Substrate Report for: Arachidonoyl-1-thioglycerol

Salinipostin A (Sal A) is a potent antiplasmodial marine natural product with an undefined mechanism of action. Using a Sal A-derived activity-based probe, we identify its targets in the Plasmodium falciparum parasite. All of the identified proteins contain alpha/beta serine hydrolase domains and several are essential for parasite growth. One of the essential targets displays a high degree of homology to human monoacylglycerol lipase (MAGL) and is able to process lipid esters including a MAGL acylglyceride substrate. This Sal A target is inhibited by the anti-obesity drug Orlistat, which disrupts lipid metabolism. Resistance selections yielded parasites that showed only minor reductions in sensitivity and that acquired mutations in a PRELI domain-containing protein linked to drug resistance in Toxoplasma gondii. This inability to evolve efficient resistance mechanisms combined with the non-essentiality of human homologs makes the serine hydrolases identified here promising antimalarial targets.

Lipids are used as cellular building blocks and condensed energy stores and also act as signaling molecules. The glycerolipid/ fatty acid cycle, encompassing lipolysis and lipogenesis, generates many lipid signals. Reliable procedures are not available for measuring activities of several lipolytic enzymes for the purposes of drug screening, and this resulted in questionable selectivity of various known lipase inhibitors. We now describe simple assays for lipolytic enzymes, including adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), sn-1-diacylglycerol lipase (DAGL), monoacylglycerol lipase, alpha/beta-hydrolase domain 6, and carboxylesterase 1 (CES1) using recombinant human and mouse enzymes either in cell extracts or using purified enzymes. We observed that many of the reported inhibitors lack specificity. Thus, Cay10499 (HSL inhibitor) and RHC20867 (DAGL inhibitor) also inhibit other lipases. Marked differences in the inhibitor sensitivities of human ATGL and HSL compared with the corresponding mouse enzymes was noticed. Thus, ATGListatin inhibited mouse ATGL but not human ATGL, and the HSL inhibitors WWL11 and Compound 13f were effective against mouse enzyme but much less potent against human enzyme. Many of these lipase inhibitors also inhibited human CES1. Results describe reliable assays for measuring lipase activities that are amenable for drug screening and also caution about the specificity of the many earlier described lipase inhibitors.

Current assays of phospholipase A1 (EC 3.1.1.32) and monoacylglycerol lipase (EC 3.1.1.23) activities in tissues are discontinuous, laborious, and expensive. Some spectrophotometric substrates were synthesized to alleviate this problem. Thioester analogs of phosphatidylcholine and phosphatidylethanolamine. rac-1,2-S,O-didecanoyl-3-phosphocholine-1-mercapto-2,3-propanediol and rac-1,2-S,O-didecanoyl-3-phosphoethanolamine-1-mercapto-2,3-propanediol, were synthesized from the diacylglycerol analog, rac-1,2-S,O--didecanoyl-1-mercapto-2,3-propanediol. The latter was prepared from triacylmercaptoglycerol by lipolysis and separation by chromatography on silica gel. Monoacylglycerol thioester analogs, 1-S-hexadecanoyl-1-mercapto-2,3-propanediol and 1-S-decanoyl-1-mercapto-2,3-propanediol, were synthesized by selective acylation of mercaptoglycerol. All of the substrates were hydrolyzed by Rhizopus delemar lipase to release sulfhydryl groups reactive towards 4,4'-dithiobispyridine. The hydrolysis could be followed continuously in a spectrophotomer with 0.1 absorbance unit corresponding to 5 nmol product. The structure and isomeric purity of the phospholipid analogs were verified by their behavior on thin-layer chromatography, elemental analyses, infrared spectra, and by the specificity of the colorimetric reaction with lipolytic enzymes. Whereas phospholipase A1 activity hydrolyzed both phospholipid analogs to release the theoretical amount of free thiol, neither phospolipases C nor A2 promoted thio release. The pH optimum, heat stability, and Ca2+ ion dependency were determined for the hydrolysis of each substrate by R. delemar lipase. The results indicate that the phospholipase A1 and monoacylglycerol lipase activities in R. delemar lipase are due to separate enzymes, and that these enzyme specific assays will be of general utility for enzyme characterization and purification studies. These substrates are useful for sensitive, convenient, and specific spectrophotometric assays for phospholipase A1 and monoacylglycerol lipase over the pH range 3 to 8.