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Impact of linker strain and flexibility in the design of a fragment-based inhibitor

Nature Chemical Biology volume 5, pages 407–413 (2009)Cite this article

Abstract

The linking together of molecular fragments that bind to adjacent sites on an enzyme can lead to high-affinity inhibitors. Ideally, this strategy would use linkers that do not perturb the optimal binding geometries of the fragments and do not have excessive conformational flexibility that would increase the entropic penalty of binding. In reality, these aims are seldom realized owing to limitations in linker chemistry. Here we systematically explore the energetic and structural effects of rigid and flexible linkers on the binding of a fragment-based inhibitor of human uracil DNA glycosylase. Analysis of the free energies of binding in combination with cocrystal structures shows that the flexibility and strain of a given linker can have a substantial impact on binding affinity even when the binding fragments are optimally positioned. Such effects are not apparent from inspection of structures and underscore the importance of linker optimization in fragment-based drug discovery efforts.

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Figure 1: Substrate fragment tethering strategy and application to human UNG2.
Figure 2: Diversification of rigid bivalent oxime linkers into flexible monoamine and diamine linkers.
Figure 3: Structures and inhibition profiles of library aldehyde fragments containing DO, MA1, MA2 and DA linkers.
Figure 4: Free energy changes (ΔΔG) arising from switching between flexible amine and rigid oxime linkages that connect the uracil and benzoic acid (30) binding fragments.

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Acknowledgements

This work was supported by US National Institutes of Health grants GM56834-12 to J.T.S. and GM066895 to L.M.A. and by a Ruth L. Kirschstein National Research Service Award (F31 GM083623) to J.B.P. The content of the publication does not necessarily reflect the views or policies of the US Department of Health and Human Services, nor does the mention of trade names, commercial products or organizations imply endorsement by the US Government.

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Authors and Affiliations

  1. Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Suhman Chung, Jared B Parker & James T Stivers

  2. Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Mario Bianchet & L Mario Amzel

Authors
  1. Suhman Chung
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  2. Jared B Parker
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  3. Mario Bianchet
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  4. L Mario Amzel
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  5. James T Stivers
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Contributions

S.C. performed chemical syntheses, binding measurements and energetic analysis; J.B.P. purified protein samples, crystallized the ligand-protein complexes and performed isothermal titration calorimetry; M.B. collected and analyzed X-ray diffraction data; L.M.A. contributed to data analysis and interpretation; J.T.S. analyzed the data and wrote the paper.

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Correspondence to James T Stivers.

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Supplementary Figures 1–4, Supplementary Schemes 1 and 2, Supplementary Table 1 and Supplementary Methods (PDF 528 kb)

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Chung, S., Parker, J., Bianchet, M. et al. Impact of linker strain and flexibility in the design of a fragment-based inhibitor. Nat Chem Biol 5, 407–413 (2009). https://doi.org/10.1038/nchembio.163

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