NMR and Docking Calculations Reveal Novel Atomistic Selectivity of a Synthetic High-Affinity Free Fatty Acid vs. Free Fatty Acids in Sudlow's Drug Binding Sites in Human Serum Albumin.

Saturation transfer difference (STD), inter-ligand NOEs (INPHARMA NMR), and docking calculations are reported for investigating specific binding sites of the high-affinity synthetic 7-nitrobenz-2-oxa-1,3-diazoyl-4-C12 fatty acid (NBD-C12 FA) with non-labeled human serum albumin (HSA) and in competition with the drugs warfarin and ibuprofen. A limited number of negative interligand NOEs between NBD-C12 FA and warfarin were interpreted in terms of a short-range allosteric competitive binding in the wide Sudlow's binding site II (FA7) of NBD-C12 FA with Ser-202, Lys-199, and Trp-214 and warfarin with Arg-218 and Arg-222. In contrast, the significant number of interligand NOEs between NBD-C12 FA and ibuprofen were interpreted in terms of a competitive binding mode in Sudlow's binding site I (FA3 and FA4) with Ser-342, Arg-348, Arg-485, Arg-410, and Tyr-411. NBD-C12 FA has the unique structural properties, compared to short-, medium-, and long-chain saturated and unsaturated natural free fatty acids, of interacting with well-defined structures with amino acids of both the internal and external polar anchor sites in Sudlow's binding site I and with amino acids in both FA3 and FA4 in Sudlow's binding site II. The NBD-C12 FA, therefore, interacts with novel structural characteristics in the drug binding sites I and II and can be regarded as a prototype molecule for drug development.

Differential sensing for the regio- and stereoselective identification and quantitation of glycerides.

Glycerides are of interest to the areas of food science and medicine because they are the main component of fat. From a chemical sensing perspective, glycerides are challenging analytes because they are structurally similar to one another and lack diversity in terms of functional groups. Furthermore, because animal and plant fat consists of a number of stereo- and regioisomeric acylglycerols, their components remain challenging analytes for chromatographic and mass spectrometric determination, particularly the quantitation of species in mixtures. In this study, we demonstrated the use of an array of cross-reactive serum albumins and fluorescent indicators with chemometric analysis to differentiate a panel of mono-, di-, and triglycerides. Due to the difficulties in identifying the regio- and stereochemistry of the unsaturated glycerides, a sample pretreatment consisting of olefin cross-metathesis with an allyl fluorescein species was used before array analysis. Using this simple assay, we successfully discriminated 20 glycerides via principal component analysis and linear discriminant analysis (PCA and LDA, respectively), including stereo- and regioisomeric pairs. The resulting chemometric patterns were used as a training space for which the structural characteristics of unknown glycerides were identified. In addition, by using our array to perform a standard addition analysis on a mixture of triglycerides and using a method introduced herein, we demonstrated the ability to quantitate glyceride components in a mixture.

Identification and Characterization of a Single High-Affinity Fatty Acid Binding Site in Human Serum Albumin.

A single high-affinity fatty acid binding site in the important human transport protein serum albumin (HSA) is identified and characterized using an NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl)-C12 fatty acid. This ligand exhibits a 1:1 binding stoichiometry in its HSA complex with high site-specificity. The complex dissociation constant is determined by titration experiments as well as radioactive equilibrium dialysis. Competition experiments with the known HSA-binding drugs warfarin and ibuprofen confirm the new binding site to be different from Sudlow-sites I and II. These binding studies are extended to other albumin binders and fatty acid derivatives. Furthermore an X-ray crystal structure allows locating the binding site in HSA subdomain IIA. The knowledge about this novel HSA site will be important for drug depot development and for understanding drug-protein interaction, which are important prerequisites for modulation of drug pharmacokinetics.

Discovery and pharmacological characterization of a novel small molecule inhibitor of phosphatidylinositol-5-phosphate 4-kinase, type II, beta.

Phosphatidylinositol-5-phosphate 4-kinase, type II, beta (PIP5K2B) is linked to the pathogenesis of obesity, insulin resistance and diabetes. Here, we describe the identification of a novel pyrimidine-2,4-diamine PIP5K2B inhibitor, designated SAR088. The compound was identified by high-throughput screening and subsequently characterized in vitro and in vivo. SAR088 showed reasonable potency, selectivity and physicochemical properties in enzymatic and cellular assays. In vivo, SAR088 lowered blood glucose levels of obese and hyperglycemic male Zucker diabetic fatty rats treated for 3 weeks. Thus, SAR088 represents the first orally available and in vivo active PIP5K2B inhibitor and provides an excellent starting point for the development of potent and selective PIP5K2B inhibitors for the treatment of insulin resistance and diabetes.

