Inhibitors of Nitric Oxide Synthase: What's up and What's Next?

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81

Inhibitors of Nitric Oxide Synthase: What’s up and What’s Next? *

Lakshmikirupa Sundaresan1,2, Suvendu Giri1,2 and Suvro Chatterjee1,2, 1 2

Vascular Biology Lab, AU-KBC Research Centre, Anna University, MIT Campus, Chennai, India Department of Biotechnology, Anna University, Chennai, India Abstract: NO is a key signaling molecule that has a wide range of biological functions including vasodilation and neurotransmission, and is implicated in several pathological conditions. Therefore, modulation of NO is a desired step in curbing the NO-associated disease processes. Targetting NOS proteins and inhibiting their activity to reduce NO production is a major approach since the inception of NOS inhibitor research. Although a series of NOS inhibitors have been developed, synthesized and practiced in experimental biology, no NOS inhibitor has been approved as yet a drug for therapeutic uses. The present review elaborates the scope of the clinical uses of NOS inhibitors and the inherent limitations of the process that makes the approach less successful so far in clinic.

Keywords: Nitric Oxide (NO), Nitric Oxide Synthase (NOS), Nitric Oxide Synthase inhibitors, NOS oxidase domain, NOS reductase domain. INTRODUCTION Mononitrogen Monoxide, commonly known as Nitric Oxide (NO) (molecular weight 30.0061 g/mol), is a heterodimer molecule, composed of two fundamental atoms, Nitrogen (1s22s22p3) and Oxygen (1s22s22p4). It is a gaseous molecule (Specific gravity =1.04) that can exhibit free radical activity as well as paramagnetic behavior due to one unpaired electron situated on the antibonding molecular orbital of the molecule [1, 2]. The bond order of NO is 2.5 and the bond distance (N-O) between double (1.15Å) and triple (1.05Å) bonds is 1.10Å. NO acts as a strong ligand while binding with metal and the M-N-O bond becomes almost linear or slightly bending [3, 4]. On inhalation, NO can react with heme group of hemoglobin similar to Carbon Monoxide (CO) making it a co-ordinate compound called methemoglobin. This cannot bind with oxygen because of the Fe3+ (ferric) state of heme iron. Thus, NO affects the oxygen carrying capacity of hemoglobin [5]. NO is a key signaling molecule that has a surprising array of biological functions including vasodilation [6], macrophage-mediated cytotoxicity [7], neurotransmission [8, 9], bronchodilation [10] and gastrointestinal smooth muscle relaxation [11] in higher animals. In plants, NO is essential for growth and development and acts as a signaling molecule in several processes including seed dormancy, root growth, germination, immune defense and stress response where the major source of NO being nitrite reduction [12]. NO IN THE MAINTENANCE OF LIFE PROCESSES Evolution The role of nitric oxide has been very crucial in evolution. The role of NO would be to provide early life with *Address correspondence to this author at the AU-KBC Research Centre, M.I.T Campus of Anna University, Chromepet, Chennai-600044, India, Tel.: +91 44 2223 4885 48 / +91 44 2223 2711 48; Fax: +91 44 2223 1034; E-mail address: [email protected] 17-/16 $58.00+.00

defense mechanisms against oxidative destruction by neutralization of ozone in primitive environment [13], providing an evolutionary advantage. Within the cellular environment, NO would have controlled the reactive oxygen species before the evolution of enzymes, thereby regulating redox mechanisms gradually NO has evolved to serve other roles as well [14]. The L-arginine based production of NO by NOS has been suggested to be a pathway of early evolutionary origin as they demonstrated the synthesis of NO from Larginine in haemocytes from an organism called horseshoe crab [15]. These organisms are considered living fossils since they have not changed much in over 500 million years of evolution [16]. Vasorelaxation Earlier in 1979, NO had been shown to exhibit vascular smooth muscle relaxant properties [17]. A year later, Furchgott and Zawadski discovered the phenomena of endothelium-dependent vasorelaxation and Endothelial Derived Relaxing Factor (EDRF) [18]. Seven years later, EDRF was identified as NO [19-21]. NO is incessantly produced in the endothelium, which activates soluble guanylyl cyclase in arteries and veins maintaining the vascular tone [22, 23]. Blood flow induced shear stress inititates the opening of calcium channels of endothelial cells, which in turn trigger the calcium-dependent tyrosine phosphorylation of endothelial Nitric Oxide Synthase (NOS) leading to increased NO production in the vicinity [24, 25]. Its unique chemical properties and the maintanence of local concentrations of NO by the endothelium make it a potent modulator of hemostasis and blood flow (Fig. 3) [26]. Immune Regulation Inducible NOS (iNOS) plays a substantial role in immune system. NO production by iNOS is induced in the presence of L-arginine in macrophages, monocytes and other cells [27] in highly toxic amounts of NO [28]. The kinetics © 2016 Bentham Science Publishers

82 Current Enzyme Inhibition, 2016, Vol. 12, No. 1

of iNOS varies greatly from that of eNOS and nNOS. NO thus produced through various mechanisms act as an inflammatory mediator [29]. NO is toxic to microorgansisms and nitrosylates proteins and other macromolecules. Reactive oxygen species produced in cells get released and produce respiratory burst in leukocytes resulting in pathogen killing [29]. Myeloid cells produce high amounts of inflammatory NO along with the generation of superoxide (O2-). Superoxide reacts with NO forming peroxynitrite (ONOO-) [30, 31]. Through peroxynitrite, NO mediates its cytotoxic effects, DNA damage, lipid oxidation, nitration of proteins and mitochondrial respiration inhibition [32]. NO and ONOO- affect several proteins that are critical for cell signalling and survival [33, 34]. NO selectively inhibits T subset proliferation resulting in humoral and allergic responses [35, 36]. NO also affects the functions of mast cells which are key players in initiating inflammatory responses [37]. NO inhibits IgE-dependent secretory functions of mast cells which play a significant role in allergic reactions [38]. NO also affects the expression of several cytokines including IL-1, TNF-, IL-6 ad IFN- which are essential in inflammatory processes [33]. The immune regulation is highly dependent on the NO concentrations and the biological environment [37]. Signaling NO due to its neutral charge can freely diffuse across the cell membranes [39]. NO produced by the NOS enzymes mediates its action mainly through soluble guanylyl cyclase (sGC) [40-42], which is the well studied target of NO. Though NO mediates its action through other targets as well, the NO/cGMP pathway is well defined. cGMP dependent pathways: Upon activation by NO [43], sGC catalyzes the conversion of guanosine 5’triphosphate (GTP) to cyclic 3’,5’guanosine monophosphate (cGMP) [44]. sGC is involved in signal transduction mediating several biological processes [45]. The increase in cGMP levels as a result of NO binding with sGC activates cGMP-gated cation channels [46], cGMPdependent protein kinase [47] and cGMP-regulated phosphodiesterase [48]. cGMP independent pathways: In vascular smooth muscle cells, NO can activate calcium-dependent potassium channels [49]. NO has been shown to activate G-proteins by increasing the rate of nucleotide exchange which results in increased bound GTP forms [50]. NO can also nitrosylate proteins including hemoglobin and cardiac calcium release channel which are S-nitrosylated in vivo. This nitrosylation can greatly modify the function of the proteins [51-53]. NO Cascade in Pathogenesis Much of the pathogenesis mediated by NO occurs through the formation of secondary intermediates including peroxynitrite and nitrogen dioxide which are highly reactive than the NO itself [54]. Certain conditions such as high blood pressure and high glucose lead to excess production of superoxide [55, 56]. Superoxide and other reactive oxygen species (ROS) react with NO to form peroxynitrite. This peroxynitrite in turn can nitrate tyrosine residues of proteins,

Sundaresan et al.

