Synthesis of beta-D-galactofuranosyl nucleoside analogues. A new type of beta-D-galactofuranosidase inhibitor.

The development of beta-D-galactofuranosidase inhibitors provides a good chemotherapeutic target for treatment of major human diseases, because beta-D-galactofuranose is a constituent of important pathogen microorganisms but is absent in mammals. With this purpose we have prepared beta-D-galactofuranosyl nucleoside analogues, derived by the addition of nucleophiles to perbenzoylated beta-D-galactofuranosyl isothiocyanate, a compound previously prepared in this laboratory. N-beta-D-Galactofuranosyl-O-ethylthiourethane, N-beta-D-galactofuranosyl-4-oxoimidazolidine-2-thione, N-beta-D-galactofuranosyl-4-imidazoline-2-thione, and N-beta-D-galactofuranosyl-4-methoxyimidazolidine-2-thione, were prepared. The biological assays showed that imidazoline and imidazolidine-2-thione derivatives act as a new type of exo beta-D-galactofuranosidase inhibitor.     

Facile synthesis of benzyl beta-D-galactofuranoside. A convenient intermediate for the synthesis of D-galactofuranose-containing molecules

Benzyl beta-D-galactofuranoside was efficiently obtained from 1,2,3,5,6-penta-O-benzoyl-alpha,beta-D-galactofuranose, via benzyl 2,3,5,6-tetra-O-benzoyl-beta-D-galactofuranoside. Conditions for the O-debenzylation were investigated in order to evaluate the synthetic application of the benzyl group as an anomeric protector of a galactofuranose moiety in synthetic strategies involving galactofuranose.     

An extracellular beta-galactofuranosidase from Aspergillus niger and its use as a tool for glycoconjugate analysis

Aspergillus niger produces an extracellular beta-galactofuranosidase, which can specifically hydrolyse beta-D-galactofuranose (Galf) from glycoconjugates. The production of this enzyme can be induced by the addition of a Galf-containing A. niger mycelial wall extract. However, on other carbon sources accumulation occurred only during the starvation conditions of the late stationary phase. Extracellular glucoamylases from this stage of cultivation possessed significantly lower levels of Galf than those from the earlier exponential growth phase when beta-galactofuranosidase is absent, suggesting in situ beta-galactofuranosidic hydrolysis. The beta-galactofuranosidase responsible was subsequently purified to homogeneity and characterised. It is a glycoprotein of 90 kDa (determined by SDS-PAGE) with activity against beta-linked Galf residues, with a Km of 4 mM against p-nitrophenyl-beta-D-galactofuranoside and a pH optimum of 3-4. The preparation did not contain other contaminating glycosidase activities; p-nitrophenyl-beta-D- and -alpha-D-galactopyranose, and alpha-D-methyl-Galf were not hydrolysed. Results are presented to show that this enzyme could be employed as a useful tool for the analysis of glycoconjugates containing biologically important Galf components.     

4-Quinolone-3-carboxylic acids as cell-permeable inhibitors of protein tyrosine phosphatase 1B

Protein tyrosine phosphatase 1B is a negative regulator in the insulin and leptin signaling pathways, and has emerged as an attractive target for the treatment of type 2 diabetes and obesity. However, the essential pharmacophore of charged phosphotyrosine or its mimetic confer low selectivity and poor cell permeability. Starting from our previously reported aryl diketoacid-based PTP1B inhibitors, a drug-like scaffold of 4-quinolone-3-carboxylic acid was introduced for the first time as a novel surrogate of phosphotyrosine. An optimal combination of hydrophobic groups installed at C-6, N-1 and C-3 positions of the quinolone motif afforded potent PTP1B inhibitors with low micromolar IC₅₀ values. These 4-quinolone-3-carboxylate based PTP1B inhibitors displayed a 2–10 fold selectivity over a panel of PTP’s. Furthermore, the bidentate inhibitors of 4-quinolone-3-carboxylic acids conjugated with aryl diketoacid or salicylic acid were cell permeable and enhanced insulin signaling in CHO/hIR cells. The kinetic studies and molecular modeling suggest that the 4-quinolone-3-carboxylates act as competitive inhibitors by binding to the PTP1B active site in the WPD loop closed conformation. Taken together, our study shows that the 4-quinolone-3-carboxylic acid derivatives exhibit improved pharmacological properties over previously described PTB1B inhibitors and warrant further preclinical studies.     

