Late stage assay provider results from the probe development effort to identify inhibitors of ABHD11: Fluorescence-based biochemical gel-based Activity-Based Protein Profiling (ABPP) inhibition of the human isoform of ABHD11
Name: Late stage assay provider results from the probe development effort to identify inhibitors of ABHD11: Fluorescence-based biochemical gel-based Activity-Based Protein Profiling (ABPP) inhibition of the human isoform of ABHD11. ..more
BioActive Compound: 1
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Benjamin Cravatt, TSRI
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R01 CA132630
Grant Proposal PI: Benjamin Cravatt, TSRI
External Assay ID: ABHD11_INH_FLUO_GEL_1XINH_HUMAN
Name: Late stage assay provider results from the probe development effort to identify inhibitors of ABHD11: Fluorescence-based biochemical gel-based Activity-Based Protein Profiling (ABPP) inhibition of the human isoform of ABHD11.
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, and identification of proteins responsible for the dynamic modulation of palmitoylation is paramount to understanding its patho/physiological roles. For example, it has been suggested by Waldmann et al. that preventing depalmitoylation of the oncogene Ras by inhibiting thioesterase activity may disrupt targeted distribution, thereby downregulating oncogenic signaling (1). As such, inhibitors of protein palmitoyl thioesterases may act as tumor suppressors by preventing aberrant growth signaling. More than a decade ago, the cytosolic serine hydrolase acyl-protein thioesterase 1 (APT1) was identified as an in vitro HRAS palmitoyl thioesterase (2). Initially classified as lysophospholipase 1 (LYPLA1) (3), the enzyme has since been demonstrated to have several hundred-fold higher activity as a protein thioesterase. Upon retroviral shRNA knockdown of LYPLA1, we found that over-expressed HRAS was hyper-palmitoylated, providing evidence that endogenous LYPLA1 a functional protein palmitoyl thioesterase in mammalian cells. LYPLA2 (a.k.a. APT2) is a close homologue of LYPLA1 (65% identical) and exhibits lysophospholipase activity in vitro, but its potential role as a thioesterase is unknown (4). Several inhibitors of LYPLA1 have been described (1,5,6), but none of these agents have proven capable of selectively inhibiting LYPLA1 activity in cells. No selective inhibitors of LYPLA2 have been reported.
As serine hydrolases, the LYPLAs are readily labeled by activity-based protein profiling (ABPP) probes bearing a fluorophosphonate (FP) reactive group (7), which can be exploited for inhibitor discovery using a competitive-ABPP platform, whereby small molecule enzyme inhibition is assessed by the ability to out-compete ABPP probe labeling (8). Competitive ABPP has also been configured to operate in a high-throughput manner via fluorescence polarization readout, FluoPol-ABPP (9). Seeking selective, in situ active chemical tools to investigate the potential roles of the LYPLAs in cancer pathogenesis, we conducted a FluoPol-ABPP HTS campaign to identify inhibitors of LYPLA1 (AIDs 2174 and 2233) and the structurally related LYPLA2 (AIDs 2177 and 2232). HTS identified a modestly potent triazole urea inhibitor (SID 7974398; IC50 795 nM vs. LYPLA1, 5200 nM vs. LYPLA2), which was successfully optimized via several rounds of SAR-by-synthesis as ML211 (SID 99445338), a low nanomolar dual inhibitor of LYPLA1 (IC50 17 nM) and LYPLA2 (IC50 30 nM). Out of more than 20 serine hydrolases profiled by gel-based competitive ABPP, ML211 is 50-fold selective for all except for one anti-target, alpha/beta hydrolase domain-containing protein 11 (ABHD11; IC50 10 nM). However, during our SAR campaign, we fortuitously discovered a selective ABHD11 inhibitor from among the synthetic triazole urea library compounds. This compound (ML226, SID 99445332) was presented as a anti-probe in the ML211 Probe Report for use as a control.
ABHD11 is a poorly characterized serine hydrolase; all that is known about its biology is that it is a mitochondrial enzyme (10) with broad tissue distribution, has little sequence homology to other proteins, and its gene is located in a region of chromosome 7 that is hemizygously deleted in Williams-Beuren syndrome, a rare genetic disease with symptoms that include vascular stenosis, mental retardation, and excessive sociability (11). However, the underlying biology and contribution of ABHD11 to the disease is unknown, as its biochemical function, substrates, and products have not been identified. A principle goal of post-genomic research is the determination of the molecular and cellular roles of uncharacterized enzymes like ABHD11. As such, potent and selective inhibitors of ABHD11 not only serve as key controls for ML211, but important chemical tools in their own right for investigation of ABHD11 biology. This AID provides ABHD11-specific characterization of the triazole urea library members, including ML226.
