3D-QSAR Studies of the Pteridine Analogues as iNOS Inhibitors

Inducible nitric oxide synthase(iNOS), which can produce nitric oxide(NO) in the induction of cytokines and other factors, has an important impact onthe physiological functions of the body for the transmission of information. However,continuous generation of NOwill produce a lot of great damages to organisms. Therefore, iNOS inhibitors with good inhibitory activity and selectivity have beenimportant means of treating a variety of diseases. Based on the public-alignment of pteridine, 3D-QSAR(Three-Dimensional Quantitative Structure-Activity Relationship) models of pteridine analogues as iNOS inhibitors were established by the 3D-QSAR protocol of Discovery Studio 3.0. Pteridine molecules divided in different groups obtained four approximate models, indicating good stability of such models, in which A3 is preferable(q~2 = 0.672, r~2 = 0.996, r_(pred)~2 . = 0.888, q~2 denotes the cross-validation coefficient, r~2 denotes the non-cross-validation coefficient). This study should be significant for the future structure design and modification of pteridine analogues as iNOS inhibitors.     

Selective monocationic inhibitors of neuronal nitric oxide synthase. Binding mode insights from molecular dynamics simulations

The reduction of pathophysiologic levels of nitric oxide through inhibition of neuronal nitric oxide synthase (nNOS) has the potential to be therapeutically beneficial in various neurodegenerative diseases. We have developed a series of pyrrolidine-based nNOS inhibitors that exhibit excellent potencies and isoform selectivities (J. Am. Chem. Soc. 2010, 132, 5437). However, there are still important challenges, such as how to decrease the multiple positive charges derived from basic amino groups, which contribute to poor bioavailability, without losing potency and/or selectivity. Here we present an interdisciplinary study combining molecular docking, crystallography, molecular dynamics simulations, synthesis, and enzymology to explore potential pharmacophoric features of nNOS inhibitors and to design potent and selective monocationic nNOS inhibitors. The simulation results indicate that different hydrogen bond patterns, electrostatic interactions, hydrophobic interactions, and a water molecule bridge are key factors for stabilizing ligands and controlling ligand orientation. We find that a heteroatom in the aromatic head or linker chain of the ligand provides additional stability and blocks the substrate binding pocket. Finally, the computational insights are experimentally validated with double-headed pyridine analogues. The compounds reported here are among the most potent and selective monocationic pyrrolidine-based nNOS inhibitors reported to date, and 10 shows improved membrane permeability.     

Facile synthesis of N-6 adenosine modified analogue toward S-adenosyl methionine derived probe for protein arginine methyltransferases

Chemically modified cellular co-factors that provide function,such as immobilization or incorporation of fluorescent dyes,are valuable probes of biological activity.A convenient route to obtain S-adenosyl methionine(AdoMet) analogues modified at N-6 adenosine to feature a linker terminating in azide functionality is described herein.Subsequent decoration of such AdoMet analogues with guanidinium terminated linkers leads to novel potential bisubstrate inhibitors for protein arginine methyltrans-ferases, PRMTs.     

Synthesis of bisubstrate analogues targeting alpha-1,3-fucosyltransferase and their activities

We designed two bisubstrate analogues targeting alpha-1,3-fucosyltransferases, based on the three dimensional structure of Lewis X, which is the product of a alpha-1,3-fucosyltransferase reaction. We selected guanosine-5'-diphospho-L-galactose as a donor mimic and 2-hydroxyethyl beta-D-galactoside as an acceptor mimic, and tethered these two mimics with a methylene or ethylene linker. For the synthesis, the 6-position of L-galactose and the 6-position of D-galactose were first tethered via a methylene or ethylene linker. The L-galactose moiety was then converted to a GDP derivative. Both bisubstrate analogues were moderate inhibitors against alpha-1,3-fucosyltransferase V and VI. The fact that they were substrates of alpha-1,3-fucosyltransferase VI suggested that these compounds bound to the donor binding site, but not to the acceptor binding site.     