Structural characterization of the antigenic O-polysaccharide in the lipopolysaccharide produced by Actinobacillus pleuropneumoniae serotype 14

The antigenic O-polysaccharide of the lipopolysaccharide produced by Actinobacillus pleuropneumoniae serotype 14 was shown by chemical analysis and 1D and 2D nuclear magnetic resonance methods to be a high-molecular-mass polymer of a repeating disaccharide unit composed of a chain of (1-->5)-linked beta-D-galactofuranose (beta-D-Galf) residues substituted at their O-2 positions by alpha-D-galactopyranose residues (D-Galp) (1:1): [formula: see text].     

Type 2 diabetes--therapy with dipeptidyl peptidase IV inhibitors

The sole application of an inhibitor of the dipeptidyl peptidase DP IV (also DP 4, CD26, DPP-IV or DPP-4) to a mammal subsequently leading to improved glucose tolerance marks a major breakthrough in metabolic research bearing the potential of a new revolutionary diabetes therapy. This was demonstrated in rat applying the specific DP IV inhibitor isoleucyl thiazolidine. It was published in 1996 for the first time that a specific DP IV inhibitor in a given dose was able to completely block glucagon-like peptide-1 (GLP-1) degradation in vivo resulting in improved insulin response accompanied, by accelerated peripheral glucose disposal. Later on, these results were confirmed by several research teams applying DP IV inhibitors intravenously or orally. Today, the DP IV inhibition for the treatment of metabolic disorders is a validated principle. Now, more than 10 years after the initial animal experiments, first DP IV inhibitors as investigational drugs are tested in phase 3 clinical trials.     

Structural analysis of the lipopolysaccharide of Actinobacillus (Haemophilus) pleuropneumoniae serotype 10.

The antigenic O-chain of the lipopolysaccharide produced by Actinobacillus (Haemophilus) pleuropneumoniae serotype 10 was shown by chemical and magnetic resonance methods to be a unique linear unbranched homopolymer of exclusively 1,2-linked beta-D-galactofuranose residues.     

Synthesis of galactofuranose disaccharides of biological significance

Methyl beta-D-galactofuranoside was readily obtained by tin(IV) chloride-catalyzed glycosylation of penta-O-benzoyl-alpha,beta-D-galactofuranose, followed by debenzoylation with sodium methoxide. Glycosylation of 1 with 2,3,5-tri-O-benzoyl-D-galactono-1,4-lactone or with the 6-O-trityl-lactone derivative 5 gave the benzoylated beta-D-galactofuranosyl-(1----6)-D-galactono-1,4-lactone 6 in excellent yield. The structure of disaccharide 6 was confirmed by borohydride reduction to the glycosyl-alditol 7. A byproduct of the condensation reaction of 1 with 4 or 5 was identified as the benzoylated (1----1)-beta,beta'-D-galactofuranosyl disaccharide 8. Compound 8 was readily prepared (88% yield) by controlled addition of water to 1, in the presence of stannic chloride. O-Debenzoylation of 8 afforded crystalline beta'-D-galactofuranosyl-(1----1)-beta-D-galactofuranoside. The glycosyl-lactone 6 constitutes a key intermediate for the synthesis of a disaccharide derivative having both units in the furanoid form. Thus, diisoamylborane reduction of the lactone function of 6 led to the disaccharide derivative 10, from which the methyl glycoside 12 was prepared. O-Debenzoylation of 12 gave the corresponding methyl beta-D-galactofuranosyl-(1----6)-beta-D-galactofuranoside. The free disaccharide beta-D-Galf-(1----6)-D-Galp and its acetylated derivative were also synthesized from 10.     

