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1.  Phenyl Ether- and Aniline-Containing 2-Aminoquinolines as Potent and Selective Inhibitors of Neuronal Nitric Oxide Synthase 
Journal of medicinal chemistry  2015;58(21):8694-8712.
Excess nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS) is implicated in neurodegenerative disorders. As a result, inhibition of nNOS and reduction of NO levels is desirable therapeutically, but many nNOS inhibitors are poorly bioavailable. Promising members of our previously reported 2-aminoquinoline class of nNOS inhibitors, although orally bioavailable and brain-penetrant, suffer from unfavorable off-target binding to other CNS receptors, and they resemble known promiscuous binders. Rearranged phenyl ether- and aniline-linked 2-aminoquinoline derivatives were therefore designed to a) disrupt the promiscuous binding pharmacophore and diminish off-target interactions, and b) preserve potency, isoform selectivity, and cell permeability. A series of these compounds was synthesized and tested against purified nNOS, endothelial NOS (eNOS), and inducible NOS (iNOS) enzymes. One compound, 20, displayed high potency, selectivity, and good human nNOS inhibition, and retained some permeability in a Caco-2 assay. Most promisingly, CNS receptor counterscreening revealed this rearranged scaffold significantly reduces off-target binding.
doi:10.1021/acs.jmedchem.5b01330
PMCID: PMC4644118  PMID: 26469213
2.  Tertiary amine pyrazolones and their salts as inhibitors of mutant superoxide dismutase 1-dependent protein aggregation for the treatment of amyotrophic lateral sclerosis 
Journal of medicinal chemistry  2015;58(15):5942-5949.
Pyrazolone derivatives have previously been found to be inhibitors of Cu/Zn superoxide dismutase 1 (SOD1)-dependent protein aggregation, which extended survival of an amyotrophic lateral sclerosis (ALS) mouse model. On the basis of ADME analysis, we describe herein a new series of tertiary amine-containing pyrazolones and their structure-activity relationships. Further conversion to the conjugate salts greatly improved their solubility. Phosphate compound 17 exhibited numerous benefits both to cellular activity and to CNS-related drug-like properties in vitro and in vivo, including microsomal stability, tolerated toxicity, and blood-brain barrier permeation. These results indicate that tertiary amine pyrazolones comprise a valuable class of ALS drug candidates.
doi:10.1021/acs.jmedchem.5b00561
PMCID: PMC4537347  PMID: 26186011
3.  2-Aminopyridines with a Truncated Side Chain to Improve Human Neuronal Nitric Oxide Synthase Inhibitory Potency and Selectivity 
Journal of medicinal chemistry  2015;58(14):5548-5560.
We have analyzed a recently obtained crystal structure of human neuronal nitric oxide synthase (nNOS), then designed and synthesized several 2-aminopyridine derivatives containing a truncated side chain to avoid the hydrophobic pocket that differentiates human and rat nNOS in an attempt to explore alternative binding poses along the substrate access channel of human nNOS. Introduction of an N-methylethane-1,2-diamine side chain and conformational constraints such as benzonitrile and pyridine as the middle aromatic linker were sufficient to increase human and rat nNOS binding affinity and inducible and endothelial NOS selectivity. We found that 14b is a potent inhibitor; the binding modes with human and rat nNOS are unexpected, inducing side chain rotamer changes in Gln478 (rat) at the top of the active site. Compound 19c exhibits Ki values of 24 and 55 nM for rat and human nNOS, respectively, with 153-fold iNOS and 1040-fold eNOS selectivity. 19c has 18% oral bioavailability.
doi:10.1021/acs.jmedchem.5b00573
PMCID: PMC4514563  PMID: 26120733
nitric oxide synthase; neurodegenerative diseases; human nNOS; selective inhibition
5.  Structure-Based Design of Bacterial Nitric Oxide Synthase Inhibitors 
Journal of Medicinal Chemistry  2014;58(2):994-1004.