Molecular basis for inhibiting human glucose transporters by exofacial inhibitors.

Human glucose transporters (GLUTs) are responsible for cellular uptake of hexoses. Elevated expression of GLUTs, particularly GLUT1 and GLUT3, is required to fuel the hyperproliferation of cancer cells, making GLUT inhibitors potential anticancer therapeutics. Meanwhile, GLUT inhibitor-conjugated insulin is being explored to mitigate the hypoglycemia side effect of insulin therapy in type 1 diabetes. Reasoning that exofacial inhibitors of GLUT1/3 may be favored for therapeutic applications, we report here the engineering of a GLUT3 variant, designated GLUT3exo, that can be probed for screening and validating exofacial inhibitors. We identify an exofacial GLUT3 inhibitor SA47 and elucidate its mode of action by a 2.3 Å resolution crystal structure of SA47-bound GLUT3. Our studies serve as a framework for the discovery of GLUTs exofacial inhibitors for therapeutic development.

Brummer lipase is an evolutionary conserved fat storage regulator in Drosophila.

Energy homeostasis, a fundamental property of all organisms, depends on the ability to control the storage and mobilization of fat, mainly triacylglycerols (TAG), in special organs such as mammalian adipose tissue or the fat body of flies. Malregulation of energy homeostasis underlies the pathogenesis of obesity in mammals including human. We performed a screen to identify nutritionally regulated genes that control energy storage in the model organism Drosophila. The brummer (bmm) gene encodes the lipid storage droplet-associated TAG lipase Brummer, a homolog of human adipocyte triglyceride lipase (ATGL). Food deprivation or chronic bmm overexpression depletes organismal fat stores in vivo, whereas loss of bmm activity causes obesity in flies. Our study identifies a key factor of insect energy homeostasis control. Their evolutionary conservation suggests Brummer/ATGL family members to be implicated in human obesity and establishes a basis for modeling mechanistic and therapeutic aspects of this disease in the fly.

Resolving Binding Events on the Multifunctional Human Serum Albumin.

Physiological processes rely on initial recognition events between cellular components and other molecules or modalities. Biomolecules can have multiple sites or mode of interaction with other molecular entities, so that a resolution of the individual binding events in terms of spatial localization as well as association and dissociation kinetics is required for a meaningful description. Here we describe a trichromatic fluorescent binding- and displacement assay for simultaneous monitoring of three individual binding sites in the important transporter and binding protein human serum albumin. Independent investigations of binding events by X-ray crystallography and time-resolved dynamics measurements (switchSENSE technology) confirm the validity of the assay, the localization of binding sites and furthermore reveal conformational changes associated with ligand binding. The described assay system allows for the detailed characterization of albumin-binding drugs and is therefore well-suited for prediction of drug-drug and drug-food interactions. Moreover, conformational changes, usually associated with binding events, can also be analyzed.

Acute and Repeated Treatment with 5-PAHSA or 9-PAHSA Isomers Does Not Improve Glucose Control in Mice.

Fatty acid esters of hydroxylated fatty acids (FAHFAs) were discovered as a novel class of endogenous mammalian lipids whose profound effects on metabolism have been shown. In the current study, in vitro and in vivo the metabolic effects of two of these FAHFAs, namely palmitic acid-5- (or -9) -hydroxy-stearic acid (5- or 9-PAHSA, respectively) were profiled. In DIO mice fed with differentially composed low- or high-fat diets, acute and subchronic treatment with 5-PAHSA and 9-PAHSA alone, or in combination, did not significantly improve the deranged metabolic status. Neither racemic 5- or 9-PAHSA, nor the enantiomers were able to: (1) increase basal or insulin-stimulated glucose uptake in vitro, (2) stimulate GLP-1 release from GLUTag cells, or (3) induce GSIS in rat, mouse, or human islets or in a human pancreatic ß cell line. Therefore, our data do not support the further development of PAHSAs or their derivatives for the control of insulin resistance and hyperglycemia.

Substrate specificity and kinetic properties of enzymes belonging to the hormone-sensitive lipase family: comparison with non-lipolytic and lipolytic carboxylesterases.