Novel 4-thiogalactofuranosyl-containing disaccharides with nitrogen in the interglycosidic linkage

The syntheses of three novel disaccharides containing a 4-thiogalactofuranosyl residue as the non-reducing unit and a nitrogen in the interglycosidic linkage are described. Acid-catalyzed condensation reactions of 4-thio-alpha/beta-D-galactofuranose with either methyl 3-amino-3-deoxy-alpha-D-mannopyranoside, methyl 2-amino-2-deoxy-alpha-D-mannopyranoside, or methyl 2-acetamido-6-amino-2,6-dideoxy-beta-D-glucopyranoside gave methyl 3-amino-3-deoxy-3-N-(4-thio-alpha/beta-D-galactofuranosyl)-alpha-D-manno pyranoside, methyl 2-amino-2-deoxy-2-N-(4-thio-alpha/beta-D-galactofuranosyl)-alpha-D-manno pyranoside, or methyl 2-acetamido-6-amino-2,6-dideoxy-6-N-(4-thio-alpha/beta-D-galactofuranosy l)-beta-D-glucopyranoside.     

Protease inhibitors. Part 8: synthesis of potent Clostridium histolyticum collagenase inhibitors incorporating sulfonylated L-alanine hydroxamate moieties

A series of hydroxamates was prepared by reaction of alkyl/arylsulfonyl halides with N-2-chlorobenzyl-L-alanine, followed by conversion of the COOH moiety to the CONHOH group, with hydroxylamine in the presence of carbodiimides. Other structurally related compounds were obtained by reaction of N-2-chlorobenzyl-L-alanine with aryl isocyanates, arylsulfonyl isocyanates or benzoyl isothiocyanate, followed by the similar conversion of the COOH into the CONHOH moiety. The new compounds were assayed as inhibitors of the Clostridium histolyticum collagenase, ChC (EC 3.4.24.3), a bacterial zinc metallo-peptidase which degrades triple helical collagen as well as a large number of synthetic peptides. The prepared hydroxamate derivatives proved to be 100-500 times more active collagenase inhibitors than the corresponding carboxylates. Substitution patterns leading to best ChC inhibitors (both for carboxylates as well as for the hydroxamates) were those involving perfluoroalkylsulfonyl- and substituted-arylsulfonyl moieties, such as pentafluorophenylsulfonyl; 3- and 4-protected-aminophenylsulfonyl-; 3- and 4-carboxyphenylsulfonyl-; 3-trifluoromethyl-phenylsulfonyl; as well as 1- and 2-naphthyl-, quinoline-8-yl- or substituted-arylsulfonylamidocarboxyl moieties among others. Similarly to the matrix metalloproteinase (MMP) hydroxamate inhibitors, ChC inhibitors of the type reported here must incorporate hydrophobic moieties at the P2' and P3' sites, in order to achieve tight binding to the enzyme. This study also proves that the 2-chlorobenzyl moiety, investigated here for the first time, is an efficient P2' anchoring moiety for obtaining potent ChC inhibitors.     

Pancreatic proteolytic enzymes of ostrich purified on immobilized protein inhibitors. Characterization of a new form of chymotrypsin (Chtr1)

Four forms of chymotrypsin (Chtr1, Chtr2, Chtr3, Chtr4), one form of trypsin and one form of elastase were purified from a slightly alkaline extract of ostrich (Struthio camelus) pancreas. The zymogens in the crude extract were activated with immobilized trypsin and then separated by affinity chromatography using immobilized inhibitors and ion exchange chromatography. One of the purified forms of chymotrypsin (Chtr1) exhibited an unusual interaction with the highly selective protein trypsin inhibitor from Cucurbita maxima (CMTI). Interactions with other protein trypsin inhibitors such as basic pancreatic trypsin inhibitor (BPTI), soybean trypsin inhibitor (STI), trypsin inhibitors from Cyclanthera pedata (CyPTI), Cucurbita pepo (CPTI), Cucurbita pepo var. giramontia (CPGTI) and Linum usitatissimum (LUTI) were also investigated. This study demonstrated the affinity of Chtr1 to inhibitors containing Arg at P1 position. Studies of substrate specificity of Chtr1 using oxidized B-chain of insulin revealed four susceptible bonds: Tyr15-Leu16, Phe24-Phe25, Phe25-Tyr26 and, surprisingly, Arg22-Gly23. The amino acid composition, as well as the first 13 residues of the N-terminal amino acid sequence, was determined. Studies of ostrich elastase showed that it can interact with immobilized CMTI in the presence of 5 M NaCl. This unusual characteristic is reported for the first time and suggests that elastase specificity depends on ionic strength. The kinetic constants K(M), k(cat) and k(cat)/K(M) for purified ostrich trypsin, chymotrypsin 4 and elastase were also determined.     

