Integrated phenotypic and activity-based profiling links Ces3 to obesity and diabetes

Abstract

Phenotypic screening is making a comeback in drug discovery as the maturation of chemical proteomics methods has facilitated target identification for bioactive small molecules. A limitation of these approaches is that time-consuming genetic methods or other means is often required to determine the biologically relevant target(s) from among multiple protein-compound interactions that are typically detected. Here, we have combined phenotypic screening of a directed small-molecule library with competitive activity-based protein profiling to map and functionally characterize the targets of screening hits. Using this approach, we identify carboxylesterase 3 (Ces3 or Ces1d) as a primary molecular target of bioactive compounds that promote lipid storage in adipocytes. We further show that Ces3 activity is dramatically elevated during adipocyte differentiation. Treatment of two mouse models of obesity-diabetes with a Ces3 inhibitor ameliorates multiple features of metabolic syndrome, illustrating the power of the described strategy to accelerate the identification and pharmacologic validation of new therapeutic targets.

ThermoMouse: An In Vivo Model to Identify Modulators of UCP1 Expression in Brown Adipose Tissue

“…Compound 4 (WWL113 in original nomenclature) was also a hit in a different, image-based screen we recently described in which white preadipocytes were treated chronically (8 days) during differentiation (Dominguez et al, 2014). WWL113 inhibits two serine hydrolases expressed in adipocytes, carboxylesterase 3 (Ces3 or Ces1d) and Ces1f (CesML1).…”

“…These results indicate that the ability of WWL113 to enhance Ucp1 expression is principally dependent on PPARα, but not PPARγ, activity. Because WWL113 is not a direct PPARα activator (Dominguez et al, 2014), we tested the extent to which co-treatment of differentiated brown adipocytes with WWL113 and a PPARα agonist (GW9578) would further boost Ucp1 mRNA levels. Co-treatment with WWL113 and GW9578 modestly but significantly increased expression of Ucp1 relative to treatment with either compound alone (Fig.…”

“…Phenotypic screens using adipocytes have proven to be a powerful method to isolate small molecules that ameliorate the symptoms of metabolic syndrome through novel mechanisms of action (Waki et al, 2007; Dominguez et al, 2014). To facilitate the identification of pharmacologic agents to modulate UCP1 expression in adipocytes, we have generated a transgenic reporter mouse, ThermoMouse, in which luciferase activity recapitulates the pattern of expression of UCP1 in vivo and allows real-time visualization and quantification of UCP1 expression in live animals.…”

Exploring Metabolic Pathways and Regulation through Functional Chemoproteomic and Metabolomic Platforms

“…This TOP-ABPP can be adapted for quantitative proteomics by incorporating an isotopically “heavy” labeled valine into the linker and measuring the ratio of heavy to light labeling on a protein or at a particular residue, a platform called isotopic TOP-ABPP (isoTOP-ABPP) (Figure 2C) (Weerapana et al, 2010). These ABPP platforms have been successfully used to identify and characterize enzyme activities in various human diseases, including cancer, obesity, neurodegenerative diseases, and microbial infection (Blais et al, 2010; Dominguez et al, 2014; Nomura et al, 2010b, 2011a; Sadler et al, 2012; Singaravelu et al, 2010). …”

Tetrazole‐Based Probes for Integrated Phenotypic Screening, Affinity‐Based Proteome Profiling, and Sensitive Detection of a Cancer Biomarker

“…[1] However,t he utility of phenotypic screening is limited by its difficulties in identifying the underlying cellular targets. [2] Common approaches for target identification, such as in vitro-based assays and affinity chromatography,m ostly rely on recombinant proteins or crude cell lysates,r endering them unsuitable for reporting genuine drug-protein interactions in native environments. [3] To address these challenges,affinity-based proteome profiling (AfBP), in which photoprobes capable of recapitulating drug-protein interactions in situ leading to subsequent protein enrichment and large-scale proteome-wide target identification, has been developed as ap owerful approach for in situ target identification during the past decade.…”