Synthesis of syn and anti isomers of trans-cyclopropyl arginine

There is currently considerable interest in arginine and its structural analogues in the context of nitric oxide synthase (NOS) substrates and inhibitors. Of particular interest are conformationally constrained arginine analogues used to probe the active sites of the three NOS isoforms. A simple procedure is described for the preparation of syn- and anti-trans-cyclopropyl arginine starting from the alpha-OBO-protected Cbz-dehydroglutamate. Cyclopropanation is effected by diazomethane addition followed by irradiation of the resulting pyrazoline and gives a 3:1 mixture of syn:anti isomers that can be separated by crystallization. Reduction of the ester to the alcohol followed by guanylation gives the fully protected cyclopropyl arginine analogues. The CBZ protecting groups are removed by hydrogenolysis and the OBO by mild acid treatment followed by base hydrolysis.     

Direct High-Throughput Screening Assay for mRNA Cap Guanine-N7 Methyltransferase Activity

In eukaryotes, mature mRNA is formed through modifications of precursor mRNA, one of which is 5’ cap biosynthesis, involving RNA cap guanine-N7 methyltransferase (N7-MTase). N7-MTases are also encoded by some eukaryotic viruses and facilitate their replication. N7-MTase inhibitors have therapeutic potential, but their discovery is difficult because long RNA substrates are usually required for activity. Herein, we report a universal N7-MTase activity assay based on small-molecule fluorescent probes. We synthesized 12 fluorescent substrate analogues (GpppA and GpppG derivatives) varying in the dye type, dye attachment site, and linker length. GpppA labeled with pyrene at the 3’-O position of adenosine acted as an artificial substrate with the properties of a turn-off probe for all three tested N7-MTases (human, parasite, and viral). Using this compound, a N7-MTase inhibitor assay adaptable to high-throughput screening was developed and used to screen synthetic substrate analogues and a commercial library. Several inhibitors with nanomolar activities were identified.     

Indications for the Occurrence of Nitric Oxide Synthases in Fungi and Plants and the Involvement in Photoconidiation of Neurospora crassa*

Indications for the occurrence of nitric oxide synthases in Dictyostelium, Neurospora, Phycomyces and the leguminous plant Mucuna hassjoo as well as a physiological role of nitric oxide in Neurospora crassa are demonstrated. An exogenous nitric oxide donor, sodium nitroprus-side, inhibited light-stimulated conidiation in N. crassa. Specific inhibitors of nitric oxide synthase, like the arginine derivatives N^G-nitro-L-arginine (L-NA) and N^G-nitro-L-arginine-methyl ester (L-NAME), enhanced conidiation in darkness and in the light, whereas the stereo-isomer D-NAME was inactive. This communication reports to our knowledge the first time the presence of enzymatic activity of nitric oxide synthase in fungi and a higher plant and an effect of nitric oxide in fungal photophysiology.     

A practical synthesis of kifunensine analogues as inhibitors of endoplasmic reticulum alpha-mannosidase I

[reaction: see text] A practical synthesis of the potent class I alpha-mannosidase inhibitor kifunensine (1) beginning from the inexpensive and readily available starting material L-ascorbic acid (15) is described. The protected amino-alcohol ((2R,3R,4R,5R)-5-amino-2,3:4,6-diisopropylidenedioxyhexanol, 11) served as a key intermediate from which several N-1 substituted kifunensine analogues (including N-methyl, N-cyclohexyl, and N-bis(hydroxymethyl)methyl) and 2-desoxakifunensine analogues (including N-H and N-methyl) were prepared and screened for inhibition of human endoplasmic reticulum alpha-mannosidase I (ER Man I) and mouse Golgi alpha-mannosidase IA (Golgi Man IA). In addition, several pseudodisaccharide kifunensine analogues in which a mannose residue was tethered to N-1 of kifunensine via a two-, three-, or four-carbon linker and an affinity-bound kifunensine analogue were also prepared and evaluated for biological activity. While the synthesized N-1 kifunesine analogues were found to be less potent inhibitors of Class I alpha-mannosidases than kifuensine itself, the bis(hydroxymethyl)methylkifunensine analogue 6 was shown to selectively inhibit ER Man I over Golgi Man IA.     

