Activity-Based Protein Profiling Identifies α-Ketoamides as Inhibitors for Phospholipase A2 Group XVI

Abstract

Phospholipase A2, group XVI (PLA2G16) is a thiol hydrolase from the HRASLS family that regulates lipolysis in adipose tissue and has been identified as a host factor enabling the cellular entry of picornaviruses. Chemical tools are essential to visualize and control PLA2G16 activity, but they have not been reported to date. Here, we show that MB064, which is a fluorescent lipase probe, also labels recombinant and endogenously expressed PLA2G16. Competitive activity-based protein profiling (ABPP) using MB064 enabled the discovery of α-ketoamides as the first selective PLA2G16 inhibitors. LEI110 was identified as a potent PLA2G16 inhibitor (Ki = 20 nM) that reduces cellular arachidonic acid levels and oleic acid-induced lipolysis in human HepG2 cells. Gel-based ABPP and chemical proteomics showed that LEI110 is a selective pan-inhibitor of the HRASLS family of thiol hydrolases (i.e., PLA2G16, HRASLS2, RARRES3 and iNAT). Molecular dynamic simulations of LEI110 in the reported crystal structure of PLA2G16 provided insight in the potential ligand–protein interactions to explain its binding mode. In conclusion, we have developed the first selective inhibitor that can be used to study the cellular role of PLA2G16.

Structure–Activity Relationship Studies of α-Ketoamides as Inhibitors of the Phospholipase A and Acyltransferase Enzyme Family

“…Compound 55 ( LEI-110 ) was identified as the most potent inhibitor of PLAAT3 and its biological characterization has previously been described in detail. 26 With regard to PLAAT2, a decrease in activity was observed for compounds 52–56 compared to 51 . Therefore, being the most potent inhibitor of PLAAT2, 51 (termed LEI-301 ) was selected for further characterization.…”

“…A focused in-house library of lipase inhibitors was screened for PLAAT inhibition using gel-based competitive ABPP. 26 This method uses an activity-based probe (ABP) containing an electrophilic group that forms a covalent bond with the catalytic nucleophile of an enzyme. 27 ABPs are also equipped with a reporter group such as a fluorophore or biotin, which allows visualization of enzymatic activity in a native biological setting.…”

“…Previously, MB064, 28 which incorporates a β-lactone electrophilic group, was validated as an effective ABP for the PLAAT enzyme family. 26 Here, cytosol fractions of human embryonic kidney HEK293T cells that overexpressed PLAAT2–5 were treated with potential inhibitors (10 μM, 30 min), followed by incubation with MB064 (250 nM, 20 min) ( Figure 1 A). The proteins were then resolved by SDS-PAGE, and the PLAAT activity was quantified by in-gel fluorescence scanning.…”

“…It was envisioned that the electrophilic ketone of 1 could bind with the PLAAT active site cysteine through a reversible covalent mechanism forming a hemithioketal adduct, similar to other reported α-ketoamide inhibitors. 26 , 29 To test this hypothesis, compound 23 was prepared, in which the ketone was replaced by an alcohol. This compound showed no activity at 10 μM ( Table 2 ), which is in line with the hypothesis.…”

“…Recently, we described the discovery of α-ketoamide LEI-110 as the first pan-active PLAAT inhibitor using a gel-based competitive activity-based protein profiling (ABPP) assay. 26 LEI-110 was able to reduce arachidonic acid levels in PLAAT3-overexpressing cells and in HepG2 cells. Here, we report the structure–activity relationship (SAR) of a library of α-ketoamides on the PLAAT family members.…”