Discovery of PqsE Thioesterase Inhibitors for Pseudomonas aeruginosa Using DNA-Encoded Small Molecule Library Screening.

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections in the United States. PqsE, a thioesterase enzyme, is vital for virulence of P. aeruginosa, making PqsE an attractive target for inhibition. Neither the substrate nor the product of PqsE catalysis has been identified. A library of 550 million DNA-encoded drug-like small molecules was screened for those that bind to the purified PqsE protein. The structures of the bound molecules were identified by high throughput sequencing of the attached DNA barcodes. Putative PqsE binders with the strongest affinity features were examined for inhibition of PqsE thioesterase activity in vitro. The most potent inhibitors were resynthesized off DNA and examined for the ability to alter PqsE thermal melting and for PqsE thioesterase inhibition. Here, we report the synthesis, biological activity, mechanism of action, and early structure-activity relationships of a series of 2-(phenylcarbamoyl)benzoic acids that noncompetitively inhibit PqsE. A small set of analogs designed to probe initial structure-activity relationships showed increases in potency relative to the original hits, the best of which has an IC50 = 5 µM. Compound refinement is required to assess their in vivo activities as the current compounds do not accumulate in the P. aeruginosa cytosol. Our strategy validates DNA-encoded compound library screening as a rapid and effective method to identify catalytic inhibitors of the PqsE protein, and more generally, for discovering binders to bacterial proteins revealed by genetic screening to have crucial in vivo activities but whose biological functions have not been well-defined.

Identification of a Fluorescent Protein from Rhacostoma Atlantica.

We have cloned a novel fluorescent protein from the jellyfish Rhacostoma atlantica. The closest known related fluorescent protein is the Phialidium yellow fluorescent protein, with only a 55% amino acid sequence identity. A somewhat unusual alanine-tyrosine-glycine amino acid sequence forms the presumed chromophore of the novel protein. The protein has an absorption peak at 466 nm and a fluorescence emission peak at 498 nm. The fluorescence quantum yield was measured to be 0.77 and the extinction coefficient is 58 200 M(-1) cm(-1) . Several mutations were identified that shift the absorption peak to about 494 nm and the emission peak to between 512 and 514 nm.

Antidiabetic and antisteatotic effects of the selective fatty acid synthase (FAS) inhibitor platensimycin in mouse models of diabetes.

Platensimycin (PTM) is a recently discovered broad-spectrum antibiotic produced by Streptomyces platensis. It acts by selectively inhibiting the elongation-condensing enzyme FabF of the fatty acid biosynthesis pathway in bacteria. We report here that PTM is also a potent and highly selective inhibitor of mammalian fatty acid synthase. In contrast to two agents, C75 and cerulenin, that are widely used as inhibitors of mammalian fatty acid synthase, platensimycin specifically inhibits fatty acid synthesis but not sterol synthesis in rat primary hepatocytes. PTM preferentially concentrates in liver when administered orally to mice and potently inhibits hepatic de novo lipogenesis, reduces fatty acid oxidation, and increases glucose oxidation. Chronic administration of platensimycin led to a net reduction in liver triglyceride levels and improved insulin sensitivity in db/+ mice fed a high-fructose diet. PTM also reduced ambient glucose levels in db/db mice. These results provide pharmacological proof of concept of inhibiting fatty acid synthase for the treatment of diabetes and related metabolic disorders in animal models.

Liver and adipose expression associated SNPs are enriched for association to type 2 diabetes.

Genome-wide association studies (GWAS) have demonstrated the ability to identify the strongest causal common variants in complex human diseases. However, to date, the massive data generated from GWAS have not been maximally explored to identify true associations that fail to meet the stringent level of association required to achieve genome-wide significance. Genetics of gene expression (GGE) studies have shown promise towards identifying DNA variations associated with disease and providing a path to functionally characterize findings from GWAS. Here, we present the first empiric study to systematically characterize the set of single nucleotide polymorphisms associated with expression (eSNPs) in liver, subcutaneous fat, and omental fat tissues, demonstrating these eSNPs are significantly more enriched for SNPs that associate with type 2 diabetes (T2D) in three large-scale GWAS than a matched set of randomly selected SNPs. This enrichment for T2D association increases as we restrict to eSNPs that correspond to genes comprising gene networks constructed from adipose gene expression data isolated from a mouse population segregating a T2D phenotype. Finally, by restricting to eSNPs corresponding to genes comprising an adipose subnetwork strongly predicted as causal for T2D, we dramatically increased the enrichment for SNPs associated with T2D and were able to identify a functionally related set of diabetes susceptibility genes. We identified and validated malic enzyme 1 (Me1) as a key regulator of this T2D subnetwork in mouse and provided support for the association of this gene to T2D in humans. This integration of eSNPs and networks provides a novel approach to identify disease susceptibility networks rather than the single SNPs or genes traditionally identified through GWAS, thereby extracting additional value from the wealth of data currently being generated by GWAS.

