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A high-throughput, multiplexed assay for superfamily-wide profiling of enzyme activity

Nature Chemical Biology volume 10, pages 656–663 (2014)Cite this article

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Abstract

The selectivity of an enzyme inhibitor is a key determinant of its usefulness as a tool compound or its safety as a drug. Yet selectivity is never assessed comprehensively in the early stages of the drug discovery process, and only rarely in the later stages, because technical limitations prohibit doing otherwise. Here, we report EnPlex, an efficient, high-throughput method for simultaneously assessing inhibitor potency and specificity, and pilot its application to 96 serine hydrolases. EnPlex analysis of widely used serine hydrolase inhibitors revealed numerous previously unrecognized off-target interactions, some of which may help to explain previously confounding adverse effects. In addition, EnPlex screening of a hydrolase-directed library of boronic acid– and nitrile-containing compounds provided structure-activity relationships in both potency and selectivity dimensions from which lead candidates could be more effectively prioritized. Follow-up of a series of dipeptidyl peptidase 4 inhibitors showed that EnPlex indeed predicted efficacy and safety in animal models. These results demonstrate the feasibility and value of high-throughput, superfamily-wide selectivity profiling and suggest that such profiling can be incorporated into the earliest stages of drug discovery.

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Figure 1: The EnPlex platform.
Figure 2: Large-scale profiling of serine hydrolase inhibitor interactions.
Figure 3: Comparative selectivity profiling of telaprevir and boceprevir.
Figure 4: JZL195 inhibits AOAH deacylation of LPS in vitro and in vivo.
Figure 5: EnPlex analysis of a boronic acid and nitrile compound library.

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Acknowledgements

We thank B. Cravatt (The Scripps Research Institute) for the FP serine hydrolase probe; B. Martin (University of Michigan), J. Long (Dana-Farber Cancer Institute), A. Lone and A. Saghatelian (both at Harvard University) for constructs; J. Davis and D. Peck (both at The Broad Institute) for technical assistance; and D. Gray and C. Yu (both at The Broad Institute) for helpful discussions. This work was supported by the National Cancer Institute (grant no. U54CA112962 to T.R.G.), Howard Hughes Medical Institute (T.R.G.), the US National Institutes of Health (NIH; grant R01 CA163930 to W.W.B.), Arisaph Pharmaceuticals (W.W.B.) and in part by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, NIH (R.S.M.).

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Authors and Affiliations

  1. The Eli and Edythe L. Broad Institute, Cambridge, Massachusetts, USA

    Daniel A Bachovchin, Luke W Koblan & Todd R Golub

  2. Department of Biochemistry, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts, USA

    Wengen Wu, Yuxin Liu, Youhua Li, Peng Zhao, Iwona Woznica, Ying Shu, Jack H Lai, Sarah E Poplawski, Sarah E Healey, Matthew DiMare, David G Sanford & William W Bachovchin

  3. Arisaph Pharmaceuticals, Boston, Massachusetts, USA

    Christopher P Kiritsy & William W Bachovchin

  4. Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

    Robert S Munford

  5. Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

    Todd R Golub

  6. Harvard Medical School, Boston, Massachusetts, USA

    Todd R Golub

  7. Howard Hughes Medical Institute, Chevy Chase, Maryland, USA

    Todd R Golub

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  1. Daniel A Bachovchin
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Contributions

D.A.B. conceived and developed the EnPlex assay, performed experiments and analyzed data; L.W.K. assisted with enzyme cloning and expression; W.W., Y. Liu, Y. Li, P.Z., I.W. and Y.S. synthesized and characterized boronic acid– and nitrile-based compounds; J.H.L. directed the synthetic efforts; S.E.P. carried out standard enzymatic substrate assays (HTRA2 and DPPs); C.P.K. directed monkey toxicity studies; S.E.H. and M.D. carried out mouse oral glucose tolerance test (OGTT) studies; D.G.S. directed OGTT studies; R.S.M. performed AOAH experiments; W.W.B. directed synthesis, enzymatic substrate assays and OGTT experiments; T.R.G. directed the project; and D.A.B. and T.R.G. wrote the paper.

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Correspondence to Todd R Golub.

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Competing interests

W.B.W. is a co-founder, advisor and board member of Arisaph Pharmaceuticals, a biotechnology company interested in developing boronic acid-based inhibitors of serine hydrolases as therapeutics. C.K. is a co-founder, CEO and board member of Arisaph Pharmaceuticals.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–11, Supplementary Tables 1–8 and Supplementary Notes 1 and 2. (PDF 14546 kb)

Supplementary Data Set

IC50 values (nM) for 55 inhibitors against 94 serine hydrolases. Blank entries indicate that the IC50 was greater than 33 mM. (XLSX 67 kb)

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Bachovchin, D., Koblan, L., Wu, W. et al. A high-throughput, multiplexed assay for superfamily-wide profiling of enzyme activity. Nat Chem Biol 10, 656–663 (2014). https://doi.org/10.1038/nchembio.1578

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