[57, 58] cause mitochondrial membrane protein thiol crosslinking and lipid peroxidation [59] and inhibit mitochondrial respiratory chain complexes [60, 61]. Peroxynitrite also causes slow modification of the properties of eNOS which is called eNOS uncoupling [62] ultimately resulting in high production of superoxide radicals [63-65]. This oxidative stress causes DNA damage and apoptosis [66, 67]. Thus increased activity of NOS enzymes has implications in various disorders ranging from neurodegeneration, inflammation, cancer and so on. Therefore NOS enzymes are regarded as pharmacological target which can be controlled by the use of inhibitors and great efforts have been done in order to meet with successful inhibition of NOS. SOURCE OF NO PRODUCTION NOS and its Isoforms The sources of NO include production by Nitric Oxide Synthase (NOS) enzymes [68], reduction of nitrite by various proteins acting as Nitrite Reductases (NR) [69] and reduction of nitrate obtained through dietary protein by nitrate reductases from the commensal bacteria present in the oral cavity [70, 71]. NO is synthesized in vivo by Nitric Oxide Synthases (NOS) from L-arginine [72]. There are three distinct enzyme isoforms namely iNOS, eNOS and nNOS which are products of different genes and have different catalytic properties with specific localizations. In physiological conditions without any immune challenge, eNOS and nNOS isoforms predominate [73]. Endothelial NO Synthase (eNOS), a 133kDa protein was the first isoform identified in the vascular endothelium. Later, neuronal NOS (nNOS) with a molecular weight of 161 kDa was identified in the nervous system, mainly in the brain and inducible NOS (iNOS), a 131 kDa enzyme; the third isoform was identified in macrophages [74]. These isoforms have many common features including dimerization property. The monomer of NOS includes C-terminal reductase domain that contain binding sites for the cofactors Flavin Adenine Dinucleotide (FAD), Flavin Mononucleotide (FMN) and Nicotininamide Adenine Dinucleotide Phosphate (NADPH) [75-77] and an Nterminal domain which contains binding sites for L-arginine, haem and tetrahydrobiopterin (BH4). The domains are linked by a Calmodulin (CaM) recognition site [68]. NADPH mediates the transfer of electrons to the reductase domain, which continues through FAD and FMN redox carriers to the oxygenase domain. At the active site, the transferred electrons interact with haem iron and tetrahydrobiopterin thereby aiding the reaction of oxygen with Larginine to generate NO and citrulline [78]. For the production of 1 mol of L-citrulline, 1.5 moles of NADPH and 2 moles of oxygen are required [79-81]. Dimerization of the monomers is aided by the haem group, without which there is no binding of BH4 or substrate analog to the enzyme [82, 83]. Both the coordination state and the spin-state of the unpaired electrons of haem iron change from low to high spin state as they bind with substrate and BH4 [84-87]. iNOS and nNOS require BH4 in addition to haem group for dimerization [88, 89] whereas eNOS does not have this requirement [90]. Binding of substrate strengthens the dimerization. eNOS and nNOS are dependent on calcium ion concentra-

Inhibitors of Nitric Oxide Synthase: What’s up and What’s Next?

tion for activation whereas iNOS is not dependent of the same since it binds with CaM in irreversibly [91]. CaM is essential for the inter-domain transfer of electron from flavins to the heme groups [92]. nNOS can transfer electrons to the haem, thereby oxidizing NADPH at a high rate than the other two isoforms [93]. Mitochondrial NOS There is another type of NOS located on the inner membrane of mitochondria, called mitochondrial NOS (mtNOS), which has an important role in cellular respiration [94], energy metabolism [95-97] and mitochondrial biogenesis [98]. NO produced by mtNOS can inhibit cytochrome oxidase by competing with oxygen and affects oxidative phosphorylation [99-101]. The mtNOS activity is also dependent on intra-mitochondrial Ca2+ concentration [102-104] that indicates that the enzyme complex binds with CaM reversibly. In this point of view, mtNOS resembles eNOS and nNOS when compared to i-NOS. Substrate analog inhibitors of eNOS and iNOS can inhibit mtNOS by competitive way of inhibition [105]. Nitrite Reductase Mediated NO Production In addition to NOS, nitrite reduction also serves as a source of NO production as nitrite epitomizes the largest tissue and intravascular storage form of NO [106]. In tissues, the nitrite levels range from 1- 20mM and in blood, the levels are between 100 and 200 nM [107, 108]. In hypoxic and acidic conditions, nitrite reduction is carried out by a number of proteins when activated including hemoglobin, myoglobin [106, 109], neuroglobin [110] Cytoglobin [111], NOS [112], Molybdenum-containing enzymes xanthine oxidoreductase [113-116], Aldehyde oxidoreductase [117], Mitochondrial electron transport chain complexes (ETC), ETC complex IV [118], ETC complex III, Cytochrome C [119-121] and mitochondrial molybdopterin enzymes [122]. The tissue specific activity of nitrite reduction by the number of enzymes depends on the differing oxygen tension thus ensuring NO generation and its function over a wide range of physiological hypoxia [71]. Biological Regulation of NOS As NO plays a diverse array of roles and is produced in a wide range of tissues. Therefore the regulation becomes very essential which is found at various levels such as transcription, through covalent modifications and feedback mechanisms. The regulation of NOS activation is mediated by several factors including Ca2+/Calmodulin binding, phosphorylation, S-nitrosylation and lipid attachment [123, 124]. Calmodulin - All the three NOS isoforms require Calmodulin (CaM) for their activities. NO production requires the presence of Ca2+ for its activity [125, 126]. eNOS and nNOS require higher concentrations of Ca2+ as compared to iNOS [91]. Phosphorylation - Phosphorylation of eNOS and nNOS affects their activity. eNOS phosphorylation at Ser1177 promotes the electron flux through the reductase domain ulti-

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mately resulting in NO production [127, 128], whereas nNOS phosphorylation at Ser847 causes decrease in nNOS activity [124, 129]. PIN - Protein Inhibitor of nNOS (PIN), which is a light chain of myosin and dynein binds to the N-terminal extension of nNOS preventing homodimerization, thereby inhibiting its activity [130]. It is a protein widely expressed in humans [130, 131]. Hsp90- Hsp90 acts as an allosteric activator of eNOS by binding to it receiving cues from VEGFR, histamine or fluid shear stress [132]. Kalirin, a cytosolic protein has been reported to confer neuroprotection during inflammation through the prevention of dimerization of iNOS thereby inhibiting its activity [133]. Caveolin - Caveolin (Cav) is a major coat protein of caveolae and they are of three types namely Cav-1, Cav-2 and Cav-3 with molecular weights ranging from 18 to 24kDa. They are microdomains of plasmalemmal membrane. eNOS binds to Cav-1 and Cav-2 are expressed in endothelial and fibrous tissues [134]. Cav-1 getting localized to caveolae in endothelial cells, binds directly to the oxygenase domain of eNOS [135, 136] whereas in cardiac myocytes, it binds to caveolin-3. With low concentrations of Ca2+/CaM, upon binding, caveloin-1 is inhibitory to eNOS activity and in skeletal muscle, caveloin-3 is inhibitory to nNOS. It has been shown that Cav-1 inhibits nNOS by regulating both intra- and inter-domain transfer of electrons [137]. Cav-1 also plays a regulatory role in angiogenesis through potential inhibition of eNOS [138]. NOSIP NOS Interacting Protein (NOSIP), a 34-kDa protein is expressed in endothelial cells, heart, lung and brain [139]. It binds to the carboxy-terminal of the eNOS oxygenase domain and promotes the translocation of eNOS from the plasma membrane to intracellular sites. This results in the inhibition of NO synthesis for which the localization of eNOS is essential to the plasma membrane [139-141]. eNOS Trafficking Inducer protein NOSTRIN, a 58 kDa protein, which is expressed in endothelium in high amounts, binds to eNOS and triggers its translocation from plasma membrane leading to attenuation of eNOS derived NO production [142]. Sub-Cellular Localization of NOS and NOS Activity eNOS is modified by the addition of both myristate and palmitate groups. Myristoylation occurs during translation at N-terminal glycine residue in an irreversible manner [143] which is essential for membrane localization and maximum activity [144], whereas palmitoylation occurs posttranslationally and this process is reversible [145]. This dual acylation is essential for the localization of eNOS to the plasmalemmal caveloae of endothelial cells [143, 146]. Apart from the two types of acylation described above, eNOS activity also depends on the intracellular localization of the enzyme [147]. Though this enzyme complex can be found in nucleus [148], mitochondria, golgi complex [149],


