Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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

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

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

References

  1. Collins, I. & Workman, P. New approaches to molecular cancer therapeutics. Nat. Chem. Biol. 2, 689–700 (2006).

    Article  CAS  PubMed  Google Scholar 

  2. Goldstein, D.M., Gray, N.S. & Zarrinkar, P.P. High-throughput kinase profiling as a platform for drug discovery. Nat. Rev. Drug Discov. 7, 391–397 (2008).

    Article  CAS  PubMed  Google Scholar 

  3. Fabian, M.A. et al. A small molecule-kinase interaction map for clinical kinase inhibitors. Nat. Biotechnol. 23, 329–336 (2005).

    Article  CAS  PubMed  Google Scholar 

  4. Davis, M.I. et al. Comprehensive analysis of kinase inhibitor selectivity. Nat. Biotechnol. 29, 1046–1051 (2011).

    Article  CAS  PubMed  Google Scholar 

  5. Anastassiadis, T., Deacon, S.W., Devarajan, K., Ma, H. & Peterson, J.R. Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity. Nat. Biotechnol. 29, 1039–1045 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Bachovchin, D.A. & Cravatt, B.F. The pharmacological landscape and therapeutic potential of serine hydrolases. Nat. Rev. Drug Discov. 11, 52–68 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Long, J.Z. & Cravatt, B.F. The metabolic serine hydrolases and their functions in mammalian physiology and disease. Chem. Rev. 111, 6022–6063 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Cravatt, B.F., Wright, A.T. & Kozarich, J.W. Activity-based protein profiling: from enzyme chemistry to proteomic chemistry. Annu. Rev. Biochem. 77, 383–414 (2008).

    Article  CAS  PubMed  Google Scholar 

  9. Liu, Y., Patricelli, M.P. & Cravatt, B.F. Activity-based protein profiling: the serine hydrolases. Proc. Natl. Acad. Sci. USA 96, 14694–14699 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Leung, D., Hardouin, C., Boger, D.L. & Cravatt, B.F. Discovering potent and selective reversible inhibitors of enzymes in complex proteomes. Nat. Biotechnol. 21, 687–691 (2003).

    Article  CAS  PubMed  Google Scholar 

  11. Bachovchin, D.A., Brown, S.J., Rosen, H. & Cravatt, B.F. Identification of selective inhibitors of uncharacterized enzymes by high-throughput screening with fluorescent activity-based probes. Nat. Biotechnol. 27, 387–394 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Bachovchin, D.A. et al. Superfamily-wide portrait of serine hydrolase inhibition achieved by library-versus-library screening. Proc. Natl. Acad. Sci. USA 107, 20941–20946 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Bachovchin, D.A. et al. Academic cross-fertilization by public screening yields a remarkable class of protein phosphatase methylesterase-1 inhibitors. Proc. Natl. Acad. Sci. USA 108, 6811–6816 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Fenteany, G. et al. Inhibition of proteasome activities and subunit-specific amino-terminal threonine modification by lactacystin. Science 268, 726–731 (1995).

    Article  CAS  PubMed  Google Scholar 

  15. Ostrowska, H., Wojcik, C., Omura, S. & Worowski, K. Lactacystin, a specific inhibitor of the proteasome, inhibits human platelet lysosomal cathepsin A–like enzyme. Biochem. Biophys. Res. Commun. 234, 729–732 (1997).

    Article  CAS  PubMed  Google Scholar 

  16. Ostrowska, H. et al. Separation of cathepsin A–like enzyme and the proteasome: evidence that lactacystin/beta-lactone is not a specific inhibitor of the proteasome. Int. J. Biochem. Cell Biol. 32, 747–757 (2000).

    Article  CAS  PubMed  Google Scholar 

  17. Fenteany, G. & Schreiber, S.L. Lactacystin, proteasome function, and cell fate. J. Biol. Chem. 273, 8545–8548 (1998).

    Article  CAS  PubMed  Google Scholar 

  18. Adams, J. The development of proteasome inhibitors as anticancer drugs. Cancer Cell 5, 417–421 (2004).

    Article  CAS  PubMed  Google Scholar 

  19. Arastu-Kapur, S. et al. Nonproteasomal targets of the proteasome inhibitors bortezomib and carfilzomib: a link to clinical adverse events. Clin. Cancer Res. 17, 2734–2743 (2011).

