De Novo Design of Potent and Selective Neuronal Nitric Oxide Synthase (nNOS) Inhibitors by a Fragment-Based Approach (original) (raw)
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Bioorganic & Medicinal Chemistry, 2013
In certain neurodegenerative diseases damaging levels of nitric oxide (NO) are produced by neuronal nitric oxide synthase (nNOS). It, therefore, is important to develop inhibitors selective for nNOS that do not interfere with other NOS isoforms, especially endothelial NOS (eNOS), which is critical for proper functioning of the cardiovascular system. While we have been successful in developing potent and isoform-selective inhibitors, such as lead compounds 1 and 2, the ease of synthesis and bioavailability have been problematic. Here we describe a new series of compounds including crystal structures of NOS-inhibitor complexes that integrate the advantages of easy synthesis and good biological properties compared to the lead compounds. These results provide the basis for additional structure-activity relationship (SAR) studies to guide further improvement of isozyme selective inhibitors.
Journal of the American Chemical Society, 2008
Experimental Section Computer Modeling Protein Structures The crystallographic coordinates for NOS from the Research Collaboratory for Structural Bioinformatics (RCSB) protein database are as follows: rat nNOS 1 :1p6h (1.98 Å resolution, R cryst = 0.231); 1p6i (1.90 Å resolution, R cryst = 0.228); 1p6j (2.00 Å resolution, R cryst = 0.225); 1qw6 (2.1 Å resolution, R cryst = 0.20), human eNOS 2 : 3nos (2.4 Å resolution, R cryst = 0.193), bovine eNOS 1 : 1p6l (2.35 Å resolution, R cryst = 0.214); 1p6m (2.27 Å resolution, R cryst = 0.215); 1p6n (2.5 Å resolution, R cryst = 0.219), human iNOS: 3 1nsi (2.55 Å resolution, R cryst = 0.209), murine iNOS 4 : 1qw4 (2.3 Å resolution, R cryst = 0.213). The amino acid sequences of NOS were retrieved from the PIR protein sequence database. The sequences are human nNOS (entry P29475), rat nNOS (entry P29476), human eNOS (entry P29474), bovine eNOS (entry P29473), human iNOS (entry P35228), and murine iNOS (entry P29477). All of the computational work was performed on Silicon Graphics Octane 2 Workstations with an IRIX 6.5 operating system. The molecular modeling was achieved with commercially available InsightII 2000 5 and SYBYL 6.8 6 software packages. The NOS model was prepared by first adding H-atoms using the Accerlyrs/InsightII 2000 software, and the protonation states of the residues were set to pH 7.0. The cvff force field of the Discover 98.0 program within Insight II was used to optimize the orientation of hydrogen atoms of the protein and of structural waters. The ligands and solvent molecules of the protein structures were removed, but the heme and H 4 B were retained near the active site. The size and spatial orientation of the active site was analyzed by the Binding Site S2 Analysis module within Insight II. The grid size for searching the proteins was set to 1 Å ×1 Å ×1 Å. All of the solvent-accessible surfaces in the proteins were filled with grid points, and only those having at least 125 grid points were accepted as possible ligand binding sites. GRID Calculations The calculations were performed with version 22 of the GRID software. 7 Hydrogens were added with the program GRIN. The GRID box dimensions were chosen to encompass all of the active site residues shown in Figure 1. This results in a box size of 31 × 28 × 31 Å. The grid spacings were set to 1 Å (directive NPLA = 1) and 0.5 Å (directive NPLA = 2), respectively. The amino acids in the active site were considered rigid (directive move = 0). The directives NETA and ALMD were set to 120 and 1, respectively, to include the atoms of heme and H 4 B in calculations and to interpret which atom(s) in the active site contribute(s) to the interaction with a specific probe atom. The following single atom probes were used in the calculation: DRY, C3, NM3, N1+, N3+, N1, NH=, O, O1, and the multi-atom probes COO-, Amidine, and ARamidine. MCSS Calculations The functional groups chosen for the MCSS calculations were benzene (all hydrogen model), cyclohexane, isobutene, N-methylacetamide, methanol, ether, acetate ion, methylammonium, trimethylamine cation, and pyrrolidine cation. The binding site area in the MCSS simulation was defined as an approximately 27 Å × 26 Å × 31 Å box, which was centered around the grid points searched by InsightII/Binding Site Analysis. Replicas of a given functional group were randomly distributed inside the binding site and then simultaneously and independently energy minimized. Pairs of molecules were considered to be identical if the root-mean-square deviation (RMSD) between them was less than 0.2 Å, and in such cases one of the pairs was eliminated. The above protocol was repeated 10 times for each of the functional groups to allow complete searching of the active site. The minima with the binding energy lower than 0 kcal/mol were retrieved in the analysis. All of the above calculations were performed using the CHARMM 22 force field and MCSS 2.1 program. 8 LUDI Ligand Design The constructed targeted fragment libraries were converted into a LUDI user library. To determine the appropriate position and orientation of the fragments from the LUDI user library in the active site of NOS, LUDI was first applied to generate the interaction sites for each pharmacophore. Four different types of the interaction sites are defined in the LUDI program: lipophilic-aliphatic, lipophilic-aromatic, hydrogen donor, and hydrogen acceptor. The residues inside an 8.0 Å radius sphere, which centered on the centroid of the minimal pharmacophoric elements, were used to generate the interaction sites. The link library switch was turned off, and the target mode switch was turned on. The LUDI scoring function was set to Energy-estimate-2. 