Inhibition of bacterial nitric oxide synthase (bNOS) has the potential to improve the efficacy of antimicrobials used to treat infections by Gram-positive pathogens Staphylococcus aureus and Bacillus anthracis. However, inhibitor specificity toward bNOS over the mammalian NOS (mNOS) isoforms remains a challenge because of the near identical NOS active sites. One key structural difference between the NOS isoforms is the amino acid composition of the pterin cofactor binding site that is adjacent to the NOS active site. Previously, we demonstrated that a NOS inhibitor targeting both the active and pterin sites was potent and functioned as an antimicrobial (Holden, , Proc. Natl. Acad. Sci. U.S.A.2013, 110, 1812724145412). Here we present additional crystal structures, binding analyses, and bacterial killing studies of inhibitors that target both the active and pterin sites of a bNOS and function as antimicrobials. Together, these data provide a framework for continued development of bNOS inhibitors, as each molecule represents an excellent chemical scaffold for the design of isoform selective bNOS inhibitors.
doi:10.1021/jm501723p
PMCID: PMC4306518  PMID: 25522110
6.  Structure-Activity Relationship of N,N′-Disubstituted Pyrimidinetriones as CaV1.3 Calcium Channel-Selective Antagonists for Parkinson’s Disease 
Journal of medicinal chemistry  2013;56(11):4786-4797.
CaV1.3 L-type calcium channels (LTCCs) have been a potential target for Parkinson’s disease since calcium ion influx through the channel was implicated in the generation of mitochondrial oxidative stress, causing cell death in the dopaminergic neurons. Selective inhibition of CaV1.3 over other LTCC isoforms, especially CaV1.2, is critical to minimize potential side effects. We recently identified pyrimidinetriones (PYTs) as a CaV1.3-selective scaffold; here we report the structure-activity relationship of PYTs with both CaV1.3 and CaV1.2 LTCCs. By variation of the substituents on the cyclopentyl and arylalkyl groups of PYT, SAR studies allowed characterization of the CaV1.3 and CaV1.2 LTCCs binding sites. The SAR also identified four important moieties that either retain selectivity or enhance binding affinity. Our study represents a significant enhancement of the SAR of PYTs at CaV1.3 and CaV1.2 LTCCs and highlights several advances in the lead optimization and diversification of this family of compounds for drug development.
Graphical abstract
doi:10.1021/jm4005048
PMCID: PMC4595045  PMID: 23651412
7.  Nitric Oxide Synthase Inhibitors That Interact with Both Heme Propionate and Tetrahydrobiopterin Show High Isoform Selectivity 
Journal of Medicinal Chemistry  2014;57(10):4382-4396.
Overproduction of NO by nNOS is implicated in the pathogenesis of diverse neuronal disorders. Since NO signaling is involved in diverse physiological functions, selective inhibition of nNOS over other isoforms is essential to minimize side effects. A series of α-amino functionalized aminopyridine derivatives (3–8) were designed to probe the structure–activity relationship between ligand, heme propionate, and H4B. Compound 8R was identified as the most potent and selective molecule of this study, exhibiting a Ki of 24 nM for nNOS, with 273-fold and 2822-fold selectivity against iNOS and eNOS, respectively. Although crystal structures of 8R complexed with nNOS and eNOS revealed a similar binding mode, the selectivity stems from the distinct electrostatic environments in two isoforms that result in much lower inhibitor binding free energy in nNOS than in eNOS. These findings provide a basis for further development of simple, but even more selective and potent, nNOS inhibitors.
doi:10.1021/jm5004182
PMCID: PMC4032192  PMID: 24758147
8.  Novel 2,4-Disubstituted Pyrimidines as Potent, Selective, and Cell-Permeable Inhibitors of Neuronal Nitric Oxide Synthase 
Journal of Medicinal Chemistry  2014;58(3):1067-1088.