Structure of a phosphoethanolamine-containing O-polysaccharide of Citrobacter freundii strain PCM 1443 from serogroup O39 and its relatedness to the Klebsiella pneumoniae O1 polysaccharide

Lipopolysaccharide was extracted from cells of Citrobacter freundii PCM 1443 from serogroup O39 and degraded by mild acid hydrolysis to give an O-polysaccharide. Based on enzymatic and methylation analyses, along with 1H and 13C nuclear magnetic resonance spectroscopy, it was found that the lipopolysaccharide studied has two different linear polysaccharide chains of d-galactan type containing 3-substituted galactose residues. One of the galactans has the disaccharide repeating units of alpha-D-galactopyranose and beta-D-galactofuranose and the other is comprised of alpha-D-galactopyranose and beta-D-galactopyranose, the latter being substituted in 25% repeats with PEtN at O-6. An immunoblotting assay demonstrated that the lipopolysaccharide of C. freundii PCM 1443 is serologically related to that of Klebsiella pneumoniae O1, which contains the same galactan chains but is devoid of phosphoethanolamine.     

Formamides mimic aldehydes and inhibit liver alcohol dehydrogenases and ethanol metabolism

Formamides are unreactive analogues of the aldehyde substrates of alcohol dehydrogenases and are useful for structure-function studies and for specific inhibition of alcohol metabolism. They bind to the enzyme-NADH complex and are uncompetitive inhibitors against varied concentrations of alcohol. Fourteen new branched chain and chiral formamides were prepared and tested as inhibitors of purified Class I liver alcohol dehydrogenases: horse (EqADH E), human (HsADH1C*2), and mouse (MmADH1). In general, larger, substituted formamides, such as N-1-ethylheptylformamide, are better inhibitors of HsADH1C*2 and MmADH1 than of EqADH, reflecting a few differences in amino acid residues that change the sizes of the active sites. In contrast, the linear, alkyl (n-propyl and n-butyl) formamides are better inhibitors of EqADH and MmADH1 than of HsADH1C*2, probably because water disrupts van der Waals interactions. These enzymes are also inhibited strongly by sulfoxides and 4-substituted pyrazoles. The structure of EqADH complexed with NADH and (R)-N-1-methylhexylformamide was determined by x-ray crystallography at 1.6 A resolution. The structure resembles the expected Michaelis complex with NADH and aldehyde, and shows for the first time that the reduced nicotinamide ring of NADH is puckered, as predicted for the transition state for hydride transfer. Metabolism of ethanol in mice was inhibited by several formamides. The data were fitted with kinetic simulation to a mechanism that describes the non-linear progress curves and yields estimates of the in vivo inhibition constants and the rate constants for elimination of inhibitors. Some small formamides, such as N-isopropylformamide, may be useful inhibitors in vivo.     

Selective PDE4 inhibitors as potent anti-inflammatory drugs for the treatment of airway diseases

