Fragments vs DsbA: targeting bacterial virulence

The quest for new antibacterial agents can seem quixotic: no sooner have you found a killer molecule than the bugs have developed resistance to it. Evolution is hard to beat, particularly when it comes down to life or death. But what if you could lower the stakes? Many bacteria express virulence factors that are not essential for survival but are important for colonizing their host. Perhaps targeting these would be less prone to generating resistance.

Virulence factors often contain disulfide bonds, and the bacterial protein DsbA is essential for catalyzing their formation. In a paper published recently in Angew. Chem. Int. Ed., Begoña Heras (formerly University of Queensland), Jamie Simpson and Martin Scanlon (Monash University) and collaborators describe a fragment-based approach against the E. coli. version of this target. (See also here for Derek Lowe’s thoughts.)

The researchers started with an STD NMR screen of an 1132 fragment library from Maybridge, with compounds in pools of 3 to 5 (each at 0.3 mM). This yielded 171 hits, 37 of which showed appreciable chemical shift perturbations (CSPs) in a two-dimensional HSQC ¹⁵N-¹H NMR assay. All of these were relatively weak, with none showing saturation at 1 mM fragment concentration, but they all appeared to be binding in a hydrophobic groove adjacent to the active site.

The 37 hits clustered into eight different structural subclasses, one of which – the phenylthiazoles – is described in detail. The Monash fragment library was designed with SAR-by-catalog in mind, and 22 commercial analogs were purchased and tested in the HSQC assay to assess the SAR. Several of the compounds were soaked into crystals of DsbA, in one case leading to a structure in which two fragments were bound stacked on top of each other in the hydrophobic groove. However, this binding mode was inconsistent with the NMR data, and indeed co-crystallography of the same fragment revealed a 1:1 complex with the protein, also in the hydrophobic groove. (As an aside, this is an interesting case of crystal soaking and co-crystallization giving different results; are readers aware of others?)

The crystal structure was used to inform fragment-growing, ultimately leading to molecules with dissociation constants around 0.2 mM as assessed by surface plasmon resonance and with similar IC₅₀ values in a functional assay. One of these compounds was also tested against E. coli. DsbA is not needed for bacterial growth in rich media but is necessary for motility, and happily the assays showed just this – the compound did not affect growth but did inhibit cell motility.

Although the molecules are still too weak to answer the question of whether targeting DsbA will be a viable antibacterial strategy in vivo, this paper presents promising starting points, along with a wealth of data (including 78 pages of supporting information!) And if you want to learn more, Martin Scanlon is one of the organizers of the FBLD symposium at Pacifichem this December – so you can ask him questions in person in Honolulu!