Chemical probes for competitive profiling of the quorum sensing signal synthase PqsD of Pseudomonas aeruginosa.

Michaela Prothiwa,Sandra Glasmacher,Thomas Böttcher,Dávid Szamosvári

doi:10.3762/bjoc.12.277

Abstract

The human pathogen Pseudomonas aeruginosa uses the pqs quorum sensing system to coordinate the production of its broad spectrum of virulence factors to facilitate colonization and infection of its host. Hereby, the enzyme PqsD is a virulence related quorum sensing signal synthase that catalyzes the central step in the biosynthesis of the Pseudomonas quinolone signals HHQ and PQS. We developed a library of cysteine reactive chemical probes with an alkyne handle for fluorescence tagging and report the selective and highly sensitive in vitro labelling of the active site cysteine of this important enzyme. Interestingly, only one type of probe, with a reactive α-chloroacetamide was capable of covalently reacting with the active site. We demonstrated the potential of our probes in a competitive labelling platform where we screened a library of synthetic HHQ and PQS analogues with heteroatom replacements and found several inhibitors of probe binding that may represent promising scaffolds for the development of customized PqsD inhibitors as well as a chemical toolbox to investigate the activity and active site specificity of the enzyme.

Highlights

The emergence of multi-drug resistant bacterial strains urges the rapid discovery of new antibiotics and the development of novel antiinfective strategies [1]
One peptide was detected with another cysteine residue (Cys138) modified by the probe compared to 64 detected peptides for CA2 labelled Cys112 underlining the selectivity of our probes (Figure 3C). These results indicate that probes CA1–3 are specific and covalently bind to Cys112 of PqsD and are to the best of our knowledge, the first account of activity-based probes targeting and selectively labelling the active site of PqsD
We have recently reported the discovery of inhibitors of the virulence factor elastase of P. aeruginosa by a library of synthetic HHQ and Pseudomonas quinolone signal (PQS) derivatives with systematic heteroatom replacements [33]

Summary

Introduction

The emergence of multi-drug resistant bacterial strains urges the rapid discovery of new antibiotics and the development of novel antiinfective strategies [1]. We were speculating that activity-based electrophilic probes may be applicable to target this enzyme in vitro which could allow to study its active site reactivity in greater detail and apply a competitive labelling platform to discover potential PqsD inhibitors. We synthesized a small library of chemical probes with electrophilic α-chloroacetamide, α,β-unsaturated amide, and α,β-unsaturated ketone moieties as protein reactive groups, which have been reported to exhibit selectivity for active site cysteines [19] (Supporting Information File 1, Figure S1).

Results

Conclusion