We have studied the kinetics of hydrolysis of triacylglycerols, vinyl esters and p-nitrophenyl butyrate by four carboxylesterases of the HSL family, namely recombinant human hormone-sensitive lipase (HSL), EST2 from Alicyclobacillus acidocaldarius, AFEST from Archeoglobus fulgidus, and protein RV1399C from Mycobacterium tuberculosis. The kinetic properties of enzymes of the HSL family have been compared to those of a series of lipolytic and non-lipolytic carboxylesterases including human pancreatic lipase, guinea pig pancreatic lipase related protein 2, lipases from Mucor miehei and Thermomyces lanuginosus, cutinase from Fusarium solani, LipA from Bacillus subtilis, porcine liver esterase and Esterase A from Aspergilus niger. Results indicate that human HSL, together with other lipolytic carboxylesterases, are active on short chain esters and hydrolyze water insoluble trioctanoin, vinyl laurate and olive oil, whereas the action of EST2, AFEST, protein RV1399C and non-lipolytic carboxylesterases is restricted to solutions of short chain substrates. Lipolytic and non-lipolytic carboxylesterases can be differentiated by their respective value of K(0.5) (apparent K(m)) for the hydrolysis of short chain esters. Among lipolytic enzymes, those possessing a lid domain display higher activity on tributyrin, trioctanoin and olive oil suggesting, then, that the lid structure contributes to enzyme binding to triacylglycerols. Progress reaction curves of the hydrolysis of p-nitrophenyl butyrate by lipolytic carboxylesterases with lid domain show a latency phase which is not observed with human HSL, non-lipolytic carboxylesterases, and lipolytic enzymes devoid of a lid structure as cutinase.

Prolonged blood glucose reduction in mrp-2 deficient rats (GY/TR(-)) by the glucose-6-phosphate translocase inhibitor S 3025.

Chlorogenic acid derivatives are potent inhibitors of hepatic glucose production by inhibition of the glucose-6-phosphate translocase component of the hepatic glucose-6-phosphatase system. The pharmacological proof of concept was clearly demonstrated during i.v. infusion of potent derivatives (S 4048, S 3483) in rats. However, the blood glucose lowering effect of S 4048 after bolus i.v. injection lasted only 60-90 min. Plasma clearance of S 4048 was very high, and the parent compound was rapidly and efficiently excreted into the bile of Wistar and GY/TR(-) rats, indicating that mrp-2 was not involved in this hepatobiliary elimination process. About 72% of the total administered radioactivity appeared in the bile within 20 min after i.v. bolus injection of the radiolabeled analogue [(3)H]S 1743 in a Wistar rat. However, in GY/TR(-) rats the dicarboxylic analogue of S 4048, S 3025, was cleared from the plasma less rapidly than its parent compound and its biliary elimination was comparatively low. In contrast, S 3025 exhibited comparable pharmacokinetics and biliary elimination profile as S 4048 in Wistar rats, suggesting that biliary elimination of S 3025 is facilitated by mrp-2, functionally absent in GY/TR(-) rats. Targeting to mrp-2 resulted in a significantly prolonged reduction of blood glucose levels in GY/TR(-) rats after i.v. bolus administration of S 3025.

Analysis of lipolysis in adipocytes using a fluorescent fatty acid derivative.

For facilitation of the experimental analysis of the mechanism and regulation of mobilization of fatty acids from adipose triacylglycerol (TAG) stores, which also represents important targets for pharmacological intervention with the pathogenesis of diabetes and obesity, we developed a convenient and reliable non-radioactive cell-based assay. Isolated rat adipocytes are incubated with the fluorescent fatty acid derivative, 12-((7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoic acid (NBD-FA), in the presence of insulin. The resulting NBD-FA-labeled TAG is efficiently cleaved by hormone-sensitive lipase (HSL) in vitro. After removal of insulin and excess of free NBD-FA, lipolysis is initiated by addition of isoproterenol and/or adenosine deaminase. The amount of NBD-FA generated in total or released into the incubation medium in the presence of modulatory hormones or compounds is then monitored by thin layer chromatography and fluorescence imaging. Release of NBD-FA, glycerol and [3H]oleic acid from TAG follows similar kinetics and concentration dependence in response to various lipolytic and anti-lipolytic stimuli as well as inhibitors of HSL. Release of NBD-FA from adipocytes correlates well to translocation of HSL from the cytosol to TAG droplets. In addition, we found that a cell-free system consisting of NBD-FA-labeled TAG droplets with endogenous associated HSL closely reflects the lipolytic state of the adipocytes used for its preparation. In conclusion, release of NBD-FA from TAG in vivo and in vitro can be used as accurate index for (regulation of) lipolysis in primary and cultured adipocytes.

The molecular mechanism of human hormone-sensitive lipase inhibition by substituted 3-phenyl-5-alkoxy-1,3,4-oxadiazol-2-ones.

