Skip to main content

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

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.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  • Adams LA, Sharma P, Mohanty B, Ilyichova OV, Mulcair MD, Williams ML, Gleeson EC, Totsika M, Doak BC, Caria S, Rimmer K, Horne J, Shouldice SR, Vazirani M, Headey SJ, Plumb BR, Martin JL, Heras B, Simpson JS, Scanlon MJ (2015) Application of fragment-based screening to the design of inhibitors of Escherichia coli DsbA. Angew Chem Int Ed 54:2179–2184

    Article  Google Scholar 

  • Ahlner A, Carlsson M, Jonsson B-H, Lundström P (2013) PINT: a software for integration of peak volumes and extraction of relaxation rates. J Biomol NMR 56:191–202

    Article  Google Scholar 

  • Brenke R, Kozakov D, Chuang G-Y, Beglov D, Hall D, Landon MR, Mattos C, Vajda S (2009) Fragment-based identification of druggable ‘hot spots’ of proteins using Fourier domain correlation techniques. Bioinformatics 25:621–627

    Article  Google Scholar 

  • Cimermancic P, Weinkam P, Rettenmaier TJ, Bichmann L, Keedy DA, Woldeyes RA, Schneidman-Duhovny D, Demerdash ON, Mitchell JC, Wells JA, Fraser JS, Sali A (2016) CryptoSite: expanding the druggable proteome by characterization and prediction of cryptic binding sites. J Mol Biol 428:709–719

    Article  Google Scholar 

  • Covington AK, Paabo M, Robinson RA, Bates RG (1968) Use of the glass electrode in deuterium oxide and the relation between the standardized pD (paD) scale and the operational pH in heavy water. Anal Chem 40:700–706

    Article  Google Scholar 

  • De Las Rivas J, Fontanillo C (2010) Protein–protein interactions essentials: key concepts to building and analyzing interactome networks. PLoS Comput Biol 6:e1000807

    Article  Google Scholar 

  • Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293

    Article  Google Scholar 

  • Dominguez C, Boelens R, Bonvin AMJJ (2003) HADDOCK: a protein–protein docking approach based on biochemical or biophysical information. J Am Chem Soc 125:1731

    Article  Google Scholar 

  • Duff AP, Wilde KL, Rekas A, Lake V, Holden PJ (2015) Robust high-yield methodologies for 2H and 2H/15N/13C labeling of proteins for structural investigations using neutron scattering and NMR. In: Kelman Z (ed) Methods in enzymology, vol. 565. Academic Press, Elsevier, pp 3–25

    Google Scholar 

  • Duncan LF, Wang G, Ilyichova OV, Scanlon MJ, Heras B, Abbott BM (2019) The fragment-based development of a benzofuran hit as a new class of Escherichia coli DsbA inhibitors. Molecules 24:3756

    Article  Google Scholar 

  • Ganesh AN, Donders EN, Shoichet BK, Shoichet MS (2018) Colloidal aggregation: from screening nuisance to formulation nuance. Nano Today 19:188–200

    Article  Google Scholar 

  • Halili MA, Bachu P, Lindahl F, Bechara C, Mohanty B, Reid RC, Scanlon MJ, Robinson CV, Fairlie DP, Martin JL (2015) Small molecule inhibitors of disulfide bond formation by the bacterial DsbA–DsbB dual enzyme system. ACS Chem Biol 10:957–964

    Article  Google Scholar 

  • Henzler-Wildman K, Kern D (2007) Dynamic personalities of proteins. Nature 450:964

    Article  ADS  Google Scholar 

  • Heras B, Shouldice SR, Totsika M, Scanlon MJ, Schembri MA, Martin JL (2009) DSB proteins and bacterial pathogenicity. Nat Rev Microbiol 7:215

    Article  Google Scholar 

  • Heras B, Scanlon MJ, Martin JL (2015) Targeting virulence not viability in the search for future antibacterials. Br J Clin Pharmacol 79:208–215

    Article  Google Scholar 

  • Hyberts SG, Milbradt AG, Wagner AB, Arthanari H, Wagner G (2012) Application of iterative soft thresholding for fast reconstruction of NMR data non-uniformly sampled with multidimensional Poisson gap scheduling. J Biomol NMR 52:315–327

    Article  Google Scholar 

  • Ireland P, McMahon R, Marshall L, Halili M, Furlong E, Tay S, Martin J, Sarkar-Tyson M (2014) Disarming Burkholderia pseudomallei: structural and functional characterization of a disulfide oxidoreductase (DsbA) required for virulence in vivo. Antioxid Redox Signal 20:606–617

    Article  Google Scholar 

  • Ishima R, Torchia DA (2003) Extending the range of amide proton relaxation dispersion experiments in proteins using a constant-time relaxation-compensated CPMG approach. J Biomol NMR 25:243–248

    Article  Google Scholar 

  • Katsuyama I, Khalil AA, Dunbar C, Zjawiony JK (2003) Concentration-dependent variation of 1 H-NMR chemical shifts of aromatic protons in sampangine derivatives. Spectrosc Lett 36:477–485

    Article  ADS  Google Scholar 

  • Keserű GM, Erlanson DA, Ferenczy GG, Hann MM, Murray CW, Pickett SD (2016) Design principles for fragment libraries: maximizing the value of learnings from pharma fragment-based drug discovery (FBDD) programs for use in academia. J Med Chem 59:8189–8206

    Article  Google Scholar 

  • Kozakov D, Grove LE, Hall DR, Bohnuud T, Mottarella SE, Luo L, Xia B, Beglov D, Vajda S (2015) The FTMap family of web servers for determining and characterizing ligand-binding hot spots of proteins. Nat Protoc 10:733–755