Selective inhibition of neuronal nitric oxide synthase (nNOS) is an important therapeutic approach to target neurodegenerative disorders. However, the majority of the nNOS inhibitors developed are arginine mimetics and, therefore, suffer from poor bioavailability. We designed a novel strategy to combine a more pharmacokinetically favorable 2-imidazolylpyrimidine head with promising structural components from previous inhibitors. In conjunction with extensive structure–activity studies, several highly potent and selective inhibitors of nNOS were discovered. X-ray crystallographic analysis reveals that these type II inhibitors utilize the same hydrophobic pocket to gain strong inhibitory potency (13), as well as high isoform selectivity. Interestingly, select compounds from this series (9) showed good permeability and low efflux in a Caco-2 assay, suggesting potential oral bioavailability, and exhibited minimal off-target binding to 50 central nervous system receptors. Furthermore, even with heme-coordinating groups in the molecule, modifying other pharmacophoric fragments minimized undesirable inhibition of cytochrome P450s from human liver microsomes.
doi:10.1021/jm501719e
PMCID: PMC4329833  PMID: 25489882
9.  Simplified 2-Aminoquinoline-Based Scaffold for Potent and Selective Neuronal Nitric Oxide Synthase Inhibition 
Journal of Medicinal Chemistry  2014;57(4):1513-1530.
Since high levels of nitric oxide (NO) are implicated in neurodegenerative disorders, inhibition of the neuronal isoform of nitric oxide synthase (nNOS) and reduction of NO levels are therapeutically desirable. Nonetheless, many nNOS inhibitors mimic l-arginine and are poorly bioavailable. 2-Aminoquinoline-based scaffolds were designed with the hope that they could (a) mimic aminopyridines as potent, isoform-selective arginine isosteres and (b) possess chemical properties more conducive to oral bioavailability and CNS penetration. A series of these compounds was synthesized and assayed against purified nNOS enzymes, endothelial NOS (eNOS), and inducible NOS (iNOS). Several compounds built on a 7-substituted 2-aminoquinoline core are potent and isoform-selective; X-ray crystallography indicates that aminoquinolines exert inhibitory effects by mimicking substrate interactions with the conserved active site glutamate residue. The most potent and selective compounds, 7 and 15, were tested in a Caco-2 assay and showed good permeability and low efflux, suggesting high potential for oral bioavailability.
doi:10.1021/jm401838x
PMCID: PMC3954451  PMID: 24472039
10.  Potent and Selective Double-Headed Thiophene-2-carboximidamide Inhibitors of Neuronal Nitric Oxide Synthase for the Treatment of Melanoma 
Journal of Medicinal Chemistry  2014;57(3):686-700.
Selective inhibitors of neuronal nitric oxide synthase (nNOS) are regarded as valuable and powerful agents with therapeutic potential for the treatment of chronic neurodegenerative pathologies and human melanoma. Here, we describe a novel hybrid strategy that combines the pharmacokinetically promising thiophene-2-carboximidamide fragment and structural features of our previously reported potent and selective aminopyridine inhibitors. Two inhibitors, 13 and 14, show low nanomolar inhibitory potency (Ki = 5 nM for nNOS) and good isoform selectivities (nNOS over eNOS [440- and 540-fold, respectively] and over iNOS [260- and 340-fold, respectively]). The crystal structures of these nNOS–inhibitor complexes reveal a new hot spot that explains the selectivity of 14 and why converting the secondary to tertiary amine leads to enhanced selectivity. More importantly, these compounds are the first highly potent and selective nNOS inhibitory agents that exhibit excellent in vitro efficacy in melanoma cell lines.
doi:10.1021/jm401252e
PMCID: PMC3983353  PMID: 24447275
11.  Target- and Mechanism-Based Therapeutics for Neurodegenerative Diseases: Strength in Numbers 
Journal of medicinal chemistry  2013;56(8):3121-3147.
The development of new therapeutics for the treatment of neurodegenerative pathophysiologies currently stands at a crossroads. This presents an opportunity to transition future drug discovery efforts to target disease modification, an area in which much still remains unknown. In this Perspective we examine recent progress in the areas of neurodegenerative drug discovery, focusing on some of the most common targets and mechanisms; N-methyl-d-aspartic acid (NMDA) receptors, voltage gated calcium channels (VGCCs), neuronal nitric oxide synthase (nNOS), oxidative stress from reactive oxygen species, and protein aggregation. These represent the key players identified in neurodegeneration and are part of a complex, intertwined signaling cascade. The synergistic delivery of two or more compounds directed against these targets, along with the design of small molecules with multiple modes of action should be explored in pursuit of more effective clinical treatments for neurodegenerative diseases.
doi:10.1021/jm3015926
PMCID: PMC3637880  PMID: 23458846
12.  Structure-guided Design of Selective Inhibitors of Neuronal Nitric Oxide Synthase 
Journal of medicinal chemistry  2013;56(7):3024-3032.