Discovery and optimization of novel 4-[(aminocarbonyl)amino]-N-[4-(2-aminoethyl)phenyl]benzenesulfonamide ghrelin receptor antagonists.

This Letter describes optimization of ghrelin receptor antagonists and inverse agonists starting from a screening hit.

Control of cell growth and survival by enzymes of the fatty acid synthesis pathway in HCT-116 colon cancer cells.

PURPOSE: For many tumor cells, de novo lipogenesis is a requirement for growth and survival. A considerable body of work suggests that inhibition of this pathway may be a powerful approach to antineoplastic therapy. It has recently been shown that inhibition of various steps in the lipogenic pathway individually can induce apoptosis or loss of viability in tumor cells. However, it is not clear whether quantitative differences exist in the ability of lipogenic enzymes to control tumor cell survival. We present a systematic approach that allows for a direct comparison of the control of lipogenic pathway enzymes over tumor cell growth and apoptosis using different cancer cells. EXPERIMENTAL DESIGN: RNA interference-mediated, graded down-regulation of fatty acid synthase (FAS) pathway enzymes was employed in combination with measurements of lipogenesis, apoptosis, and cell growth. RESULTS: In applying RNA interference titrations to two lipogenic enzymes, acetyl-CoA carboxylase 1 (ACC1) and FAS, we show that ACC1 and FAS both significantly control cell growth and apoptosis in HCT-116 cells. These results also extend to PC-3 and A2780 cancer cells. CONCLUSIONS: Control of tumor cell survival by different steps in de novo lipogenesis can be quantified. Because ACC1 and FAS both significantly control tumor cell growth and apoptosis, we propose that pharmacologic inhibitors of either enzyme might be useful agents in targeting cancer cells that critically rely on fatty acid synthesis. The experimental approach described here may be extended to other targets or disease-relevant pathways to identify steps suitable for therapeutic intervention.

A fluorescence-based thiol quantification assay for ultra-high-throughput screening for inhibitors of coenzyme A production.

Here we report the development and miniaturization of a cell-free enzyme assay for ultra-high-throughput screening (uHTS) for inhibitors of two potential drug targets for obesity and cancer: fatty acid synthase (FAS) and acetyl-coenzyme A (CoA) carboxylase (ACC) 2. This assay detects CoA, a product of the FAS-catalyzed condensation of malonyl-CoA and acetyl-CoA. The free thiol of CoA can react with 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM), a profluorescent coumarin maleimide derivative that becomes fluorescent upon reaction with thiols. FAS produces long-chain fatty acid and CoA from the condensation of malonyl-CoA and acetyl-CoA. In our FAS assay, CoA released in the FAS reaction forms a fluorescence adduct with CPM that emits at 530 nm when excited at 405 nm. Using this detection method for CoA, we measured the activity of sequential enzymes in the fatty acid synthesis pathway to develop an ACC2/FAS-coupled assay where ACC2 produces malonyl-CoA from acetyl-CoA. We miniaturized the FAS and ACC2/FAS assays to 3,456- and 1,536-well plate format, respectively, and completed uHTSs for small molecule inhibitors of this enzyme system. This report shows the results of assay development, miniaturization, and inhibitor screening for these potential drug targets.

Deficiency in cytosolic malic enzyme does not increase acetaminophen-induced hepato-toxicity.

Cytosolic malic enzyme (ME-1) is a nicotinamide adenine dinucleotide phosphate (NADP)-dependent enzyme that generates NADPH. The activity of this enzyme, the reversible oxidative decarboxylation of malate to yield pyruvate, links glycolytic pathway to citric acid cycle. The high level of ME-1 expression in liver, and its involvement in NADPH production, suggests reduced ME-1 activity might compromise hepatic production of reduced glutathione (GSH) by the NADPH-dependent enzyme glutathione reductase, and hence affect xenobiotic detoxification. The role of ME-1 in liver detoxification was evaluated in Mod1 deficient mice (mod1(-/-)) by evaluating their sensitivity to acetaminophen-induced liver injury. The results show that mod1(-/-) mice are not more sensitive to acetaminophen hepato-toxicity. Although GSH levels were initially depleted more in the mod1(-/-) liver than in wild-type controls, the GSH levels recovered quickly. In conclusion, our data indicate that ME-1 deficiency does not adversely affect GSH-dependent detoxification.

