There is interest in developing inhibitors of human group III secreted phospholipase A2 (hGIII-sPLA2) because this enzyme plays a role in mast cell maturation. There are no potent inhibitors for hGIII-sPLA2 reported to date, so we adapted a fluorescence-based enzyme activity monitoring method to a high-throughput screening format. We opted to use an assay based on phospholipid substrate present in phospholipid vesicles since this matrix more closely resembles the natural substrate of hGIII-sPLA2, as opposed to phospholipid/detergent mixed micelles. The substrate is a phospholipid analogue containing BODIPY fluorophores dispersed as a minor component in vesicles of nonfluorescent phospholipids. Action of hGIII-sPLA2 liberates a free fatty acid from the phospholipid, leading to a reduction in quenching of the fluorophore and hence an increase in fluorescence. The assay uses optical detection in a 1536-well plate format with an excitation wavelength far away from the UV range so as to minimize false-positive library hits that result from quenching of the fluorescence. The high-throughput screen was successfully carried out on a library of 370,276 small molecules. Several hits were discovered, and data have been uploaded to PubChem. This study describes the first high-throughput optical screening assay for secreted phospholipase A2 inhibitors based on a phospholipid vesicle substrate.
Fat and muscle lipolysis involves functional interactions of adipose triglyceride lipase (ATGL), alpha-beta hydrolase domain-containing protein 5 (ABHD5), and tissue-specific perilipins 1 and 5 (PLIN1 and PLIN5). ABHD5 potently activates ATGL, but this lipase-promoting activity is suppressed when ABHD5 is bound to PLIN proteins on lipid droplets. In adipocytes, protein kinase A (PKA) phosphorylation of PLIN1 rapidly releases ABHD5 to activate ATGL, but mechanisms for rapid regulation of PLIN5-ABHD5 interaction in muscle are unknown. Here, we identify synthetic ligands that release ABHD5 from PLIN1 or PLIN5 without PKA activation and rapidly activate adipocyte and muscle lipolysis. Molecular imaging and affinity probe labeling demonstrated that ABHD5 is directly targeted by these synthetic ligands and additionally revealed that ABHD5-PLIN interactions are regulated by endogenous ligands, including long-chain acyl-CoA. Our results reveal a new locus of lipolysis control and suggest ABHD5 ligands might be developed into novel therapeutics that directly promote fat catabolism.
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. Lysophospholipase 1 (LYPLA1) has been identified 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) HTS campaign to identify inhibitors of LYPLA1 and the structurally related LYPLA2. HTS identified a lead triazole urea micromolar inhibitor, which we optimized as dual LYPLA1/LYPLA2 inhibitor ML211, and reversible compounds ML348 and ML349 that act as selective LYPLA1 and LYPLA2 inhibitors, respectively. Using an advanced competitive ABPP strategy employing ABPP probes with controlled reactivity rates, we successfully confirmed potent and selective target engagement of these reversible compounds in living systems as detailed here for ML348 and in the accompanying ML349 Probe Report. Together, these compounds should greatly aid investigations into the biological function of LYPLA1 and LYPLA2
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. Lysophospholipase 1 (LYPLA1) has been identified as a candidate protein palmitoyl thioesterase responsible for HRAS depalmitoylation in mammalian cells. LYPLA1 has a close homolog, LYPLA2 (65% sequence identity), whose substrate specificity and biochemical roles are, as yet, uncharacterized. Seeking chemical tools to investigate biochemical pathway involvement and potential roles in cancer pathogenesis of these enzymes, we conducted a fluorescence polarization-based competitive activity-based protein profiling (fluopol-ABPP) HTS campaign to identify inhibitors of LYPLA1 and LYPLA2. HTS identified a lead triazole urea micromolar inhibitor, which we optimized as dual LYPLA1/LYPLA2 inhibitor ML211, and reversible compounds ML348 and ML349 that act as selective LYPLA1 and LYPLA2 inhibitors, respectively. Using an advanced competitive ABPP strategy employing ABPP probes with controlled reactivity rates, we successfully confirmed potent and selective target engagement of these reversible compounds in living systems as detailed here for ML349 and in the accompanying ML348 Probe Report. Together, these compounds should greatly aid investigations into the biological function of LYPLA1 and LYPLA2.