cytoskeleton and even in caveolae [146], the highest activity has been observed from NOS situated in plasma membrane and golgi complex [150]. Local availability of substrate for the enzyme might be a probable reason for this observation [151]. Platelet activating factor and lysophosphatidic acid play a surprising role in NO production as they are involved in sub-cellular localization of the NOS reported by Costa et al. [152]. Some reactive nitrogen and oxygen species that are differentially distributed among the sub-cellular compartments can also affect the localized activity of NOS by oxidative modification of the enzyme by redox reaction [153]. It has been reported that sub-cellular distribution of eNOS is regulated based on its phosphorylation status [154, 155] since phosphorylation aids extracellular signaling & Akt dependent pathway of NO production [156]. NOS Inhibitors The NOS isoforms share a general structure [68]. The variations in their activities are believed to be as a result of their cellular localization and regulation with different and even opposing physiological functions. This difference in functions is observed in pathological conditions as well. For instance, in lungs, all the three isoforms from different cell sources co-ordinate to maintain the function, whereas during asthmatic conditions, there is an increase in iNOS and decrease in nNOS and eNOS. And in brain, nNOS and eNOS play complementary roles in maintaining the hippocampal synaptic plasticity [157], however during ischemia, eNOS seems to be protective and nNOS has detrimental effects [158, 159]. The number of NOS inhibitors reported so far is overwhelming. A list of notable inhibitors along with their selectivity and mechanism of action has been provided in Table 1. NOS inhibitors can be classified based on their chemical properties, isoform selectivity and the binding site. Based on the mechanism of action, they can be categorized into four groups. The first group of inhibitors is arginine analogs which bind to the substrate binding site. This group includes many arginine based analogs such as L-NMA, L-NAME and so on [72, 160]. The second group of inhibitors includes agents that resemble tetrahydrobiopterin in structure e.g.: 4amino pteridine derivatives [161]. The third group of inhibitors binds to the haem interfering with the dimerization of the monomer and enzyme activity. e.g., S-Methyl-L thiocitrulline (SMTC) [162]. The fourth group has compounds that interact with flavin cofactors including DiphenyleneioTable 1.

Sundaresan et al.

donium chloride (DPI) [163]. The different types of inhibitors based on the binding site have been summarized in Table 2. Based on their chemical properties, the inhibitors can be divided into two types. One group is amino acid based which mostly are derivatives and analogs of arginine whereas the other group has a range of compounds with structures differing from that of arginine. Arginine Based Inhibitors Inhibitors that bind to the arginine binding site, thus competitively inhibiting the NOS such as aminoguanidine, S-ethylisourea, thiocitrulline are quite high in number. The most studied compounds include L- N - Nitroarginine, LN- Nitroarginine methyl ester and NG-propyl-L-arginine. Though many L-arginine based analogs have been described and studied [166-169], the amino acid moiety is neither a requirement for selectivity nor potency. These inhibitors are sensitive to local concentrations of arginine since they are similar in structure to L-arginine. An extensive review of arginine-base inhibitor has been done by Víteek et al. [170]. Natural NOS Inhibitors

LN -Methylarginine (L-NMA), naturally occurring in living organisms, is the first reported inhibitor [72, 171], which acts as a competitive inhibitor of all NOS isoforms [84, 167, 172]. It is quite stable and possesses low toxicity and has a Ki value ranging from 10-100 M. It has been used to study the role of NO in different physiological systems in animal models. In several studies, L-NMA has been useful in treating septic shock associated with hypotension and was also found to increase systemic vascular resistance [174, 175]. But decreased cardiac output and oxygen delivery with increased mortality were observed as side effects indicating controversies associated with different dosage, treatment period and animal models [173-175]. ADMA - L-N, N- Dimethylargnine (ADMA) - This is another naturally present inhibitor which is also a product of degradation of arginine-methylated proteins and an equal potency as that of L-NMA [176]. Several studies with animal models [176-178] and human studies [179, 180] have showed that NOS inhibition by ADMA poses cardiovascular risks, making the applicability of ADMA for therapeutics very limited.

NOS inhibitors based on binding sites with pharmacological examples.

S. No.

Binding site

Pharmacological inhibitors as examples

1.

Substrate binding site

L-N- Methylarginine [72]

2.

Pterin binding site

4-amino pteridine derivatives [161] 2,4-diamino-5-(3’,4’dichlorophenyl)pyrimidine [164] 7-Nitroindazole [165]

3.

Inhibitor that binds with heam group

S-Methyl-L thiocitrulline [162]

4.

flavin/NADPH site

Diphenyleneiodonium chloride [163]


Table 2.

No.


Different Inhibitors with their selectivity & inhibitory mechanism.

Inhibitors

Chemical Name

Ki Value/ IC50

Mechanism

iNOS

nNOS

eNOS

(Binding site)

7nM

2M

50 M

(human)

(human)

(human)

4.2 nM

34 μM

150nM

(Mouse)

(Rat)

(Bovine)

iNOS

86 nM

17 M

162 M

iNOS

17nM

29nM

36nM

Selectivity

N'-[[3-(Aminomethyl)phenyl]methyl] 1.

1400W dihydrochloride

ethanimidamide;dihydrochloride

iNOS

PubChem CID: 2733515 6-Methyl-5,6-dihydro-4H-1,32.

85

AMT hydrochloride

thiazin-2-amine;hydrochloride

iNOS

PubChem CID: 2733501

Refs.

Glu-371/592 in of [187, 195, oxygenase domainloss of 196] heme cofactor

Competitive with the substrate L-arginine

[197,198]

Reversible l-argininecompetitive inhibitor

[199, 200, 201].

2-[2-(4-Methoxypyridin-2-yl)ethyl]3.

BYK 191023 dihydrochloride

1H-imidazo[4,5-b]pyridine;dihydrochloride PubChem CID: 56972216

4.

EIT hydrobromide

5.

Aminoguanidine hydrochloride

6.

L-NIL hydrochloride

Ethyl carbamimidothioate;hydrobromide PubChem CID: 200213

2-Aminoguanidine;hydrochloride

iNOS

PubChem CID: 2734687 (2S)-2-Amino-6-(1aminoethylideneamino)hexanoic acid;hydrochloride

iNOS

0.14M

3.3 M

(human)

(human)

0.4-3.3 M

17-92 M

8-38 M

(Rat)

(Mouse)


7.

9.

2-Iminopiperidine hydrochloride

S-Isopropylisothiourea hydrobromide

2,3,4,5-Tetrahydropyridin-6amine;hydrochloride

L-NIO dihydrochloride

Propan-2-yl carbamimidothioate;hydrobromide

12.

pentanoic acid;dihydrochloride

Methyl-L-NIO (hydrochloride)

Ethyl-L-NIO

0.63 M

(human)

(human)

9.8nm

37nm

22nm

(human)

(human)

(human)

iNOS

3.9 M

1.7 M

3.9 M

nNOS

(mouse)

(rat)

(Bovine)

9.5 M

3.0 M

10.0 M

(mouse)

(rat)

(bovine)

iNOS



11.

0.24 M

PubChem CID: 85236

(2S)-2-Amino-5-(1-aminoethylideneamino) 10.

0.3M (human)

iNOS

nNOS

Chloro (2S)-2-amino-5-(1aminobutylideneamino)pentanoate

nNOS

PubChem CID: 35028729 13.

Propenyl-L-NIO (hydrochloride)

14.

Vinyl-L-NIO

nNOS (2S)-2-Amino-5-(1-aminobut-3enylideneamino)pentanoic acid;hydrochloride

nNOS

PubChem CID: 71433867 Methyl (2S)-2-amino-5-[[amino(nitramido) 15.

L-NAME hydrochloride

methylidene]amino]pentanoate;hydrochloride

Non-selective


3-Bromo-7-nitroindazole

3-Bromo-7-nitro-2H-indazole

nNOS

PubChem CID: 1649

12 M

5.3 M

18 M

(mouse)

(rat)

(bovine)

17 M

10.3 M

58.2 M

(mouse)

(rat)

(bovine)

60 μM

100 nM

12 μM

(mouse)

(rat)

(bovine)

4.4 M

15 nM

39 nM

(mouse),

(bovine)

(human)

0.29 M

0.17 M

0.86 M

(rat)

(rat)

(Bovine)

Competitive inhibitors of iNOS at the L-arginine binding site

[184, 185]

Irreversible inhibitor of iNOS

[202-204]


[205]

[206, 207]


[185]


[208]

Reversible l-argininecompetitive

[209]

inhibitor Reversible l-argininecompetitive inhibitor

[209]

l-Argininecompetitive inhibitor

[210]

Heme cofactor modified

[209]


[211, 212]

Binds with heam group

[213-215]

N'-[4-[2-[(3-chlorophenyl)methylamino] 17. ARL 17477 dihydrochloride

ethyl]phenyl]thiophene-2carboximidamide;dihydrochloride PubChem CID: 9824646

nNOS

1 μM

17 μM

(human)

(human)

[216-218]


Sundaresan et al.

