Convergent synthesis and properties of photoactivable NADPH mimics targeting nitric oxide synthases.

A new series of photoactivable NADPH mimics bearing one or two O-carboxymethyl groups on the adenosine moiety have been readily synthesized using click chemistry. These compounds display interesting one- or two-photon absorption properties. Their fluorescence emission wavelength and quantum yields (Φ) are dependent on the solvent polarity, with a red-shift in a more polar environment (λmax,em = 460-467 nm, Φ > 0.53 in DMSO, and λmax,em = 475-491 nm, Φ < 0.17 in Tris). These compounds show good binding affinity towards the constitutive nNOS and eNOS, confirming for the first time that the carboxymethyl group can be used as a surrogate of phosphate. Two-photon fluorescence imaging of nanotriggers in living cells showed that the presence of one carboxymethyl group (especially on the 3' position of the ribose) strongly favors the addressing of nanotriggers to eNOS in the cell context.

Recruitment of governing elements for electron transfer in the nitric oxide synthase family.

At least three building blocks are responsible for the molecular basis of the modulation of electron transfer in nitric oxide synthase (NOS) isoforms: the calmodulin-binding sequence, the C-terminal extension, and the autoregulatory loop in the reductase domain. We have attempted to impart the control conferred by the C termini of NOS to cytochrome P450 oxidoreductase (CYPOR), which contains none of these regulatory elements. The effect of these C termini on the properties of CYPOR sheds light on the possible evolutionary origin of NOS and addresses the recruitment of new peptides on the development of new functions for CYPOR. The C termini of NOSs modulate flavoprotein-mediated electron transfer to various electron acceptors. The reduction of the artificial electron acceptors cytochrome c, 2,6-dichlorophenolindophenol, and ferricyanide was inhibited by the addition of any of these C termini to CYPOR, whereas the reduction of molecular O(2) was increased. This suggests a shift in the rate-limiting step, indicating that the NOS C termini interrupt electron flux between flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) and/or the electron acceptors. The modulation of CYPOR by the addition of the NOS C termini is also supported by flavin reoxidation and fluorescence-quenching studies and antibody recognition of the C-terminal extension. These experiments support the origin of the NOS enzymes from modules consisting of a heme domain and CYPOR or ferredoxin-NADP(+) reductase- and flavodoxin-like subdomains that constitute CYPOR, followed by further recruitment of smaller modulating elements into the flavin-binding domains.

Bioactive nitric oxide concentration does not increase during reactive hyperemia in human skin.

This study examined whether nitric oxide (NO) is involved in the cutaneous response to reactive hyperemia (RH) in the human forearm. We enrolled seven healthy volunteers. NO concentrations were monitored using a NO selective amperometric electrode (ISO-NOP200, World Precision Instruments) inserted into the skin of the forearm. Laser-Doppler flowmetry (Moor Instruments) was used for monitoring skin blood flow (SkBF) at the same site. SkBF and NO levels were monitored and recorded continuously throughout the experiment. An intradermal microdialysis probe was inserted adjacent to the NO electrode for drug delivery. Data collection began 140 min after the NO electrodes and microdialysis probes were inserted. RH was achieved by the inflation of a blood pressure cuff to 25 mmHg above systolic pressure for 7 min after which the pressure in the cuff was abruptly released. Acetylcholine (ACh) was given by microdialysis probe at the end of RH study to verify the ability of the electrode system to detect changes in the NO concentration. SkBF and NO data before RH and immediately, 2, 5, 7, and 10 min after cuff deflation were used for analysis. SkBF increased immediately after release of the occlusion (P < 0.0001) and remained elevated for 2 min. No significant NO changes occurred with the increases in LDF. ACh induced increases in both SkBF and NO (P < 0.000 and P < 0.037, respectively). We conclude that RH increases SkBF by mechanisms that do not require a measurable increase in NO concentrations.

Nitric oxide concentration increases in the cutaneous interstitial space during heat stress in humans.