Solid-phase synthesis of aspartic peptidase inhibitors: 3-alkoxy-4-aryl piperidines

[reaction: see text]. The 3-alkoxy-4-aryl piperidines are non-peptide peptidomimetic inhibitors of several aspartic peptidases. The solid-phase functionalization of 3,4-disubstituted piperidine scaffolds using a traceless linker strategy is described. Synthesis of diverse analogues based on this scaffold provides the potential to generate selective inhibitors of this important class of peptidase.     

Design and synthesis of C5 methylated L-arginine analogues as active site probes for nitric oxide synthase

The role of nitric oxide (NO) as a biological signaling molecule is well established. NO is produced by the nitric oxide synthases (NOSs, EC 1.14.13.39), a class of heme proteins capable of converting l-arginine to NO and l-citrulline. Despite the large body of knowledge associated with the NOSs, mechanistic details relating to the unique oxidative chemistry performed by these enzymes remain to be fully elucidated. Furthermore, a number of disease states are associated with either the over- or underproduction of NO, making the NOS pathway an attractive target for the development of therapeutics. For these reasons, molecular tools capable of providing mechanistic insights into the production of NO and/or the inhibition of the NOSs remain of interest. We report here the stereospecific synthesis and testing of a number of new l-arginine analogues bearing a minimal substitution, methylation at position 5 of the amino acid side chain (such analogues have not been previously reported). The synthetic approach employed a modified photolysis procedure whereby irradiation of the appropriate diacylperoxide precursors at 254 nm gave access to the required unnatural amino acids in good yields. A heme domain construct of the inducible NOS isoform (iNOSheme) was used to assess the binding of each compound to the enzyme active site. The compounds were also investigated as either inhibitors of, or alternate substrates for, the inducible NOS isoform. The results obtained provide new insight into the steric and stereochemical tolerance of the enzyme active site. These findings also further support the role of a conserved active site water molecule previously proposed to be necessary for NOS catalysis.     

Mechanism of nitric oxide loss in 3,6-diphenyl and 3,5,6-triphenyl-4-nitro cyclohexenes under electron ionization

Possible mechanisms have been proposed for the loss of nitric oxide from the molecular ions of 3,6-diphenyl- and 3,5,6-triphenyl-4-nitro cyclohexenes. Elimination of nitric oxide in 3,6-diphenyl nitro cyclohexenes involves oxygen migration to one of the double bond carbon atoms. In the 3,5,6-triphenyl analogues, it involves a 3-membered cyclic transition state.     

Conformationally constrained analogues of bleomycin A5

The bleomycin (BLM) group antitumor antibiotics are glycopeptide-derived natural products shown to cause sequence selective lesions in DNA. Prior studies have indicated that the linker region, composed of the methylvalerate and threonine residues, may be responsible for a conformational bend in the agent required for efficient DNA cleavage. We have synthesized a number of conformationally constrained methylvalerate analogues and incorporated them into deglycobleomycin A(5) congeners using our recently reported procedure for the solid phase construction of (deglyco)bleomycin and its analogues. These analogues were designed to probe the effects of conformational constraint of the native valerate moiety. Initial experiments indicated that the constrained molecules, none of which mimic the conformation proposed for the natural valerate linker, possessed DNA cleavage activity, albeit with potencies less than that of (deglyco)BLM and lacking sequence selectivity. Further experiments demonstrated that these analogues failed to produce alkali-labile lesions in DNA or sequence selective oxidative damage in RNA. However, two of the conformationally constrained deglycoBLM analogues were shown to mediate RNA cleavage in the absence of added Fe(2+). The ability of the analogues to mediate the oxygenation of small molecules was also assayed, and it was shown that they were as competent in the transfer of oxygen to low molecular weight substrates as the parent compound.     

