Poster Presentation The 44th Lorne Conference on Protein Structure and Function 2019

Visualising “invisible” states of proteins to support fragment-based drug design (#156)

Biswaranjan Mohanty 1 , Robert B. Fenwick 2 , Wesam S. Alwan 1 , Tony Wang 3 , Gaurav Sharma 1 , Bradley C. Doak 1 , Begoña Heras 3 , Peter E. Wright 2 , Martin J. Scanlon 1
  1. Monash Institute of Pharmaceutical Sciences, Parkville, VIC, Australia
  2. Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, United States
  3. La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia

Bacterial resistance to antibiotics has become a major global concern [1]. Therefore there is a pressing need to discover new antibacterials with novel mechanisms of action. Targeting bacterial virulence rather than bacterial growth is one approach to tackling the problem of bacterial resistance. DsbA is an oxidoreductase enzyme found in the periplasm of many Gram-negative bacteria that is required for correct folding of many secreted proteins. Without a functional DsbA, bacteria display attenuated virulence, and thereby diminished capacity to cause infection [2]. Therefore DsbA is a novel anti-virulence target [3].

Escherichia coli DsbA (EcDsbA) is a challenging target to inhibit with small molecules due to the nature of it’s substrate binding site which is a large, shallow and flexible groove [4]. In the current work we have characterised an “invisible” state of EcDsbA using proton relaxation dispersion NMR experiments [5] that represents a minor population of the protein [6]. Using fragment screening by NMR and X-ray crystallography, we have identified a small-molecule that binds in a cryptic pocket adjacent to the residues showing slow dynamics in the dispersion data. Furthermore, we have demonstrated that this compound inhibits the activity of EcDsbA in vitro. This is a novel binding pocket distal to the main binding site of EcDsbA, which has not been previously reported. We hypothesise that the minor conformation identified in the dispersion data allows access of the fragment to the cryptic pocket. Efforts to optimize the cryptic-pocket binders as inhibitors of EcDsbA are underway.

  1. WHO, “Antimicrobial resistance: global report on surveillance”, 2014.
  2. Heras et al., “Targeting virulence not viability in the search for future antibacterials”, Br J Clin Pharmacol. 79(2): 208–215, 2015.
  3. Ireland et al, “Disarming Burkholderia pseudomallei: structural and functional characterization of a disulfide oxidoreductase (DsbA) required for virulence in vivo”, Antioxid. Redox. Signal., 20(4), 606-617, 2014.
  4. Adams et al., “Application of fragment-based screening to the design of inhibitors of Escherichia coli DsbA”, Angew. Chem. Int. Ed., 54, 2179-2184, 2015.
  5. Fenwick et al., Multi-probe relaxation dispersion measurements increase sensitivity to protein dynamics, Phys. Chem. Che. Phys., 18 (8), 5789-5798, 2016.
  6. Vallurupalli et al., “Structures of invisible, excited protein states by relaxation dispersion NMR spectroscopy”, Proc. Natl. Acad. Sci., 105, 11766-11771, 2008.