To examine the role of nitric oxide (NO) in cutaneous active vasodilation, we measured the NO concentration from skin before and during whole body heat stress in nine healthy subjects. A forearm site was instrumented with a NO-selective, amperometric electrode and an adjacent intradermal microdialysis probe. Skin blood flow (SkBF) was monitored by laser-Doppler flowmetry (LDF). NO concentrations and LDF were measured in normothermia and heat stress. After heat stress, a solution of ACh was perfused through the microdialysis probe to pharmacologically generate NO and verify the electrode's function. During whole body warming, both SkBF and NO concentrations began to increase at the same internal temperature. Both SkBF and NO concentrations increased during heat stress (402 +/- 76% change from LDF baseline, P < 0.05; 22 +/- 5% change from NO baseline, P < 0.05). During a second baseline condition after heat stress, ACh perfusion led to increases in both SkBF and NO concentrations (496 +/- 119% change from LDF baseline, P < 0.05; 16 +/- 10% change from NO baseline, P < 0.05). We conclude that NO does increase in skin during heat stress in humans, attendant to active vasodilation. This result suggests that NO has a role beyond that of a permissive factor in the process; rather, NO may well be an effector of cutaneous vasodilation during heat stress.

Synthesis and evaluation of dipeptide amides containing N omega-nitroarginine and D-2,4-diaminobutyric acids as inhibitors of neuronal nitric oxide synthase.

Selective inhibition of the isoforms of nitric oxide synthase (NOS) could be beneficial in the treatment of certain disease states arising from the overproduction of nitric oxide by NOS. Recently, we reported dipeptide amides containing a basic amine side chain as potent and selective inhibitors of neuronal NOS (Huang H. et al. (1999) J. Med. Chem., 42, 3147). The most potent nNOS inhibitor among these compounds is L-ArgNO2-L-Dbu-NH2 (1) (Ki = 130 nM), which also exhibits the highest selectivity over eNOS (> 1500-fold). The D,D-dipeptide, D-Lys-D-ArgNO2-NH2 (3) also shows high potency and selectivity. Here the dipeptide amides containing ArgNO2 and D-Dbu (9-12) were synthesized and evaluated. They are all modest inhibitors of nNOS, but poor inhibitors of eNOS and iNOS. D-Dbu-D-ArgNO2-NH2 (12) exhibits decreased inhibitory potency as compared with 3. A hypothesis regarding the binding at the active site of nNOS is proposed to explain the potency differences between the L- and D-form dipeptide amides.

Expressed CYP4A4 metabolism of prostaglandin E(1) and arachidonic acid.

Cytochrome P4504A4 (CYP4A4) is a hormonally induced pulmonary cytochrome P450 which metabolizes prostaglandins and arachidonic acid (AA) to their omega-hydroxylated products. Although the physiological function of this enzyme is unknown, prostaglandins play an important role in the regulation of reproductive, vascular, intestinal, and inflammatory systems and 20-hydroxyeicosatetraenoic acid, the omega-hydroxylated product of arachidonate, is a potent vasoconstrictor. Therefore, it is important to obtain sufficient quantities of the protein for kinetic and biophysical characterization. A CYP4A4 construct was prepared and expressed in Escherichia coli. The enzyme was purified, and its activity with substrates prostaglandin E(1) (PGE(1)) and AA was examined in the presence and absence of cytochrome b(5) (cyt b(5)) and with a heme-depleted form of cyt b(5) (apo b(5)). The stimulatory role played by cyt b(5) in this system is not dependent on electron transfer from cyt b(5) to the CYP4A4 as similar stimulation was observed with apo b(5). Rapid kinetic measurement of CYP4A4 electron transfer rates confirmed this result. Both flavin and heme reduction rates were constant in the absence and presence of cyt b(5) or apo b(5). CD spectroscopy demonstrated that a conformational change occurred in CYP4A4 protein upon binding of cyt b(5) or apo b(5). Finally, acetylenic fatty acid inhibitors 17-octadecynoic acid, 12-hydroxy-16-heptadecynoic acid, 15-hexadecynoic acid, and 10-undecynoic acid (10-UDYA) were used to probe the substrate-binding pocket of CYP4A4. The short-chain fatty acid inhibitor 10-UDYA was unable to inhibit either PGE(1) or AA metabolism. All but 10-UDYA were effective inhibitors of CYP4A4.