Synthesis and Antitumor Evaluation of Novel Hybrids of Phenylsulfonylfuroxan and Estradiol Derivatives

Fifteen novel furoxan-based nitric oxide (NO) releasing hybrids of estradiol derivatives were synthesized and evaluated in vitro anti-proliferative activity in MDA-MB-231, A2780, Hela and HUVEC cell lines. Most of them displayed potent anti-proliferative effects. Among the compounds, 4-bromo-3-((phenylsulfonyl)-1,2,5-oxadiazole 2-oxide)-oxy-propoxy-estradiol ( 11 b ) exhibited the best activity with IC₅₀ values of 3.58–0.0008 μM. Preliminary pharmacological studies showed that 11 b induced apoptosis and hardly affected the cell cycle of MDA-MB-231 cell line. NO-releasing capacity and inhibition of ERK/MAPK pathway signaling might explain the potent antineoplastic activity of these compounds. The preliminary structure-activity relationship (SAR) showed that steroidal scaffolds with a linker in 3-position were favorable moieties to evidently increase the bioactivities of these hybrids. Overall, these results implied that 11 b merited to be further investigated as a promising anti-cancer candidate.     

Betulinic acid derivatives: a new class of α-glucosidase inhibitors and LPS-stimulated nitric oxide production inhibition on mouse macrophage RAW 264.7 cells

Chemical manipulation studies were conducted on betulinic acid (1), twenty-one new rationally designed analogues of 1 with modifications at C-28 were synthesized for their evaluation of inhibitory effects on α-glucosidase and LPS-stimulated nitric oxide production in mouse macrophage RAW 264.7 cells. Compound 2 (IC₅₀ = 5.4 μM) exhibited an almost 1.4-fold increase in α-glucosidase inhibitory activity on yeast α-glucosidase while analogues 5 (IC₅₀ 16.4 μM) and 11 (IC₅₀ 16.6 μM) exhibited a 2-fold enhanced inhibitory activity on NO-production than betulinic acid.     

Efficient discovery and capture of new neuronal nitric oxide synthase–postsynaptic density protein‐95 uncouplers from herbal medicines using magnetic molecularly imprinted polymers as artificial antibodies

In the scope of stroke treatment, new neuronal nitric oxide synthase–postsynaptic density protein‐95 uncouplers from herbal medicines were discovered and captured. To do so, highly selective magnetic molecularly imprinted polymers with a core–shell structure were prepared as artificial antibodies. According to the results of computational simulations, we designed and synthesized various polymers with varying amounts and types of template, functional monomer, cross‐linker, and solvent. Characterization and performance tests revealed that the most appropriate artificial antibodies showed uniform spherical morphologies, large adsorption capacities, fast‐binding kinetics, high selectivity, and quick separation. These artificial antibodies were then used as sorbents for dispersive magnetic solid‐phase extraction coupled with high‐performance liquid chromatography and mass spectrometry to capture and identify structural analogs to ZL006 from extracts of Scutellariae radix, Psoraleae fructus, and Trifolium pratense. Furthermore, according to the neuroprotective effect and coimmunoprecipitation test, Baicalein, Neobavaisoflavone, Corylifol A, and Biochanin A can be the potential uncouplers of neuronal nitric oxide synthase–postsynaptic density protein‐95. Therefore, this present study contributes valuable information for the discovery of neuronal nitric oxide synthase–postsynaptic density protein‐95 uncouplers from herbal medicines.     

Synthesis of new pyrimidine nucleoside derivatives with nitric oxide donors for antiviral activity

New pyrimidine nucleoside derivatives with nitric oxide(NO) donor were systematically synthesized.The antivirus activities of these nucleoside analogues against vesicular stomatitis virus(VSV) in Wish cell were evaluated.It was demonstrated that most of compounds had stronger antiviral acitivity than acyclovir,while their toxicities were similar or lower to acyclovir.     

