Inhibitor Report for: Compound-7600

BACKGROUND: Human lymphocyte antigen B-associated transcript 5 (BAT5, also known as ABHD16A) is a poorly characterized 63 kDa protein belonging to the alpha/beta-hydrolase domain (ABHD) containing family of metabolic serine hydrolases. Its natural substrates and biochemical properties are unknown. METHODOLOGY/PRINCIPAL FINDINGS: Amino acid sequence comparison between seven mammalian BAT5 orthologs revealed that the overall primary structure was highly (>/=95%) conserved. Activity-based protein profiling (ABPP) confirmed successful generation of catalytically active human (h) and mouse (m) BAT5 in HEK293 cells, enabling further biochemical characterization. A sensitive fluorescent glycerol assay reported hBAT5-mediated hydrolysis of medium-chain saturated (C14ratio0), long-chain unsaturated (C18ratio1, C18ratio2, C20ratio4) monoacylglycerols (MAGs) and 15-deoxy-Delta12,14-prostaglandin J2-2-glycerol ester (15d-PGJ2-G). In contrast, hBAT5 possessed only marginal diacylglycerol (DAG), triacylglycerol (TAG), or lysophospholipase activity. The best MAG substrates were 1-linoleylglycerol (1-LG) and 15d-PGJ2-G, both exhibiting low-micromolar Km values. BAT5 had a neutral pH optimum and showed preference for the 1(3)- vs. 2-isomers of MAGs C18ratio1, C18ratio2 and C20ratio4. Inhibitor profiling revealed that beta-lactone-based lipase inhibitors were nanomolar inhibitors of hBAT5 activity (palmostatin B > tetrahydrolipstatin > ebelactone A). Moreover, the hormone-sensitive lipase inhibitor C7600 (5-methoxy-3-(4-phenoxyphenyl)-3H-[1], [3], [4]oxadiazol-2-one) was identified as a highly potent inhibitor (IC50 8.3 nM). Phenyl and benzyl substituted analogs of C7600 with increased BAT5 selectivity were synthesized and a preliminary SAR analysis was conducted to obtain initial insights into the active site dimensions. CONCLUSIONS/SIGNIFICANCE: This study provides an initial characterization of BAT5 activity, unveiling the biochemical and pharmacological properties with in vitro substrate preferences and inhibitor profiles. Utilization of glycerolipid substrates and sensitivity to lipase inhibitors suggest that BAT5 is a genuine lipase with preference for long-chain unsaturated MAGs and could in this capacity regulate glycerolipid metabolism in vivo as well. This preliminary SAR data should pave the way towards increasingly potent and BAT5-selective inhibitors.

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.

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.

BACKGROUND: Human lymphocyte antigen B-associated transcript 5 (BAT5, also known as ABHD16A) is a poorly characterized 63 kDa protein belonging to the alpha/beta-hydrolase domain (ABHD) containing family of metabolic serine hydrolases. Its natural substrates and biochemical properties are unknown. METHODOLOGY/PRINCIPAL FINDINGS: Amino acid sequence comparison between seven mammalian BAT5 orthologs revealed that the overall primary structure was highly (>/=95%) conserved. Activity-based protein profiling (ABPP) confirmed successful generation of catalytically active human (h) and mouse (m) BAT5 in HEK293 cells, enabling further biochemical characterization. A sensitive fluorescent glycerol assay reported hBAT5-mediated hydrolysis of medium-chain saturated (C14ratio0), long-chain unsaturated (C18ratio1, C18ratio2, C20ratio4) monoacylglycerols (MAGs) and 15-deoxy-Delta12,14-prostaglandin J2-2-glycerol ester (15d-PGJ2-G). In contrast, hBAT5 possessed only marginal diacylglycerol (DAG), triacylglycerol (TAG), or lysophospholipase activity. The best MAG substrates were 1-linoleylglycerol (1-LG) and 15d-PGJ2-G, both exhibiting low-micromolar Km values. BAT5 had a neutral pH optimum and showed preference for the 1(3)- vs. 2-isomers of MAGs C18ratio1, C18ratio2 and C20ratio4. Inhibitor profiling revealed that beta-lactone-based lipase inhibitors were nanomolar inhibitors of hBAT5 activity (palmostatin B > tetrahydrolipstatin > ebelactone A). Moreover, the hormone-sensitive lipase inhibitor C7600 (5-methoxy-3-(4-phenoxyphenyl)-3H-[1], [3], [4]oxadiazol-2-one) was identified as a highly potent inhibitor (IC50 8.3 nM). Phenyl and benzyl substituted analogs of C7600 with increased BAT5 selectivity were synthesized and a preliminary SAR analysis was conducted to obtain initial insights into the active site dimensions. CONCLUSIONS/SIGNIFICANCE: This study provides an initial characterization of BAT5 activity, unveiling the biochemical and pharmacological properties with in vitro substrate preferences and inhibitor profiles. Utilization of glycerolipid substrates and sensitivity to lipase inhibitors suggest that BAT5 is a genuine lipase with preference for long-chain unsaturated MAGs and could in this capacity regulate glycerolipid metabolism in vivo as well. This preliminary SAR data should pave the way towards increasingly potent and BAT5-selective inhibitors.

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.

Monoglyceride lipase (MGL) is a serine hydrolase that terminates the signaling of the primary endocannabinoid, 2-arachidonoyl glycerol (2-AG). Versatile high-throughput screening methods allowing the testing of MGL inhibitors are rare, thereby limiting the development and analysis of novel inhibitors. Here we describe an improved fluorescence-based technique that is capable of determining time- and dose-dependent inhibition of MGL with one or multiple binding sites and, at the same time, is capable of revealing the reversibility of inhibitor binding in a simple kinetic assay format. Known reference compounds as well as novel inhibitors, such as JZL184 and CAY10499, were evaluated for their MGL-binding properties and potency.

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.