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Discovery of small-molecule enzyme activators by activity-based protein profiling

Nature Chemical Biology volume 16, pages 997–1005 (2020)Cite this article

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Abstract

Activity-based protein profiling (ABPP) has been used extensively to discover and optimize selective inhibitors of enzymes. Here, we show that ABPP can also be implemented to identify the converse—small-molecule enzyme activators. Using a kinetically controlled, fluorescence polarization-ABPP assay, we identify compounds that stimulate the activity of LYPLAL1—a poorly characterized serine hydrolase with complex genetic links to human metabolic traits. We apply ABPP-guided medicinal chemistry to advance a lead into a selective LYPLAL1 activator suitable for use in vivo. Structural simulations coupled to mutational, biochemical and biophysical analyses indicate that this compound increases LYPLAL1’s catalytic activity likely by enhancing the efficiency of the catalytic triad charge-relay system. Treatment with this LYPLAL1 activator confers beneficial effects in a mouse model of diet-induced obesity. These findings reveal a new mode of pharmacological regulation for this large enzyme family and suggest that ABPP may aid discovery of activators for additional enzyme classes.

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Fig. 1: Structure of a small-molecule activator of LYPLAL1.
Fig. 2: Characterization of a small-molecule activator of LYPLAL1.
Fig. 3: Summary of findings from structure–activity study.
Fig. 4: Optimization of LYPLAL1 activators.
Fig. 5: Key noncatalytic residues regulate LYPLAL1 activity and pharmacological activator efficacy.
Fig. 6: LYPLAL1 activation imparts metabolic benefits in vivo.

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Data availability

All data generated or analyzed during this study are included in this article and its Supplementary Information files, or are available from the corresponding authors upon request. Source data for Figs. 2 and 4–6 and Extended Data Figs. 1–6 are provided with the paper. Structures of 4 (CCDC 1825320), 12 (CCDC 1825321), 34 (CCDC 1825319), 37 (CCDC 1825322) and 78 (CCDC 1825323) established by single-crystal X-ray structure determinations and accompanying data have been deposited in the Cambridge Crystallographic Data Center (CCDC). Data and reagents requests should be addressed to E. Saez (esaez@scripps.edu) or D.L. Boger (boger@scripps.edu).

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Acknowledgements

We thank C. Moore and A. Rheingold of the Crystallography Facility at the University of California, San Diego for X-ray structure determinations of 4, 12, 34, 37 and 78, C. Vernochet, T.V. Magee and C. Hong at Pfizer for compounds C11 and C12, and M. Petrassi at Calibr for discussions. This work was supported by National Institutes of Health grant nos. DA015648 (to D.L.B.), GM069832 (to S.F.), DK099810 (to E.S. and B.F.C.) and DK114785 (to E.S. and B.F.C.). B.P.K. was supported in part by fellowship no. 15POST25100007 from the American Heart Association.

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  1. These authors contributed equally: Bernard P. Kok, Srijana Ghimire.

Authors and Affiliations

  1. Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, USA

    Bernard P. Kok, Woojoo Kim, Tyler Johns, Seiya Kitamura, Janice Xu, Ara Sukiasyan, Sean M. Kim, Cristina Godio, Julia M. Bittencourt, Andrea Galmozzi, Dennis W. Wolan & Enrique Saez

  2. Department of Chemistry, The Scripps Research Institute, La Jolla, CA, USA

    Srijana Ghimire, Shreyosree Chatterjee, Daisuke Ogasawara, Benjamin F. Cravatt & Dale L. Boger

  3. Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA

    Jerome Eberhardt & Stefano Forli

  4. Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL, USA

    Michael Cameron

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Contributions

E.S. conceived the project. D.W.W., S.F., B.F.C., D.L.B. and E.S. designed and directed research. T.J. and J.X. purified recombinant proteins. T.J., W.K., B.P.K. and C.G. developed and ran Fluopol-ABPP HTS screen, completed initial activator characterization and supported SAR studies. S.G. and S.C. carried out medicinal chemistry and SAR-guided synthesis. B.P.K., W.K., T.J., A.S., S.M.K. and C.G. ran ABPP assays, mutagenesis experiments and protein analyses. S.K. performed nanoDSF assays. B.P.K., T.J. and A.S. performed substrate hydrolysis assays with help from J.X. J.E. ran MD simulations. D.O. carried out proteomic studies. M.C. performed in vitro ADME studies. B.P.K., A.G., A.S., S.M.K. and J.M.B. performed mouse studies and cell-based assays. All authors analyzed the data. D.L.B. and E.S. wrote the manuscript with input from other authors.

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Correspondence to Dale L. Boger or Enrique Saez.

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Extended data

Extended Data Fig. 1 Screen to identify modulators of LYPLAL1 activity.

