Discovery of Triterpenoids as Reversible Inhibitors of α/β hydrolase Domain Containing 12 (ABHD12)

Peer reviewe

Suppression of Saprolegnia infections in rainbow trout (Oncorhynchus mykiss) eggs using protective bacteria and ultraviolet irradiation of the hatchery water

201

Biochemical and Pharmacological Characterization of the Human Lymphocyte Antigen B-Associated Transcript 5 (BAT5/ABHD16A)

<div><p>Background</p><p>Human lymphocyte antigen B-associated transcript 5 (BAT5, also known as ABHD16A) is a poorly characterized 63 kDa protein belonging to the α/β-hydrolase domain (ABHD) containing family of metabolic serine hydrolases. Its natural substrates and biochemical properties are unknown.</p><p>Methodology/Principal Findings</p><p>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 (C14∶0), long-chain unsaturated (C18∶1, C18∶2, C20∶4) monoacylglycerols (MAGs) and 15-deoxy-Δ^12,14-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 K_m values. BAT5 had a neutral pH optimum and showed preference for the 1(3)- vs. 2-isomers of MAGs C18∶1, C18∶2 and C20∶4. Inhibitor profiling revealed that β-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-<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109869#pone.0109869-Simon1" target="_blank">[1]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109869#pone.0109869-Lord1" target="_blank">[3]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109869#pone.0109869-Hoover1" target="_blank">[4]</a>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.</p><p>Conclusions/Significance</p><p>This study provides an initial characterization of BAT5 activity, unveiling the biochemical and pharmacological properties with <i>in vitro</i> 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 <i>in vivo</i> as well. This preliminary SAR data should pave the way towards increasingly potent and BAT5-selective inhibitors.</p></div

K_m and V_max values for hBAT5-dependent hydrolysis of 1-LG and 15d-PGJ₂-G.

<p>HEK293 cells were transiently transfected with the cDNA encoding hBAT5, as detailed in the Methods section. After 48 h, cells were harvested and lysates prepared for hydrolase activity measurements. Cellular lysates (0.3 µg/well) were incubated together with the indicated concentrations of 1-LG in assay mixes containing (+ BSA) or not (– BSA) 0.5% (w/v) BSA (<b>A</b>) or with 15d-PGJ₂-G in assay mix containing BSA (<b>B</b>). The K_m and V_max values (<b>C</b>) were determined at time-point 90 min, where substrate utilization was <10%, and were calculated as non-linear regressions using GraphPad Prism 5.0 for Windows. As crude cellular lysates were used, total protein concentration was used instead of true enzyme concentration. The Lineweaver-Burk plots are shown inside rectangles. Statistical comparison of the K_m and V_max values for 1-LG between the + and - BSA conditions was done using unpaired t-test and the significance is indicated with an asterix (*, p<0.05). Values are mean ± SEM from three independent experiments.</p

Activity-based protein profiling (ABPP) to visualize catalytically active BAT5 protein in lysates of HEK293 cells after transient expression of human (h) or mouse (m) BAT5 orthologs.

<p>Serine hydrolases were labeled using the active site serine targeting fluorescent probe TAMRA-FP. After separation in SDS-electrophoresis gel (10%), serine hydrolase activity was visualized by in-gel fluorescent gel scanning as detailed in the Methods section. Molecular weight markers (MW) are indicated at left. Transient transfections with the cDNAs encoding hBAT5 (<b>A</b>) or mBAT5 (<b>B</b>) results in robust labeling of a ∼63 kDa protein band that is absent from parental HEK293 cells. Data are from one typical transfection, transfections were repeated twice with similar outcome.</p

Competitive ABPP unveiling palmostatin B targets among the serine hydrolases in mouse brain membrane proteome.

<p>Membranes (100 µg) were pretreated for 1 h with DMSO or the indicated concentrations of the serine hydrolase inhibitors, after which TAMRA-FP labelling was conducted for 1 hour at RT. The reaction was stopped, 10 µg protein (10 µl) was loaded per lane and the proteins were separated in SDS-electrophoresis gel (10%). TAMRA-FP labeling was visualized after in-gel fluorescence imaging as described in the Methods section. Molecular weight markers are indicated at left. Reference inhibitors JJKK-048, WWL70, THL and JZL195 were used at the indicated concentrations to identify the following serine hydrolases from the gel: fatty acid amide hydrolase (FAAH), inhibited by JZL195 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109869#pone.0109869-Long2" target="_blank">[21]</a>; MAGL doublet, inhibited by JJKK-048 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109869#pone.0109869-Aaltonen1" target="_blank">[22]</a> and JZL195 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109869#pone.0109869-Long2" target="_blank">[21]</a>; ABHD6, inhibited by WWL70 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109869#pone.0109869-Li1" target="_blank">[23]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109869#pone.0109869-Patel1" target="_blank">[24]</a>, THL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109869#pone.0109869-Parkkari1" target="_blank">[18]</a>, and also by JZL195 at the used concentration; BAT5, inhibited by THL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109869#pone.0109869-Hoover1" target="_blank">[4]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109869#pone.0109869-Parkkari1" target="_blank">[18]</a>. Note that palmostatin B dose-dependently inhibits probe labeling of BAT5, ABHD12 and LYPLA1/2, two closely-related proteins migrating at ∼25 kDa <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109869#pone.0109869-Adibekian1" target="_blank">[25]</a>. At the highest concentration, probe labeling of MAGL and ABHD6 is also inhibited. Note the absence of additional visible targets for palmostatin B at the tested concentrations. Data are representative from three ABPP experiments with similar outcome.</p