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NMR fragment screening reveals a novel small molecule binding site near the catalytic surface of the disulfide–dithiol oxidoreductase enzyme DsbA from Burkholderia pseudomallei

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Abstract

The presence of suitable cavities or pockets on protein structures is a general criterion for a therapeutic target protein to be classified as ‘druggable’. Many disease-related proteins that function solely through protein–protein interactions lack such pockets, making development of inhibitors by traditional small-molecule structure-based design methods much more challenging. The 22 kDa bacterial thiol oxidoreductase enzyme, DsbA, from the gram-negative bacterium Burkholderia pseudomallei (BpsDsbA) is an example of one such target. The crystal structure of oxidized BpsDsbA lacks well-defined surface pockets. BpsDsbA is required for the correct folding of numerous virulence factors in B. pseudomallei, and genetic deletion of dsbA significantly attenuates B. pseudomallei virulence in murine infection models. Therefore, BpsDsbA is potentially an attractive drug target. Herein we report the identification of a small molecule binding site adjacent to the catalytic site of oxidized BpsDsbA. 1HN CPMG relaxation dispersion NMR measurements suggest that the binding site is formed transiently through protein dynamics. Using fragment-based screening, we identified a small molecule that binds at this site with an estimated affinity of KD ~ 500 µM. This fragment inhibits BpsDsbA enzymatic activity in vitro. The binding mode of this molecule has been characterized by NMR data-driven docking using HADDOCK. These data provide a starting point towards the design of more potent small molecule inhibitors of BpsDsbA.

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Acknowledgements

This work was supported in part by funding from NHMRC Grant GNT1061241. Some of the data reported herein were acquired by the Monash Fragment Platform. Some of the materials used in this study were provided by the National Deuteration Facility, which is supported by the Australian Government through the National Collaborative Research Infrastructure Strategy (NCRIS) program. This study made use of NMRbox: National Center for Biomolecular NMR Data Processing and Analysis, a Biomedical Technology Research Resource (BTRR), which is supported by NIH Grant P41GM111135 (NIGMS). BM would like to thank the NMR facility at Tata Institute of Fundamental Research (TIFR), Hyderabad, India for hosting him during his visit in 2019.

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Correspondence to Biswaranjan Mohanty or Martin J. Scanlon.

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Nebl, S., Alwan, W.S., Williams, M.L. et al. NMR fragment screening reveals a novel small molecule binding site near the catalytic surface of the disulfide–dithiol oxidoreductase enzyme DsbA from Burkholderia pseudomallei. J Biomol NMR 74, 595–611 (2020). https://doi.org/10.1007/s10858-020-00339-5

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