(Table 2) contd… Ki Value/ IC50 No.

Inhibitors

Chemical Name

iNOS 18.

L-NNA

N-Nitro-L-arginine

nNOS,eNOS


7-NINA

sodium;7-nitroindazol-1-ide

Non-selective

PubChem CID: 16760594 5-[[{2}-Azanylidene(methylamino)methyl] 20.

L-NMMA acetate

amino]pentanoic acid

Non-selective


TRIM

1-[2-(Trifluoromethyl)phenyl]imidazole

Mechanism

Selectivity

nNOS

PubChem CID: 1359

nNOS

Refs.

eNOS

(Binding site) Reversible l-argininecompetitive inhibitor

[169, 218]

Binds with heam group

[189, 220].


[160]

Arginine & tetrahydrobiopterin binding site

[221, 222]


[223]


[224, 225]

l-Argininecompetitive inhibitor

[226-228]

Flavin/NADPH binding inhibitor

[163, 229, 230]

Heme-binding inhibitor

[161, 181, 231]

4.4 M

15 nM

39 nM

(mouse)

(bovine)

(human)

5.8 M

0.71 M

0.78 M

(rat)

(rat)

(Bovine)

6 M

0.18 M

0.4 M

(mouse)

(rat)

(Human)

27.0 M

28.2 M

1.06 mM

(rat)

(mouse)

(bovine)

Ethyl N'-[4-(trifluoromethyl)phenyl] 22.

EPIT/TFPI Hydrochloride

carbamimidothioate;hydrochloride

nNOS

0.32 M

PubChem CID: 2733516 (2S)-2-Amino-4-(diaminomethylideneamino) 23.

L-Canavanine sulfate

oxybutanoic acid;sulfuric acid

60 μM

Non-selective

(Murine)

PubChem CID: 11957500 Methyl (2S)-2-amino-5-[[amino(nitramido) 24.

L-NAME

methylidene]amino]pentanoate; hydrochloride

Non-selective


26.

DPI

SMTC S-Methyl-L thiocitrulline

27.

Gallotannin

28.

GW274150

Diphenyleneiodonium chloride PubChem CID: 16219231

iNOS

2-amino-5-[[amino(methylsulfanyl) methylidene] amino]pentanoic acid

nNOS

PubChem CID: 5148969 TANNIC ACID PubChem CID: 16129778 (2S)-2-Amino-4-[2-(1-aminoethylideneamino) ethylsulfanyl]butanoic acid

4.4 M

15 nM

39 nM

(mouse)

(bovine)

(human)

50 nM

0.3 M

(mouse)

(porcine)

40 nM

1.2nM

11 nM

(human)

(human)

(human)

eNOS

2.2 μM

[232]

iNOS

2.19 μM

177 μM

544 μM

Arginine competitive

[233, 234]

iNOS

8.0M

630 M

1000 M

Arginine competitive

[233]


GW273629

(2S)-2-Amino-3-[2-(1-aminoethylideneamino) ethylsulfonyl]propanoic acid PubChem CID: 44370729

30.

N-PLA

2-Amino-5-[(N'-propylcarbamimidoyl) amino]pentanoic acid

180 M

57 nM

8.5μM

Competitive inhibitor of

(murine)

(bovine)

(bovine)

L-Arginine

Non-selective

3 M

0.3M

2.5 μM

6 nM

350nM

[241]

1.1 μM

[241]

nNOS


L-NAA

NG-Amino-L-arginine

32.

N-(3(aminomethyl)phenyl)-2furanylamidine

nNOS

160 nM

33.

-Fluoro-N-(3(aminomethyl)phenyl) acetamidine

nNOS

480nM

34.

35.

ADMA

(2S)-2-Amino-5-[[amino(dimethylamino)

(NG,NG-DimethylLarginine)

methylidene] amino]pentanoic acid

Agmatine

11nM (rat)

[235-237]

Covalent alteration of the [238-240] heme prosthetic group

[176, 242244]

PubChem CID: 123831 2-(4-Aminobutyl)guanidine PubChem CID: 199

Non-selective

220μM

660 M

7.5 mM

(human)

(rat)

(bovine)

Competitive inhibitor of L-Arginine

[245, 246]



87

(Table 2) contd… Ki Value/ IC50 No.

Inhibitors

Chemical Name

iNOS

Acetic acid;4-methyl-2,3,4,5-tetra 36.

2-Imino-4-methylpiperidine acetate

hydropyridin-6-amine

iNOS


37.

38.

39.

1,3 (S,S'-(1,3Phenylenebis(1,2ethanediyl))bisisothiourea)

2-[3-(2-Carbamimidoylsulfanylethyl)

1,4-PBIT (S,S'-(1,4Phenylenebis(1,2ethanediyl))bisisothiourea)

2-[4-(2-Carbamimidoylsulfanylethyl)

-Guanidinoglutaric acid

phenyl]ethyl carbamimidothioate

iNOS

PubChem CID: 1331

phenyl]ethyl carbamimidothioate

iNOS & nNOS

PubChem CID: 1337 2-(Diaminomethylideneamino)pentanedioic acid PubChem CID: 2105

Synthetic NOS Inhibitors

L- N - Nitroarginine (L-NNA) [160] is the first synthetic inhibitor developed following the discovery of L-NAME (LN- Nitroarginine methyl ester). This compound is readily hydrolyzed into L-NNA under physiological conditions, thereby provides a means of applicability in both in vivo and in vivo experiments. In several studies, the role of NO in both heath and disease has been explored with the use of L-NNA and L-NAME [170]. L-citrulline analogs such as L-Thiocitrulline, S-methyl and S-ethyl derivatives despite having increased potency for nNOS in vivo did not show selectivity in vivo [168]. Non amino acid-based inhibitors - Aminoguanidine belongs to the class of guanidines [181, 182], which has been tested in many in vivo and preclinical models, however high doses of the inhibitor is required for achieving the desired effects but at this concentrations, it inhibits all the isoforms [183]. S-substituted isothioureas reported to be highly potent inhibitors act by interfering with the heme moiety [184]. S-methyl-isothiourea has been found to be protective in rodent model of septic shock [185]; however it showed cardiovascular effects as well. Bis-isothioureas also have been shown to be potent and selective for iNOS inhibition but their high toxicity and cell impermeable characteristics prevent them from being candidates for therapeutic use [184]. S-substituted isothioureas could potentially inhibit NO synthesis in cultured bovine chondrocytes and patients with osteoarthritis [186]. N-(3-(aminomethyl) benzyl) acetamidine (1400W) is one among the group, which binds slowly but in a tight manner with human iNOS both in vivo and in vivo [187]. Indazole - 7-Nitroindazole (7-NI), the best studied among the Indazole class of inhibitors binds to the heme group altering both pterin and substrate binding [188]. Though it was reported to be equipotent for all the NOS isoforms, in vivo studies demonstrated that it exhibits a remarkable selectivity for nNOS with no cardiovascular effects observed [189, 190]. 7-NI has excellent antinociceptive action

Mechanism

Selectivity

nNOS

0.1 M (human)

nNOS

eNOS

0.2 M

1.1 M

47nM

0.25 μM

9μM

(human)

(human)

(human)

7.4 nM

16 nM

360 nM

(human)

(human)

(human)

2.69 M (rat)

Refs.