3-Aryl-4-hydroxyquinolin-2(1H)-one derivatives as type I fatty acid synthase inhibitors.

A series of 3-aryl-4-hydroxyquinolin-2(1H)-ones with fatty acid synthase inhibitory activity was prepared. Starting from a derivative with an IC(50) = 1.4 microM, SAR studies led to compounds with more than 70-fold increase in potency (IC(50) < 20 nM).

Dynamic activation of cystic fibrosis transmembrane conductance regulator by type 3 and type 4D phosphodiesterase inhibitors.

The diseases of cystic fibrosis, chronic obstructive pulmonary disease (COPD), and chronic bronchitis are characterized by mucus-congested and inflamed airways. Anti-inflammatory agents that can simultaneously restore or enhance mucociliary clearance through cystic fibrosis transmembrane conductance regulator (CFTR) activation may represent new therapeutics in their treatment. Herein, we report the activation of CFTR-mediated chloride secretion by phosphodiesterase (PDE) 4 inhibitors in T84 monolayer using (125)I anion as tracer. In the absence of forskolin, the iodide secretion was insensitive to PDE4 inhibitor L-826,141 [4-[2-(3,4-bis-difluoromethoxyphenyl)-2-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]-ethyl]-3-methylpyridine-1-oxide], roflumilast, or to PDE3 inhibitor trequinsin. However, these inhibitors potently augmented iodide secretion after forskolin stimulation, with efficacy coupled to the activation states of adenylyl cyclase. The iodide secretion from PDE3 or PDE4 inhibition was characterized at first by a prolonged efflux duration, followed by progressively elevated peak efflux rates at higher inhibitor concentrations. Paralleled with an increased phosphor-cAMP response element-binding protein formation, the CFTR activation dissociated from a global cAMP elevation and was blocked by H89 [N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide]. 2-(4-Fluorophenoxy)-N-[(1S)-1-(4-methoxyphenyl)ethyl]nicotinamide, a stereoselective PDE4D inhibitor, augmented iodide efflux more efficiently than its less potent (R)-isomer. The peak efflux from maximal PDE4 and PDE3 inhibition matched that from full adenylyl cyclase activation. These data suggest that PDE3 and PDE4 (mainly PDE4D) form the major cAMP diffusion barrier in T84 cells to ensure a compartmentalized CFTR signaling. Together with their potent anti-inflammatory properties, the potentially enhanced airway mucociliary clearance from CFTR activation may have contributed to the efficacy of PDE4 inhibitors in COPD and asthmatic patients. PDE4 inhibitors may represent new opportunities to combat cystic fibrosis and other respiratory diseases in future.

Expression, refolding, and purification of recombinant human phosphodiesterase 3B: definition of the N-terminus of the catalytic core.

We have developed an expression, refolding, and purification protocol for the catalytic domain of human Phosphodiesterase 3B (PDE3B). High level expression in Escherichia coli has been achieved with yields of up to 20mg/L. The catalytic domain of the enzyme was purified by affinity chromatography utilizing a novel affinity ligand. PDE3B, purified by affinity chromatography, with no single impurity #10878;1% as determined by SDS-PAGE, has a specific activity of 2210+/-442nmol/min/mg and a KM for cAMP of 44+/-4.5nM. Reducing the size of the expressed catalytic domain from residues 387-1112 to residues 654-1086 greatly reduced the aggregation phenomena observed with the affinity purified PDE3B. The definition of the N-terminus of the catalytic core was examined through the generation of several truncation mutants spanning amino acid residues 636-674. Constructs starting at E665 and M674 were fully active and devoid of activity, respectively. A construct starting at D668 had a Vmax reduced by approximately 10-fold relative to the longer constructs, yet the KM was not affected. This indicates the minimal N-terminus of the catalytic core lies between E665 and Y667. Refolding and affinity purification of the 654-1073 catalytic core of PDE3B has been employed to produce large quantities of highly pure enzyme for structural studies.

Crystal structure of human phosphodiesterase 3B: atomic basis for substrate and inhibitor specificity.

