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

Abstract

Type IV secretion systems are multiprotein complexes that mediate the translocation of macromolecules across the bacterial cell envelope. In Helicobacter pylori a type IV secretion system encoded by the cag pathogenicity island encodes 27 proteins and most are essential for virulence. We here present 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 suggesting that they bind Cagα. Several molecules affect binding of ADP and four of them inhibit the ATPase activity. Analysis of enzyme kinetics suggests that their mode of action is non-competitive, suggesting that they do not bind to the active site. Cross-linking suggests that the active molecules change protein conformation and gel filtration and transmission electron microscopy show that molecule 1G2 dissociates the Cagα hexamer. 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.

Type IV secretion systems: Advances in structure, function, and activation

“…In some cases, inhibitors are being identified through high throughput screens for molecules blocking a T4SS‐mediated process, for example, conjugation (Cabezon et al., 2017; Casu et al., 2017; Shaffer et al., 2016). In others, specific requirements for machine assembly or function are targeted for inhibition, such as VirB8 dimerization (Casu et al., 2016; Paschos et al., 2011; Smith, 2012 #3395), or catalytic activities of the VirD4, VirB4, or VirB11 ATPases (Arya et al., 2019; Garcia‐Cazorla et al., 2018; Ripoll‐Rozada et al., 2016). This latter approach, in particular, will benefit through structural definition of intersubunit contacts critical to T4SS integrity, as these are prime targets for small‐molecule disruption of machine assembly.…”

Inhibition of Type IV Secretion Activity and Growth of Helicobacter pylori by Cisplatin and Other Platinum Complexes

“…One study reported the identification of compounds that were able to reduce formation of secretion system-associated pili, and also type IV secretion itself (Shaffer et al, 2016). Other studies have described small-molecule inhibitors that are able to inhibit one of the ATPases, Caga (Hilleringmann et al, 2006;Sayer et al, 2014;Arya et al, 2019).…”

Phillygenin Inhibits Helicobacter pylori by Preventing Biofilm Formation and Inducing ATP Leakage

“…ATP was found to provide energy in many key cellular processes and reactions (Quinn et al, 2021 ). The decrease of ATP level suggested that the functions of mitochondria were impaired, and the cells would therefore undergo apoptosis and necrosis (Arya et al, 2019 ). The Lpt protein family was found to be required in the export of lipopolysaccharide (LPS) to the cell surface (Turkina et al, 2015 ).…”