Induction of rabbit lung CYP4A4 prostaglandin omega-hydroxylase by various steroid hormones.

Cytochrome P4504A4 (CYP4A4) is expressed at low basal levels in adult rabbit lungs, but is significantly induced during pregnancy by an unknown mechanism. As the gradual rise in CYP4A4 levels appears to coincide with the progressive increase in several steroid hormones throughout pregnancy, we examined the induction of CYP4A4 after treatment with various steroid hormones by monitoring both the CYP4A4 mRNA level and the CYP4A4-specific prostaglandin E(1) (PGE(1)) omega-hydroxylation reaction in rabbit lung microsomes. Treatment with progesterone and/or a synthetic glucocorticoid (dexamethasone) resulted in a significant increase in PGE(1) omega-hydroxylase activity, whereas estradiol, aldosterone, dehydroepiandrosterone, and dehydroepiandrosterone sulfate did not. These studies indicated that dexamethasone was a more potent inducer of CYP4A4 than progesterone. Simultaneous injection of dexamethasone and glucocorticoid/progesterone antagonists (RU38486, RU40555, or RU43044) inhibited the increase in PGE(1) omega-hydroxylase activity as well as mRNA levels by approximately 50%. In addition, simultaneous treatment with both dexamethasone and progesterone did not result in an additive or synergistic effect on PGE(1) omega-hydroxylase activity. These data indicate that, while distinctive receptors for glucocorticoid and/or progesterone are involved, induction may also require common or interacting regulatory elements (yet to be determined) in the CYP4A4 gene. These findings implicate both of these steroid receptors (PR/GR) in the induction of CYP4A4 in rabbit lung.

Reduced amide bond peptidomimetics. (4S)-N-(4-amino-5-[aminoakyl]aminopentyl)-N'-nitroguanidines, potent and highly selective inhibitors of neuronal nitric oxide synthase.

Selective inhibition of the isoforms of nitric oxide synthase (NOS) could be therapeutically useful in the treatment of certain disease states arising from the overproduction of nitric oxide. Recently, we reported nitroarginine-containing dipeptide amides (Huang, H; Martasek, P.; Roman, L. J.; Masters, B. S. S.; Silverman, R. B. J. Med. Chem. 1999, 42, 3147.) and some peptidomimetic analogues (Huang, H; Martasek, P.; Roman, L. J.; Silverman, R.B. J. Med Chem. 2000, 43, 2938.) as potent and selective inhibitors of neuronal NOS (nNOS). Here, reduced amide bond pseudodipeptide analogues are synthesized and evaluated for their activity. The deletion of the carbonyl group from the amide bond either preserves or improves the potency for nNOS. Significantly, the selectivities for nNOS over eNOS (endothelial NOS), and iNOS (inducible NOS) are greatly increased in these series. The most potent nNOS inhibitor among these compounds is (4S)-N-(4-amino-5-[aminoethyl]aminopentyl)-N'-nitroguanidine (7) (K(i) = 120 nM), which also shows the highest selectivity over eNOS (greater than 2500-fold) and 320-fold selectivity over iNOS. The reduced amide bond is an excellent surrogate of the amide bond, and it will facilitate the design of new potent and selective inhibitors of nNOS.

The kinetic and spectral characterization of the E. coli-expressed mammalian CYP4A7: cytochrome b5 effects vary with substrate.