Phosphodiesterases (PDEs) are enzymes that modulate cyclic nucleotide signaling and as such are clinical targets for a range of disorders including congestive heart failure, erectile dysfunction, and inflammation. The PDE3 family comprises two highly homologous subtypes expressed in different tissues, and inhibitors of this family have been shown to increase lipolysis in adipocytes. A specific PDE3B (the lipocyte-localized subtype) inhibitor would be a very useful tool to evaluate the effects of PDE3 inhibition on lipolysis and metabolic rate and might become a novel tool for treatment of obesity. We report here the three-dimensional structures of the catalytic domain of human PDE3B in complex with a generic PDE inhibitor and a novel PDE3 selective inhibitor. These structures explain the dual cAMP/cGMP binding capabilities of PDE3, provide the molecular basis for inhibitor specificity, and can supply a valid platform for the design of improved compounds.

Crystallization and preliminary X-ray diffraction analysis of the catalytic domain of recombinant human phosphodiesterase 3B.

The catalytic domain of human phosphodiesterase 3B has been cloned, expressed in Escherichia coli and purified in the presence of the PDE3 inhibitors IBMX (3-isobutylmethylxanthine) or MERCK1 by affinity chromatography. Initial screening of crystallization conditions for these complexes in the hanging-drop vapor-diffusion mode resulted in three different crystal forms, all characterized by quite large unit-cell parameters, elevated solvent content and poor diffraction quality. Subsequent optimization of these conditions led to crystals that diffract to 2.4 A and belong to space group C2, with unit-cell parameters a = 146.7, b = 121.5, c = 126.3 A, beta = 100.6 degrees. Rotation-function analysis indicates that the asymmetric unit contains four copies of the monomeric enzyme, corresponding to a solvent content of 64%. To solve the structure of the PDE3B catalytic domain, molecular replacement as well as multiple isomorphous replacement methods are currently being utilized.

Benzyl vinylogous amide substituted aryldihydropyridazinones and aryldimethylpyrazolones as potent and selective PDE3B inhibitors.

Aryldihydropyridazinones and aryldimethylpyrazolones with 2-benzyl vinylogous amide substituents have been identified as potent PDE3B subtype selective inhibitors. Dihydropyridazinone 8a (PDE3B IC(50)=0.19 nM, 3A IC(50)=1.3 nM) was selected for in vivo evaluation of lipolysis induction, metabolic rate increase, and cardiovascular effects.

The role of tryptophan 1072 in human PDE3B inhibitor binding.

The catalytic domain of recombinant human PDE3B was expressed in Escherichia coli as inclusion bodies and refolded to form active enzyme. A mutation at tryptophan 1072 in PDE3B disrupts inhibitor binding, but has minimal effect on cAMP hydrolysis. The W1072A mutation caused a 158-fold decrease in affinity for cilostamide, a 740-fold decrease for cGMP, and a 15-fold decrease in affinity for IBMX. The corresponding tyrosine mutation had a smaller effect. However, the K(m) of cAMP for the W1072A mutation was only increased by about 7-fold. The data indicate that the inhibitor binding region is not completely coincident with the substrate binding region. The homologous residue in PDE4B is located on helix 16 within 7A of the predicted bound substrate. A model of PDE3B was constructed based on the X-ray crystal structure of PDE4B.

A role for the melanocortin 4 receptor in sexual function.

By using a combination of genetic, pharmacological, and anatomical approaches, we show that the melanocortin 4 receptor (MC4R), implicated in the control of food intake and energy expenditure, also modulates erectile function and sexual behavior. Evidence supporting this notion is based on several findings: (i) a highly selective non-peptide MC4R agonist augments erectile activity initiated by electrical stimulation of the cavernous nerve in wild-type but not Mc4r-null mice; (ii) copulatory behavior is enhanced by administration of a selective MC4R agonist and is diminished in mice lacking Mc4r; (iii) reverse transcription (RT)-PCR and non-PCR based methods demonstrate MC4R expression in rat and human penis, and rat spinal cord, hypothalamus, brainstem, pelvic ganglion (major autonomic relay center to the penis), but not in rat primary corpus smooth muscle cavernosum cells; and (iv) in situ hybridization of glans tissue from the human and rat penis reveal MC4R expression in nerve fibers and mechanoreceptors in the glans of the penis. Collectively, these data implicate the MC4R in the modulation of penile erectile function and provide evidence that MC4R-mediated proerectile responses may be activated through neuronal circuitry in spinal cord erectile centers and somatosensory afferent nerve terminals of the penis. Our results provide a basis for the existence of MC4R-controlled neuronal pathways that control sexual function.