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Discovering potent and selective reversible inhibitors of enzymes in complex proteomes

Nature Biotechnology volume 21, pages 687–691 (2003)Cite this article

Abstract

To realize the promise of genomics-based therapeutics, new methods are needed to accelerate the discovery of small molecules that selectively modulate protein activity. Toward this end, advances in combinatorial synthesis have provided unprecedented access to large compound libraries of considerable structural complexity and diversity1,2, shifting the bottleneck in drug discovery to the development of efficient screens for protein targets3. Screening for reversible enzyme inhibitors typically requires extensive target-specific work, including protein expression and purification, as well as the development of specific substrate assays. Here we report a proteomic method for the discovery of reversible enzyme inhibitors that avoids these steps. We show that competitive profiling of a library of candidate serine hydrolase inhibitors in complex proteomes with activity-based chemical probes4,5,6 identifies nanomolar reversible inhibitors of several enzymes simultaneously, including the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH)7, triacylglycerol hydrolase (TGH)8 and an uncharacterized membrane-associated hydrolase that lacks known substrates. The strategy tests inhibitors against numerous enzymes in parallel, assigning both potency and selectivity factors to each agent. In this way, promiscuous inhibitors were readily rejected in favor of equally potent compounds with 500-fold or greater selectivity for their targets.

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Figure 1: ABPP of complex proteomes with the serine hydrolase–directed probe FP-rhodamine.
Figure 2: Identification and characterization of serine hydrolase targets of the inhibitor library.
Figure 3: Cluster analysis of the inhibitor sensitivity profiles of FAAH, TGH and KIAA1363.

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Acknowledgements

We thank members of the Cravatt group for helpful discussions, G. Adam for sulfonate probes and J. Leszyk and J. Wu for assistance with MS analysis. This work was supported by National Institutes of Health grants CA87660 (B.F.C.), DA13173 (B.F.C.) and DA15648 (D.L.B.), by Activx Biosciences and by the Skaggs Institute for Chemical Biology.

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  1. The Skaggs Institute for Chemical Biology and the Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, 92037, California, USA

    Donmienne Leung, Christophe Hardouin, Dale L Boger & Benjamin F Cravatt

  2. The Skaggs Institute for Chemical Biology and the Department of Cell Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, 92037, California, USA

    Donmienne Leung & Benjamin F Cravatt

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  1. Donmienne Leung
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Correspondence to Benjamin F Cravatt.

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B.F.C. is a founder of Activx Biosciences, Inc., which has the right to license patents held by the Scripps Research Institute based on the technology described in this paper.

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Leung, D., Hardouin, C., Boger, D. et al. Discovering potent and selective reversible inhibitors of enzymes in complex proteomes. Nat Biotechnol 21, 687–691 (2003). https://doi.org/10.1038/nbt826

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