(Binding site)

[247]

Competitive inhibitors

[185]

Competitive inhibitors

[185]

[248]

and reduces behavioral disturbances related to opioid withdrawal [189], [190-192] and cocaine administration [193]. Imidazolde - derivatives have a similar inhibitor profile as that of indazoles among which trimethylphenylfluoroimidazole (TRIM) was widely used to test its efficacy in preclinical and clinical studies. TRIM resembling 7-NI exhibits nNOS selectivity and has similar binding mechanisms [188, 189, 194]. Isoform Specific Inhibitors Based on the isoform selectivity, NOS inhibitors can be classified as eNOS, nNOS and iNOS selective inhibitors. eNOS Inhibitors The major protective barrier against vascular disease is endothelial Nitric Oxide Synthase (eNOS), the enzyme which is responsible for the production [249]. Tannin is a potent natural polyphenol to inhibit eNOS with an IC50 value of 2.2M whereas other isoforms are inhibited at much higher concentrations. Quercetin is also an inhibitor of eNOS with IC50 value of 220M, however less potent than tannin [232]. Since eNOS is a key regulator of vascular tissues and blood pressure, eNOS inhibition leads to high blood pressure and other vascular effects posing detrimental effects [68]. iNOS Inhibitors iNOS inhibition has been found to be fruitful in the treatment of septic shock and chronic inflammatory diseases including arthritis. One of the first iNOS inhibitors to be developed is L-N-iminoethyl lysine (L-NIL) with an IC50 value of 4.2nM [197, 250]. 1400W, a bis-isothioureas has been reported to be highly selective for iNOS with a Ki of less than 7nM [187] and to be highly efficient in in vivo models and animal models as well [187, 251-254] but is acutely toxic at high doses. GW273629 and GW274150 are acetamidine amino acids that are two highly selective inhibitors with selectivity for human iNOS of more than 80- and 100- fold than that for nNOS and eNOS respectively [255257]. Their potency is intact in cells and tissues and another


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advantage they possess is safety unlike 1400W and hence they have been used to understand the roles of iNOS in preclinical models [256, 257].

In the present review, we have tried to categorize the uses of pharmacological inhibitors of NOS in pathology of different systems.

iNOS Dimerization Inhibitors

Respiratory System

Most of the iNOS dimerization inhibitors have an Nsubstituted imidazole group which is believed to directly bind to the heme iron in the enzyme’s active site [258-261] but a peptide-derived compound [262]. KLYP956 is a quinolinone based compound which does not interact with heme group for the destabilization of dimers. It has a 1900-fold selectivity for iNOS over eNOS and 260-fold over nNOS with an IC50 value of 0.01M for human iNOS (Fig. 1) [263].

Lung consists of at least 40 different types of cells including vascular smooth muscle cells, bronchial smooth muscle cells, endothelial cells, macrophages and epithelial cells [271] with each type capable of producing NO through one or more NOS isoforms [272-276]. The NO thus produced has a variety of roles to play including vasodilation at endothelium, phagocytosis in macrophages, and bronchodilation at inhibitory nerve terminals and mucin production in epithelial cells [227, 272, 275]. NO production should be controlled under normal and in inflammatory conditions as well such as an allergen attack [275]. During septic shock, iNOS expression is induced in endothelial cells, leading to the production of high amounts of NO, which is associated to vasoplegia and persistent hypotension [278-280]. NO and nitrogen reactive species have been implicated in a range of inflammatory pulmonary diseases [281]. Elevated NO levels are observed in the exhaled air of asthma patients [276, 282] which also correlate with the increased expression of iNOS [276, 283].

Several Pyrimidine imidazole-based inhibitors (PID) have been developed and tested since they inhibit iNOS dimerization with high selectivity and low toxicity as well [258, 259, 264, 265]. These derivatives inhibit the NOS by preventing the heme group insertion [258] by forming an irreversible iNOS-monomer-inhibitor complex [264, 266] and these inhibitors can disrupt the fully formed iNOS dimers as well [267]. PID has a very high affinity for iNOS monomer than free imidazoles [268] and the potency of substituted pyrimidine imidazoles for iNOS has been reported to be over 1000-fold and 200-fold as compared to eNOS and nNOS respectively [259]. Through the application of ultra-high throughput screen of a novel series of quinolinone small molecule, Bonnefous et al. have identified 2 compounds from the lead compound 4-((2-Cyclobutyl-1H-imidazo[4,5-b]pyrazin-1-yl)methyl)-7, 8-difluoroquinolin-2(1H)-one (KD7332) which were potent inhibitors of human and mouse iNOS and were also highly selective over eNOS. These compounds were found to significantly reduce pain behavior in formalin mouse model and also effectively reduced neuropathic pain in chronic constriction injury model [269]. Another study by the same group discovered dual iNOS/nNOS inhibitors, derivatives of Benzimidazole- Quinolinone, that were orally active and effective in pain models [270].

Non-selective inhibition of NOS in patients with septic shock originating from respiratory tract was found to be linked with an increase in mortality in a phase 3 clinical trial [280]. In ovine acute lung injury model, aminoguanidine treatment to inhibit iNOS was ineffective in the attenuation of septic changes [284], whereas other studies involving nNOS inhibition by 7-nitroindazole [285] and iNOS inhibition [286] have reported partial efficacy of inhibition. Early inhibition of nNOS and delayed inhibition of iNOS with BBS-2 and 7-nitroindazole respectively was found to have reduced degree of airway obstruction and better pulmonary gas exchange [287]. In burn and inhalation injury, both iNOS and nNOS activity was found to be increased [288, 289]. In ovine model of smoke inhalation and severe burn injury, iNOS and nNOS inhibition by BBS-2 and 7-

Fig. (1). Structure of NOS dimer: Each monomer is comprised of an oxygenase and reductase domain along with the cofactor (FAD, FMN, Haem and BH4) binding sites. Electron transfer occurs from the reductase domain to oxygenase domain through the cofactors; FAD to FMN which donates electrons to haem, where the oxygen is reduced and its reaction with L-arginine results in the production of NO.


nitroindazole could effectively reduce the development of burn and smokeinduced acute lung injury [290]. In Chronic Obstructive Pulmonary Disease (COPD), a lung disorder linked with pulmonary hypertension [291], the exhaled air of patients had a two-fold increased levels of NO than normal subjects but one third lower than the levels found in asthma patients [292]. The levels directly correlated with the severity of the disease [292] and COPD has been linked with high oxidative stress in lung [293, 294]. Asthma patients have high exhaled NO levels; however their airways are affected by bronchoconstriction. This is because increased iNOS expression in bronchial epithelia and macrophages in alveoli leads to increased lung mucus secretion; however decrease in eNOS and nNOS in pulmonary vessels and bronchial smooth muscle results in bronchoconstriction and pulmonary hypotension. This is a condition where therapeutic intervention by NOS inhibitors is challenged since reduction of iNOS alone is desired whereas the inhibitors should not affect the nNOS and eNOS whose activity has already been affected in bronchial and vascular smooth muscle relaxation processes [295]. In chronic traumatic tetraplegia, the levels of exhaled NO were found to be higher than the healthy volunteers and were comparable to that in mild asthma [296]. In a mice model of emphysema that was exposed to tobacco smoke chronically, iNOS and peroxynitrite were shown to play roles in the progression of the disease. When L-NIL was used to treat the mice, the tobacco smoke-induced disease could be reversed emphasizing the role of iNOS in COPD and the significance of inhibition as well [297]. Cardiovascular System Continuous production of NO by vascular endothelial cells is essential for the normal blood pressure maintenance and blood homeostasis [278]. Nitric oxide mediates both beneficial and damaging effects on the cardiovascular system which are determined by the concentrations of NO produced, localization and interactions of NO with the proteins and the cellular environment [295, 298]. Any imbalance in the production of NO has been implicated in various disorders of vascular endothelium including heart failure, hypertension, atherosclerosis and diabetes [299]. Hypertension, diabetes mellitus, chronic smoking, hypercholesterolemia are some of the cardiovascular risk factors that result in the stimulation of reactive oxygen species production in vascular endothelium. Superoxide reacts with vascular NO to form peroxynitrite. BH4, one of the cofactors of eNOS is very sensitive to oxidation by peroxynitrite and as it gets depleted [300, 301], the ferrous-dioxygen complex dissociates ultimately superoxide production by eNOS which is known as eNOS uncoupling ultimately resulting in the abolishment of protective function, and in fact enhancement of the risk. Monomers of NOS and even reductase domains alone are sufficient for the production of superoxide [259]. iNOS overexpression is observed in the myocardium of patients with myocardial infarction (MI). In rats with MI, long-term gavage administration of iNOS inhibitor S-methylisothiourea (SMT) was found to improve the car-


