Fragment library screening identifies hits that bind to the non-catalytic surface of Pseudomonas aeruginosa DsbA1

Abstract

At a time when the antibiotic drug discovery pipeline has stalled, antibiotic resistance is accelerating with catastrophic implications for our ability to treat bacterial infections. Globally we face the prospect of a future when common infections can once again kill. Anti-virulence approaches that target the capacity of the bacterium to cause disease rather than the growth or survival of the bacterium itself offer a tantalizing prospect of novel antimicrobials. They may also reduce the propensity to induce resistance by removing the strong selection pressure imparted by bactericidal or bacteriostatic agents. In the human pathogen Pseudomonas aeruginosa, disulfide bond protein A (PaDsbA1) plays a central role in the oxidative folding of virulence factors and is therefore an attractive target for the development of new anti-virulence antimicrobials. Using a fragment-based approach we have identified small molecules that bind to PaDsbA1. The fragment hits show selective binding to PaDsbA1 over the DsbA protein from Escherichia coli, suggesting that developing species-specific narrow-spectrum inhibitors of DsbA enzymes may be feasible. Structures of a co-complex of PaDsbA1 with the highest affinity fragment identified in the screen reveal that the fragment binds on the non-catalytic surface of the protein at a domain interface. This biophysical and structural data represent a starting point in the development of higher affinity compounds, which will be assessed for their potential as selective PaDsbA1 inhibitors.

A high-throughput cell-based assay pipeline for the preclinical development of bacterial DsbA inhibitors as antivirulence therapeutics

“…Measuring virulence target inhibition reliably and at large-scale is often difficult when using microbiological assays, so when the target is known, inhibitor screening and early evaluation typically relies on biochemical/biophysical approaches. This is the case for DsbA inhibitors that have been reported to date, with hits from several chemical classes having been identified as part of fragment-based screening campaigns primarily using saturation transfer difference NMR spectroscopy 12 , 21 – 23 . Later-stage microbiological evaluation has validated some but not all hits, and in some cases, even chemically elaborated analogues have failed to show activity in cell-based assays 12 , 30 .…”

High-throughput cell-based assays for the preclinical development of DsbA inhibitors as antivirulence therapeutics

“…Measuring virulence target inhibition reliably and at large-scale is often difficult when using microbiological assays, so when the target is known, inhibitor screening and early evaluation typically relies on biochemical/biophysical approaches. This is the case for DsbA inhibitors that have been reported to date, with hits from several chemical classes having been identified as part of fragment-based screening campaigns primarily using saturation transfer distance NMR spectroscopy (12, 21–23). Later-stage microbiological evaluation has validated some but not all hits, and in some cases, even chemically elaborated analogues have failed to show activity in cell-based assays (12, 29 and unpublished data).…”

Disulfide Bond Formation in the Periplasm of Escherichia coli

“…In parallel, scientists have started to expand their research into oxidative folding of proteins in clinically relevant strains, such as Mycobacterium (15,175,176), Pseudomonas (136,177,178), Helicobacter (179,180), and Vibrio (181,182). The discovery of Dsb machinery in these clinically relevant strains has resulted in the discovery of potential biopharmaceuticals with the ability to target and inhibit disulfide bond formation in pathogenic strains (183)(184)(185). The targeting of Dsb machinery also addresses the need for new approaches to overcome the problem of rapidly developing antibiotic resistance.…”