Nitric oxide synthases (NOSs) comprise three closely related isoforms that catalyze the oxidation of l-arginine to l-citrulline and the important second messenger nitric oxide (NO). Pharmacological selective inhibition of neuronal NOS (nNOS) has the potential to be therapeutically beneficial in various neurodegenerative diseases. Here we present a structure-guided, selective nNOS inhibitor design based on the crystal structure of lead compound 1 in nNOS. The best inhibitor, 7, exhibited low nanomolar inhibitory potency and good isoform selectivities (nNOS over eNOS and iNOS are 472-fold and 239-fold, respectively). Consistent with the good selectivity, 7 binds to nNOS and eNOS with different binding modes. The distinctly different binding modes of 7, driven by the critical residue Asp597 in nNOS, offers compelling insight to explain its isozyme selectivity, which should guide future drug design programs.
doi:10.1021/jm4000984
PMCID: PMC3632207  PMID: 23451760
13.  Arylazanylpyrazolone derivatives as inhibitors of mutant superoxide dismutase 1-dependent protein aggregation for the treatment of amyotrophic lateral sclerosis 
Journal of medicinal chemistry  2013;56(6):2665-2675.
The arylsulfanyl- and aryloxanylpyrazolone scaffolds previously were reported to inhibit Cu/Zn superoxide dismutase 1 dependent protein aggregation and to extend survival in the ALS mouse model. However, further evaluation of these compounds indicated weak pharmacokinetic properties and a relatively low maximum tolerated dose. On the basis of an ADME analysis, a new series of compounds, the arylazanyl pyrazolones, has been synthesized, and structure-activity relationships were determined. The SAR results showed that the pyrazolone ring is critical to cellular protection. The NMR, IR, and computational analyses suggest that phenol-type tautomers of the pyrazolone ring are the active pharmacophore with the arylazanylpyrazolone analogues. A comparison of experimental and calculated IR spectra is shown to be a valuable method to identify the predominant tautomer.
doi:10.1021/jm400079a
PMCID: PMC3627359  PMID: 23445362
15.  CPP-115, a Potent γ-Aminobutyric Acid Aminotransferase Inactivator for the Treatment of Cocaine Addiction 
Journal of Medicinal Chemistry  2011;55(1):357-366.
Vigabatrin, a GABA aminotransferase (GABA-AT) inactivator, is used to treat infantile spasms and refractory complex partial seizures and is in clinical trials to treat addiction. We evaluated a novel GABA-AT inactivator (CPP-115) and observed that it does not exhibit other GABAergic or off-target activities and is rapidly and completely orally absorbed and eliminated. Using in vivo microdialysis techniques in freely moving rats and micro-PET imaging techniques, CPP-115 produced similar inhibition of cocaine-induced increases in extracellular dopamine and in synaptic dopamine in the nucleus accumbens at 1/300–1/600th the dose of vigabatrin. It also blocks expression of cocaine-induced conditioned place preference at a dose 1/300th that of vigabatrin. Electroretinographic (ERG) responses in rats treated with CPP-115, at doses 20–40 times higher than those needed to treat addiction in rats, exhibited reductions in ERG responses, which were less than the reductions observed in rats treated with vigabatrin at the same dose needed to treat addiction in rats. In conclusion, CPP-115 can be administered at significantly lower doses than vigabatrin, which suggests a potential new treatment for addiction with a significantly reduced risk of visual field defects.
doi:10.1021/jm201231w
PMCID: PMC3257419  PMID: 22128851
GABA aminotransferase; Enzyme inactivator; Addiction; Cocaine; Visual field defect; Pharmacokinetics; Micro-PET imaging; Conditioned place preference
16.  Improved Synthesis of Chiral Pyrrolidine Inhibitors and Their Binding Properties to Neuronal Nitric Oxide Synthase 
Journal of medicinal chemistry  2011;54(18):6399-6403.
