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A detergent-based assay for the detection of promiscuous inhibitors

Nature Protocols volume 1, pages 550–553 (2006)Cite this article

Abstract

At micromolar concentrations, many small molecules self-associate into colloidal aggregates that non-specifically inhibit enzymes and other proteins. Here we describe a protocol for identifying aggregate-based inhibitors and distinguishing them from small molecules that inhibit via specific mechanisms. As a convenient proxy for promiscuous, aggregate-based inhibition, we monitor inhibition of β-lactamase in the absence and presence of detergent. Inhibition that is attenuated in the presence of detergent is characteristic of an aggregate-based mechanism. In the 96-well-format assay described here, about 200 molecules can be tested, in duplicate, per hour for detergent-dependent sensitivity. Furthermore, we also describe simple experiments that can offer additional confirmation of aggregate-based inhibition.

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Figure 1: Transmission electron micrograph of aggregates of tetraiodophenolphthalein (dark edged circles) associated with β-galactosidase.

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References

  1. Hajduk, P.J., Huth, J.R. & Fesik, S.W. Druggability indices for protein targets derived from NMR-based screening data. J. Med. Chem. 48, 2518–2525 (2005).

    Article  CAS  Google Scholar 

  2. Rishton, G.M. Reactive compounds and in vitro false positives in HTS. Drug Discov. Today 2, 382–384 (1997).

    Article  CAS  Google Scholar 

  3. Walters, W.P. & Namchuk, M. Designing screens: how to make your hits a hit. Nat. Rev. Drug Discov. 2, 259–266 (2003).

    Article  CAS  Google Scholar 

  4. Feng, B. & Shoichet, B.K. Synergy and antagonism of promiscuous inhibition in multiple-compound mixtures. J. Med. Chem. 49, 2151–2154 (2006).

    Article  CAS  Google Scholar 

  5. Feng, B.Y. et al. High-throughput assays for promiscuous inhibitors. Nat. Chem. Biol. 1, 146–148 (2005).

    Article  CAS  Google Scholar 

  6. McGovern, S.L., Caselli, E., Grigorieff, N. & Shoichet, B.K. A common mechanism underlying promiscuous inhibitors from virtual and high-throughput screening. J. Med. Chem. 45, 1712–1722 (2002).

    Article  CAS  Google Scholar 

  7. McGovern, S.L., Helfand, B.T., Feng, B.Y. & Shoichet, B.K. A specific mechanism of nonspecific inhibition. J. Med. Chem. 46, 4265–4272 (2003).

    Article  CAS  Google Scholar 

  8. McGovern, S.L. & Shoichet, B.K. Kinase inhibitors: not just for kinases anymore. J. Med. Chem. 46, 1478–1483 (2003).

    Article  CAS  Google Scholar 

  9. Seidler, J., McGovern, S.L., Doman, T. & Shoichet, B.K. Identification and prediction of promiscuous aggregating inhibitors among known drugs. J. Med. Chem. 46, 4477–4486 (2003).

    Article  CAS  Google Scholar 

  10. Seidler, J., McGovern, S.L., Doman, T.N. & Shoichet, B.K. Identification and prediction of promiscuous drugs. J. Med. Chem. 46, 4477–4486 (2003).

    Article  CAS  Google Scholar 

  11. Weston, G.S., Blazquez, J., Baquero, F. & Shoichet, B.K. Structure-based enhancement of boronic acid-based inhibitors of AmpC β-lactamase. J. Med. Chem. 41, 4577–4586 (1998).

    Article  CAS  Google Scholar 

  12. Ryan, A.J., Gray, N.M., Lowe, P.N. & Chung, C. Effect of detergent on 'promiscuous' inhibitors. J. Med. Chem. 46, 3448–3451 (2003).

    Article  CAS  Google Scholar 

  13. Straus, O.H. & Goldstein, A. Zone behavior of enzymes: illustrated by the effect of dissociation constant and dilution on the system cholinesterase–physostigmine. J. Gen. Physiol. 26, 559–585 (1943).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank A. McReynolds and K. Coan for reading this manuscript, K.C. for thoughtful discussions and GM71630.

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  1. Department of Pharmaceutical Chemistry, Graduate Group in Chemistry and Chemical Biology, University of California–San Francisco, 1700 4th Street, California, 94158-2330, USA

    Brian Y Feng & Brian K Shoichet

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  1. Brian Y Feng
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  2. Brian K Shoichet
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Correspondence to Brian K Shoichet.

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The authors declare no competing financial interests.

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Feng, B., Shoichet, B. A detergent-based assay for the detection of promiscuous inhibitors. Nat Protoc 1, 550–553 (2006). https://doi.org/10.1038/nprot.2006.77

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