Imaging of nitric oxide generation in the rat brain

Nitric oxide radicals produced in the rat brain subjected to ischemia-hypoxia were trapped by the systemically administered diethyldithiocarbamate and iron. The right hemisphere of the brain was then removed and frozen with liquid nitrogen. Three-dimensional spatial distribution of the nitric oxide radicals in this hemisphere was performed using electron paramagnetic resonance imaging techniques. The results suggest that nitric oxide radicals were produced and trapped in the areas which are known to have high nitric oxide synthase activity, such as piriform cortex, hippocampus, hypothalamus, amygdala, and substantia nigra. In this model, which did not interrupt the posterior circulation, the production and trapping of nitric oxide in the cerebellum was relatively weak.     

Synthesis and anticancer properties of RGD peptides conjugated to nitric oxide releasing functional groups and abiraterone

A series of analogues of the integrin binding aspartic acid-glycine-arginine (RGD) peptide sequence were synthesised conjugated to nitric oxide (NO) donating functional groups. Also the cytotoxicity of abiraterone, a prostate cancer drug, was explored when it was conjugated in three part constructs to RGD sequences and NO releasing heterocycles. In general the analogues showed integrin binding affinity comparable to RGD reference compounds, and all released NO by the Griess test assay. Two analogues exhibited significant cytotoxic effects against PC3 and MCF7 cell lines.     

Solid-phase parallel synthesis of 17alpha-substituted estradiol sulfamate and phenol libraries using the multidetachable sulfamate linker

We report an application of the multidetachable sulfamate linker in the synthesis of two model libraries of N-derivatized 17alpha-piperazinomethyl estradiols (phenols and sulfamates) by solid-phase parallel chemistry. The solid-phase precursor, a 3-sulfamoyl-17alpha-(N-trifluoroacetyl-piperazinomethyl) estradiol, was synthesized in solution from estrone and loaded efficiently onto trityl chloride resin as polymeric support. After cleavage of the trifluoroacetyl protecting group, sequential acylation reactions with five Fmoc-protected amino acids and five carboxylic acids were performed to introduce two levels of molecular diversity. Finally, the resins were split into two parts, and acidic (5% trifluoroacetic acid in dichloromethane) and nucleophilic (piperazine in tetrahydrofuran) cleavages were used to generate libraries A (5 x 5 sulfamates) and B (5 x 5 phenols) members in overall yields of 18-66% and high HPLC purities (87-96%) without purification steps. A preliminary screening test for inhibition of steroid sulfatase showed that the phenols were clearly weaker inhibitors, as compared to their sulfamate analogues. The most potent inhibitors were those with suitable hydrophobic amino acid and carboxylic acid substituents. Thus, compounds with a phenylalanine residue as the first element of diversity inhibited over 90% of steroid sulfatase activity at a concentration of 1 nM in homogenates of HEK-293 transfected cells, being as potent as the leading inhibitor 17alpha-tert-butylbenzyl estradiol 3-O-sulfamate previously reported. These results suggest that the steroid sulfatase inhibitory potency of estradiol derivatives, sulfamoylated or not, can be increased by the hydrophobic effect of a suitable substituent introduced in the proximity of the D ring of the steroid. The present work also demonstrated the efficiency and the cleavage versatility of the sulfamate linker to generate libraries of compounds with relevant biological importance, phenols and sulfamates.     

A Sub-Micromolar MraYAA Inhibitor with an Aminoribosyl Uridine Structure and a (S,S)-Tartaric Diamide: Synthesis,Biological Evaluation and Molecular Modeling

New inhibitors of the bacterial tranferase MraY are described. Their structure is based on an aminoribosyl uridine scaffold, which is known to be important for the biological activity of natural MraY inhibitors. A decyl alkyl chain was introduced onto this scaffold through various linkers. The synthesized compounds were tested against the MraY_AA transferase activity, and the most active compound with an original (S,S)-tartaric diamide linker inhibits MraY activity with an IC₅₀ equal to 0.37 µM. Their antibacterial activity was also evaluated on a panel of Gram-positive and Gram-negative strains; however, the compounds showed no antibacterial activity. Docking and molecular dynamics studies revealed that this new linker established two stabilizing key interactions with N190 and H325, as observed for the highly potent inhibitors carbacaprazamycin, muraymycin D2 and tunicamycin.