We report an efficient synthetic route to chiral pyrrolidine inhibitors of neuronal nitric oxide synthase (nNOS) and crystal structures of the inhibitors bound to nNOS and to endothelial NOS. The new route enables versatile structure activity relationship studies on the pyrrolidine-based scaffold, which can be beneficial for further development of nNOS inhibitors. The X-ray crystal structures of three new fluorine-containing inhibitors bound to nNOS provide insights into the effect of the fluorine atoms on binding.
doi:10.1021/jm200411j
PMCID: PMC3174355  PMID: 21809851
17.  Symmetric Double-Headed Aminopyridines, A Novel Strategy for Potent and Membrane-Permeable Inhibitors of Neuronal Nitric Oxide Synthase 
Journal of medicinal chemistry  2011;54(7):2039-2048.
We report novel neuronal nitric oxide synthase (nNOS) inhibitors based on a symmetric double-headed aminopyridine scaffold. The inhibitors were designed from crystal structures of leads 1 and 2 (Delker, S. L.; Ji, H.; Li, H.; Jamal, J.; Fang, J.; Xue, F.; Silverman, R. B.; Poulos, T. L. Unexpected binding modes of nitric oxide synthase inhibitors effective in the prevention of cerebral palsy. J. Am. Chem. Soc. 2010, 132, 5437–5442) and synthesized using a highly efficient route. The best inhibitor, 3j, showed low nanomolar inhibitory potency and modest isoform selectivity. It also exhibited enhanced membrane permeability. Inhibitor 3j binds to both the substrate site and the pterin site in nNOS but only to the substrate site in eNOS. These compounds provide a basis for further development of novel, potent, isoform selective, and bioavailable inhibitors for nNOS.
doi:10.1021/jm101071n
PMCID: PMC3072459  PMID: 21410186
18.  Pyrimidine-2,4,6-trione Derivatives and Their Inhibition of Mutant SOD1-dependent Protein Aggregation. Toward a Treatment for Amyotrophic Lateral Sclerosis 
Journal of medicinal chemistry  2011;54(7):2409-2421.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons, leading to muscle weakness, paralysis, and death, most often from respiratory failure. The only FDA approved drug for the treatment of ALS, riluzole, only extends the median survival in patients by 2–3 months. There is an urgent need for novel therapeutic strategies for this devastating disease. Using a high-throughput screening assay targeting an ALS cultured cell model (PC12-G93A-YFP cell line), we previously identified three chemotypes that were neuroprotective. We present a further detailed analysis of one promising scaffold from that group, pyrimidine-2,4,6-triones (PYTs), characterizing a number of PYT analogs using SAR and ADME. The PYT compounds show good potency, superior ADME data, low toxicity, brain penetration, and excellent oral bioavailability. Compounds from this series show 100% efficacy in the protection assay with a good correlation in activity between the protection and protein aggregation assays. The modifications of the PYT scaffold presented here suggest that this chemical structure may be a novel drug candidate scaffold for use in clinical trials in ALS.
doi:10.1021/jm101549k
PMCID: PMC3074252  PMID: 21375347
Pyrimidine-2,4,6-triones (PYT); amyotrophic lateral sclerosis; mutant G93A SOD1; blood-brain barrier penetration; ADME
19.  Exploration of the Active Site of Neuronal Nitric Oxide Synthase by the Design and Synthesis of Pyrrolidinomethyl 2-Aminopyridine Derivatives 
Journal of medicinal chemistry  2010;53(21):7804-7824.
