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.

Assay of isoforms of Escherichia coli-expressed nitric oxide synthase.

The techniques described herein have added to our repertoire of experimental approaches for the characterization of the NOSs. These procedures have reinforced our conviction that the NOSs are structurally suited to perform unique functions in their cellular milieux and that these differences have physiological consequences.

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.

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.

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.

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.

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.

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.

Characterization of the helicase activity of the Escherichia coli RecBCD enzyme using a novel helicase assay.

We describe an assay to measure the extent of enzymatic unwinding of DNA by a DNA helicase. This assay takes advantage of the quenching of the intrinsic protein fluorescence of Escherichia coli SSB protein upon binding to ssDNA and is used to characterize the DNA unwinding activity of recBCD enzyme. Unwinding in this assay is dependent on the presence of recBCD enzyme and linear dsDNA, is consistent with the known properties of recBCD enzyme, and closely parallels other methods for measuring recBCD enzyme helicase activity. The effects of varying temperature, substrate concentrations, enzyme concentration, and mono- and divalent salt concentrations on the helicase activity of recBCD enzyme were characterized. The apparent Km values for recBCD enzyme helicase activity on linear M13 dsDNA molecules at 25 degrees C are 0.6 nM dsDNA molecules and 130 microM ATP, respectively. The apparent turnover number for unwinding is approximately 15 microM base pairs s-1 (microM recBCD enzyme)-1. When this rate is corrected for the observed stoichiometry of recBCD enzyme binding to dsDNA, kcat for helicase activity corresponds to an unwinding rate of approximately 250 base pairs of DNA s-1 (functional recBCD complex)-1 at 25 degrees C. At 37 degrees C, the apparent Km value for dsDNA molecules was the same as that at 25 degrees C, but the apparent turnover number became 56 microM base pairs s-1 (microM recBCD enzyme)-1 [or 930 base pairs s-1 (functional recBCD complex)-1 when corrected for observed stoichiometry]. With increasing NaCl concentration, kcat peaks at 100 mM, and the apparent Km value for dsDNA increases by 3-fold at 200 mM NaCl. In the presence of 5 mM calcium acetate, the apparent Km value is increased by 3-fold, and kcat decreased by 20-30%. We have also shown that recBCD enzyme molecules are able to catalytically unwind additional dsDNA substrates subsequent to initiation, unwinding, and dissociation from a previous dsDNA molecule.

Characterization of the adenosinetriphosphatase activity of the Escherichia coli RecBCD enzyme: relationship of ATP hydrolysis to the unwinding of duplex DNA.

We find that the rate of dsDNA-dependent ATPase activity is biphasic, with a fast component which represents the unwinding of the dsDNA and a slow component which results from the ssDNA-dependent ATPase activity of recBCD enzyme. Comparison of the ATPase and helicase activities permits evaluation of the efficiency of ATP hydrolysis during unwinding. This efficiency can be calculated from the maximum rates of ATPase and helicase activities and is found to range between 2.0 and 3.0 ATP molecules hydrolyzed per base pair of DNA unwound. The number of ATP molecules hydrolyzed per base pair unwound is not altered by temperature but does increase at low concentrations of DNA and high concentrations of sodium chloride and magnesium acetate. The apparent Km values for the DNA and ATP substrates of recBCD enzyme dsDNA-dependent ATPase activity at 25 degrees C were determined to be 0.13 nM DNA molecules and 85 microM ATP, respectively. The observed kcat value is approximately 45 microM ATP s-1 (microM recBCD enzyme)-1. If this rate is corrected for the measured stoichiometry of recBCD enzyme binding to dsDNA, the kcat for ATPase activity corresponds to an ATP hydrolysis rate of approximately 740 ATP molecules s-1 (functional recBCD complex)-1 at 25 degrees C.

Relationship of the physical and enzymatic properties of Escherichia coli recA protein to its strand exchange activity.

We have shown that performing the recA protein catalyzed strand exchange reaction in the presence of acetate anions, rather than chloride which is commonly used, greatly increases the rate of the reaction. The initial rate of the reaction in an acetate-based buffer is approximately 3-4 times higher in the presence of Escherichia coli single-stranded DNA binding protein (SSB protein) and 2 times higher in its absence than the initial rate in chloride. To determine the enzymatic basis for this stimulatory effect of acetate buffer, we investigated the relationship between a number of physical and enzymatic properties of recA protein and the strand exchange reaction. We have found that although the acetate anion has some effect on the aggregation properties and the single-stranded DNA-dependent ATPase activity of recA protein, these effects cannot explain the enhanced strand exchange activity in an acetate-based buffer. We do find, however, that two aspects of recA protein activity closely parallel the ability of this protein to catalyze strand exchange. The first is the ability of recA protein to displace SSB protein from single-stranded DNA, an event critical to presynaptic complex formation. RecA protein is able to resist displacement by SSB protein at a lower magnesium concentration in acetate than in chloride buffer. The magnesium ion concentration dependence of strand exchange coincides exactly with this behavior. The second activity correlated to strand exchange is the duplex DNA-dependent ATPase activity of recA protein. We find that over a wide variety of sodium chloride and sodium acetate concentrations, this duplex DNA-dependent ATPase activity is linearly related to the amount of product formed in the strand exchange reaction. We postulate that this duplex DNA-dependent ATPase activity is important in the denaturation of the duplex DNA during the branch migration step of strand exchange and have also determined that this reaction is quite efficient, with the number of ATP molecules hydrolyzed per base pair exchanged being 0.75 +/- 0.25. In addition, recA protein catalyzed strand exchange between circular single-strand and linear duplex DNA molecules is shown to be irreversible, and a possible explanation for this irreversibility is presented.