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diac function significantly whereas short term administration did not [302]. Mycocardial infarction following iNOS and PARP1 overexpression along with apoptosis leads to deterioration of cardiac function [303]. iNOS knock-out provides protection against myocardial damage [304] and cardioprotection in induced hypertension models as well [305]. Use of 1400W along with poly (ADP-ribose) polymerase 1 (PARP1) inhibitor, DPQ was able to protect against myocardial damage in ischemia by reduction of apoptosis [306]. Cardiogenic shock is one of the complications occurring during or after myocardial infarction and is a common and lethal disorder despite the extensive attempts of early revascularization [307]. Systemic inflammation and iNOS derived NO is thought to contribute to the process of persistent cardiogenic shock [307]. A randomized, double-blind, placebo controlled clinical trial using L-NMMA (tilarginine acetate) for the treatment of cardiogenic shock was conducted in which Tilarginine infusion did not reduce the mortality in patients with refractory cardiogenic shock following acute MI. The study was therefore terminated [308]. Cardiac allograft survival was found to be significantly improved in rats that had undergone heterotrpopic abdominal heart transplantation and were subsequently treated with pytimidylimidazole- based iNOS selective inhibitors BBS-1 and BBS-2 [309] reducing myocardial inflammation and cardiomyocyte damage as well in cardiac allograft rejection [310]. Though decreased eNOS expression and activity could be the reason for endothelial dysfunction, research suggests that rather than decrease, the increase in eNOS expression is associated with cardiovascular risk factors [311] since the production of NO becomes less as the perxoxynitrite production is high. Inflammation The activity of iNOS increases by 3-5 fold in an inflamed endothelium [312, 313]. Under inflammatory conditions, high levels of NO are produced by iNOS, however inflamed endothelium promotes eNOS to act like iNOS resulting in the production of NO at high levels [314]. Increased iNOS mRNA expression has been observed in the gastric antrum in gastric ulcer patients and in the duodenum in the case of duodenal ulcer patients who are Helicobacter pyroli (Hp) positive. When Hp was eradicated, the iNOS expression decreased too [315]. Hp infection is complicated and it upregulates eNOS expression inducing angiogenesis which leads to gastric mucosa inflammation [316]. NO promotes tumor growth during chronic inflammation. NO levels are higher in Hp positive patients and pre-neoplastic diseases. Along with Hp infection, NO contributes to the gastric malignancy transformation since increased iNOS expression and nitrotyrosine levels are observed [324, 325]. High expression of iNOS was found in colon adenomas and colon cancer and in metastases, it has been found and the progression from colon adenoma to colon cancer may be the result of increased NO levels [326]. During the transplantation of rat liver grafts from cardiac death donors, iNOS expression is found to be more resulting in failure of grafting, whereas 1400W effectively blocked the


production of RNS species and also improved the transplantation outcome of grafts from cardiac death donors [317]. NO also plays a significant role in the development of Inflammatory Bowel Diseases (IBD). Increased mucosal iNOS expression was observed in addition to increase in rectal lumen along with increased nitrotysoine levels in IBD patients [318-321]. iNOS inhibitor, L-NIL had no effect whereas non selective inhibitor L-NNA reduced colitis colonic blood flow suggesting that increased colonic mucosal blood flow could be due to increased eNOS activity [322]. The activity of nNOS and iNOS in colonic mucosa may be a helpful tool in the assessment of progression of ulcerative colitis [323]. In a mice model of collagen-induced arthritis, GW274150, iNOS inhibitor showed efficacy in reducing the degree of chronic inflammation and tissue damage [327]. Aminoguanidine was found to improve heat tolerance, protective against hypertension and cerebral ischemic injury in an experimental heatstroke rat model [328]. Lim et al. reported the application of dual inhibitors of iNOS and COX-2, namely OQ1 6-(4-fluorophenyl)-amino-5, 8-quinolinedione and OQ21, 6-(2, 3, 4- trifluorophenyl)amino-5, 8-quinolinedione; derivatives of quinolinedione for the treatment of inflammatory conditions. These inhibitors affected iNOS expression and activity as well in RAW264.7 cells. OQ1 showed anti-inflammatory effects in mouse ear oedema [329]. A clinical study was conducted on Rheumatoid Arthritis for examining the efficacy of GW274150. GW274150 treated group showed a trend towards decrease in synovial thickness and synovial vascularity though this was not statistically significant [330]. Another double-blind multicentre study examining the effects of oral iNOS selective inhibitor, cindunistat hydrochlroide maleate (SD-6010) on the progression of osteoarthritis reported that it was not effective in slowing the progression of the disease [331]. 1400W has been reported to exhibit anti-inflammatory activity by modulating the release of cytokines during inflammation and has the ability to modify the host immune response by reduction in release of TNF- in J774A.1 macrophages [332]. IL-2 production was as well inhibited as well in stimulated J774A.1 cells by 1400W [333]. Shock iNOS expression is up-regulated during sustained shock which contributes to vascular decompensation [279]. In hemorrhagic shock, selective inhibition of iNOS and scavenging of peroxynitrite with mercaptoethylguanidine prevented the delayed vascular decompensation and cellular energetic failure related with hemorrhagic shock [280]. N6-(iminoethyl)-L-lysine mediated inhibition of iNOS results in significant reduction of shockinduced liver and lung injury. iNOS, in addition to promotion of peroxynitrite formation mediate the inflammatory response worsening the conditions [334]. Pulmonary flow in endotoxic shock varies at different stages of the condition [335]. At early stages of the shock, there is increased pulmonary flow whereas at later stages, since iNOS is overexpressed massive pulmonary vasodilation occurs.

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This should be prevented since it makes other vital organs devoid of oxygen since pulmonary vessels can act as a sink for a large amount of systemic blood [279]. L-NMA was used to treat septic shock in three levels of human trials with varying concentrations [279, 336], however, a drop in cardiac output was observed and the overall mortality rate was higher as compared to that of placebo group due to cardiovascular complications revealing the adverse effects of chronic administration of L-NMA. Therefore, 546C88 (L-NMA) trial against septic shock was terminated [280]. The application of NOS inhibitors has been done to prevent extensive systemic vasodilation; however, uncontrolled vasoconstriction leads to ischemia of major organs. This results in high mortality rate among the patients treated with NOS inhibitor since it had effects on eNOS as well [280]. Sepsis and Kidney Injury In patients of sepsis, increased iNOS expression in kidney has been shown to contribute to Acute Kidney Injury (AKI) [337-339]. In rodent models of sepsis as well, high iNOS activity has been observed [340-342]. Animal studies have showed that the renal function could be improved and survival rate could be better on application of iNOS selective inhibitors [339]. In models of sepsis-induced AKI, L-NIL was found to be effective and RNS generation by renal tubules was less [341, 343, 344]. A Phase I clinical trial was conducted to study the effects of selective iNOS inhibition by aminoguanidine during endotoxemia. It was suggested that iNOS inhibition could be a potential target for the treatment of septic acute renal failure and proximal tubule injury could be prevented by iNOS inhibition [338]. Migraine NO has been shown to cause immediate headache in migraineurs [345-349]. A double-blind placebo controlled clinical study with L-NMMA treatment was found to be effective and well- tolerated in migraine attacks [350] and could remarkably reduce pain in chronic tension-type headache patients [351]. However L-NMMA affects cardiovascular system adversely because of the non-selective inhibition [350]. In rat, L-NAME, a pan NOS inhibitor and nNOS inhibitor, SMTC and not DPI, L-NIO or SMT were able to inhibit neurogenic dural vasodilation, the mechanism through which migraine attack is believed to occur [352]. GW274150 and GW273629 were effective in the treatment of migraine in a placebo controlled study [353] and in an open-label pharmacokinetic study as well [354]. In another double-blind placebo controlled phase 2 trial, GW274150 was ineffective as a prophylactic agent for acute migraine [355]. Nervous System NOS enzymes have major role to play in nervous system and are expressed widely in neurons [356, 357]. NOS enzymes are expressed widely in the cerebellum, the midbrain, the cortex, the hippocampus and striatum with nNOS being the predominant enzyme in addition to which eNOS is expressed as well [358-360]. Normally iNOS has no known


role in the brain whereas conditions such as trauma, infection or ischemia provoke the production of iNOS leading to several complications [361, 362]. Increased production of NO by nNOS or iNOS has been demonstrated to be one of the fundamental causes of neuron degeneration which leads to neuropathic pain [363] and neurodegenerative diseases including stroke [364], Alzheimer’s [365, 366], Parkinson’s [367, 368] amyotrophic lateral sclerosis (ALS) [369], depression [370] and Schizophrenia [371]. The toxic effects are thought to be mediated by peroxynitrite which can modify proteins and is involved in excitoxicity, a process of neuronal cell death [372]. Several preclinical studies have demonstrated that NOS inhibition results in neuroprotection. nNOS Inhibitors Vinyl-1-N-5-(imino-3-butenyl-)-1-ornithine (L-VNIO), N -Propyl-L-Arginine (NPA) and 1400W seem to be potent and relatively selective nNOS inhibitors. L-VNIO has a very low Ki of 0.1M for nNOS (rat) and has been reported to be a highly selective nNOS inhibitor [209]. NPA having a Ki for isolated bovine nNOS of 0.06M is potent and selective [373], whereas 1400W is a potent inhibitor of iNOS with Ki of 7nM and Ki 2M for nNOS possessing moderate specificity for nNOS (Fig. 2) [187]. NG-nitro-L-arginine (L-NA), an L-arginine analog was one of the nNOS inhibitors discovered with a Ki value of 15nM for bovine nNOS (169). N- propyl-L-arginine has a Ki of 57nM, a very high selectivity over iNOS and modest selectivity over eNOS [373]. L-MIT (L-Smethylisothiocitrulline [374] is more selective for nNOS over eNOS whereas little selectivity for nNOS over iNOS. Dipeptide-based inhibitor, D-Phe-D-Arg-NO2-OMe was reported to have ~ 1800-fold selectivity for nNOS over eNOS with a Ki of 2 M [375].