    Article  CAS  PubMed  Google Scholar 

  20. Roujeau, J.C. et al. Telaprevir-related dermatitis. JAMA Dermatol. 149, 152–158 (2013).

    Article  PubMed  Google Scholar 

  21. Schlütter, J. Therapeutics: new drugs hit the target. Nature 474, S5–S7 (2011).

    Article  PubMed  CAS  Google Scholar 

  22. Talas, U., Dunlop, J., Khalaf, S., Leigh, I.M. & Kelsell, D.P. Human elastase 1: evidence for expression in the skin and the identification of a frequent frameshift polymorphism. J. Invest. Dermatol. 114, 165–170 (2000).

    Article  CAS  PubMed  Google Scholar 

  23. Tani, T., Ohsumi, J., Mita, K. & Takiguchi, Y. Identification of a novel class of elastase isozyme, human pancreatic elastase III, by cDNA and genomic gene cloning. J. Biol. Chem. 263, 1231–1239 (1988).

    Article  CAS  PubMed  Google Scholar 

  24. Sawyer, L. et al. The atomic structure of crystalline porcine pancreatic elastase at 2.5-Å resolution: comparisons with the structure of α-chymotrypsin. J. Mol. Biol. 118, 137–208 (1978).

    Article  CAS  PubMed  Google Scholar 

  25. Lindenbach, B.D. & Rice, C.M. Unravelling hepatitis C virus replication from genome to function. Nature 436, 933–938 (2005).

    Article  CAS  PubMed  Google Scholar 

  26. Liverton, N.J. et al. MK-7009, a potent and selective inhibitor of hepatitis C virus NS3/4A protease. Antimicrob. Agents Chemother. 54, 305–311 (2010).

    Article  CAS  PubMed  Google Scholar 

  27. Soisson, S.M. et al. Structural definition and substrate specificity of the S28 protease family: the crystal structure of human prolylcarboxypeptidase. BMC Struct. Biol. 10, 16 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Zhou, C. et al. Design and synthesis of prolylcarboxypeptidase (PrCP) inhibitors to validate PrCP as a potential target for obesity. J. Med. Chem. 53, 7251–7263 (2010).

    Article  CAS  PubMed  Google Scholar 

  29. Wallingford, N. et al. Prolylcarboxypeptidase regulates food intake by inactivating alpha-MSH in rodents. J. Clin. Invest. 119, 2291–2303 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Mallela, J., Yang, J. & Shariat-Madar, Z. Prolylcarboxypeptidase: a cardioprotective enzyme. Int. J. Biochem. Cell Biol. 41, 477–481 (2009).

    Article  CAS  PubMed  Google Scholar 

  31. Long, J.Z. et al. Dual blockade of FAAH and MAGL identifies behavioral processes regulated by endocannabinoid crosstalk in vivo. Proc. Natl. Acad. Sci. USA 106, 20270–20275 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Munford, R.S. & Hall, C.L. Purification of acyloxyacyl hydrolase, a leukocyte enzyme that removes secondary acyl chains from bacterial lipopolysaccharides. J. Biol. Chem. 264, 15613–15619 (1989).

    Article  CAS  PubMed  Google Scholar 

  33. Long, J.Z., Jin, X., Adibekian, A., Li, W. & Cravatt, B.F. Characterization of tunable piperidine and piperazine carbamates as inhibitors of endocannabinoid hydrolases. J. Med. Chem. 53, 1830–1842 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Lu, M., Varley, A.W., Ohta, S., Hardwick, J. & Munford, R.S. Host inactivation of bacterial lipopolysaccharide prevents prolonged tolerance following Gram-negative bacterial infection. Cell Host Microbe 4, 293–302 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Lu, M. et al. Lipopolysaccharide deacylation by an endogenous lipase controls innate antibody responses to Gram-negative bacteria. Nat. Immunol. 6, 989–994 (2005).

    Article  CAS  PubMed  Google Scholar 

  36. Munford, R.S. & Erwin, A.L. Eukaryotic lipopolysaccharide deacylating enzyme. Methods Enzymol. 209, 485–492 (1992).

    Article  CAS  PubMed  Google Scholar 

  37. Perrier, J., Durand, A., Giardina, T. & Puigserver, A. Catabolism of intracellular N-terminal acetylated proteins: involvement of acylpeptide hydrolase and acylase. Biochimie 87, 673–685 (2005).