Hormone-sensitive lipase (HSL) plays an important role in the mobilization of free fatty acids (FFA) from adipocytes. The inhibition of HSL may offer a pharmacological approach to reduce FFA levels in plasma and diminish peripheral insulin resistance in type 2 diabetes. In this work, the inhibition of HSL by substituted 3-phenyl-5-alkoxy-1,3,4-oxadiazol-2-ones has been studied in vitro. 5-methoxy-3-(3-phenoxyphenyl)-1,3,4-oxadiazol-2(3H)-one (compound 7600) and 5-methoxy-3-(3-methyl-4-phenylacetamidophenyl)-1,3,4-oxadiazol-2(3H)-one (compound 9368) were selected as the most potent HSL inhibitors. HSL is inhibited after few minutes of incubation with compound 7600, at a molar excess of 20. This inhibition is reversed in the presence of an emulsion of lipid substrate. The reactivation phenomenon is hardly observed when incubating HSL with compound 9368. The molecular mechanism underlying the reversible inhibition of HSL by compound 7600 was investigated using high performance liquid chromatography and tandem mass spectrometry. The stoichiometry of the inhibition reaction revealed that specifically one molecule of inhibitor was bound per enzyme molecule. The inhibition by compound 7600 involves a nucleophilic attack by the hydroxy group of the catalytic Ser of the enzyme on the carbon atom of the carbonyl moiety of the oxadiazolone ring of the inhibitor, leading to the formation of covalent enzyme-inhibitor intermediate. This covalent intermediate is subsequently hydrolyzed, releasing an oxadiazolone decomposition product, carbon dioxide and the active HSL form. On the basis of this study, a kinetic model is proposed to describe the inhibition of HSL by compound 7600 in the aqueous phase as well as its partial reactivation at the lipid-water interface.

Use of an inhibitor to identify members of the hormone-sensitive lipase family.

Hormone-sensitive lipase (HSL) contributes importantly to the mobilization of fatty acids from the triacylglycerols stored in adipocytes, which provide the main source of energy in mammals. On the basis of amino acid sequence alignments and three-dimensional structures, this enzyme was previously found to be a suitable template for defining a family of serine carboxylester hydrolases. In this study, the HSL family members are characterized rather on the basis of their inhibition by 5-methoxy-3-(4-phenoxyphenyl)-3H-[1,3,4]oxadiazol-2-one (compound 7600). This compound inhibits mammalian HSL as well as other HSL family members, such as EST2 from the thermophilic eubacterium Alicyclobacillus acidocaldarius and AFEST from the hyperthermophilic archaeon Archaeoglobus fulgidus. Various carboxylester hydrolases that are not members of the HSL family were found not to be inhibited by compound 7600 under the same experimental conditions. These include nonlipolytic hydrolases such as Torpedo californica acetylcholinesterase and pig liver esterase, as well as lipolytic hydrolases such as human pancreatic lipase, dog gastric lipase, Thermomyces lanuginosus lipase, and Bacillus subtilis LipA. When vinyl esters were used as substrates, the residual activity of HSL, AFEST, and EST2 decreased with an increase in compound 7600 concentration in the incubation mixture. The inhibitor concentration at which the enzyme activity decreased to 50% after incubation for 5 min was 70, 20, and 15 nM with HSL, AFEST, and EST2, respectively. Treating EST2 and AFEST with the inhibitor resulted in an increase in the molecular mass, as established by performing matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis. This increase in the molecular mass, which corresponds approximately to the molecular mass of the inhibitor, indicates that a covalent enzyme-inhibitor complex has been formed. Surface-enhanced laser desorption ionization time-of-flight mass spectrometry analysis of a trypsin digest of AFEST treated with the inhibitor or not treated showed the occurrence of an increase in the molecular masses of the "GESAGG"-containing peptide, which is compatible with the formation of a covalent complex with the inhibitor.

6,8-Difluoro-4-methylumbiliferyl phosphate: a fluorogenic substrate for protein tyrosine phosphatases.

The fluorogenic substrate 6,8-difluoro-4-methylumbiliferyl phosphate (DIFMUP) has been widely used for the detection of serine and threonine phosphatase activities. Here we describe the use of this substrate for the characterization of protein tyrosine phosphatases (PTPs) and for the screening for PTP inhibitors. The measured kinetic and inhibitor constants for DIFMUP cleavage were comparable with those of the widely used but less discriminative and practicable substrates, para-nitrophenylphosphate and phosphotyrosine-containing peptides, respectively. Furthermore, the continuous and highly sensitive assay allows fast and accurate investigations of the type, kinetic behavior, and binding mode of small-molecule inhibitors. We discuss the validation of this assay system for various PTPs and its use in inhibitor screening for PTP1B.

