Small-Molecule Inhibitors of the Pseudaminic Acid Biosynthetic Pathway: Targeting Motility as a Key Bacterial Virulence Factor

Abstract

Helicobacter pylori is motile by means of polar flagella, and this motility has been shown to play a critical role in pathogenicity. The major structural flagellin proteins have been shown to be glycosylated with the nonulosonate sugar, pseudaminic acid (Pse). This glycan is unique to microorganisms, and the process of flagellin glycosylation is required for H. pylori flagellar assembly and consequent motility. As such, the Pse biosynthetic pathway offers considerable potential as an antivirulence drug target, especially since motility is required for H. pylori colonization and persistence in the host. This report describes screening the five Pse biosynthetic enzymes for small-molecule inhibitors using both high-throughput screening (HTS) and in silico (virtual screening [VS]) approaches. Using a 100,000-compound library, 1,773 hits that exhibited a 40% threshold inhibition at a 10 M concentration were identified by HTS. In addition, VS efforts using a 1.6-million compound library directed at two pathway enzymes identified 80 hits, 4 of which exhibited reasonable inhibition at a 10 M concentration in vitro. Further secondary screening which identified 320 unique molecular structures or validated hits was performed. Following kinetic studies and structureactivity relationship (SAR) analysis of selected inhibitors from our refined list of 320 compounds, we demonstrated that three inhibitors with 50% inhibitory concentrations (IC 50 s) of approximately 14 M, which belonged to a distinct chemical cluster, were able to penetrate the Gram-negative cell membrane and prevent formation of flagella.

Targeting Bacillosamine Biosynthesis in Bacterial Pathogens: Development of Inhibitors to a Bacterial Amino-Sugar Acetyltransferase from Campylobacter jejuni

“…53 Closely related to the work in this paper, the Sulea and Logan research groups have reported an inhibitor that targets the pseudaminic acid biosynthetic pathway found in H. pylori and C. jejuni . 54 The “sialic-acid like” nonulosonate sugar product is required for the glycosylation of structural flagellin proteins and exposure to inhibitors attenuated flagellin production at concentrations of ≥100 μM. The flagella are required for persistent infections of H. pylori and this work demonstrates the link between bacterial glycosylation and pathogenicity using chemical tools (as opposed to genetic modification).…”

The Helicobacter pylori J99 jhp0106 Gene, under the Control of the CsrA/RpoN Regulatory System, Modulates Flagella Formation and Motility

“…Currently, carbohydrate-based therapies and diagnostics in cancer research and infectious disease have received considerable attention. Menard et al identified three inhibitors of the pseudaminic acid biosynthetic enzymes which show activity in inhibiting the flagellin proteins on the C. jejuni cell surface, by bacterial cell-based assays (Menard et al, 2014). In this study, we found the prevalence of the jhp0106 gene among 95 clinical isolates of H. pylori in Taiwan was 100%.…”

“…H. pylori flagellin proteins are synthesized, then post-translationally modified intracellularly by glycosylation with a nine carbon pseudaminic acid sugar derivative that resembles sialic acid (Schirm et al, 2003; Logan, 2006). The enzymes of the pseudaminic acid biosynthetic pathway in H. pylori , in order, are PseB, PseC, PseH, PseG, and PseI (Schirm et al, 2003; Menard et al, 2014), and the glycosylation process is essential for assembly of functional flagellar filaments and consequent bacterial motility (Schoenhofen et al, 2006). …”

Novel Serine/Threonine-O-glycosylation with N-Acetylneuraminic acid and 3-Deoxy-D-manno-octulosonic acid by Maf glycosyltransferases

“…In pathogens such as Campylobacter jejuni and Helicobacter pylori, flagellin glycosylation is required for flagellum assembly, bacterial motility, and colonization ability, and therefore 1 9 associated with virulence 8 . Flagellin glycosylation therefore offers potential as an antivirulence target, and hence small molecule inhibitors of nonulosonic acid biosynthesis 91 Albeit these systems are available for studying putative legionaminic acid and pseudaminic acid transferases, our demonstration of donor substrate promiscuity (especially with respect to KDO transferase activity) has the implication that Maf proteins may be easily subjected to high-throughput screening for small molecule inhibitors or to other biochemical characterization using a common lab strain of E. coli.…”