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Oxime-based linker libraries as a general approach for the rapid generation and screening of multidentate inhibitors

Nature Protocols volume 7, pages 686–702 (2012)Cite this article

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Abstract

The described oxime-based library protocol provides detailed procedures for the linkage of aminooxy functionality with aldehyde building blocks that result in the generation of libraries of multidentate inhibitors. Synthesis of inhibitors for protein tyrosine phosphatases (PTPs) and antagonists directed against the human tumor susceptibility gene 101 (TSG101) are shown as examples. Three steps are involved: (i) the design and synthesis of aminooxy platforms; (ii) tethering with aldehydes to form oxime-based linkages with sufficient purity; and (iii) direct in vitro biological evaluation of oxime products without purification. Each coupling reaction is (i) performed in capped microtubes at room temperature (20–23 °C); (ii) diluted for inhibitory evaluation; and (iii) screened with targets in microplates to provide IC50 or Kd values. The synthesis of the aminooxy platforms takes 3–5 d; tethering with the aldehydes takes 24 h; and inhibition assay of enzymes and protein-protein interactions takes 30 min and 2 h, respectively.

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Figure 1: Oxime-based tethering.
Figure 2: Design of YopH inhibitors.
Figure 3
Figure 4: Structure of peptide oxime 9.
Figure 5: Synthesis of aminooxy-containing 1.
Figure 6: Synthesis of aminooxy-protected proline 18.
Figure 7: Synthesis of 8d.
Figure 8
Figure 9
Figure 10: Analytical data for inhibitors 3 and 9.

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Acknowledgements

This work was supported in part by the Intramural Research Program and under the Contract No. HHSN261200800001E of the US National Institutes of Health, Center for Cancer Research, National Cancer Institute–Frederick and the National Cancer Institute, National Institutes of Health. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government.

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  1. Fa Liu

    Present address: Present address: Lilly Research Laboratories, Indianapolis, Indiana, USA.,

Authors and Affiliations

  1. Chemical Biology Laboratory, Molecular Discovery Program, Center for Cancer Research, National Cancer Institute, US National Institutes of Health, National Cancer Institute-Frederick, Frederick, Maryland, USA

    Medhanit Bahta, Fa Liu, Sung-Eun Kim & Terrence R Burke Jr

  2. Protein Chemistry Laboratory, Advanced Technology Program, SAIC-Frederick, Inc., Frederick, Maryland, USA

    Andrew G Stephen & Robert J Fisher

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Contributions

M.B., F.L., S.-E.K. and A.G.S. carried out the experimental procedures; M.B., F.L., S.-E.K., A.G.S., R.J.F. and T.R.B. planned the work, interpreted the data and wrote the manuscript.

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Correspondence to Terrence R Burke Jr.

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Bahta, M., Liu, F., Kim, SE. et al. Oxime-based linker libraries as a general approach for the rapid generation and screening of multidentate inhibitors. Nat Protoc 7, 686–702 (2012). https://doi.org/10.1038/nprot.2012.007

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