rule out the use of nNOS inhibitors in, for example, stroke or when administered prior to surgery to treat postoperative pain. In addition, further experiments are also required to determine the ability of the presently available nNOS inhibitors (e.g. 7-NI, TRIM) to influence NANC transmission. Given the current interest of the pharmaceutical industry in the development of NOS inhibitors it seems likely that additional, even more potent and more selective, nNOS inhibitors than are currently known will become available in the next few years. It must be hoped that the arrival of such compounds for pharmacological evaluation will provide the fundamental knowledge on which matters of clinical usage can be based. Selected references 1 Marletta. M. (1994) Cell 78.927-930 2 Forstermann; U. and KIeinert, H. (1995) Nuunyn-SchmiudeberX’s Arch. Pharmacol. 352,351-364 3 Anggard, E. (1994) Laticet 343,1199-1206 4 Dalkara, T. and Moskowitz, M. A. (1994) Brain Pathol. 4,49-57 5 Lyons, C. R. (1995) Adv. htm~umol. 60,32kL371 6 Griffiths. 0. W. and Kilboum. R. G. (1996) Methods Enzwmol. ~ _I 268, 375-392 7 Dwyer, M. A., Bredt, D. S. and Snyder, S. H. (1991) Biocllem. Biophys. Res. Commun. 176,11361141 8 Rees, D. D., PaImer, R. M. J., Schulz, R., Hodson, H. F. and Moncada, S. (1990) Br. 1. Pharmacol. 101,74&752
R 9 Rees, D. D., Palmer, R. M. J. and Moncada, S. (1990) Proc. Natl. Acad. Sci. U. S. A. 86,3375-3378 10 Gardiner, S. M., Compton, A. M., Bennett, T., Palmer, R. M. J. and Moncada, S. (1990) Eur. J. Pharmacol. 213,449-451 11 Moore, P. K., Oluyomi, A. O., Babbedge, R. C., Wallace, P. and Hart, S. L. (1991) Br. I. Pharmucol. 102,198-202 12 Gibson, A., Mirzazadeh, S., Hobbs, A. J. and Moore, l’. K. (1990) Br. J. Pharmacol. 99,602-606 13 Nava, E., Palmer, R. M. J. and Moncada, S. (1991) Lancet 338, 1555-1557 14 McCall, T. H., Feelisch, M., Palmer, R. M. J. and Moncada, S. (1991) Br. 1. Pharmacol. 102,234-238 1.5 Moore, W. M. et al. (1994) J. Med. Chem. 37,388&3888 16 Furfine, E. S. ct al. (1994) 1. Biof. Chem. 269,26677-26683 17 Nagafuji, T. et RI. (1995) NeuroReport 6,1541-1545 18 Narayanan, K. et al. (1995) 1. Biol. Chem. 270,11103-11110 19 Joly, G. A., Narayanan, K., Griffith, 0. W. and Kilboum, R. G. (1995) Br. J. Pharmacol. 115,491-497 20 Moore, P. K., Wallace, P., Gaffen, Z., Hart, S. L. and Babbedge, R. C. (1993) Br. 1. Pharmacol. 110,219-224 21 Bland-Ward, I’. A. and Moore, P. K. (1995) L$e Sci. 57,131-135 22 Wolff, D. J., Datto, G. A., Samatovicz, R. A. and Tempsick, R. A. (1993) Proc. Nntl. Acad. Sci. U. S. A. 268,9425-9429 23 Wolff, D. J., Datto, G. A. and Samatovicz, R. A. (1993) 1, Biol. Chem. 268,9430-9436 24 Handy, R. L. C. et al. (1996) Br. 1. Pharmacol. 119,423431 25 Klatt, P. et RI (1994) 1. Bio2.Chenr. 269,13861-13866 26 Mayer, B., Klatt, I’., Werner, R. and Schmidt, K. (1994) Neurophnrmncology 33,1253-1259 27 Babbedge, R. C., Bland-Ward, P. A., Hart, S. L. and Moore, P. K. (1993) Br. 1. Pharmncol. 110,225226 28 Wolff, D. J. and Gribin, B. J. (1994) Arch. Biochem. Biophys. 311, 300-306 29 Beierwaltes, W. H. (1995) Am. J. Physiol. 38, F134-F139 30 Zagvazdin, Y. S., Fitzgerald, M. E. C., Sancesario, G. and Reiner, A. (1996) Inrwf. O$zfhdmol. Vis. Sri. 37,666-672 31 Stanfa, L. C., Misra, C. and Dickenson, A. H. (1996) Brain Res. 737, 92-98 32 Kelly, I’. A. T., Ritchie, 1. M. and Arbuthnott, G. W. (1995) J. Cereb. Blood Flozc M&b. 15, 766-773
Gene targeting - homing in on qadrenoceptorsubtype function Ewen
MacDonald,
Brian K. Kobilka and Mika Scheinin
The qadrenoceptorwas a2A-1 a2El-
subdivided into three subtypes:
and cl,,-adrenoceptors
Since then, the search develop subtype-selective
agonists and antagonists,
as yet no major breakthrough
and but
has been made. In the past
year, several strains of genetically become available,
almost ten years ago.
has been on to discover
engineered
either overexpressing,
mice have
totally lacking
or expressing heavily modified c*,-adrenoceptor
subtypes.