    Article  Google Scholar 

  • Lee SY (1996) High cell-density culture of Escherichia coli. Trends Biotechnol 14:98–105

    Article  Google Scholar 

  • Mabonga L, Kappo AP (2019) Protein–protein interaction modulators: advances, successes and remaining challenges. Biophys Rev 11:559–581

    Article  Google Scholar 

  • Marley J, Lu M, Bracken C (2001) A method for efficient isotopic labeling of recombinant proteins. J Biomol NMR 20:71–75

    Article  Google Scholar 

  • Mayer M, Meyer B (2001) Group epitope mapping by saturation transfer difference NMR to identify segments of a ligand in direct contact with a protein receptor. J Am Chem Soc 123:6108

    Article  Google Scholar 

  • McMahon RM, Premkumar L, Martin JL (2014) Four structural subclasses of the antivirulence drug target disulfide oxidoreductase DsbA provide a platform for design of subclass-specific inhibitors. BBA Proteins Proteomics 1844:1391

    Article  Google Scholar 

  • McMahon RM, Ireland PM, Sarovich DS, Petit G, Jenkins CH, Sarkar-Tyson M, Currie BJ, Martin JL (2018) Virulence of the melioidosis pathogen Burkholderia pseudomallei requires the oxidoreductase membrane protein DsbB. Infect Immun 86:e00938-17

    Article  Google Scholar 

  • Mitra A, Seaton PJ, Ali Assarpour R, Williamson T (1998) Unprecedented concentration dependent chemical shift variation in 1H-NMR studies: a caveat in the investigations of molecular recognition and structure elucidation. Tetrahedron 54:15489–15498

    Article  Google Scholar 

  • Mohanty B, Serrano P, Pedrini B, Jaudzems K, Geralt M, Horst R, Herrmann T, Elsliger MA, Wilson IA, Wüthrich K (2010) Comparison of NMR and crystal structures for the proteins TM1112 and TM1367. Acta Crystallogr Sect F 66:1381–1392

    Article  Google Scholar 

  • Mohanty B, Williams ML, Doak BC, Vazirani M, Ilyichova O, Wang G, Bermel W, Simpson JS, Chalmers DK, King GF, Mobli M, Scanlon MJ (2016) Determination of ligand binding modes in weak protein–ligand complexes using sparse NMR data. J Biomol NMR 66:195–208

    Article  Google Scholar 

  • Mohanty B, Rimmer K, McMahon RM, Headey SJ, Vazirani M, Shouldice SR, Coincon M, Tay S, Morton CJ, Simpson JS, Martin JL, Scanlon MJ (2017) Fragment library screening identifies hits that bind to the non-catalytic surface of Pseudomonas aeruginosa DsbA1. PLoS One 12:e0173436

    Article  Google Scholar 

  • Niklasson M, Otten R, Ahlner A, Andresen C, Schlagnitweit J, Petzold K, Lundström P (2017) Comprehensive analysis of NMR data using advanced line shape fitting. J Biomol NMR 69:93–99

    Article  Google Scholar 

  • Shah DM, Eiso AB, Diercks T, Hass MAS, van Nuland NAJ, Siegal G (2012) Rapid protein–ligand costructures from sparse NOE data. J Med Chem 55:10786

    Article  Google Scholar 

  • Shouldice SR, Heras B, Jarrott R, Sharma P, Scanlon MJ, Martin JL (2010) Characterization of the DsbA oxidative folding catalyst from Pseudomonas aeruginosa reveals a highly oxidizing protein that binds small molecules. Antioxid Redox Signal 12:921

    Article  Google Scholar 

  • Shouldice SR, Heras B, Walden PM, Totsika M, Schembri MA, Martin JL (2011) Structure and function of DsbA, a key bacterial oxidative folding catalyst. Antioxid Redox Signal 14:1729–1760

    Article  Google Scholar 

  • Smith R, Paxman JJ, Scanlon MJ, Heras B (2016) Targeting bacterial Dsb proteins for the development of anti-virulence agents. Molecules 21:811

    Article  Google Scholar 

  • Vajda S, Beglov D, Wakefield AE, Egbert M, Whitty A (2018) Cryptic binding sites on proteins: definition, detection, and druggability. Curr Opin Chem Biol 44:1–8

    Article  Google Scholar 

  • Vom A, Headey S, Wang G, Capuano B, Yuriev E, Scanlon MJ, Simpson JS (2013) Detection and prevention of aggregation-based false positives in STD-NMR-based fragment screening. Aust J Chem 66:1518–1524

    Article  Google Scholar 

  • Williamson MP (2013) Using chemical shift perturbation to characterise ligand binding. Prog Nucl Magn Reson Spectrosc 73:1

    Article  Google Scholar 

  • Ziarek JJ, Peterson FC, Lytle BL, Volkman BF (2011) Binding site identification and structure determination of protein–ligand complexes by NMR a semiautomated approach. Methods Enzymol 493:241

    Article  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Biswaranjan Mohanty or Martin J. Scanlon.

Ethics declarations

Conflict of interest

The authors declare no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 2077 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10858-020-00339-5

Keywords

  • Disulfide oxidoreductase
  • Burkholderia pseudomallei
  • BpsDsbA
  • Fragment-based drug design (FBDD)
  • Structure-based drug design (SBDD)
  • Protein–ligand complexes
  • Protein dynamics
  • CPMG relaxation dispersion
  • Methyl-NMR
  • Non-uniform sampling (NUS)
  • High ambiguity driven biomolecular docking (HADDOCK)