The design, synthesis and biological evaluation of novel URB602 analogues as potential monoacylglycerol lipase inhibitors

We have synthesised an extensive series of URB602 analogues as inhibitors of monoacylglycerol lipase (MAGL), which is the major enzyme responsible for metabolising the endocannabinoid 2-arachidonylglycerol. The recently identified crystal structure of MAGL was used in the design strategy and revealed three possible binding sites for URB602 and the proposed analogues. A test series of carbamate analogues were docked into the identified sites to predict the most favourable binding location. The synthesised analogues of URB602 explored the biological effects of isosteric replacement, ring size and substitution, para substitution of the biphenyl moiety and the incorporation of a bicyclic element. The compounds were tested for their ability to inhibit human MAGL. The carbamate analogue 16 displayed the most significant inhibitory activity, reducing MAGL activity to 26% of controls at 100 μM compared to 73% for the parent compound URB602.     

High-performance liquid chromatography assay with fluorescence detection for the evaluation of inhibitors against human recombinant monoacylglycerol lipase

A fluorescent assay for the evaluation of inhibitors of monoacylglycerol lipase (MAGL) is described. 1,3-Dihydroxypropan-2-yl 4-pyren-1-ylbutanoate was designed and synthesized as novel fluorogenic substrate. Activity of human recombinant MAGL was determined in the presence of the surfactant Triton X-100 without further sample cleanup by measuring the amount of 4-pyren-1-ylbutanoic acid released by the enzyme with reversed-phase high-performance liquid chromatography (HPLC) and fluorescence detection. The known covalent binding MAGL inhibitors methyl arachidonyl fluorophosphonate (MAFP), 4-nitrophenyl 4-[bis(1,3-benzodioxol-5-yl)hydroxymethyl]piperidine-1-carboxylate (JZL184), and [4-(5-methoxy-2-oxo-1,3,4-oxadiazol-3-yl)-2-methylphenyl]carbamic acid benzyl ester (CAY10499) were used to validate the test system. Applying an incubation time of 15 min, the IC₅₀ values obtained for these compounds were 0.16, 3.7, and 1.1 μM, respectively. A prolongation of the incubation to 45 min results in a two- to threefold decrease of the IC₅₀ values.     

Pyridoxine as a template for the design of antiplatelet agents

The B(6) vitamers have been shown to display beneficial therapeutic effects in cardiovascular related disorders. The design of novel antiplatelet agents using pyridoxine as a template has led to the discovery of a class of novel cardio- and cerebro-protective agents. The present study describes the synthesis of several of these derivatives along with the antiplatelet and antiischemic activity of derivative 16.     

Proteomics-based target identification: bengamides as a new class of methionine aminopeptidase inhibitors

LAF389 is a synthetic analogue of bengamides, a class of marine natural products that produce inhibitory effects on tumor growth in vitro and in vivo. A proteomics-based approach has been used to identify signaling pathways affected by bengamides. LAF389 treatment of cells resulted in altered mobility of a subset of proteins on two-dimensional gel electrophoresis. Detailed analysis of one of the proteins, 14-3-3gamma, showed that bengamide treatment resulted in retention of the amino-terminal methionine, suggesting that bengamides directly or indirectly inhibited methionine aminopeptidases (MetAps). Both known MetAps are inhibited by LAF389. Short interfering RNA suppression of MetAp2 also altered amino-terminal processing of 14-3-3gamma. A high resolution structure of human MetAp2 co-crystallized with a bengamide shows that the compound binds in a manner that mimics peptide substrates. Additionally, the structure reveals that three key hydroxyl groups on the inhibitor coordinate the di-cobalt center in the enzyme active site.     

Perspectives in the design of small molecule enzyme inhibitors as useful drugs

An irreversible inhibitor is often assessed as a potential drug on the basis of its specificity towards a target enzyme in vitro. This is inadequate because in vivo the enzyme may be re-synthesized and the inhibitor may react with other major body constituents, e.g. glutathione.Guidelines are given for selecting the class of inhibitor to be studied for use in a particular clinical area.     