Neuronal nitric oxide synthase (nNOS) represents an important therapeutic target for the prevention of brain injury and the treatment of various neurodegenerative disorders. A series of trans substituted amino pyrrolidinomethyl 2-aminopyridine derivatives (8–34) was designed and synthesized. A structure-activity relationship analysis led to the discovery of low nanomolar nNOS inhibitors [(±)-32 and (±)-34] with more than 1000-fold selectivity for nNOS over eNOS. Four enantiomerically pure isomers of 3′-[2″-(3‴-fluorophenethylamino)ethoxy]pyrrolidin-4′-yl}methyl}-4-methylpyridin-2-amine (4) also were synthesized. It was found that (3′R, 4′R)-4 can induce enzyme elasticity to generate a new “hot spot” for ligand binding. The inhibitor adopts a unique binding mode, the same as that observed for (3′R, 4′R)-3′-[2″-(3‴-fluorophenethylamino)ethylamino]pyrrolidin-4′-yl}methyl}-4-methylpyridin-2-amine ((3′R, 4′R)-3) (J. Am. Chem. Soc. 2010, 132(15), 5437–5442). On the basis of structure-activity relationships of 8–34 and different binding conformations of the cis and trans isomers of 3 and 4, critical structural requirements of the NOS active site for ligand binding are revealed.
doi:10.1021/jm100947x
PMCID: PMC2978073  PMID: 20958055
20.  Crystal Structures of Constitutive Nitric Oxide Synthases in Complex with De Novo Designed Inhibitors 
Journal of medicinal chemistry  2009;52(7):2060-2066.
New nitric oxide synthase (NOS) inhibitors were designed de novo with knowledge gathered from the studies on the nNOS-selective dipeptide inhibitors. Each of the new inhibitors consists of three fragments: an aminopyridine ring, a pyrrolidine, and a tail of various length and polarity. The in vitro inhibitory assays indicate good potency and isoform selectivity for some of the compounds. Crystal structures of these inhibitors bound to either wild type or mutant nNOS and eNOS have confirmed design expectations. The aminopyridine ring mimics the guanidinium group of L-arginine and functions as an anchor to place the compound in the NOS active site where it hydrogen bonds to a conserved Glu. The rigidity of the pyrrolidine ring places the pyrrolidine ring nitrogen between the same conserved Glu and the selective residue nNOS Asp597/eNOS Asn368 which results in similar interactions observed with the α-amino group of dipeptide inhibitors bound to nNOS. These structures provide additional information to help in the design of inhibitors with greater potency, physico-chemical properties, and isoform selectivity.
doi:10.1021/jm900007a
PMCID: PMC3103786  PMID: 19296678
21.  L337H Mutant of Rat Neuronal Nitric Oxide Synthase Resembles Human Neuronal Nitric Oxide Synthase Toward Inhibitors 
Journal of medicinal chemistry  2009;52(14):4533-4537.
A common dichotomy exists in inhibitor design: should the compounds be designed to block the enzymes of animals in the preclinical studies or to inhibit the human enzyme? We report that a single mutation of Leu-337 in rat neuronal nitric oxide synthase (nNOS) to His makes the enzyme resemble human nNOS more than rat nNOS. We expect that the approach used in this study can unite the dichotomy and speed up the process of inhibitor design and development.
doi:10.1021/jm900380j
PMCID: PMC2758698  PMID: 19537690
22.  Discovery of Highly Potent and Selective Inhibitors of Neuronal Nitric Oxide Synthase by Fragment Hopping 
Journal of medicinal chemistry  2009;52(3):779-797.
Selective inhibition of neuronal nitric oxide synthase (nNOS) has been shown to prevent brain injury and is important for the treatment of various neurodegenerative disorders. This study shows that not only greater inhibitory potency and isozyme selectivity, but more drug-like properties can be achieved by fragment hopping. Based on the structure of lead molecule 6, fragment hopping effectively extracted the minimal pharmacophoric elements in the active site of nNOS for ligand hydrophobic and steric interactions and generated appropriate lipophilic fragments for lead optimization. More potent and selective inhibitors with better drug-like properties were obtained within the design of 20 derivatives (compounds 7-26). Our structure-based inhibitor design for nNOS and SAR analysis reveal the robustness and efficiency of fragment hopping in lead discovery and structural optimization, which implicates a broad application of this approach to many other therapeutic targets for which known drug-like small-molecule modulators are still limited.
doi:10.1021/jm801220a
PMCID: PMC2664101  PMID: 19125620
23.  Fluorinated Conformationally-Restricted γ-Aminobutyric Acid Aminotransferase Inhibitors 
Journal of medicinal chemistry  2006;49(25):7404-7412.
