Anti-virulence targets have been suggested as a viable alternative to bacteriostatic or bactericidal therapies in order to combat multi-drug resistant bacteria. DsbA is a key regulator of virulence integral for the correct folding of many virulence factors and is found in many gram-negative bacteria. Bacteria lacking DsbA display an avirulent phenotype making it an interesting antibacterial target. While fragment screening yielded multiple starting points for fragment development, the shallow and hydrophobic binding groove found in Escherichia coli DsbA (EcDsbA) has made it difficult to develop high affinity small molecule inhibitors of EcDsbA.
Astex Therapeutics recently described an new approach to finding binding sites and binding pockets using high-concentration X-ray crystallography of a small library of 5-8 heavy atom “Mini Frags”(1). Their studies found these screens identified novel binding sites that were not found using their normal fragment screening library.
We have designed a similar library of small fragments (5-8 heavy atoms) to cover as many 2-point pharmacophores, scaffolds and topologies as possible. In addition we are extending this approach to be amenable to screening by 15N-1H HSQC NMR in addition to X-ray crystallography. Here we describe the application of this to identify novel binding sites on EcDsbA that correlate with protein dynamics and novel ligands discovered binding to EcDsbA.