Phosphodiesterases (PDEs) are responsible for the breakdown of intracellular cyclic nucleotides, from which PDE4 are the major cyclic AMP metabolizing isoenzymes found in inflammatory and immune cells. This generated greatest interest on PDE4 as a potential target to treat lung inflammatory diseases. For example, cigarette smoke-induced neutrophilia in BAL was dose and time dependently reduced by cilomilast. Beside the undesired side effects associated with the first generation of PDE4 inhibitors, the second generation of selective inhibitors such as cilomilast and roflumilast showed clinical efficacy in asthma and chronic obstructive pulmonary diseases trials, thus re-enhancing the interest on these classes of compounds. However, the ability of PDE4 inhibitors to prevent or modulate the airway remodelling remains relatively unexplored. We demonstrated that selective PDE4 inhibitor RP 73-401 reduced matrix metalloproteinase (MMP)-9 activity and TGF-beta1 release during LPS-induced lung injury in mice and that CI-1044 inhibited the production of MMP-1 and MMP-2 from human lung fibroblasts stimulated by pro-inflammatory cytokines. Since inflammatory diseases of the bronchial airways are associated with destruction of normal tissue structure, our data suggest a therapeutic benefit for PDE4 inhibitors in tissue remodelling associated with chronic lung diseases.     

Conformational flexibility of mammalian cytochrome P450 2B4 in binding imidazole inhibitors with different ring chemistry and side chains. Solution thermodynamics and molecular modeling

Recent x-ray structures of cytochrome P450 2B4 (CYP2B4) reveal an open form that undergoes a large-scale structural transition to a closed form upon binding to 4-(4-chlorophenyl)imidazole (4-CPI). Here, we report for the first time a complete solution thermodynamic study using isothermal titration calorimetry supported by spectroscopic studies to elucidate the conformational flexibility of CYP2B4 in binding imidazole inhibitors with different ring chemistry and side chains: 4-CPI, 1-benzylimidazole (1-BI), 1-CPI, 4-phenylimidazole (4-PI), 1-(2-(benzyloxy)ethyl)imidazole (BEI), and 1-PI. Each of the inhibitors induced type II spectral changes, and IC50 values for enzyme inhibition ranged from 0.1 to 2.4 microM, following the order 1-BI < 4-CPI < 1-CPI < 4-PI < BEI < 1-PI. Calorimetric titrations using monomeric enzyme yielded a 1:1 binding stoichiometry, with the associated KD values ranging from 0.3 to 4.8 microM and following the same rank order as the IC50 values. Changes in enthalpy at 25 degrees C ranged from -6.5 to -8.8 kcal mol(-1). The largest difference in binding entropy (+5.9 versus -4.1 cal mol(-1) K(-1)) was observed between 4-CPI and BEI, respectively, with a 2-fold difference in heat capacity changes (-604 versus -331 cal mol(-1) K(-1)), which is inferred to result from the reduction of apolar surface area of the enzyme ensuing from a conformational change upon 4-CPI binding. Accessibility to acrylamide of the only tryptophan (Trp121), which is located in helix C, was greatly decreased only in protein bound to 4-CPI. Steric restrictions hindered the perfect docking of only BEI to the closed conformation of the enzyme. The thermodynamic signature obtained for structurally similar inhibitors suggests remarkable plasticity of CYP2B4.     

Serpins of oat (Avena sativa) grain with distinct reactive centres and inhibitory specificity

Most proteinase inhibitors from plant seeds are assumed to contribute to broad-spectrum protection against pests and pathogens. In oat (Avena sativa L.) grain the main serine proteinase inhibitors were found to be serpins, which utilize a unique mechanism of irreversible inhibition. Four distinct inhibitors of the serpin superfamily were detected by native PAGE as major seed albumins and purified by thiophilic adsorption and anion exchange chromatography. The four serpins OSZa-d are the first proteinase inhibitors characterized from this cereal. An amino acid sequence close to the blocked N-terminus, a reactive centre loop sequence, and the second order association rate constant (ka') for irreversible complex formation with pancreas serine proteinases at 24 degrees C were determined for each inhibitor. OSZa and OSZb, both with the reactive centre scissile bond P1-P1' Thr downward arrow Ser, were efficient inhibitors of pancreas elastase (ka' > 105M-1 s-1). Only OSZb was also an inhibitor of chymotrypsin at the same site (ka' = 0.9 x 105M-1 s-1). OSZc was a fast inhibitor of trypsin at P1-P1' Arg downward arrow Ser (ka' = 4 x 106M-1 s-1); however, the OSZc-trypsin complex was short-lived with a first order dissociation rate constant kd = 1.4 x 10-4 s-1. OSZc was also an inhibitor of chymotrypsin (ka' > 106M-1 s-1), presumably at the overlapping site P2-P1 Ala downward arrow Arg, but > 90% of the serpin was cleaved as substrate. OSZd was cleaved by chymotrypsin at the putative reactive centre bond P1-P1' Tyr downward arrow Ser, and no inhibition was detected. Together the oat grain serpins have a broader inhibitory specificity against digestive serine proteinases than represented by the major serpins of wheat, rye or barley grain. Presumably the serpins compensate for the low content of reversible inhibitors of serine proteinases in oats in protection of the grain against pests or pathogens.     