Rational design of alpha-keto triglyceride analogues as inhibitors for Staphylococcus hyicus lipase.

We have synthesized a series of alpha-keto triglyceride analogues as inhibitors for the lipase from Staphylococcus hyicus (SHL). Hydrolysis at positions 1 and 2 was prevented by replacement of the ester bonds by nonhydrolyzable ether, carbamoyl, or amide bonds, and an alpha-keto fatty acid was introduced at position 3. Such compounds act as competitive inhibitors of SHL. Inhibition must be caused by the presence of the alpha-keto functions, since the compounds containing an ester or a hydroxyl group in position 3 did not inhibit the enzyme. We propose that our inhibitors react with the active site Ser of the lipase, hereby mimicking the tetrahedral intermediate occurring in substrate hydrolysis. We conclude that the localization of the alpha-keto triglycerides is very important for inhibition because only those compounds which are insoluble in water are lipase inhibitors. In addition, other specific protein-inhibitor interactions, probably with the carbonyl oxygen at position 1 and/or 2, improve inhibitor binding. This makes the compounds with amide or carbamoyl groups at positions 1 and 2 better inhibitors than their ether counterparts. The inhibitory power could be improved further by replacing the oxygen at position 3 by an amido group. The resulting inhibitor 1, 2-diethylcarbamoyl-3-amido-alpha-ketododecanoylglycerol has a Ki value of 0.008 mol %, indicating that it binds approximately 125-fold tighter than the substrate. These results illustrate that effective lipase inhibitors can be designed by combining an alpha-keto group with good micellar solubility and optimal protein-inhibitor interactions.     

Hormone-sensitive lipase and monoacylglycerol lipase are both required for complete degradation of adipocyte triacylglycerol

The respective roles of monoacylglycerol lipase and hormone-sensitive lipase in the sequential hydrolysis of adipose tissue triacylglycerols have been examined. An adipose tissue preparation, containing both lipases in approximately the same proportion as in the intact tissue, hydrolyzed emulsified tri- or dioleoylglycerol to fatty acids and glycerol, with little accumulation of di- or monooleoylglycerol. Selective removal of the monoacylglycerol lipase by immunoprecipitation markedly reduced the glycerol release. Isolated hormone-sensitive lipase hydrolyzed acylglycerols with a marked accumulation of monoacylglycerol in accordance with the positional specificity of this enzyme (Fredrikson, G. and Belfrage, P. (1983) J. Biol. Chem. 258, 14253-14256). Addition of increasing amounts of isolated monoacylglycerol lipase led to a corresponding increase in glycerol release, due to hydrolysis of the monoacylglycerols formed. The reaction proceeded to completion when the relative proportion of the two lipases was similar to that in the intact tissue. These findings indicate that hormone-sensitive lipase catalyzes the hydrolysis of triacylglycerol in the rate-limiting step of adipose tissues lipolysis, and of the resulting diacylglycerol, whereas the action of monoacylglycerol lipase is required in the final hydrolysis of the 2-monoacylglycerols produced.     

A new target for shigellosis: rational design and crystallographic studies of inhibitors of tRNA-guanine transglycosylase

Eubacterial tRNA-guanine transglycosylase (TGT) is involved in the hyper-modification of cognate tRNAs leading to the exchange of G34 at the wobble position in the anticodon loop by preQ1 (2-amino-5-(aminomethyl)pyrrolo[2,3-d]pyrimidin-4(3H)-one) as part of the biosynthesis of queuine (Q). Mutation of the tgt gene in Shigella flexneri results in a significant loss of pathogenicity of the bacterium, revealing TGT as a new target for the design of potent drugs against Shigellosis. The X-ray structure of Zymomonas mobilis TGT in complex with preQ1 was used to search for new putative inhibitors with the computer program LUDI. An initial screen of the Available Chemical Directory, a database compiled from commercially available compounds, suggested several hits. Of these, 4-aminophthalhydrazide (APH) showed an inhibition constant in the low micromolar range. The 1.95 A crystal structure of APH in complex with Z. mobilis TGT served as a starting point for further modification of this initial lead.     