9 Other settings were kept with the standard default parameters. To convert the above determined fragments into a molecule, the side chain library in Supporting Information Figure 5 was converted into a LUDI link library and was used further in the connection operation. The hydrogen atoms in the above fragment structure were replaced by a link fragment to create a new substructure. The switch for the target mode was turned off, but the switch for the link library was turned on. The linkage parameter can be set to 1 (the link fragment fit at least one link site), 2 (the link fragment simultaneously fit at least two link sites), or be specified (the link site was S3 specifically assigned) according to the actual requirements. The other settings are the standard default parameters. AutoDock Analysis AutoDock 3.0 was employed to perform the docking calculations, as reported in our previous paper. 10 The ligands and solvent molecules of the protein structures were removed, but the cofactors heme and H 4 B were retained near the active site. For each protein structure, polar hydrogen atoms were added, and Kollman united atom charges were assigned. 11 Hydrogens were also added to the heme and H 4 B, and charges were calculated by the Gasteiger-Marsili method. 12 The charge of the Fe atom bound to heme was assigned +3. The nonpolar hydrogen atoms of heme and H 4 B were removed manually and their charges were united with the bonded carbon atoms. Atomic solvation parameters and fragmental volumes were assigned using the AddSol utility. The 3D structures of the ligands were built using SYBYL 6.8. Partial atomic charges were also calculated using the Gasteiger-Marsili method. The rotatable bonds in the ligands were defined using another AutoDock 3.0 auxiliary program, AutoTors, which also unites the nonpolar hydrogens and partial atomic charges to the bonded carbon atoms. The grid maps were calculated using AutoGrid. The dimensions of the grid box was 27 × 26 × 31 Å and the grid spacing was set to 0.375 Å. Docking was performed using the Lamarckian genetic algorithm (LGA), and the pseudo-Solis and Wets method were applied for the local search. Each docking experiment was performed 100 times, yielding 100 docked conformations. Parameters for the docking experiments were as follows: initial population size of 200; random starting position and conformation; maximal mutation of 0.2 Å in translation and 5º in orientation and rotation; elitism of 5; mutation rate of 0.02 and crossover rate of 0.8; local search rate of 0.06 and maximal iteration per local search of 300. Simulations were performed with a maximum of 1.5 × 10 6 energy evaluations and a maximum of 27,000 generations. Other settings were the standard default parameters. All of the ligands followed the same docking protocol. The results of the docking experiments were evaluated by calculating the positional root-mean-square deviation (RMSD) of the corresponding atoms of each conformation 13 and/or by a visual comparison to determine if the orientation of the inhibitor matches the pharmacophores determined. Chemical Synthesis General Methods, Reagents and Materials All reagents were purchased from Aldrich, Acros Organics, and Fisher Scientific and were used without further purification unless stated otherwise. NADPH, calmodulin, and human ferrous hemoglobin were obtained from Sigma Chemical Co. Tetrahydrobiopterin (H 4 B) was purchased from Alexis Biochemicals. 1 H NMR and 13 C NMR spectra were recorded on a Varian Mercury 400 MHz or a Varian Inova 500 MHz spectrometer (100.6, or 125.7 MHz, for 13 C NMR spectra, respectively) in CDCl 3 , DMSO-d 6 , CD 3 OD or D 2 O. Chemical shifts are reported as values in parts per million and the reference resonance peaks set at 0 ppm [TMS(CDCl 3)], 2.50 ppm [(CD 2 H) 2 SO], 3.31 ppm (CD 2 HOD), and 4.80 ppm (HOD) respectively for 1 H NMR and 77.23 ppm (CDCl 3), 39.52 ppm (DMSO-d 6) and 49.00 ppm (CD 3 OD) for 13 C NMR spectra. An Orion research model 701H pH meter with a general combination electrode was used for pH measurements. Mass spectra were performed on a Micromass Quattro II triple quadrupole HPLC/MS mass spectrometer with an electrospray ionization (ESI) source or atmospheric pressure chemical ionization (APCI) source. High-resolution mass spectra were carried out
Potent and selective neuronal nitric oxide synthase inhibitors with improved cellular permeability
Bioorganic & Medicinal Chemistry Letters, 2010
Recently, a series of potent and selective neuronal nitric oxide synthase inhibitors containing two basic nitrogen atoms was reported (Ji, H.; Stanton, B. Z.; Igarashi, J.;, Li, H.; Martásek, P.; Roman, L. J.; Poulos, T. L.; Silverman, R. B. J. Am. Chem. Soc. 2008, 130(12), 3900-3914). In an effort to improve their bioavailability, three compounds (2a-c) were designed with electron-withdrawing groups near one of the basic nitrogen atoms to lower its pK a . Inhibition studies with these compounds showed that two of them not only retained most of the potency and selectivity of the best analogue of the earlier series, but also showed improved membrane permeability based on data from a cellbased assay.