Formation of heteroduplex DNA promoted by the combined activities of Escherichia coli recA and recBCD proteins.

We have established an in vitro reaction in which heteroduplex DNA formation is dependent on the concerted actions of recA and recBCD proteins, the major components of the recBCD pathway of genetic recombination in vivo. We find that heteroduplex DNA formation requires three distinct enzymatic functions: first, the helicase activity of recBCD enzyme initiates heteroduplex DNA formation by unwinding the linear double-stranded DNA molecule to transiently form single-stranded DNA (ssDNA); second, recA protein traps this ssDNA before it reanneals; third, recA protein catalyzes the pairing of this ssDNA molecule with another homologous ssDNA molecule, followed by the renaturation of these molecules to form heteroduplex DNA. The first two functions should be important to all in vitro reactions involving recA and recBCD proteins, whereas the third will be specific to the DNA substrates used. The rate-limiting step of heteroduplex DNA formation is the trapping by recA protein of the ssDNA produced by recBCD enzyme. A model for this reaction is described.

RecBCD-dependent joint molecule formation promoted by the Escherichia coli RecA and SSB proteins.

We describe the formation of homologously paired joint molecules in an in vitro reaction that is dependent on the concerted actions of purified RecA and RecBCD proteins and is stimulated by single-stranded DNA-binding protein (SSB). RecBCD enzyme initiates the process by unwinding the linear double-stranded DNA to produce single-stranded DNA, which is trapped by SSB and RecA. RecA uses this single-stranded DNA to catalyze the invasion of a supercoiled double-stranded DNA molecule, forming a homologously paired joint molecule. At low RecBCD enzyme concentrations, the rate-limiting step is the unwinding of duplex DNA by RecBCD, whereas at higher RecBCD concentrations, the rate-limiting step is RecA-catalyzed strand invasion. The behavior of mutant RecA proteins in this in vitro reaction parallels their in vivo phenotypes, suggesting that this reaction may define biochemical steps that occur during homologous recombination by the RecBCD pathway in vivo.

Processivity of the DNA helicase activity of Escherichia coli recBCD enzyme.

A fluorescence assay was used to measure the processivity of Escherichia coli recBCD enzyme helicase activity. Under standard conditions, recBCD enzyme unwinds an average of 30 +/- 3.2 kilobase pairs (kb)/DNA end before dissociating. The average processivity (P obs) of DNA unwinding under these conditions is 0.99997, indicating that the probability of unwinding another base pair is 30,000-fold greater than the probability of dissociating from the double-stranded DNA. The average number of base pairs unwound per binding event (N) is sensitive to both mono- and divalent salt concentration and ranges from 36 kb at 80 mM NaCl to 15 kb at 280 mM NaCl. The processivity of unwinding increases in a hyperbolic manner with increasing ATP concentration, yielding a KN value for ATP of 41 +/- 9 microM and a limiting value of 32 +/- 1.8 kb/end for the number of base pairs unwound. The importance of the processivity of recBCD enzyme helicase activity to the recBCD enzyme-dependent stimulation of recombination at Chi sites observed in vivo is discussed.

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.

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.

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.

Involvement of the reductase domain of neuronal nitric oxide synthase in superoxide anion production.

Neuronal nitric oxide synthase (nNOS) is a modular enzyme which consists of a flavin-containing reductase domain and a heme-containing oxygenase domain, linked by a stretch of amino acids which contains a calmodulin (CaM) binding site. CaM binding to nNOS facilitates the transfer of NADPH-derived electrons from the reductase domain to the oxygenase domain, resulting in the conversion of L-arginine to L-citrulline with the concomitant formation of a guanylate cyclase activating factor, putatively nitric oxide. Numerous studies have established that peroxynitrite-derived nitrogen oxides are present following nNOS turnover. Since peroxynitrite is formed by the diffusion-limited reaction between the two radical species, nitric oxide and O2.-, we employed the adrenochrome assay to examine whether nNOS was capable of producing O2.- during catalytic turnover in the presence of L-arginine. To differentiate between the role played by the reductase domain and that of the oxygenase domain in O2.- production, we compared its production by nNOS against that of a nNOS mutant (CYS-331), which was unable to transfer NADPH-derived electrons efficiently to the heme iron under special conditions, and against that of a flavoprotein module construct of nNOS. We report that O2.- production by nNOS and the CYS-331 mutant is CaM-dependent and that O2.- production can be modulated by substrates and inhibitors of nNOS. O2.- was also produced by the reductase domain of nNOS; however, it did not display the same CaM dependency. We conclude that both the reductase and oxygenase domains of nNOS produce O2.-, but that the reductase domain is both necessary and sufficient for O2.- production.

Electron paramagnetic resonance spectroscopy of the heme domain of inducible nitric oxide synthase: binding of ligands at the arginine site induces changes in the heme ligation geometry.

The electron paramagnetic resonance spectra of the heme domain of inducible nitric oxide synthase (iNOS) demonstrate a close relationship to the corresponding spectra of the neuronal isoform (nNOS). The binding of ligands to the iNOS arginine site perturbs the environment of the high-spin ferriheme in a highly ligand-specific manner. The iNOS forms five-coordinate, high-spin complexes with arginine analogs which are clearly related to the corresponding complexes of nNOS. Studies indicate that the binding of L-arginine, N(omega)-hydroxy-L-arginine (NHA), and N(omega)-methyl-L-arginine (NMA) produces various spectroscopic species closely corresponding to the equivalent complexes of nNOS, while N(omega)-nitro-L-arginine (NNA) binding produces a state which appears intermediate in character between the nNOS NNA and arginine complexes. These spectroscopic studies have permitted the determination of ligand-specific high-spin states which reveal similarities and differences between iNOS and nNOS.