Based on the structures of several potent inhibitor molecules for γ-aminobutryric acid aminotransferase (GABA-AT) that were previously reported, six modified fluorine-containing conformationally-restricted analogues were designed, synthesized, and tested as GABA-AT inhibitors. The syntheses of all six molecules followed from a readily synthesized ketone intermediate. Three of the molecules were found to be irreversible inhibitors of GABA-AT with comparable or larger kinact/KI values than that of vigabatrin, a clinically used antiepilepsy drug, and the other three were reversible inhibitors. A possible mechanism for inactivation by one of the inactivators is proposed.
doi:10.1021/jm0608715
PMCID: PMC2577068  PMID: 17149870
24.  Structure-Based Design and Synthesis of Nω-Nitro-L-Arginine-Containing Peptidomimetics as Selective Inhibitors of Neuronal Nitric Oxide Synthase. Displacement of the Heme Structural Water 
Journal of medicinal chemistry  2007;50(9):2089-2099.
The neuronal isoform of nitric oxide synthase (nNOS), the enzyme responsible for the production of nitric oxide in the central nervous system, represents an attractive target for the treatment of various neurodegenerative disorders. X-ray crystal structures of complexes of nNOS with two nNOS-selective inhibitors, (4S)-N-{4-amino-5-[(2-aminoethylamino]pentyl}-N′-nitroguanidine (1) and 4-N-(Nω-nitro-L-argininyl)-trans-4-amino-L-proline amide (2), led to the discovery of a conserved structural water molecule that was hydrogen bonded between the two heme propionates and the inhibitors (Figure 2). Based on this observation, we hypothesized that by attaching a hydrogen bond donor group to the amide nitrogen of 2 or to the secondary amine nitrogen of 1, the inhibitor molecules could displace the structural water molecule and obtain a direct interaction with the heme cofactor. To test this hypothesis, peptidomimetic analogues 3–5, which have either an N-hydroxyl (3 and 5) or N-amino (4) donor group, were designed and synthesized. X-ray crystal structures of nNOS with inhibitors 3 and 5 bound verified that the N-hydroxyl group had, indeed, displaced the structural water molecule and provided a direct interaction with the heme propionate moiety (Figures 4 and 5). Surprisingly, in vitro activity assay results indicated that the addition of a hydroxyl group (3) only increased the potency slightly against the neuronal isoform over the parent compound (1). Rationalizations for the small increase in potency are consistent with other changes in the crystal structures.
doi:10.1021/jm061305c
PMCID: PMC2562355  PMID: 17425297
25.  Conformationally-Restricted Dipeptide Amides as Potent and Selective Neuronal Nitric Oxide Synthase Inhibitors 
Journal of medicinal chemistry  2006;49(21):6254-6263.
Four new conformationally-restricted analogues of the potent and selective neuronal nitric oxide synthase inhibitor, L-nitroargininyl-L-2,4-diaminobutyramide (1), have been synthesized. Nα-Methyl and Nα-benzyl derivatives (3 and 4, respectively) of 4N-(L-ArgNO2)-trans-4-amino-L-prolineamide (2) are also selective inhibitors, but the potency and selectivity of 3 are weak. Analogue 4 has only one-third the potency and one-half to one-third the selectivity of 2 against iNOS and eNOS, respectively. 3-N-(L-ArgNO2)-trans-3-amino-L-prolineamide (6) is as potent an inhibitor of nNOS as is 2; selectivity for nNOS over iNOS is half of that for 2 but the selectivity for nNOS over eNOS is almost double that for 2. The corresponding cis-isomer (5) is a weak inhibitor of nNOS. These results are supported by computer modeling.
doi:10.1021/jm0604124
PMCID: PMC2517862  PMID: 17034131

Results 1-25 (25)