The crystal structure of transthyretin in complex with diethylstilbestrol: a promising template for the design of amyloid inhibitors

Transthyretin (TTR) is a homotetrameric plasma protein that, in conditions not yet completely understood, may aggregate, forming the fibrillar material associated with TTR amyloidosis. A number of reported experiments indicate that dissociation of the TTR tetramer occurs prior to fibril formation, and therefore, studies aiming at the discovery of compounds that stabilize the protein quaternary structure, thereby acting as amyloid inhibitors, are being performed. The ability of diethylstilbestrol (DES) to act as a competitive inhibitor for the thyroid hormone binding to TTR indicated a possible stabilizing effect of DES upon binding. Here we report the crystallographic study of DES binding to TTR. The structural data reveal two different binding modes, both located in the thyroxine binding channel. In both cases, DES binds deeply in the channel and establishes interactions with the equivalent molecule present in the adjacent binding site. The most remarkable features of DES interaction with TTR are its hydrophobic interactions within the protein halogen binding pockets, where its ethyl groups are snugly fitted, and the hydrogen bonds established at the center of the tetramer with Ser-117. Experiments concerning amyloid formation in vitro suggest that DES is effectively an amyloid inhibitor in acid-mediated fibrillogenesis and may be used for the design of more powerful drugs. The present study gave us further insight in the molecular mechanism by which DES competes with thyroid hormone binding to TTR and highlights key interactions between DES and TTR that oppose amyloid formation.     

Fluorescence correlation spectroscopy as a new method for the investigation of aptamer/target interactions

Fluorescence correlation spectroscopy is an attractive tool for monitoring molecular interactions in solution. We report here a new and highly sensitive method for studying the interaction of aptamers with their targets using this technique. In vitro selection technology is a combinatorial method for the generation of nucleic acid receptors (aptamers) that are capable of binding to various target molecules. Using the in vitro selection approach we isolated RNAs which bind to the antibiotic moenomycin with high affinity. The formation of RNA-moenomycin complexes was studied by fluorescence correlation spectroscopy with a tetramethylrhodamine-labeled derivative of moenomycin.     

Hydrolysis of tri- and monoacylglycerol by lipoprotein lipase: evidence for a common active site.

The relationship between triacylglycerol and monoacylglycerol hydrolyzing activities of purified rat heart lipoprotein lipase was studied using emulsified trioleoylglycerol and micellar or albumin-bound monooleoylglycerol as substrates. The maximal reaction rates obtained with the two substrates were similar (650 and 550 nmol of fatty acid released per min per mg of protein, respectively). Addition of apolipoprotein C-II or serum increased the maximal reaction rate for the trioleolyglycerol hydrolyzing activity about four-fold, but had no effect on the monooleolyglycerol hydrolyzing activity. Hydolysis of the two substrates apparently takes place at the same active site of the enzyme since (1) mutual competitive inhibition between the substrates could be demonstrated; (2) the rate of inactivation of enzymatic activity with the two substrates in 1.2 M NaCl was the same; (3) similar losses of hydrolytic activity with tri- and monooleoylglycerol were observed in the presence of low concentrations of n-butyl (p-nitrophenyl) carbamide; (4) inhibition of both hydrolytic activities by this compound could be prevented by prior exposure of lipoprotein lipase to either substrate.     

Crystal structure of lumazine synthase from Mycobacterium tuberculosis as a target for rational drug design: binding mode of a new class of purinetrione inhibitors