The CYP4A gene subfamily is composed of a number of genes that encode cytochromes P450 from various species, including human, which catalyze the hydroxylation of various saturated and unsaturated fatty acids, including arachidonic acid and prostaglandins. CYP4A7, a fatty acid metabolizing cytochrome P450 from rabbit kidney, was expressed in E. coli by adding the first 10 codons of CYP17alpha producing final yields of 20 nmol/L in order to perform detailed kinetic and spectral studies. CYP4A7 metabolized arachidonate, laurate, and myristate, with maximum turnover numbers of 152, 130, and 64.5 min(-1) and corresponding Km values of 74.5, 27, and 16.7 microM, respectively, in the presence of cytochrome b5. In the absence of cytochrome b5, CYP4A7 metabolized laurate and myristate with turnover numbers of 27.4 and 33.6 min(-1) and corresponding Km values of 3.9 and 33 microM, respectively. Arachidonate was not metabolized in the absence of cytochrome b5. Saturation kinetics studies performed with heme-depleted cytochrome b5 (apo cytochrome b5) yielded turnover numbers of 118 and 74 min(-1) and Km values of 74 and 25 microM with laurate and myristate, respectively, indicating that cytochrome b5 is not involved in electron transfer but rather plays a conformational role. Laurate perturbation of the visible absorption spectrum of CYP4A7 allowed for determination of the spectral binding constant (KS) in the absence and presence of cytochrome b5 (13 and 43 microM, respectively). In stopped-flow kinetics experiments, the flavin reduction (approximately 90 s(-1)) and heme reduction (approximately 9 s(-1)) phases of the monooxygenase reaction of CYP4A7 were not altered by the presence of cytochrome b5. Estimations of the rate of CPR (0.3 s(-1)) or cytochrome b5 (9.1 s(-1)) binding with CYP4A7 were also determined.

Kinetics of CO and NO ligation with the Cys(331)-->Ala mutant of neuronal nitric-oxide synthase.

Nitric-oxide synthases (NOS) catalyze the conversion of l-arginine to NO, which then stimulates many physiological processes. In the active form, each NOS is a dimer; each strand has both a heme-binding oxygenase domain and a reductase domain. In neuronal NOS (nNOS), there is a conserved cysteine motif (CX(4)C) that participates in a ZnS(4) center, which stabilizes the dimer interface and/or the flavoprotein-heme domain interface. Previously, the Cys(331) --> Ala mutant was produced, and it proved to be inactive in catalysis and to have structural defects that disrupt the binding of l-Arg and tetrahydrobiopterin (BH(4)). Because binding l-Arg and BH(4) to wild type nNOS profoundly affects CO binding with little effect on NO binding, ligand binding to the mutant was characterized as follows. 1) The mutant initially has behavior different from native protein but reminiscent of isolated heme domain subchains. 2) Adding l-Arg and BH(4) has little effect immediately but substantial effect after extended incubation. 3) Incubation for 12 h restores behavior similar but not quite identical to that of wild type nNOS. Such incubation was shown previously to restore most but not all catalytic activity. These kinetic studies substantiate the hypothesis that zinc content is related to a structural rather than a catalytic role in maintaining active nNOS.

Identification of unique amino acids that modulate CYP4A7 activity.

A multifamily sequence alignment of the rabbit CYP4A members with the known structure of CYP102 indicates amino acid differences falling within the so-called substrate recognition site(s) (SRS). Chimeric proteins constructed between CYP4A4 and CYP4A7 indicate that laurate activity is affected by the residues within SRS1 and prostaglandin activity is influenced by SRS2-3. Site-directed mutant proteins of CYP4A7 found laurate and arachidonate activity markedly diminished in the R90W mutant (SRS1) and somewhat decreased in W93S. While PGE(1) activity was only slightly increased, the mutant proteins H206Y and S255F (SRS2-3), on the other hand, exhibited remarkable increases in laurate and arachidonate metabolism (3-fold) above wild-type substrate metabolism. Mutant proteins H206Y, S255F, and H206Y/S255F but not R90W/W93S, wild-type CYP4A4, or CYP4A7 metabolized arachidonic acid in the absence of cytochrome b(5). Stopped-flow kinetic experiments were performed in a CO-saturated environment performed to estimate interaction rates of the monooxygenase reaction components. The mutant protein H206Y, which exhibits 3-fold higher than wild-type substrate activity, interacts with CPR at a rate at least 10 times faster than that of wild-type CYP4A7. These experimental results provide insight regarding the residues responsible for modulation of substrate specificity, affinity, and kinetics, as well as possible localization within the enzyme structure based on comparisons with homologous, known cytochrome P450 structures.