    Article  CAS  PubMed  Google Scholar 

  38. Woitach, J.T., Zhang, M., Niu, C.H. & Thorgeirsson, S.S. A retinoblastoma-binding protein that affects cell-cycle control and confers transforming ability. Nat. Genet. 19, 371–374 (1998).

    Article  CAS  PubMed  Google Scholar 

  39. Shields, D.J. et al. RBBP9: a tumor-associated serine hydrolase activity required for pancreatic neoplasia. Proc. Natl. Acad. Sci. USA 107, 2189–2194 (2010).

    Article  CAS  PubMed  Google Scholar 

  40. Bachovchin, D.A. et al. Oxime esters as selective, covalent inhibitors of the serine hydrolase retinoblastoma-binding protein 9 (RBBP9). Bioorg. Med. Chem. Lett. 20, 2254–2258 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Mentlein, R., Gallwitz, B. & Schmidt, W.E. Dipeptidyl-peptidase IV hydrolyses gastric inhibitory polypeptide, glucagon-like peptide-1(7–36)amide, peptide histidine methionine and is responsible for their degradation in human serum. Eur. J. Biochem. 214, 829–835 (1993).

    Article  CAS  PubMed  Google Scholar 

  42. Choy, M. & Lam, S. Sitagliptin: a novel drug for the treatment of type 2 diabetes. Cardiol. Rev. 15, 264–271 (2007).

    Article  PubMed  Google Scholar 

  43. Thareja, S. et al. Saxagliptin: a new drug for the treatment of type 2 diabetes. Mini Rev. Med. Chem. 10, 759–765 (2010).

    Article  CAS  PubMed  Google Scholar 

  44. Flentke, G.R. et al. Inhibition of dipeptidyl aminopeptidase IV (DP-IV) by Xaa-boroPro dipeptides and use of these inhibitors to examine the role of DP-IV in T-cell function. Proc. Natl. Acad. Sci. USA 88, 1556–1559 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Connolly, B.A. et al. Dipeptide boronic acid inhibitors of dipeptidyl peptidase IV: determinants of potency and in vivo efficacy and safety. J. Med. Chem. 51, 6005–6013 (2008).

    Article  CAS  PubMed  Google Scholar 

  46. Lankas, G.R. et al. Dipeptidyl peptidase IV inhibition for the treatment of type 2 diabetes: potential importance of selectivity over dipeptidyl peptidases 8 and 9. Diabetes 54, 2988–2994 (2005).

    Article  CAS  PubMed  Google Scholar 

  47. Li, W., Blankman, J.L. & Cravatt, B.F. A functional proteomic strategy to discover inhibitors for uncharacterized hydrolases. J. Am. Chem. Soc. 129, 9594–9595 (2007).

    Article  CAS  PubMed  Google Scholar 

  48. Ahn, K. et al. Discovery and characterization of a highly selective FAAH inhibitor that reduces inflammatory pain. Chem. Biol. 16, 411–420 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Alexander, J.P. & Cravatt, B.F. Mechanism of carbamate inactivation of FAAH: implications for the design of covalent inhibitors and in vivo functional probes for enzymes. Chem. Biol. 12, 1179–1187 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Hoover, H.S., Blankman, J.L., Niessen, S. & Cravatt, B.F. Selectivity of inhibitors of endocannabinoid biosynthesis evaluated by activity-based protein profiling. Bioorg. Med. Chem. Lett. 18, 5838–5841 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Yang, X. et al. A public genome-scale lentiviral expression library of human ORFs. Nat. Methods 8, 659–661 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Martin, B.R., Giepmans, B.N., Adams, S.R. & Tsien, R.Y. Mammalian cell-based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity. Nat. Biotechnol. 23, 1308–1314 (2005).

    Article  CAS  PubMed  Google Scholar 

  53. Munford, R., Lu, M. & Varley, A. Chapter 2: Kill the bacteria...and also their messengers? Adv. Immunol. 103, 29–48 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

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.).

Author information

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

Authors
  1. Daniel A Bachovchin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Luke W Koblan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Wengen Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yuxin Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Youhua Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Peng Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Iwona Woznica
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ying Shu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jack H Lai
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Sarah E Poplawski
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Christopher P Kiritsy
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Sarah E Healey
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Matthew DiMare
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. David G Sanford
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Robert S Munford
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. William W Bachovchin
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Todd R Golub
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Corresponding author

Correspondence to Todd R Golub.

Ethics declarations

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)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.1578

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing