Fragments vs DsbA: towards a chemical probe

Despite its ubiquitous use as a model organism, Escherichia coli causes nearly a million deaths each year worldwide. Antibiotics provoke rapid selection for resistance and are becoming ineffective. An interesting alternative is to inhibit virulence factors. Doing so won’t directly kill the bacteria but instead reduce its infectivity, a trait that might be subject to less evolutionary selection.

 

The oxidoreductase DsbA facilitates disulfide bond formation in other bacterial proteins and is a key regulator of resistance. Martin Scanlon’s lab at Monash University has been pursuing this enzyme for some two decades; we described some of their work in 2015. In two recent papers, he and his colleagues describe progress towards a chemical probe.

 

DsbA has more than 300 protein substrates that bind in a shallow, hydrophobic groove. The lack of deep pockets or specific recognition elements makes finding small molecule ligands particularly challenging. Three years ago we highlighted fragment screens that identified two dozen hits in this groove. Intriguingly, that screen also identified a couple fragments that bind in a cryptic pocket close to the groove. This pocket is the focus of a paper published in Angew. Chem. late last year by Martin and collaborators at Monash University, La Trobe University Bundoora, and Scripps.  

 

Crystallography revealed that compound 1 binds in a pocket that is completely enclosed by DsbA. Twenty commercial analogs were purchased and tested by protein-observed [¹⁵N,¹H]-HSQC NMR. Six bound to the protein, but NMR suggested all bound in the hydrophobic groove, not in the cryptic pocket. Undeterred, the researchers made and tested a few dozen analogs, some of which did indeed bind the cryptic pocket and also had slightly higher affinities as measured by NMR and SPR.

 

How do the fragments get inside a pocket with no apparent entrances? Computational, protein-observed NMR, SPR, and HDX experiments suggested that DsbA is dynamic and one region can open up to allow access of the fragments. Interestingly, the fragments bind preferentially to the oxidized (active) form of DsbA, a fact that makes sense given that this state is more dynamic, allowing readier access to the pocket.

 

Unfortunately, the affinity of the best fragments is only around 150 micromolar. The small size of the cryptic pocket makes further affinity improvements unlikely, so the researchers sought to break the bounds of this pocket to gain added affinity. This is the focus of a paper just published in J. Med. Chem. by Martin, Bradley Doak, and collaborators at Monash, Vernalis, University of Western Australia, and The University of Sydney.

 

The researchers first built a small set of compounds that would break out of the pocket. Compound 5 had slightly worse affinity, as measured by SPR, but crystallography confirmed that the alkyne does in fact protrude as designed. A small set of analogs led to compound 13, with mid micromolar affinity. This compound was nearly 30-fold more potent than its enantiomer, with the hydroxyl moiety displacing a conserved water to make hydrogen bond interactions with the protein.

 

To gain additional interactions in the hydrophobic groove, the researchers chose direct-to-biology, screening crude reaction mixtures without purification, an increasingly popular strategy as we noted last week. In this case the researchers used automated flow reactors, allowing air- and moisture-sensitive organometallic chemistry. A set of 92 compounds was made and tested by off-rate screening (ORS) SPR and affinity-selected mass spectrometry (ASMS). Four crude hits were remade, purified, and tested, and compound 17 came in as a low micromolar binder both by SPR and ITC. This molecule also inhibited the enzyme in a functional assay and even showed some activity in a bacterial swarming motility assay.

 

Further improvements in potency will be needed to obtain a chemical probe, let alone a drug, but these two papers describe meaningful progress. They also provide a useful reminder that proteins are far from static. Cryptic pockets are surprisingly common, and even if they are too small and enclosed to support high affinity binding, they can be used as footholds to build larger molecules.