Deuterium isotope effects and product studies for the oxidation of N(omega)-allyl-L-arginine and N(omega)-allyl-N(omega)-hydroxy-L-arginine by neuronal nitric oxide synthase.

The nitric oxide synthases (NOS), which require heme, tetrahydrobiopterin, FMN, FAD, and NADPH, catalyze the O2-dependent conversion of L-arginine to L-citrulline and nitric oxide. N(omega)-Allyl-L-arginine, a mechanism-based inactivator of neuronal NOS, also is a substrate, producing L-arginine, acrolein, and H2O (Zhang, H. Q.; Dixon, R. P., Marletta, M. A.; Nikolic, D.; Van Breemen, R.; Silverman, R. B. J. Am. Chem. Soc. 1997, 119, 10888). Two possible mechanisms for this turnover are proposed, one initiated by allyl C-H bond cleavage and the other by guanidino N H cleavage, and these mechanisms are investigated with the use of N(omega)-allyl-L-arginine (1), N(omega)-[1,1-(2)H2]allyl-L-arginine (7), N(omega)-allyl-N(omega)-hydroxy-L-arginine (2) and N(omega)-[1,1-(2)H2]allyl-N(omega)-hydroxy-L-arginine (8) as substrates. Significant isotope effects on the two kinetic parameters, kcat and kcat/Km, are observed in case of 1 and 7 during turnover, but not with 2 and 8. No kinetic isotope effects are observed for either compound in their role as inactivators. These results support a mechanism involving initial C-H bond cleavage of N(omega)-allyl-L-arginine followed by hydroxylation and breakdown to products.

Synthesis and evaluation of peptidomimetics as selective inhibitors and active site probes of nitric oxide synthases.

Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to L-citrulline and nitric oxide (NO). Selective inhibition of the isoforms of NOS could have great therapeutic potential in the treatment of certain disease states arising from pathologically elevated synthesis of NO. Recently, we reported dipeptide amides containing a basic amine side chain as potent and selective inhibitors of neuronal NOS (Huang, H.; Martasek, P.; Roman, L. J.; Masters, B. S. S.; Silverman, R. B. J. Med. Chem. 1999, 42, 3147). The most potent nNOS inhibitor among these compounds is L-ArgNO2-L-Dbu-NH2 (1) (Ki = 130 nM), which also exhibits the highest selectivity over eNOS (>1,500-fold) with excellent selectivity over iNOS (190-fold). Here we describe the design and synthesis of a series of peptidomimetic analogues of this dipeptide as potential selective inhibitors of nNOS. The biochemical evaluation of these compounds also revealed the binding requirements of the dipeptide inhibitors with NOS. Incorporation of protecting groups at the N-terminus of the dipeptide amide 1 (compounds 4 and 5) resulted in dramatic decreases in the inhibitory potency of nNOS. Masking the NH group of the peptide bond (peptoids 6-8 and N-methylated compounds 9-11) also gave much poorer nNOS inhibitors than 1. Both of the results demonstrate the importance of the alpha-amine of the dipeptide and the NH moiety of the peptide bond for binding at the active site. Modifications at the C-terminus of the peptide included converting the amide to the methyl ester (12), tert-butyl ester (13), and carboxylic acid (14) and also descarboxamide analogues (15-17), which revealed less restricted binding requirements for the C-terminus of the dipeptide. Further optimization should be possible when we learn more about the binding requirements at the active sites of NOSs.

The C termini of constitutive nitric-oxide synthases control electron flow through the flavin and heme domains and affect modulation by calmodulin.

