Thiol-benzo-triazolo-quinazolinone Inhibits Alg44 Binding to c-di-GMP and Reduces Alginate Production by Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that affects a large proportion of cystic fibrosis (CF) patients. CF patients have dehydrated mucus within the airways that leads to the inability of the mucociliary escalator to expel inhaled microbes. Once inhaled, P. aeruginosa can persist in the lungs of the CF patients for the remainder of their lives. During this chronic infection, a phenomenon called mucoid conversion can occur in which P. aeruginosa can mutate and inactivate their mucA gene. As a consequence, transcription of the alg operon is highly expressed, leading to the copious secretion of the alginate exopolysaccharide, which is associated with decreased lung function and increased CF patient morbidity and mortality. Alginate biosynthesis by P. aeruginosa is post-translationally regulated by bis(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), which binds to the receptor protein Alg44 to activate alginate production. The identification of small molecules that disrupt the binding of c-di-GMP to Alg44 could inhibit the ability of P. aeruginosa to produce alginate. In this work, a class of thiol-benzo-triazolo-quinazolinone compounds that inhibited Alg44 binding to c-di-GMP in vitro was identified after screening chemical libraries consisting of ∼50 000 chemical compounds. Thiol-benzo-triazolo-quinazolinones were shown to specifically inhibit Alg44-c-di-GMP interactions by forming a disulfide bond with the cysteine residue in the PilZ domain of Alg44. The more potent thiol-benzo-triazolo-quinazolinone had the ability to reduce P. aeruginosa alginate secretion by up to 30%. These compounds serve as leads in the development of novel inhibitors of alginate production by P. aeruginosa after mucoid conversion.

Small Molecule Anti-biofilm Agents Developed on the Basis of Mechanistic Understanding of Biofilm Formation

“…(D) 29a discovered by the Sintim group, and further investigated the importance of the benzoisothiazolinone moiety by testing 29b and 29c (Zheng et al, 2016). (E) H19 30a and 925 30b ; two Alg44 (pilZ domain) inhibitors identified by Wang et al, and structural features further investigated by (Zhou et al, 2017). (F) The small molecules terrein 31 and 32 used by Kim et al in studying the correlation between regulation of c-di-GMP levels and QS.…”

“…In 2017, Wang et al identified two compounds, H19 30a and 925 30b (Figure 6) that interrupted c-di-GMP binding to Alg44 (PilZ domain) in P. aeruginosa , thereby inhibiting its ability to produce alginate, an exopolysaccharide polymer which is part of the biofilm matrix (Zhou et al, 2017). The sulfhydryl group, forming a covalent disulfide bond with Cys-98 in Alg44 (Figure 6), was found to be of high importance for activity.…”

“…The sulfhydryl group, forming a covalent disulfide bond with Cys-98 in Alg44 (Figure 6), was found to be of high importance for activity. Furthermore, a broad range of alkyl substituents was tested without any improvement of inhibitory activity (Zhou et al, 2017).…”

Differential Radial Capillary Action of Ligand Assay (DRaCALA)

“…A reduction in the F B is indicative of lead compounds in the chemical library that prevent binding of the protein to the nucleic acid. An example of a screen for small molecule inhibitors of Pseudomonas aeruginosa Alg44 binding to 32 P-c-di-GMP is depicted in Figure 6 (Zhou et al, 2017). The use of positive and negative controls in every plate provides a measure of assay quality and reproducibility.…”

“…The compound identified from screening this plate was AC1LFHI9 (HI9), a derivative of thiol-benzo-triazolo-quinazolinone. Adapted with permission fromZhou et al (2017).…”