Continuous monitoring of cholesterol oleate hydrolysis by hormone-sensitive lipase and other cholesterol esterases.

Hormone-sensitive lipase (HSL) contributes importantly to the hydrolysis of cholesteryl ester in steroidogenic tissues, releasing the cholesterol required for adrenal steroidogenesis. HSL has broad substrate specificity, because it hydrolyzes triacylglycerols (TAGs), diacylglycerols, monoacylglycerols, and cholesteryl esters. In this study, we developed a specific cholesterol esterase assay using cholesterol oleate (CO) dispersed in phosphatidylcholine and gum arabic by sonication. To continuously monitor the hydrolysis of CO by HSL, we used the pH-stat technique. For the sake of comparison, the hydrolysis of CO dispersion was also tested using other cholesteryl ester-hydrolyzing enzymes. The specific activities measured on CO were found to be 18, 100, 27, and 3 micromol/min/mg for HSL, cholesterol esterase from Pseudomonas species, Candida rugosa lipase-3, and cholesterol esterase from bovine pancreas, respectively. The activity of HSL on CO is approximately 4- to 5-fold higher than on long-chain TAGs. In contrast, with all other enzymes tested, the rates of TAG hydrolysis were higher than those of CO hydrolysis. The relatively higher turnover of HSL on CO observed in vitro adds further molecular insight on the physiological importance of HSL in cholesteryl ester catabolism in vivo. Thus, HSL could be considered more as a cholesteryl ester hydrolase than as a TAG lipase.

Sensitive assay for hormone-sensitive lipase using NBD-labeled monoacylglycerol to detect low activities in rat adipocytes.

The recent finding that p-nitrobenzofurazan (NBD)-FA is incorporated into and released from the acylglycerols of isolated rat adipocytes in an insulin-sensitive manner [G. Muller, H. Jordan, C. Jung, H. Kleine, and S. Petry. 2003. Biochimie. 85: 1245-1246] suggests that NBD-FA-labeled acylglycerols are cleaved by rat adipocyte hormone-sensitive lipase (HSL) in vivo. In the present study, we developed a continuous, sensitive in vitro lipase assay using a monoacylglycerol (MAG) containing NBD (NBD-MAG). NBD-MAG was found to provide an efficient substrate for rat adipocyte and human recombinant HSL. Ultrasonic treatment applied in the presence of phospholipids leads to the incorporation of NBD-MAG into the phospholipid liposomes and to a concomitant change of its spectrophotometric properties. The enzymatic release of NBD-FA and its dissociation from the carrier liposomes is accompanied by the recovery of the original spectrophotometric characteristics. The rate of lipolysis was monitored by measuring the increase in optical density at 481 nm, which was found to be linear with time and linearly proportional to the amount of lipase added. To assess the specific activity of recombinant HSL, we determined the molar extinction coefficient of NBD-FA under the assay conditions. This convenient assay procedure based on NBD-MAG should facilitate the search for small molecule HSL inhibitors.

Might the kinetic behavior of hormone-sensitive lipase reflect the absence of the lid domain?

Hormone-sensitive lipase (HSL) is thought to contribute importantly to the mobilization of fatty acids from the triacylglycerols (TAGs) stored in adipocytes, providing the main source of energy in mammals. To investigate the HSL substrate specificity more closely, we systematically assessed the lipolytic activity of recombinant human HSL on solutions and emulsions of various vinyl esters and TAG substrates, using the pH-stat assay technique. Recombinant human HSL activity on solutions of partly soluble vinyl esters or TAG was found to range from 35 to 90% of the maximum activity measured with the same substrates in the emulsified state. The possible existence of a lipid-water interface due to the formation of small aggregates of vinyl esters or TAG in solution may account for the HSL activity observed below the solubility limit of the substrate. Recombinant human HSL also hydrolyzes insoluble medium- and long-chain acylglycerols such as trioctanoylglycerol, dioleoylglycerol, and olive oil, and can therefore be classified as a true lipase. Preincubation of the recombinant HSL with a serine esterase inhibitor such as diethyl p-nitrophenyl phosphate in 1:100 molar excess leads to complete HSL inhibition within 15 min. This result indicates that the catalytic serine of HSL is highly reactive and that it is readily accessible. Similar behavior was also observed with lipases with no lid domain covering their active site, or with a deletion in the lid domain. The 3-D structure of HSL, which still remains to be determined, may therefore lack the lid domain known to exist in various other lipases.