The sequences of nitric-oxide synthase flavin domains closely resemble that of NADPH-cytochrome P450 reductase (CPR). However, all nitric-oxide synthase (NOS) isoforms are 20-40 residues longer in the C terminus, forming a "tail" that is absent in CPR. To investigate its function, we removed the 33 and 42 residue C termini from neuronal NOS (nNOS) and endothelial NOS (eNOS), respectively. Both truncated enzymes exhibited cytochrome c reductase activities without calmodulin that were 7-21-fold higher than the nontruncated forms. With calmodulin, the truncated and wild-type enzymes reduced cytochrome c at approximately equal rates. Therefore, calmodulin functioned as a nonessential activator of the wild-type enzymes and a partial noncompetitive inhibitor of the truncated mutants. Truncated nNOS and eNOS plus calmodulin catalyzed NO formation at rates that were 45 and 33%, respectively, those of their intact forms. Without calmodulin, truncated nNOS and eNOS synthesized NO at rates 14 and 20%, respectively, those with calmodulin. By using stopped-flow spectrophotometry, we demonstrated that electron transfer into and between the two flavins is faster in the absence of the C terminus. Although both CPR and intact NOS can exist in a stable, one-electron-reduced semiquinone form, neither of the truncated enzymes do so. We propose negative modulation of FAD-FMN interaction by the C termini of both constitutive NOSs.

The C terminus of mouse macrophage inducible nitric-oxide synthase attenuates electron flow through the flavin domain.

The sequences of nitric-oxide synthase (NOS) flavin domains closely resemble that of NADPH-cytochrome P450 reductase (CPR), with the exception of a few regions. One such region is the C terminus; all NOS isoforms are 20-40 amino acids longer than CPR, forming a "tail" that is absent in CPR. To investigate its function, we removed the 21-amino acid C-terminal tail from murine macrophage inducible NOS (iNOS) holoenzyme and from a flavin domain construct. Both the truncated holoenzyme and reductase domain exhibited cytochrome c reductase activities that were 7-10-fold higher than the nontruncated forms. The truncated holoenzyme catalyzed NO formation approximately 20% faster than the intact form. Using stopped-flow spectrophotometry, we demonstrated that electron transfer into and between the two flavins and from the flavin to the heme domain is 2-5-fold faster in the absence of the C-terminal tail. The heme-nitrosyl complex, formed in all NOS isoforms during NO catalysis, is 5-fold less stable in truncated iNOS. Although both CPR and intact NOS can exist in a stable, one electron-reduced semiquinone form, neither the truncated holoenzyme nor the truncated flavin domain demonstrate such a form. We propose that this C-terminal tail curls back to interact with the flavin domain in such a way as to modulate the interaction between the two flavin moieties.

Enhanced electron flux and reduced calmodulin dissociation may explain "calcium-independent" eNOS activation by phosphorylation.

Bovine endothelial nitric oxide synthase (eNOS) is phosphorylated directly by the protein kinase Akt at serine 1179. Mutation of this residue to the negatively charged aspartate (S1179D eNOS) increases nitric oxide (NO) production constitutively, in the absence of agonist challenge. Here, we examine the potential mechanism of how aspartate at 1179 increases eNOS activity using purified proteins. Examination of NO production and cytochrome c reduction resulted in no substantial changes in the K(m)/EC(50) for L-arginine, calmodulin, and calcium, whereas there was a 2-fold increase in the rate of NO production for S1179D and a 2-4-fold increase in reductase activity (based on cytochrome c reduction). The observed increase in activity for both assays of NOS function indicates that a faster rate of electron flux through the reductase domain is likely the rate-limiting step in NO formation from eNOS. In addition, S1179D eNOS did show an increased resistance to inactivation by EGTA compared with wild type eNOS. These results suggest that a negative charge imposed at serine 1179, either by phosphorylation or by replacement with aspartate, increases eNOS catalytic activity by increasing electron flux at the reductase domain and by reducing calmodulin dissociation from activated eNOS when calcium levels are low.

1H-pyrazole-1-carboxamidines: new inhibitors of nitric oxide synthase.

1H-Pyrazole-1-carboxamidines were prepared as potential inhibitors of the three isozymes of nitric oxide synthase. All of the compounds were found to be competitive inhibitors of all three isoforms. The most selective compound prepared was 1H-pyrazole-N-(3-aminomethylanilino)-1-carboxamidine (14), which is 100-fold selective for nNOS over eNOS with a Ki value of 2 microM.

