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Facile target validation in an animal model with intracellularly expressed monobodies


Rapidly determining the biological effect of perturbing a site within a potential drug target could guide drug discovery efforts, but it remains challenging. Here, we describe a facile target validation approach that exploits monobodies, small synthetic binding proteins that can be fully functionally expressed in cells. We developed a potent and selective monobody to WDR5, a core component of the mixed lineage leukemia (MLL) methyltransferase complex. The monobody bound to the MLL interaction site of WDR5, the same binding site for small-molecule inhibitors whose efficacy has been demonstrated in cells but not in animals. As a genetically encoded reagent, the monobody inhibited proliferation of an MLL-AF9 cell line in vitro, suppressed its leukemogenesis and conferred a survival benefit in an in vivo mouse leukemia model. The capacity of this approach to readily bridge biochemical, structural, cellular characterization and tests in animal models may accelerate discovery and validation of druggable sites.

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Fig. 1: Monobody Mb(S4) inhibits MLL1 enzyme activity and targets the Win motif-binding site on WDR5.
Fig. 2: The crystal structure of the WDR5–Mb(S4) complex.
Fig. 3: Mb(S4) inhibits HoxA9 expression and proliferation of MLL-AF9 cells.
Fig. 4: Expression of Mb(S4) in MLL-AF9 cells inhibits leukemia progression in a mouse model.


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We thank J. Andrade and B. Ueberheide (NYU Langone Medical Center) for proteomics analysis; S. Gräslund and C. Arrowsmith (Structural Genomics Consortium) for expression vectors; J. Schmollerl and F. Grebien (Ludwig Boltzmann Institute of Cancer Research) for a retroviral vector and guidance. Research reported in this publication was supported by the National Institutes of Health under award numbers R01 DA036887 and R01 CA194864 (to S.K.) and R01 GM082856 (to Y.D.). Results shown in this report are derived from work performed at Argonne National Laboratory, Structural Biology Center (Beamline 19ID) at the Advanced Photon Source. Argonne is operated by UChicago Argonne, LLC, for the US Department of Energy, Office of Biological and Environmental Research under contract DE-AC02-06CH11357. The mass spectrometric experiments were supported in part by the Laura and Isaac Perlmutter Cancer Center support grant P30CA016087 from the National Cancer Institute.

Author information




A.G., Y.D. and S.K. designed the study; A.G., A.K. and S.K. generated monobodies; A.G., S.T.T. and A.K. performed binding measurements; A.G. and S.L. tested monobody effects on the MLL1 complex; A.G. and S.K. determined and analyzed the X-ray crystal structure; A.G. and K.K. constructed degron-controlled vectors; A.G. performed immunoprecipitation experiments; A.G. and S.T.T. performed cell proliferation assays; A.G., J.X., B.Z., M.S.W., A.J.R. and Y.D. performed transcription and ChIP analyses; A.G., J.X. and B.Z. performed mouse experiments and histological analyses; A.G., Y.D. and S.K. wrote the manuscript, and all authors commented and approved the manuscript.

Corresponding authors

Correspondence to Yali Dou or Shohei Koide.

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Competing interests

S.K. and A.K. are inventors on patent applications filed by the University of Chicago and Novartis Ag that cover monobody library designs (WO2012016245A3 and equivalent nationalized applications).

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Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–2, Supplementary Figures 1–11

Reporting Summary

Supplementary Dataset 1

Mass spectrometry analyses of proteins captured by Mb(S4) or Mb(S4mut) from the whole-cell lysate or nuclear extract of HEK293T cells

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Gupta, A., Xu, J., Lee, S. et al. Facile target validation in an animal model with intracellularly expressed monobodies. Nat Chem Biol 14, 895–900 (2018).

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