Ewen MacDonald,
Scheinin
Brian
Kobilka
and Mika
describe how these mice may be utilized to elucidate the physiological
functions of the receptor subtypes and the
properties of future subtype-selective
CD1947, Elsevier Science Ltd
drugs.
E
V
E
I
W-
33 Vaupel, D. B., Kines, A. S. and London, E. D. (1995) Ps!/chopharmncology 18,361-368 34 Itzhak, Y. and Ali, S. F. (1996) J. Neurochem. 67,1770-1773 35 Przegalinski, E., Baran, L. and Siwanowicz, J. (1996) Nnrrosci. Left. 217,145-148 36 Dalkara, T., Yoshida, T., Irikura, K. and Moskowitz, M. A. (1994) Neuropharmacology 33,1447-1452 37 Dimonte, D. A., Royland, J. E., Jakowec, M. W. and Langston, J. W (1996) J. Neurochem. 67,244s2450 38 O’Neill, M. J., Hicks, C. and Ward, M. (1996) Eur. 1. Phnrmacol. 310. 115-122 39 Schulz, J. B., Matthews, R. T., Muqit, M. M. K., Browne, S. E. and Beal, M. F. (1995) J. krrochem. 64,936-939 40 Hantraye, P. et a/. (1996) Nat. Med. 2, 1017-1021 41 Mesenge, C., Verrecchia, C., Allix, M., Boulu, R. R. and Plotkine, M. (1996) J. Neurotrauma 13,209-214 42 Kiss, J. P., Sershen, H., Lajtha, A. and Vizi, E. S. (1996) Nrurosci. Lrff. 215,115-118 43 Silva, M. T. et al. (1995) Br. 1. Pharmncof. 114,257-258 44 Rand, M. J. and Li, C. J. (1995) AH~I~. RCP Physiol 57, 659-682 45 Valiance, I’., Leone, A., Claver, A., Collier, J. and Moncada, S. (1992) J. Cardiovusc. Pharmacol. 20,560-562 46 Moore, P. K., al-Swayeh, 0. A. and Evans, R. (1990) Br. 1. Pharmacoi’. 101,865+368 47 Jaffrey, S. R. and Snyder, S. H. (1996) Sciencr 274,771777
r Chemical names HF2035: 2-[2-aminoethyl]-N-[2,4,5-trichlorobenzenesulphonyl] amino-N-[4-chlorocinnamyl]N-methylbenzylamine LY83583: 6-anilino-5,Squinolinedione HF2035: 2-[2-aminoethyl]-N-[2,4,Strichlorobenzenesulphonyllamino-N-[4-chlorocinnamyl]N-methylbenzylamine
One of the greatest contributions that molecular biology has made to pharmacology is the clarification of receptor classification, particularly the functional contributions of varying receptor subtypes. Before molecular cloning as a means for receptor identification, classification of a new receptor subtype relied on pharmacological methods (e.g. order of agonist potency) that were often confounded in interpretation due to variable receptor reserve in different tissues or the exploitation of differing effecters in different settings by receptor subpopulations. Currently, receptor subtypes are often cloned before specific ligands binding to them are available, i.e. so-called orphan receptors posing a new set of challenges for pharmacologists and the pharmaceutical industry. In many ways, the a,-adrenoceptors exemplify the above scenario. There are now three well characterized receptor subtypes; aZA, aZB and (tic (Ref. 1; Table 1). However, available ligands have only marginal subtype selectivity. This review will describe strategies other than the evaluation of subtype-selective ligands to allocate physiological effects to distinct g-adrenoceptor subtypes and subsequently predict the pharmacological and therapeutic properties of subtype-selective drugs under development.
1’11:5016%6147(97)01063-8
TIPS-June
1997 (VOI 18)
E. MacDonald.
Lecturer, Departmentof Pharmacologyand Toxicology, Urwers~ty of Ku&o. PL 1627, FIN-7021 1 Kuoplo, Finland, B. K. Kobilka. Associate Professor, Department of Molecular and Cellular Physiology. AssIstant Investigator, Howard Hughes MedIcal Institute, Stanford Unwerslty, Stanford, CA 94305, USA. and M. Scheinin. Professor of Clwcal Pharmacology, Department of Pharmacology and Climcal Pharmacology, Unwerslty of Turku, FIN-20520 Turku. Finland
'11