The HIV Protease Inhibitor Saquinavir Inhibits HMGB1-Driven Inflammation by Targeting the Interaction of Cathepsin V with TLR4/MyD88

Extracellular high-mobility group box 1 (HMGB1) (disulfide form), via activation of toll-like receptor 4 (TLR4)-dependent signaling, is a strong driver of pathologic inflammation in both acute and chronic conditions. Identification of selective inhibitors of HMGB1-TLR4 signaling could offer novel therapies that selectively target proximal endogenous activators of inflammation. A cell-based screening strategy led us to identify first generation HIV-protease inhibitors (PI) as potential inhibitors of HMGB1-TLR4 driven cytokine production. Here we report that the first-generation HIV-PI saquinavir (SQV), as well as a newly identified mammalian protease inhibitor STO33438 (334), potently block disulfide HMGB1-induced TLR4 activation, as assayed by the production of TNF-α by human monocyte-derived macrophages (THP-1). We further report on the identification of mammalian cathepsin V, a protease, as a novel target of these inhibitors. Cellular as well as recombinant protein studies show that the mechanism of action involves a direct interaction between cathepsin V with TLR4 and its adaptor protein MyD88. Treatment with SQV, 334 or the known cathepsin inhibitor SID26681509 (SID) significantly improved survival in murine models of sepsis and reduced liver damage following warm liver ischemia/reperfusion (I/R) models, both characterized by strong HMGB1-TLR4 driven pathology. The current study demonstrates a novel role for cathepsin V in TLR4 signaling and implicates cathepsin V as a novel target for first-generation HIV-PI compounds. The identification of cathepsin V as a target to block HMGB1-TLR4-driven inflammation could allow for a rapid transition of the discovery from the bench to the bedside. Disulfide HMGB1 drives pathologic inflammation in many models by activating signaling through TLR4. Cell-based screening identified the mammalian protease cathepsin V as a novel therapeutic target to inhibit TLR4-mediated inflammation induced by extracellular HMGB1 (disulfide form). We identified two protease inhibitors (PIs) that block cathepsin V and thereby inhibit disulfide HMGB1-induced TLR4 activation: saquinavir (SQV), a first-generation PI targeting viral HIV protease and STO33438 (334), targeting mammalian proteases. We discovered that cathepsin V binds TLR4 under basal and HMGB1-stimulated conditions, but dissociates in the presence of SQV over time. Thus cathepsin V is a novel target for first-generation HIV PIs and represents a potential therapeutic target of pathologic inflammation.     

Selective inhibitors of the glycosylphosphatidylinositol biosynthetic pathway of Trypanosoma brucei.