(a) ABPP-based fluorescence polarization assay (Fluopol-ABPP) amenable for high-throughput screening. Purified mLYPLAL1 is incubated with compounds prior to addition of the FP-rhodamine (FP-Rh) probe. FP-biotin probe (a non-fluorescent FP probe, 25 μM) serves as a control for inhibition, while the PPARγ ligand rosiglitazone (25 μM) is used as an inactive control. Data are shown as mean ± s.d., n = 12, where n represents independent samples. (b) A 16,000-compound screen (single point, 10 μM) identified hits that reduced the fluorescence polarization signal of FP-rhodamine labeling of mLYPLAL1 (putative inhibitors) and some that increased it (potential activators). Percent mLYPLAL1 activity was calculated relative to DMSO (100% activity) and FP-biotin (0% activity) wells. 4 is shown as a red dot.

Source data

Extended Data Fig. 2 Validation of primary HTS hits.

Candidate LYPLAL1 inhibitors and activators were analyzed by gel-based ABPP. Hit picks (10 μM) were incubated with 100 nM of purified mLYPLAL1 for 1.5 h at 37 °C prior to the addition of FP-rhodamine probe (1 μM). After 1 h at 37 °C, reactions were quenched, separated by SDS–PAGE and in-gel fluorescence scanned. LYPLAL1 activity was calculated relative to DMSO (100%). Designations above lanes correspond to compound location on the plate. Red asterisk denotes 4. Representative results from two independent experiments; similar results were obtained in both experiments. Uncropped gels are shown in Supplementary Fig. 6.

Source data

Extended Data Fig. 3 Derivatized thermal melt curves for compound-treated mLYPLAL1.

Temperature-dependent fluorescence shifts in purified mLYPLAL1 incubated with increasing concentrations of 4 and 12. Increased protein flexibility is noted upon compound interaction. In contrast, an inactive compound, 80, does not shift the derivatized thermal melt curve of mLYPLAL1. Representative results from three independent experiments; similar results were obtained in all experiments.

Source data

Extended Data Fig. 4 Evolution of the global Root Mean Square Deviation (RMSD) during Molecular Dynamics (MD) trajectories.

(a) WT hLYPLAL1 and R80 mutants, and (b) WT hLYPLAL1 with chlorobenzene as cosolvent. The RMSD was calculated considering only the backbone atoms and using the starting frames as the reference structures. Results from 10 independent simulations.

Source data

Extended Data Fig. 5 Effects of chronic treatment of DIO mice with a pharmacological LYPLAL1 activator.

DIO mice were treated with 12 for 28 days. (a) Body weights (left panel: n = 8 for veh, n = 9 for 12; right panel: n = 7 for veh, 12 and 12 + C11 groups, n = 8 for C11) prior to OGTT (left panel: d 11, right panel: d 8) and ITT (left panel: d 15, right panel: d 12); cumulative food intake per mouse (left panel: n = 3 cages for veh and 12, right panel: n = 2 cages for all groups) of vehicle and compound-treated DIO mice. (b) Indicated tissues from DIO mice were analyzed by western blot. The intensity of the LYPLAL1 signal was normalized to that of HSP90, the loading control. Quantification is shown on the right; n = 8 for veh and 7 for 12. n represents individual mice (body weights and western blot) or cages (food intake). Error bars represent mean ± s.e.m. Representative results from two independent experiments; similar results were obtained in both experiments. Uncropped blots for b are shown in Supplementary Fig. 6.

Source data

Extended Data Fig. 6 Glucose production in primary mouse hepatocytes treated with LYPLAL1 modulators.

Basal (20 mM lactate/2 mM pyruvate) (left panel) and glucagon-stimulated (middle panel) glucose production was measured in primary mouse hepatocytes treated for 6 h with 12, C11, or both compounds in combination; results were normalized to protein content. Gel-based ABPP analysis of 50 nM purified mLYPLAL1 treated for 1 h with 12, C11, and the combination (right panel). Protein levels are shown in the bottom panel. Error bars represent mean ± s.d.; n = 5 per group where n=biologically independent samples; *p = 0.02, **p = 0.003, ***p = 0.0003 by two-tailed t-test. Representative results from two independent experiments; similar results were obtained in each experiment. Uncropped gels/blots are shown in Supplementary Fig. 6.

Source data

Supplementary information

Supplementary information

Supplementary Tables 1–8, Figs. 1–6 and Notes.

Reporting Summary

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Extended Data Fig. 1

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Extended Data Fig. 6

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Extended Data Fig. 6

Unprocessed gels.

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Kok, B.P., Ghimire, S., Kim, W. et al. Discovery of small-molecule enzyme activators by activity-based protein profiling. Nat Chem Biol 16, 997–1005 (2020). https://doi.org/10.1038/s41589-020-0555-4

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