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N-(3-chlorophenyl)-N-((8-fluoro-2-oxo-1,2-dihydroquinolin-4-yl)methyl)-4-methylthiazole-5-carboxamide, also known as KLYP956 is the first nonimidazole-based iNOS inhibitor, acts by inhibiting the dimerization of iNOS and nNOS [376]. A series of pyrroline-based inhibitors have been reported to show high selectivity for nNOS among which a compound called FX-5043 showed 780-fold selectivity for nNOS over eNOS [377]. A series of inhibitors called NOpiates, with dual action exhibiting nNOS inhibition and -opioid binding activity as well were reported which could prove as analgesics for the treatment of pain [378]. Parkinson’s Disease NO has been implicated in the pathogenesis of Parkinson’s disease (PD). NO, in excess reacting with superoxide forms peroxynitrite which in turn can nitrate tyrosine residues or inhibit tyrosine phosphorylation, alter protein formation and can target protein for proteolysis [379, 380]. In PD patients and 1-methyl-4-phenyl-1, 2, 3, 6tetrahydropyridine (MPTP) treated mice, increased nitrotyrosine levels were observed in Lewy bodies [381] indicating the formation of peroxynitrite. In PD patients, higher NOS expression in the nigrostiatal regions of brain was observed through post-mortem analysis [382]. The role of NO in the pathogenesis has been confirmed by several studies in which up-regulation of NOS enzymes was seen and the inhibition of which provided neuroprotection [383387]. In MPTP-induced PD model of baboons, nNOS inhibition by 7-nitroindazole was shown to block MPTP neurotoxicity and to protect against MPTP-induced motor and frontal-type cognitive defects [388]. In a mice model of MPTP-induced PD, 7-nitroindaole was found to exhibit neuroprotective and the neuronal loss was significantly reduced, when compared to iNOS inhibitor, minocycline which showed no protective effect [389].

Fig. (2). The sources and downstream events of NO: NO can be enzymatically produced by NOS, reduction of nitrite which serves as NO reservoir by several nitrite reductases and dietary uptake of nitrate which is reduced to NO eventually. NO can activate sGC or mediate nitrosylation and nitrosation reactions further effecting downstream processes.


Neurotoxicity The nNOS inhibitor, S-methyl-L-thiocitrulline dihydrochloride (L-MIN) has been reported to reduce the neurotoxic effects of quinolinic acid on chronic administration to the rat striatum whereas 7-nitroindazole monosodium salt (7-NINA) did not have any significant effect on neuroprotection [390]. A 2-(((3R,4R)-4-(Allyloxy)-1-benzylpyrrolidin-3-yl) methyl)-6-(2,5-dimethyl-1H-pyrrol-1-yl)-4-methylpyridine derivative , developed through technique called fragment hopping with minimal pharmacophoric elements was administered to pregnant rabbit models of Cerebral Palsy (CP) and the activity of neuronal NOS was found to be inhibited in vivo reducing the NO concentrations without cardiovascular effects [391]. An aromatic peptide-bond nitroarginine inhibitor was found to protect fetal rabbit brains from neurodegeneration [392] in the same rabbit CP model. In a rabbit model of hypoxia-ischemia fetal neurodegeneration, compound 13 and 14 were found to remarkably reduce symptoms of neurodegeneration and no systemic toxicity was observed [393]. A 6-phenyl-2-aminopyridine-derivative, a potent inhibitor of human nNOS with Ki of 140 nM was administered subcutaneously to rats exhibited very weak cardiovascular side effects even with high doses of the inhibitor [394, 395]. In rabbits, nNOS inhibitors, HJ619 and JI-8 were tested in a perinatal model of hypoxia-ishcemia, which were found to be non-toxic [396]. They inhibited fetal brain NOS activity in vivo and significantly reduced the number of deaths in a CP rabbit model [391]. Combination of 7-NI and aminoguanidine targeted to inhibit nNOS and iNOS respectively in hypoxia-ischemic model, proved to give long-lasting protection [397] whereas 1-(2-trifluoromethylphenyl) imidazole, a compound that inhibits both nNOS and iNOS demonstrated devastating results in a whole-body ischemic model [398]. 2-iminobiotin, a dual inhibitor of both iNOS and nNOS showed gender-specific protection in female rat pups, however this mode of action was independent of NO signalling pathway [399, 400].

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4-mino-tetrahydrobiopterin (VAS203) has been reported to prevent edema formation and intracranial hypertension following traumatic brain injury in mice. In a Phase II clinical study, VAS203 has been shown to improve moderate and severe traumatic brain injury and was not found to be associated with heptic, cardiac or hematologic toxic effects, however higher doses posed the risk of acute kidney injury [401]. Stroke ARL17477 dihydrochloride, a thiopene-2carboximidamido compound is a potent inhibitor of nNOS having a Ki of 35nM reduced the infarct volume after ischemia in a rat focal model of stroke [217] and also reduction in neuronal death was observed in an ischemic model of dog [402]. It has been reported to exhibit neuroprotective effects in the treatment of global and focal cerebral ischemia [216] whereas in a gerbil model of global cerebral ischemia, 7nitroindazole or ARL17477 alone did not offer significant neuroprotection, however both in combination with NMDA or AMPA receptor antagonists were found to be effective against ischemic cell death [217, 403]. AR-R18512 also had similar neuroprotective effects, in a rat ischemic model [404]. Neuropathic Pain Nitric Oxide has been implicated in the pain generation process in the nervous system. 7-Nitroindazole (7-NI), a non-selective inhibitor of nNOS has been used in several animal studies, where it has been shown to inhibit rat cerebrellar nitric oxide synthases and hence have antinociceptive effects [189, 190, 405]. TRIM (1-(2-trifluroethyphenyl) imidazole), a nitrogen-containing heterocyclic compound is a potent inhibitor that selectively inhibits neuronal NOS and iNOS both in vivo as well as in vivo and showing antinociceptive effects in mouse [406] and cerebroprotective effects in rat [221]. In a preclinical study with non selective inhibitors, L-NAME, nNOS selective inhibitors including 7-NI and TRIM along with iNOS selective inhibitor 1400W en-

Fig. (3). Caveloin-1 inhibits both intra and inter-domain electron transfer in endothelial cells. Within the reductase domain, Cav-1 prevents the electron flow from FAD to FMN. Cav-1 also blocks the transfer of electrons from reductase to oxygenase domain, thereby inhibiting the electron flow at both intra- and inter-domain levels.


hanced the antinociceptive effects of morphine in acute pain and in chronic constriction injury (CCI) pain models. This data suggest that both neuronal and non-neuronal derived NO production have implications in the behavioral pain mechanisms initiated by nerve injury [407]. KLYP956, an nNOS and iNOS dimerization inhibitor has been found to be effective alleviating the pain behavior in formalin model of nociception when administered orally [263]. Ramnauth et al. reported the discovery of Compound 33 obtained from an extensive refinement of several leads for the production of selective nNOS inhibitors. This compound showed remarkable results in reducing pain in Chung spinal ligation model of neuropathic pain [408]. Administration of compound 33 could reverse thermal hyperalgesia in rats [409]. In a rat migraine model, compound 33 reduced the development of allodynia [409]. Compound 35 on oral administration offered neuroprotection in neuropathic pain model in rodents [410]. Psychiatric Disorders Aberrant nitric oxide signaling has been implicated in bipolar disorder [411], depression [412, 413] suicide [414]. LNNA, L-NAME, L-NMMA have been reported to show antidepressant effects and in rats [415-417]. Agmatine, a decarboxylated arginine showed promising antidepressant action in preclinical models of depression [418-420] as well in humans [421, 422] however agmatine has also been reported to have off-targets not linked to NOS [423-425]. 7-NI, 7-NIBr and TRIM have been shown to have antidepressant effects in rats [426-429]. Aminoguanidine, a hydrazine derivative has been shown to be neuroprotective in chronically stressed rats [430] and also had anti-depressant effects [411].


