ABHD11 (alpha/beta-hydrolase domain containing 11) is a non-annotated enzyme belonging to the family of metabolic serine hydrolases (mSHs). Its natural substrates and products are unknown. Using competitive activity-based protein profiling (ABPP) to identify novel inhibitors of human (h)ABHD11, three compounds from our chemical library exhibited low nanomolar potency towards hABHD11. Competitive ABPP of various proteomes revealed fatty acid amide hydrolase (FAAH) as the sole off-target among the mSHs. Our fluorescent activity assays designed for natural lipase substrates revealed no activity of hABHD11 towards mono- or diacylglycerols. A broader profiling using para-nitrophenyl (pNP)-linked substrates indicated no amidase/protease, phosphatase, sulfatase, phospholipase C or phosphodiesterase activity. Instead, hABHD11 readily utilized para-nitrophenyl butyrate (pNPC4), indicating lipase/esterase-type activity that could be exploited in inhibitor discovery. Additionally, a homology model was created based on the crystal structure of bacterial esterase YbfF. In contrast to YbfF, which reportedly hydrolyze long-chain acyl-CoA, hABHD11 did not utilize oleoyl-CoA or arachidonoyl-CoA. In conclusion, the present study reports the discovery of potent hABHD11 inhibitors with good selectivity among mSHs. We developed substrate-based activity assays for hABHD11 that could be further exploited in inhibitor discovery and created the first homology-based hABHD11 model, offering initial insights into the active site of this poorly characterized enzyme.
Protein palmitoylation is an essential post-translational modification necessary for trafficking and localization of regulatory proteins that play key roles in cell growth and signaling. Multiple oncogenes, including HRAS and SRC, require palmitoylation for malignant transformation. We and others have previously identified lysophospholipase 1 (LYPLA1) as a candidate protein palmitoyl thioesterase responsible for HRAS depalmitoylation in mammalian cells. Seeking chemical tools to investigate biochemical pathway involvement and potential roles in cancer pathogenesis, we conducted a fluorescence polarization-based competitive activity-based protein profiling (FluoPol ABPP) high throughput screening (HTS) campaign to identify inhibitors of LYPLA1 and the structurally related LYPLA2. HTS identified a micromolar triazole urea inhibitor, that we successfully optimized via several rounds of SAR-by-synthesis as ML211 (SID 99445338), a low nanomolar dual inhibitor of LYPLA1 and LYPLA2. The reported probe operates by a covalent mechanism of action and is active both in vitro and in situ. Out of more than 20 serine hydrolases (SHs) profiled by gel-based competitive activity-based protein profiling (ABPP), ML211 was observed to have one anti-target, alpha/beta hydrolase domain-containing protein 11 (ABHD11). Fortuitously, one of the triazole urea library members synthesized during the course of probe optimization was found to be a potent and selective inhibitor of ABHD11, a poorly characterized SH that is hemizygously deleted in Williams-Beuren syndrome [4], and was presented as a control anti-probe (SID 99445332) in the ML211 Probe Report. The optimized ABHD11 probe ML226 is a potent inhibitor of ABHD11, with an IC50 of 15 nM, and exhibits at least 100-fold selectivity for all other SHs (~20) assessed by gel-based competitive ABPP. The probe is also active in situ, completely and selectively inhibiting ABHD11 at sub-nanomolar concentrations. As with ML211, the probe operates by a covalent mechanism of action, carbamoylating the active site serine of ABHD11. The complete properties, characterization, and synthesis of ML226 are detailed in this Probe Report.