Design, synthesis, and evaluation of potential inhibitors of nitric oxide synthase
Bioorganic & Medicinal Chemistry, 2008
Selective inhibitors of neuronal nitric oxide synthase (nNOS) were shown to protect brain and may be useful in the treatment of neurodegenerative diseases. In this context, our purpose has been to design and synthesize a new family of derivatives of thiadiazoles as possible inhibitors of nNOS. To achieve it a supervised artificial neural network model has been developed for the prediction of inhibition of Nitric Oxide Synthase using a dataset of 119 nNOS inhibitors. The definition of the molecules was achieved from a not-supervised neural network using a home made program named CODES. Also, thiadiazole-based heterocycles, previously predicted, were prepared as conformationally restricted analogues of a selective nNOS inhibitor, S-ethyl N-phenylisothiourea.
A new class of selective and potent inhibitors of neuronal nitric oxide synthase
Bioorganic & Medicinal Chemistry Letters, 1999
The synthesis and SAR of a series of 6-(4-(substituted)phenyl)-2-aminopyridines as inhibitors of nitric oxide synthase are described. Compound 3a from this series shows potent and selective inhibition of the human nNOS isoform, with pharmacokinetics sufficient to provide in vivo inhibition of nNOS activity.
International journal of molecular sciences, 2014
Neuronal nitric oxide synthase (nNOS) plays an important role in neurotransmission and smooth muscle relaxation. Selective inhibition of nNOS over its other isozymes is highly desirable for the treatment of neurodegenerative diseases to avoid undesirable effects. In this study, we present a workflow for the identification and prioritization of compounds as potentially selective human nNOS inhibitors. Three-dimensional pharmacophore models were constructed based on a set of known nNOS inhibitors. The pharmacophore models were evaluated by Pareto surface and CoMFA (Comparative Molecular Field Analysis) analyses. The best pharmacophore model, which included 7 pharmacophore features, was used as a search query in the SPECS database (SPECS®, Delft, The Netherlands). The hit compounds were further filtered by scoring and docking. Ten hits were identified as potential selective nNOS inhibitors.
Advances in Design and Development of Inhibitors of Nitric Oxide Synthases
Nitric oxide synthase (NOS) is a dimeric enzyme that catalyses the production of nitric oxide (NO) in the human body. The nitric oxide has been identified as the most interesting and vital mediators for normal and pathological processes, including the regulation of blood pressure, neurotransmission, and macrophage defence system, but the over production of it can be toxic, hence their inhibitors are desired. In this article, the various isoforms of NOS and their roles are described and a detail of the development of their inhibitors has been presented. The inhibitors studied include aminopyridines, iminopiperidines, N-phenylamidines, benzoxazolones, isothioureas, oxazepanes, thiazepanes, diazepanes, 4,5-disubstituted-1,3-oxazolidin-2-imine derivatives, thiazolidines, pyrazoles, pyrazolines, and some others
Novel inhibitors of neuronal nitric oxide synthase with potent antioxidant properties
Bioorganic & Medicinal Chemistry Letters, 2003
A series of hybrid compounds possessing an nNOS pharmacophore linked to an antioxidant fragment has been synthesized. Among them, compound 8d, a propofol derivative, displayed the greatest dual potencies against nNOS (IC 50 =0.12 mM) and lipid peroxidation (IC 50 =0.4 mM) accompanied with e/nNOS selectivity (67.5). This shows that nNOS was able to accommodate very bulky groups such as di-tert-butyl or di-iso-propyl phenol in its active site. #
Journal of Medicinal Chemistry, 2009
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.