The enzymes involved in the biosynthesis of riboflavin represent attractive targets for the development of drugs against bacterial pathogens, because the inhibitors of these enzymes are not likely to interfere with enzymes of the mammalian metabolism. Lumazine synthase catalyzes the penultimate step in the riboflavin biosynthesis pathway. A number of substituted purinetrione compounds represent a new class of highly specific inhibitors of lumazine synthase from Mycobacterium tuberculosis. To develop potent antibiotics for the treatment of tuberculosis, we have determined the structure of lumazine synthase from M. tuberculosis in complex with two purinetrione inhibitors and have studied binding via isothermal titration calorimetry. The structures were determined by molecular replacement using lumazine synthase from Saccharomyces cerevisiae as a search model and refined at 2 and 2.3 A resolution. The R-factors were 14.7 and 17.4%, respectively, and the R(free) values were 19.3 and 26.3%, respectively. The enzyme was found to be a pentamer consisting of five subunits related by 5-fold local symmetry. The comparison of the active site architecture with the active site of previously determined lumazine synthase structures reveals a largely conserved topology with the exception of residues Gln141 and Glu136, which participate in different charge-charge interactions in the core space of the active site. The impact of structural changes in the active site on the altered binding and catalytic properties of the enzyme is discussed. Isothermal titration calorimetry measurements indicate highly specific binding of the purinetrione inhibitors to the M. tuberculosis enzyme with dissociation constants in micromolar range.     

N-(1,3-Diaryl-3-oxopropyl)amides as a new template for xanthine oxidase inhibitors

A series of forty two N-(1,3-diaryl-3-oxopropyl)amides were synthesized via an efficient, modified Dakin-West reaction and were evaluated for in vitro xanthine oxidase inhibitory activity for the first time. Structure-activity relationship analyses have been presented. Selected active xanthine oxidase inhibitors (3r, 3s, and 3zh) were assessed in vivo to study their anti-hyperuricemic effect in potassium oxonate induced hyperuricemic mice model. Compound 3s emerged as the most potent xanthine oxidase inhibitor (IC(50)=2.45 μM) as well as the most potent anti-hyperuricemic agent. The basis of significant inhibition of xanthine oxidase by 3s was rationalized by its molecular docking into catalytic site of xanthine oxidase.     

Distribution and function of monoacylglycerol lipase in the gastrointestinal tract

The endogenous cannabinoid system plays an important role in the regulation of gastrointestinal function in health and disease. Endocannabinoid levels are regulated by catabolic enzymes. Here, we describe the presence and localization of monoacylglycerol lipase (MGL), the major enzyme responsible for the degradation of 2-arachidonoylglycerol. We used molecular, biochemical, immunohistochemical, and functional assays to characterize the distribution and activity of MGL. MGL mRNA was present in rat ileum throughout the wall of the gut. MGL protein was distributed in the muscle and mucosal layers of the ileum and in the duodenum, proximal colon, and distal colon. We observed MGL expression in nerve cell bodies and nerve fibers of the enteric nervous system. There was extensive colocalization of MGL with PGP 9.5 and calretinin-immunoreactive neurons, but not with nitric oxide synthase. MGL was also present in the epithelium and was highly expressed in the small intestine. Enzyme activity levels were highest in the duodenum and decreased along the gut with lowest levels in the distal colon. We observed both soluble and membrane-associated enzyme activities. The MGL inhibitor URB602 significantly inhibited whole gut transit in mice, an action that was abolished in cannabinoid 1 receptor-deficient mice. In conclusion, MGL is localized in the enteric nervous system where endocannabinoids regulate intestinal motility. MGL is highly expressed in the epithelium, where this enzyme may have digestive or other functions yet to be determined.     

Pyrimido-oxazepine as a versatile template for the development of inhibitors of specific kinases

Pyrimido-oxazepine based sub-micromolar inhibitors (2–4) for Aurora and FLT-3 were designed and synthesized. These preliminary results supported the potential use of pyrimido-oxazepines as a versatile template for developing specific kinase inhibitors.     

Carbonic anhydrase inhibitors: the beta-carbonic anhydrase from Helicobacter pylori is a new target for sulfonamide and sulfamate inhibitors

DNA clones for the beta-class carbonic anhydrase (CA, EC 4.2.1.1) of Helicobactor pylori (hpbetaCA) were obtained. A recombinant hpbetaCA protein lacking the N-terminal 15-amino acid residues was produced and purified, representing a catalytically efficient CA. hpbetaCA was strongly inhibited (K(I)s in the range of 24-45 nM) by many sulfonamides/sulfamates, among which acetazolamide, ethoxzolamide, topiramate, and sulpiride, all clinically used drugs. The dual inhibition of alpha- and/or beta-class CAs of H. pylori might represent a useful alternative for the management of gastritis/gastric ulcers, as well as gastric cancer. This is also the first study showing that a bacterial beta-CA can be a drug target.     