Imidazole-containing amino acids as selective inhibitors of nitric oxide synthases.

Two series of imidazole-containing amino acids (1a-e and 2a-c), all larger homologues and analogues of L-histidine, were prepared. Since imidazole and phenyl substituted imidazoles have been reported to be inhibitors of NOS and the mode of action of these compounds as heme ligands is a potential mechanism of inhibitory action, we designed imidazole-containing amino acids as combined inhibitors at both the amino acid as well as heme binding sites. To study the influence of the distance between the amino acid moiety and the imidazole moiety on inhibitory potency, the number of carbons between these two functional groups was varied from two to six. The structure-activity relationships of this class of inhibitors can be correlated with the distance between the heme and the amino acid binding sites of the enzyme. Two of the compounds (1b and 1d) with three and five methylenes between the imidazole and amino acid functional groups, respectively, were found to be potent and selective inhibitors for nNOS and iNOS over eNOS. When phenyl was substituted on the nitrogen of the imidazole, both the potency and isoform selectivity diminished.

N(omega)-Nitroarginine-containing dipeptide amides. Potent and highly selective inhibitors of neuronal nitric oxide synthase.

Selective inhibition of the isoforms of nitric oxide synthase (NOS) could be therapeutically useful in the treatment of certain disease states arising from the overproduction of nitric oxide (NO). Recently, we reported the dipeptide methyl ester, D-Phe-D-Arg(NO)()2-OMe (19), as a modest inhibitor of nNOS (K(i) = 2 microM), but with selectivity over iNOS as high as 1800-fold (Silverman, R. B.; Huang, H.; Marletta, M. A.; Martasek, P. J. Med. Chem. 1997, 40, 2813-2817). Here a library of 152 dipeptide amides containing nitroarginine and amino acids other than Phe are synthesized and screened for activity. Excellent inhibitory potency and selectivity for nNOS over eNOS and iNOS is achieved with the dipeptide amides containing a basic amine side chain (20-24), which indicates a possible electrostatic (or hydrogen bonding) interaction at the enzyme active site. The most potent nNOS inhibitor among these compounds is L-Arg(NO)()2-L-Dbu-NH(2) (23) (K(i) = 130 nM), which also exhibits the highest selectivity over eNOS (>1500-fold) with a 192-fold selectivity over iNOS. These compounds do not exhibit time-dependent inhibition. The order and the chirality of the amino acids in the dipeptide amides have profound influences on the inhibitory potency as well as on the isoform selectivity. These dipeptide amide inhibitors open the door to the design of potent and highly selective inhibitors of nNOS.

Conformationally-restricted arginine analogues as alternative substrates and inhibitors of nitric oxide synthases.

Conformationally restricted arginine analogues (1-5) were synthesized and found to be alternative substrates or inhibitors of the three isozymes of nitric oxide synthase (NOS). A comparison of k(cat)/Km values shows that (E)-3,4-didehydro-D,L-arginine (1) is a much better substrate than the corresponding (Z)-isomer (2) and 3-guanidino-D,L-phenylglycine (3), although none is as good a substrate as is arginine; 5-keto-D,L-arginine (4) is not a substrate, but is an inhibitor of the three isozymes. Therefore, it appears that arginine binds to all of the NOS isozymes in an extended (E-like) conformation. None of the compounds exhibits time-dependent inhibition of NOS, but they are competitive reversible inhibitors. Based on the earlier report that N(omega)-propyl-L-arginine is a highly selective nNOS inhibitor (Zhang, H. Q.; Fast, W.; Marletta, M.; Martasek, P.; Silverman, R. B. J. Med. Chem. 1997, 40, 3869), (E)-N(omega)-propyl-3,4-didehydro-D,L-arginine (5) was synthesized, but it was shown to be weakly potent and only a mildly selective inhibitor of NOS. Imposing conformational rigidity on an arginine backbone does not appear to be a favorable approach for selective NOS inhibition.