Fragment-based screening identifies inhibitors of the ATPase activity and of hexamer formation of Cagα from the Helicobacter pylori type IV secretion system

Abstract

Type IV secretion systems are membrane-bound multiprotein complexes that mediate the translocation of macromolecules across the bacterial cell envelope. In Helicobacter pylori a type IV secretion system is encoded by the cag pathogenicity island that encodes 27 Cag proteins and most of these are essential for bacterial virulence. We here present our work on the identification and characterization of inhibitors of Cagα, a hexameric ATPase and member of the family of VirB11-like proteins that is essential for translocation of the CagA cytotoxin into mammalian cells. We conducted fragment-based screening using a differential scanning fluorimetry assay and identified 16 molecules that stabilize the protein during thermal denaturation suggesting that they bind Cagα. Several of these molecules affect binding of ADP and four of them inhibit the ATPase enzyme activity of Cagα.Analysis of enzyme kinetics suggests that their mode of action is non-competitive, suggesting that they do not bind to the ATPase active site. Cross-linking analysis suggests that the active molecules change the conformation of the protein and gel filtration and transmission electron microscopy show that molecule 1G2 dissociates the Cagα hexamer. Analysis by X-ray crystallography reveals that molecule 1G2 binds at the interface between Cagα subunits. Addition of the molecule 1G2 inhibits the induction of interleukin-8 production in gastric cancer cells after co-incubation with H. pylori suggesting that it inhibits Cagα in vivo. Our results reveal a novel mechanism for the inhibition of the ATPase activity of VirB11-like proteins and the identified molecules have potential for the development into antivirulence drugs. Author summaryWe here report the results of a small-molecule screening approach to identify inhibitors of an essential virulence factor from the gastrointestinal pathogen Helicobacter pylori. We identifed novel chemical entities that bind to the Cagα protein and inhibit its function. We discovered a novel inhibitor binding site that disrupts the hexameric quaternary structure and inhibits ATPase enzyme activity of Cagα. Based on the structural information on the binding site, these molecules could be developed into high-affinity inhibitors that may have potential as anti-virulence drugs. This approach represents a generally applicable strategy for the inhibition of bacterial virulence factors for which structural information is available that could be applied to target similar proteins from many bacterial pathogens.

Type IV Coupling Proteins as Potential Targets to Control the Dissemination of Antibiotic Resistance

“…Unfortunately, in both studies (Hilleringmann et al, 2006;Sayer et al, 2014), there are concerns about the specificity of these compounds since they may also inhibit other bacterial or mammalian ATPases. Alternatively, an approach that does not specifically target Cagα ATPase activity has been tested (Arya et al, 2019), finding molecules that bind and stabilize Cagα, some of which inhibited Cagα ATPase activity and also dissociated Cagα hexamers.…”

Targeting bacterial pathogenesis by inhibiting virulence-associated Type III and Type IV secretion systems

“…The binding of 1G2 to the ATPase led to alterations in Caga conformation and appeared to interfere with Caga hexamer formation. The inhibition appears to be partial since treatment of bacteria with 1G2 reduced CagA-induced IL-8 production in cells upon infection, but intracellular phosphorylation of CagA within the host cells remained unaffected (Arya et al, 2019).…”

Therapeutic effect of demethylated hydroxylated phillygenin derivative on Helicobacter pylori infection

“…In addition, PHI-Der could also regulate ATP leakage of H. pylori (Arya et al, 2019) and downregulate virulence factors (Xia et al, 2022) (see Supplementary materials for details). Some pathogens that have evolved virulence factors highly resistant to oxidative stress can adhere and form biofilms on cell surfaces.…”