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Retinal Degeneration nNOS has been shown to play key role in the pathogenesis of N-methyl-N-nitrosourea-induced retinal degeneration in mice. Inhibition of nNOS by ethyl[4-(trifluoromethyl) phenyl] carbamimidothioate (ETPI) significantly decreased the number of apoptotic cells, thus suggesting that nNOS inhibitors can be candidates for the treatment of retinal degeneration [432]. nNOS Inhibitors and Challenges Research on nNOS inhibitors with chemical modifications promoting isoform selectivity, improving potency and bioavailability with reduced toxicity is taking place continuously. A recent study by Pigott et al. intended to demonstrate the selectivity of nNOS inhibitors has challenged market captions such as “potent, selective inhibitor(s) of nNOS” [433]. Lack of selectivity of classic nNOS inhibitors, 1400W, NPA and L-VNIO and the relatively novel pyrroline-based inhibitors tested in rodent hippocampal slices and rat aorta did not show selectivity over eNOS or iNOS that has been reported in most of the studies. The IC50 values for L-VNIO were 0.27M and 0.9 M for rat nNOS and eNOS respectively and for 1400W, IC50 were 154 M and 744 M for rat nNOS and eNOS respectively whereas NPA IC50 values were 1.3 M and 4.3 M respectively [433] which are in discordance with the values reported from cellfree systems [187, 231, 209], whereas similar values of selectivity for the nNOS inhibitors have been reported by few studies (Figs. 4 and 5) [198, 434-436]. BBS-1, a potent imidazole-derivative obtained through a combinatorial library screen showed high selectivity for nNOS versus eNOS. It was shown to inhibit nNOS by

Fig. (4). NOS inhibition strategies: NOS can be inhibited in different ways: By regulation of cellular Calcium ion level, through caveolin, NOSIP and other natural inhibitors, phosphorylation of NOS, sub-cellular localization of NOS etc.


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Fig. (5). Different inhibition sites of NOS - The inhibitors can bind to any of the sites in NOS inhibiting its activity 1. Substrate binding site 2. Pterin binding site 3. Heam binding site 4. Flavin/NADPH binding site

preventing dimerization in DLD-1 cells to be effective in vivo as well [259]. 7-Nitroindazole, a widely used inhibitor has been showed to be non-selective for isolated nNOS [68, 435] and also has capacity to interact with monoamine oxidase-B as well (Castagnoli et al., 1997). Other nNOS inhibitors such as S,S 1,4-phenylene-bis(1,2-ethanediyl) bis-isothiourea, L-MIT, TRIM, bis-isothiourea also have selectivity issues [433]. Development of 3,6-Disubstituted indole derivatives, N(3-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-indol-6-yl) thiophene-2-carboximidamide for the treatment of neuropathic pain, one compound showed promising results also apparently without eNOS inhibition [437, 438]. Following this, modifications in the substitutions lead to the discovery of a 1,6-disubstituted indole derivative, which has been demonstrated to be highly selective for human nNOS inhibitor, where the compound showed efficacy in reversing thermal hyperalgesia in an in vivo model of neuropathic pain with remarkable safety profile without mediating any cardiovascular effects [439]. Inhibitors with cyclopropyl- and methyl- groups of inhibitors show high selectivity in in vivo enzyme systems [440]. -amino functionalized aminopyridine derivatives that interact with BH4 and heme propionate as well have been reported to show high isoform selectivity having Ki of 24 nM for nNOS and over 2822-fold and 273-fold selectivity over eNOS and iNOS respectively [441]. To obtain highly selective and potent inhibitors, investigations through different modern approaches such as anchored plasticity approach [442], exploratory chemistry [263], Structure-Activity Relationship (SAR) studies, fragment hopping which resulted in highly selective inhibitors than then available inhibitors [443, 444], combination of virtual screening, molecular docking with pharmacophore modelling methods [445], strategies to improve the bioavailability [446] such as design of polar nNOS inhibitors that are suitable for crossing blood-brain barrier [392] have been carried out. Modifications through chiral linker with thiophenecarboximidamide have also shown to improve the selectivity and bioavailability of the inhibitors [447]. Aminoquinoline-based compounds have been shown to have better blood-brain-barrier (BBB) penetration and oral availability. 7-subsitiuted aminoquinoline derivatives have been reported to be more selective in Caco-2 cells with low efflux [448]. Recently, a report investigating the efficacy of

novel 2,4-disubstituted pyrimidines in nNOS inhibition demonstrated that these inhibitors bind to the hydrophobic pocket exhibiting high potency and isoform selectivity. These compounds showed excellent oral bioavailability and good permeability in Caco-2 cells [449]. The applicability of NOS inhibitors in neuroprotection has not been fruitful, however inhibitors obtained by recent approaches seem to show high selectivity and potency for nNOS, further preclinical and clinical studies may prove their efficacy in treating the disorders. Selectivity: An Inherent Problem with NOS Inhibitor Pharmacology Despite the availability of a number of selective inhibitors, none of them are successful in a clinical setup and search and identification of inhibitors with useable selectivity in cells and tissues is highly essential. The major pitfalls in obtaining therapeutically useful inhibitor are potency and high isoform selectivity NOS. Non-selective inhibition leads to other complications as the processes mediated by NO derived from other isoforms are affected crucially as well. Though there are several inhibitors available readily, the clinical trials conducted so far have not been successful as anticipated because of this challenge [170, 445]. High selectivity of the agents to inhibit a single isoform is an important criterion in order to achieve the desired targeted therapeutic outcome. Often inhibitors are not selective to a particular isoform since NOS isoforms have high similarity with respect to structure especially the active site region is highly conserved. Therefore, developing inhibitors specific to a particular isoform is particularly challenging. Cell and tissue type specificity plays a major role in selection and qualification of an inhibitor as a pharmacological agent. Also it is very essential to know which NOS isoform is involved at a particular state of disorder in order to selectively target the isoform. Combined inhibition at proper time points may be the most effective way to circumvent this issue [287]. CONCLUSION Inhibition of NOS as a therapeutic strategy remained as a favorite research topic for decades, and also accumulated plenty of data. However, no NOS inhibitor has been approved so far by FDA. Only two iNOS inhibitors, GW274150 and SD6010 have been listed in NIH orphan drug list. This is mainly due to the reason that no conserted efforts have been put forward to optimize the testing plat-


forms to test the efficacy of the newly designed NOS inhibitors. Researchers used a wide range of screening platforms using different models such as cell-free systems [187, 231, 209] cell-based systems [157, 451, 452] and tissues [453, 454, 433] which made the conclusion elusive and weak. We also observe significant variations in the uses of the model systems, source of enzyme, methods of preparations of enzyme, the pre-incubation duration of inhibitors and concentrations of substrate, which may account for the discrepancy in the values obtained by the researchers [433]. Therefore, a conserted and strategic approach should be taken to use the existing NOS inhibitors and newly developed NOS inhibitors for therapeutic purposes. We envisage that the emerging concepts in the areas of biomaterial science and Nano medicine would make it possible for targeted delivery of NOS inhibitors and controlled inhibition of NO production by NOS and NR. CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest.


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ACKNOWLEDGEMENTS This work was partially supported by FP7-PEOPLE2011-IRSES; Grant No 295181 - Acronym: INTERBONE, and a grant from University Grant Commission (UGC) Faculty Recharge Program, Government of India to SC. LS is grateful for the financial support from Department of Biotechnology-Junior Research Fellowship (DBT-JRF) programme, Government of India. SG is thankful for financial support from Department of Science & Technology- INSPIRE Fellowship programme, Government of India.

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Received: July 13, 2015


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Revised: October 17, 2015

Accepted: October 18, 2015