Lipoprotein-associated phospholipase A(2) (Lp-PLA(2) or PLA(2)G7) binds to low-density lipoprotein (LDL) particles, where it is thought to hydrolyze oxidatively truncated phospholipids. Lp-PLA(2) has also been implicated as a pro-tumorigenic enzyme in human prostate cancer. Several inhibitors of Lp-PLA(2) have been described, including darapladib, which is currently in phase 3 clinical development for the treatment of atherosclerosis. The selectivity that darapladib and other Lp-PLA(2) inhibitors display across the larger serine hydrolase family has not, however, been reported. Here, we describe the use of both general and tailored activity-based probes for profiling Lp-PLA(2) and inhibitors of this enzyme in native biological systems. We show that both darapladib and a novel class of structurally distinct carbamate inhibitors inactivate Lp-PLA(2) in mouse tissues and human cell lines with high selectivity. Our findings thus identify both inhibitors and chemoproteomic probes that are suitable for investigating Lp-PLA(2) function in biological systems.
Endocannabinoids (ECs) are a unique group of lipids that function as chemical messengers in the nervous system. The two principle ECs thus far identified in mammals are N-arachidonoyl-ethanolamine (anandamide) and 2-arachidonoyl-glycerol (2-AG). These compounds have been implicated in various physiological and pathological functions including appetite, pain, sensation, memory, and addiction. Because ECs are synthesized and released on demand and then rapidly degraded to terminate signaling, the metabolic pathways that govern EC turnover directly influence the magnitude and duration of neuronal signaling events. There is strong evidence that two serine hydrolases, diacylglycerol lipase-alpha and -beta (DAGL-alpha and -beta) function as 2-AG synthetic enzymes both in vitro and in vivo. However, because constitutive gene disruption, the only currently available means to investigate DAGL-alpha/beta biology due to a lack of selective chemical inhibitors, can result in compensatory effects and network-wide changes, there is still uncertainty surrounding the extent to which DAGL-alpha/beta contribute to 2-AG-mediated signaling. In an effort to provide chemical tools for manipulation of DAGL-beta activity, we initiated a competitive activity-based protein profiling (ABPP) screen of triazole urea compounds to identify selective enzyme inhibitors. This campaign, made possible by previous inhibitor development efforts for LYPLA1/2 (ML211), PAFAH2 (ML225), and ABHD11 (ML226) based on the triazole urea scaffold, yielded the medchem optimized probe ML294 (SID 125269120). ML294 is highly potent against its target enzyme (IC50 = 56 nM in vitro; 12 nM in situ), and is active in vivo, showing both oral bioavailability and blood-brain barrier penetration. Out of more than 20 serine hydrolases (SHs) profiled by gel-based competitive ABPP, ML294 is observed to have one anti-target, alpha/beta hydrolase domain-containing protein 6 (ABHD6). Otherwise, ML294 is at least 35-fold selective for all other brain SHs (approximately 20) assessed by gel-based competitive ABPP and 7-fold selective vs. its closest homolog, DAGL-alpha. To control for ABHD6-directed activity in biological studies, we also developed a structurally related ABHD6-selective control "anti-probe", ML295, also based on the triazole urea scaffold. The complete properties, characterization, and synthesis of ML294 are detailed in this report, and full details of ABHD6 inhibitors are detailed in the Probe Report for ML295 and ML296.
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.
Oxidative stress has been implicated as an underlying inflammatory factor in several disease pathologies, including cancer, atherosclerosis, aging, and various neurodegenerative disorders. Phospholipids in particular are susceptible to oxidative damage, and it is thought that the cytosolic enzyme type II platelet-activating factor acetylhydrolase (PAFAH2) may facilitate turnover of oxidized phospholipids via hydrolysis of their oxidatively truncated acyl chains. In support of this theory, over-expression of PAFAH2 has been shown to reduce oxidative stress-induced cell death. However, no selective inhibitors of PAFAH2 are known for investigation of PAFAH2 biology. We initiated a fluorescence polarization activity-based protein profiling (FluoPol-ABPP) HTS campaign to identify potential inhibitors of PAFAH2 (AIDs 492956 and 493030). The assay also served as a counterscreen for inhibitor discovery for the related enzyme, plasma PAFAH (pPAFAH; AIDs 463082, 463230).