1. Dekker, F.J., et al., Small-molecule inhibition of APT1 affects Ras localization and signaling. Nat. Chem. Biol., 2010. 6(6): p. 449-56.
2. Duncan, J.A. and A.G. Gilman, A cytoplasmic acyl-protein thioesterase that removes palmitate from G protein alpha subunits and p21(RAS). J. Biol. Chem., 1998. 273(25): p. 15830-7.
3. Sugimoto, H., H. Hayashi, and S. Yamashita, Purification, cDNA cloning, and regulation of lysophospholipase from rat liver. J. Biol. Chem., 1996. 271(13): p. 7705-11.
4. Toyoda, T., H. Sugimoto, and S. Yamashita, Sequence, expression in Escherichia coli, and characterization of lysophospholipase II. Biochim. Biophys. Acta, 1999. 1437(2): p. 182-93.
5. Biel, M., et al., Synthesis and evaluation of acyl protein thioesterase 1 (APT1) inhibitors. Chemistry, 2006. 12(15): p. 4121-43.
6. Deck, P., et al., Development and biological evaluation of acyl protein thioesterase 1 (APT1) inhibitors. Angew. Chem. Int. Ed. Engl., 2005. 44(31): p. 4975-80.
7. Jessani, N., et al., Enzyme activity profiles of the secreted and membrane proteome that depict cancer cell invasiveness. Proc. Natl. Acad. Sci. U. S. A., 2002. 99(16): p. 10335-40.
8. Leung, D., et al., Discovering potent and selective reversible inhibitors of enzymes in complex proteomes. Nat. Biotechnol., 2003. 21(6): p. 687-91.
9. Bachovchin, D.A., et al., Identification of selective inhibitors of uncharacterized enzymes by high-throughput screening with fluorescent activity-based probes. Nat. Biotechnol., 2009. 27(4): p. 387-94.
10. Forner, F., et al., Quantitative proteomic comparison of rat mitochondria from muscle, heart, and liver. Mol. Cell. Proteomics, 2006. 5(4): p. 608-19.
11. Schubert, C., The genomic basis of the Williams-Beuren syndrome. Cell. Mol. Life Sci., 2009. 66(7): p. 1178-97.
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The purpose of this assay is to determine whether powder samples of test compounds can inhibit the human isoform of ABHD11 in a complex proteomic lysate. In this assay, a complex proteome containing recombinant human ABHD11 is incubated with test compound followed by reaction with a rhodamine-conjugated fluorophosphonate (FP-Rh) activity-based probe. The reaction products are separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. The percentage activity remaining is determined by measuring the integrated optical density (IOD) of the bands. As designed, test compounds that act as ABHD11 inhibitors will prevent enzyme-probe interactions, thereby decreasing the proportion of bound fluorescent probe, giving lower fluorescence intensity in the band in the gel. Percent inhibition is calculated relative to a DMSO (no compound) control.
Recombinant human ABHD11 (50 ug/mL) was spiked into mouse brain membrane proteome (1 mg/mL in DPBS, 50 uL reaction volume) and treated with 0 (DMSO only), 3, 10, 30, or 150 nM or 20 uM test compound (1 uL of a 50x stock in DMSO). (Note: mouse membrane proteome alone is used as a control for visualization and quantification of the human ABHD11 band). Test compounds were incubated for 30 minutes at 25 C. FP-Rh (1 uL of 50x stock in DMSO) was added to a final concentration of 2 uM. The reaction was incubated for 30 minutes at 25 C, quenched with 2x SDS-PAGE loading buffer, separated by SDS-PAGE and visualized by in-gel fluorescent scanning. The percentage activity remaining was determined by measuring the integrated optical density of the human ABHD11 band.
%_Inhibition = ( 1 - ( IOD_Test_Compound - IOD_Low_Control ) / ( IOD_High_Control - IOD_Low_Control ) ) * 100
Test_Compound is defined as target treated with test compound.
High_Control is defined as target treated with DMSO only (no compound).
Low_Control is defined as background in the "no enzyme" control (mouse membrane proteome only, no added ABHD11).
PubChem Activity Outcome and Score:
Compounds with greater than or equal to 50% inhibition at 0.030 uM were considered active. Compounds with less than 50% inhibition at 30 nM were considered inactive.
The reported PubChem Activity Score has been normalized to 100% observed inhibition at 0.030 uM. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 0-0.
List of Reagents:
Mouse brain membrane proteome (provided by the Assay Provider)
Recombinant human ABHD11 (provided by the Assay Provider)
FP-Rh (provided by the Assay Provider)
DPBS (Cellgro 20-031-CV)
This assay was performed by the assay provider with powder samples of synthetic compounds.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)