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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 are often required to determine the biologically relevant target (or targets) 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, also known as Ces1d) as a primary molecular target of bioactive compounds that promote lipid storage in adipocytes. We further show that Ces3 activity is markedly 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.

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Figure 1: Discovery of serine hydrolase inhibitors with adipogenic activity and identification of their molecular target.
Figure 2: WWL229, a selective Ces3 inhibitor, recapitulates the effects of WWL113 in adipocytes.
Figure 3: WWL113 treatment corrects multiple features of metabolic syndrome in db/db mice.
Figure 4: WWL113 treatment enhances insulin sensitivity and glucose tolerance in a model of diet-induced obesity.
Figure 5: hCES1 is more active in adipose tissue of obese and type 2 diabetic individuals.

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Acknowledgements

We thank N. Kralli and P. Tontonoz for valuable discussions. E.S. is supported by a Career Development Award from the American Diabetes Association, the McDonald's Center for Type 2 Diabetes and Obesity and US National Institutes of Health grants DK081003 and DK099810. B.F.C. is supported by NIH grant DK099810. M.J.W. is supported by grants and a Senior Research Fellowship from the National Health and Medical Research Council of Australia. A.P.R. is supported by a Career Development Fellowship from the National Health and Medical Research Council of Australia. E.D. was supported by an Anxeles Alvariño fellowship from the Xunta de Galicia, Spain. K.-L.H. was supported by a Hewitt Foundation postdoctoral fellowship.

Author information

Authors and Affiliations

Authors

Contributions

E.S., E.D. and B.F.C. designed experiments. E.S., E.D., J.P., C.G. and A.G. performed cell-based, biochemical and in vivo experiments. W.L. and J.W.C. synthesized compounds. J.T. and D.K.N. performed lipidomic analysis. A.G., E.D. and K.-L.H. performed proteomic experiments. D.P. provided technical help. A.P.R., M.J.W. and P.E.O. provided human samples. E.D., J.P., C.G., A.G., J.T., D.K.N., K.-L. H., S.N., B.F.C. and E.S. analyzed data. E.D., B.F.C. and E.S. wrote the manuscript.

Corresponding authors

Correspondence to Benjamin F Cravatt or Enrique Saez.

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Competing interests

B.F.C. is a cofounder and advisor for a biotechnology company interested in developing inhibitors for serine hydrolases as therapeutic targets.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–14, Supplementary Table 1 and Supplementary Note. (PDF 21322 kb)

Supplementary Data Set 1

Quantification of protein activities in predifferentiated and mature 10T1/2 adipocytes. (XLSX 167 kb)

Supplementary Data Set 2

Quantification of protein activities in predifferentiated and mature 3T3-L1 adipocytes; quantification of protein activities in competitive ABPP-MudPIT analysis of 3T3-L1 adipocyte proteome pre-incubated with WWL38 (10 μM). (XLSX 179 kb)

Supplementary Data Set 3

Quantification of protein activities in competitive ABPP-MudPIT analysis of 10T1/2 adipocyte proteome pre-incubated with WWL113 (10 μM). (XLSX 52 kb)

Supplementary Data Set 4

Quantification of protein activities in competitive ABPP-MudPIT analysis of 10T1/2 adipocyte proteome pre-incubated with WWL229 (10 μM). (XLSX 53 kb)

Supplementary Data Set 5

Quantification of protein activities labeled by JW972 (1 μM) in 10T1/2 adipocyte proteome. (XLSX 56 kb)

Supplementary Data Set 6

Quantification of protein activities labeled by JW972 (1 μM) in 10T1/2 adipocyte proteome pre-incubated with WWL113 (10 μM). (XLSX 57 kb)

Supplementary Data Set 7

Quantification of protein activities in competitive ABPP-MudPIT analysis of white adipose tissue of mice treated with 30 mg/kg WWL113 4 hr prior to analysis. (XLSX 76 kb)

Supplementary Data Set 8

Quantification of protein activities in competitive ABPP-MudPIT analysis of liver of mice treated with 30 mg/kg WWL113 4 hr prior to analysis. (XLSX 69 kb)

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Dominguez, E., Galmozzi, A., Chang, J. et al. Integrated phenotypic and activity-based profiling links Ces3 to obesity and diabetes. Nat Chem Biol 10, 113–121 (2014). https://doi.org/10.1038/nchembio.1429

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