Interestingly, the triazole urea SID 7974398a top lead in the lysophospholipase (LYPLA1) inhibitor screen from which we derived a dual inhibitor of LYPLA1/LYPLA2 (ML211) and inhibitor of ABHD11 (ML226) was also a top hit in the PAFAH2 HTS assay. Given that triazole ureas were previously found to have tunable potency and selectivity, low cytotoxicity, and good activity in situ, we endeavored to derive a PAFAH2-selective probe from the triazole urea scaffold. The medchem optimized probe (ML225, SID 103913572) is highly potent against its target enzyme (IC50 = 3 nM), and is active in situ at sub-nanomolar concentrations. ML225 is at least 333-fold selective for all other serine hydrolases (~20) assessed by gel-based competitive activity-based protein profiling, and is selective for other PAFAH enzymes. ML225 inhibits PAFAH2 by carbamoylating the active site serine. The complete properties, characterization, and synthesis of ML225 are detailed in this Probe Report.
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, which we successfully optimized via several rounds of structure activity relationship (SAR)-by-synthesis to produce 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 ABPP, ML211 is observed to have one anti-target, alpha/beta hydrolase domain-containing protein 11 (ABHD11). However, during our SAR campaign, we fortuitously discovered a selective ABHD11 inhibitor from among the synthetic triazole urea library compounds. This compound, ML226, is presented as an anti-probe for control studies.
The retinoblastoma (RB) tumor suppressor protein controls cell cycle progression by regulating the activity of the transcription factor E2F, which activates genes essential for DNA replication. Thus, factors that bind and regulate RB activity are considered valuable targets for preventing tumorigenesis. The enzyme RB binding protein 9 (RBBP9) is widely expressed in different tissues and upregulated in certain tumors. As a result, the identification of compounds that selectively inhibit RBBP9 activity would serve as potentially valuable probes for the study of apoptosis, cell cycle, and tumorigenesis. We previously reported a modestly potent, RBBP9 reversible inhibitor, ML081 (CID-6603320). However, ML081 exhibits high cytotoxicity. We, therefore, have now identified a newer probe, ML114 (CID-5934766), which is 10-fold more potent than ML081, exhibits no cytotoxicity, and is from an entirely different structural and mechanistic class of compounds that covalently inhibit RBBP9. This new probe will be useful for in vitro assays in which it is desirable to specifically block RBBP9 activity for primary research purposes.
Recent findings have identified protein phosphatase methylesterase-1 (PME-1) as a protector of sustained ERK pathway activity in malignant gliomas. PME-1 is a protein methylesterase that functions in the regulation of protein phosphatase 2A (PP2A) by reversible methylation. Biochemical elucidation of PME-1 would thus greatly benefit from the development of potent and selective chemical inhibitors. The probe compound ML136 (CID-44607965), containing a sulfonyl acrylonitrile core, represents the first potent, selective inhibitor of PME-1. Moreover, the probe does not appear to exhibit cytotoxicity. Thus, ML136 should serve as a useful tool for in vitro and in situ research assays in which it is desirable to specifically block PME-1 activity.
The retinoblastoma (RB) tumor suppressor protein controls cell cycle progression by regulating the activity of the transcription factor E2F, which in turn activates genes essential for DNA replication. Thus, factors that bind and regulate RB activity provide for valuable targets for preventing tumorigenesis. The enzyme, RB binding protein 9 (RBBP9), is widely expressed in a number of different tissues and is upregulated in certain tumors. As a result, the identification of compounds that selectively inhibit RBBP9 activity would serve as potentially valuable probes for the study of apoptosis, cell cycle, and tumorigenesis. The probe ML081 (CID- 6603320; emetine hydrochloride) represents the first non-covalent, selective RBBP9 inhibitor, and will be useful for exploring the enzymatic functions of RBBP9 in biological systems. Moreover, the tight structure-activity relationship of the emetine-RBBP9 interaction suggests that only minor modifications to the emetine structure will improve its activity. As a result, future studies will involve semi-synthetic addition of small moieties to the emetine and cephaeline scaffolds.