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

“Open sesame”: fragments crack open a cryptic pocket on a challenging protein-protein interaction target (#14)

Geqing Wang 1 , Wesam Alwan 2 , Biswaranjan Mohanty 2 , Bradley Doak 2 , Rabeb Dhouib 3 , Makrina Totsika 3 , Róisín McMahon 4 , Benvenuto Capuano 2 , Peter Scammells 2 , Jennifer Martin 4 , Martin Scanlon 2 , Begoña Heras 1
  1. La Trobe Institute for Molecular Science, Bundoora, VIC, Australia
  2. Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Parkville, VIC, Australia
  3. Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, QLD, Australia
  4. Griffith Institute for Drug Discovery, Griffith University, Nathan, QLD

Disulfide bond formation protein A (DsbA) is a thiol-disulfide oxidoreductase enzyme which catalyzes disulfide bond formation in the periplasm of Gram-negative bacteria. DsbA facilitates folding of multiple virulent factors that bacteria use for host cell manipulation, host colonization and spread. Bacteria lacking a functional DsbA display reduced virulence, increased sensitivity to antibiotics and diminished capacity to cause infection.1 The critical role of DsbA in bacterial virulence makes it an attractive drug target to combat multi-drug-resistant (MDR) bacteria.

The active site of DsbA is flanked by an extended hydrophobic groove, which binds to various unfolded substrates. Previously we carried out a fragment screening campaign against Escherichia coli DsbA (EcDsbA) and identified the first inhibitors that bind to the hydrophobic groove and inhibit EcDsbA activity in vitro and cell-based assays.2,3 By exploiting an array of biophysical/biochemical tools (NMR, SPR, X-ray crystallography and in vitro assays), two chemical classes of inhibitors, phenythiazole and diaryl ether, were optimized in a structure-based approach in this work. 200+ analogues were synthesized and 100+ co-structures were solved by X-ray crystallography, but only limited affinity gain was obtained. This shows that the hydrophobic groove is highly difficult to target. More recently through structural, biochemical and NMR dynamics studies we postulated a cryptic pocket on EcDsbA that could be exploited for inhibitor development. A second fragment screen campaign using smaller and more polar fragments allows the identification of binders for the predicated cryptic pocket. These fragment hits showed promising structure-activity relationship (SAR) and higher binding affinity. More importantly, binding of fragments to the cryptic pocket inhibit EcDsbA oxidation activity both in vitro and in cell-based assay. This work shows that identification of cryptic sites on proteins can convert poorly druggable or undruggable targets into druggable targets, potentially expanding the druggable proteome.

  1. 1. B. Heras, et al. (2009), DSB proteins and bacterial pathogenicity, Nat. Rev. Microbiol., 7,215-225 2. L.A. Adams, et al. (2015), Application of fragment-based screening to the design of inhibitors of Escherichia coli DsbA, Angew. Chem. Int. Ed., 54, 2179-2184 3. M.Totsika, et al. (2018), Inhibition of diverse DsbA enzymes in multi-DsbA encoding pathogens. Antioxid. Redox. Signal., In press.