Synthetic analogues of D-GlcNalpha1-6D-myo-inositol-1-HPO(4)-3(sn-1, 2-diacylglycerol) (GlcN-PI), with the 2-position of the inositol residue substituted with an O-octyl ether [D-GlcNalpha1-6D-(2-O-octyl)myo-inositol-1-HPO(4)-3-sn-1, 2-dipalmitoylglycerol; GlcN-(2-O-octyl) PI] or O-hexadecyl ether [D-GlcNalpha1-6D-(2-O-hexadecyl)myo-inositol-1-HPO(4)-3-sn-1, 2-dipalmitoylglycerol; GlcN-(2-O-hexadecyl)PI], were tested as substrates or inhibitors of glycosylphosphatidylinositol (GPI) biosynthetic pathways using cell-free systems of the protozoan parasite Trypanosoma brucei (the causative agent of human African sleeping sickness) and human HeLa cells. Neither these compounds nor their N-acetyl derivatives are substrates or inhibitors of GPI biosynthetic enzymes in the HeLa cell-free system but are potent inhibitors of GPI biosynthesis in the T.brucei cell-free system. GlcN-(2-O-hexadecyl)PI was shown to inhibit the first alpha-mannosyltransferase of the trypanosomal GPI pathway. The N-acetylated derivative GlcNAc-(2-O-octyl)PI is a substrate for the trypanosomal GlcNAc-PI de-N-acetylase and this compound, like GlcN-(2-O-octyl)PI, is processed predominantly to Man(2)GlcN-(2-O-octyl)PI by the T.brucei cell-free system. Both GlcN-(2-O-octyl)PI and GlcNAc(2-O-octyl)PI also inhibit inositol acylation of Man(1-3)GlcN-PI and, consequently, the addition of the ethanolamine phosphate bridge in the T.brucei cell-free system. The data establish these substrate analogues as the first generation of in vitro parasite GPI pathway-specific inhibitors.     

Novel human mPGES-1 inhibitors identified through structure-based virtual screening

Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible prostaglandin E synthase after exposure to pro-inflammatory stimuli and, therefore, represents a novel target for therapeutic treatment of acute and chronic inflammatory disorders. It is essential to identify mPGES-1 inhibitors with novel scaffolds as new leads or hits for the purpose of drug design and discovery that aim to develop the next-generation anti-inflammatory drugs. Herein we report novel mPGES-1 inhibitors identified through a combination of large-scale structure-based virtual screening, flexible docking, molecular dynamics simulations, binding free energy calculations, and in vitro assays on the actual inhibitory activity of the computationally selected compounds. The computational studies are based on our recently developed three-dimensional (3D) structural model of mPGES-1 in its open state. The combined computational and experimental studies have led to identification of new mPGES-1 inhibitors with new scaffolds. In particular, (Z)-5-benzylidene-2-iminothiazolidin-4-one is a promising novel scaffold for the further rational design and discovery of new mPGES-1 inhibitors. To our best knowledge, this is the first time a 3D structural model of the open state mPGES-1 is used in structure-based virtual screening of a large library of available compounds for the mPGES-1 inhibitor identification. The positive experimental results suggest that our recently modeled trimeric structure of mPGES-1 in its open state is ready for the structure-based drug design and discovery.     

A new model of Pde4d deficiency: genetic knock-down of PDE4D enzyme in rats produces an antidepressant phenotype without spatial cognitive effects

Phosphodiesterases (PDEs) are a superfamily of intracellular second messenger cyclic nucleotide hydrolyzing enzymes composed of 12 families. The Pde4 family has been implicated in depression and cognition, and PDE4 inhibitors have been evaluated as antidepressants and possible cognitive enhancers. Pde4d(-/-) mice show an antidepressant phenotype and learning enhancement on some tests, but not others as do mice treated with PDE4 inhibitors. Here, we report for the first time the behavioral phenotype of a new Pde4d knock-down (KD) rat model of PDE4D deficiency. Consistent with other data on PDE4D deficiency, Pde4d KD rats showed depression resistance in the Porsolt forced swim test and hyperreactivity of the acoustic startle response with no differential response on prepulse inhibition, suggesting no sensorimotor gating defect. Pde4d KD rats also exhibited a small exploratory activity reduction but no difference following habituation, and no enhanced spatial learning or reference memory in the Morris water maze. A selective improvement in route-based learning in the Cincinnati water maze was seen as well as enhanced contextual and cued fear conditioning and a more rapid rate of cued extinction from their higher freezing level that declined to wild-type (WT) levels only after ～20 extinction trials. The rat model confirms Pde4d's role in depression but not in spatial learning or memory enhancement and shows for the first time higher fear conditioning and altered extinction compared with controls. The new model provides a tool by which to better understand the role of PDE4D in neuropsychiatric disorders and for the development of alternate treatment approaches.