Endothelial lipase: a new lipase on the block

Endothelial lipase (EL) is a newly described member of the triglyceride lipase gene family. It has a considerable molecular homology with lipoprotein lipase (LPL) (44%) and hepatic lipase (HL) (41%). Unlike LPL and HL, this enzyme is synthesized by endothelial cells and functions at the site where it is synthesized. Furthermore, its tissue distribution is different from that of LPL and HL. As a lipase, EL has primarily phospholipase A1 activity. Animals that overexpress EL showed reduced HDL cholesterol levels. Conversely, animals that are deficient in EL showed a marked elevation in HDL cholesterol levels, suggesting that it plays a physiologic role in HDL metabolism. Unlike LPL and HL, EL is located in the vascular endothelial cells and its expression is highly regulated by cytokines and physical forces, suggesting that it may play a role in the development of atherosclerosis. However, there is only a limited amount of information available about this enzyme. Some of our unpublished data in addition to previously published data support the possibility that the enzyme plays a role in the formation of atherosclerotic lesion.     

Recruiting a new substrate for triacylglycerol synthesis in plants: the monoacylglycerol acyltransferase pathway

Background

Monoacylglycerol acyltransferases (MGATs) are predominantly associated with lipid absorption and resynthesis in the animal intestine where they catalyse the first step in the monoacylglycerol (MAG) pathway by acylating MAG to form diacylglycerol (DAG). Typical plant triacylglycerol (TAG) biosynthesis routes such as the Kennedy pathway do not include an MGAT step. Rather, DAG and TAG are synthesised de novo from glycerol-3-phosphate (G-3-P) by a series of three subsequent acylation reactions although a complex interplay with membrane lipids exists.

Methodology/Principal Findings

We demonstrate that heterologous expression of a mouse MGAT acyltransferase in Nicotiana benthamiana significantly increases TAG accumulation in vegetative tissues despite the low levels of endogenous MAG substrate available. In addition, DAG produced by this acyltransferase can serve as a substrate for both native and coexpressed diacylglycerol acyltransferases (DGAT). Finally, we show that the Arabidopsis thaliana GPAT4 acyltransferase can produce MAG in Saccharomyces cerevisiae using oleoyl-CoA as the acyl-donor.

Conclusions/Significance

This study demonstrates the concept of a new method of increasing oil content in vegetative tissues by using MAG as a substrate for TAG biosynthesis. Based on in vitro yeast assays and expression results in N. benthamiana, we propose that co-expression of a MAG synthesising enzyme such as A. thaliana GPAT4 and a MGAT or bifunctional M/DGAT can result in DAG and TAG synthesis from G-3-P via a route that is independent and complementary to the endogenous Kennedy pathway and other TAG synthesis routes.     

Sequence-based design of kinase inhibitors applicable for therapeutics and target identification

A platform for specifically modulating kinase-dependent signaling using peptides derived from the catalytic domain of the kinase is presented. This technology, termed KinAce, utilizes the canonical structure of protein kinases. The targeted regions (subdomain V and subdomains IX and X) are analyzed and their sequence, three-dimensional structure, and involvement in protein-protein interaction are highlighted. Short myristoylated peptides were derived from the target regions of the tyrosine kinases c-Kit and Lyn and the serine/threonine kinases 3-phosphoinositide-dependent kinase-1 (PDK1) and Akt/protein kinase B (PKB). For each kinase an active designer peptide is shown to selectively inhibit the signaling of the kinase from which it is derived, and to inhibit cancer cell proliferation in the micromolar range. This technology emerges as an applicable tool for deriving sequence-based selective inhibitors for a broad range of protein kinases as hits that may be further developed into drugs. Moreover, it enables identification of novel kinase targets for selected therapeutic indications as demonstrated in the KinScreen application.