Designed Small-Molecule Inhibitors of the Anthranilyl-CoA Synthetase PqsA Block Quinolone Biosynthesis in Pseudomonas aeruginosa

Abstract

The Gram-negative bacterial pathogen Pseudomonas aeruginosa uses three interconnected intercellular signaling systems regulated by the transcription factors LasR, RhlR, and MvfR (PqsR), which mediate bacterial cell–cell communication via small-molecule natural products and control the production of a variety of virulence factors. The MvfR system is activated by and controls the biosynthesis of the quinolone quorum sensing factors HHQ and PQS. A key step in the biosynthesis of these quinolones is catalyzed by the anthranilyl-CoA synthetase PqsA. To develop inhibitors of PqsA as novel potential antivirulence antibiotics, we report herein the design and synthesis of sulfonyladeonsine-based mimics of the anthranilyl-AMP reaction intermediate that is bound tightly by PqsA. Biochemical, microbiological, and pharmacological studies identified two potent PqsA inhibitors, anthranilyl-AMS (1) and anthranilyl-AMSN (2), that decreased HHQ and PQS production in P. aeruginosa strain PA14. However, these compounds did not inhibit production of the virulence factor pyocyanin. Moreover, they exhibited limited bacterial penetration in compound accumulation studies. This work provides the most potent PqsA inhibitors reported to date and sets the stage for future efforts to develop analogues with improved cellular activity to investigate further the complex relationships between quinolone biosynthesis and virulence factor production in P. aeruginosa and the therapeutic potential of targeting PqsA.

Discovery of scmR as a global regulator of secondary metabolism and virulence in Burkholderia thailandensis E264

“…Interference with the function of MvfR results in lowered virulence by P. aeruginosa (52,53). Consequently, MvfR has advanced to a target for the development of strategies to combat P. aeruginosa infections.…”

Structures of the N‐Terminal Domain of PqsA in Complex with Anthraniloyl‐ and 6‐Fluoroanthraniloyl‐AMP: Substrate Activation in Pseudomonas Quinolone Signal (PQS) Biosynthesis

“…In summary, the crystal structures of PqsA NTD discussed here broaden our knowledge of anthranilate‐CoA ligases in general. The structural insight into the active site architecture of PqsA NTD might also help rationally guided improvement of the only moderately active AMP‐based sulfonyladenosine PqsA inhibitors recently published by Ji et al., and this in turn might ultimately lead to alternative therapeutic approaches targeting the quinolone signaling cascade of P. aeruginosa .…”

“…It has previously been shown that AQ production in P. aeruginosa can be strongly reduced by halogenated anthranilate derivatives: this is a consequence of incorporation of these compounds by PqsA, probably followed by inhibition of one of the downstream steps in HHQ/PQS biosynthesis. The most efficient inhibitor was 6‐fluoroanthranilate (6FABA), which was turned over to the corresponding thioester with efficiency similar to that of the natural substrate anthranilate but abolished PQS production completely when applied at concentrations in the millimolar range . We therefore performed cocrystallization experiments with 6FABA/ATP⋅Mg 2+ and were able to obtain diffraction data from a crystal in form 1.…”

“…[46] In addition to PqsD, severala ttempts to develop small-molecule inhibitors of PqsA were undertakend espite the absence of structural insight. [24,51,52] Here we reportc ocrystal structures of the N-terminal domain of PqsA (PqsA NTD )i nc omplex with anthraniloyl-AMP and also with its analogue 6-fluoroanthraniloyl-AMP( 6FABA-AMP). The overall structure of PqsA NTD is described, and the adenylate-binding pocket of PqsA NTD is analyzed by comparison with other acyl-CoAl igases.…”

“…One of the reasons for the preference for PqsD as a potential drug target is the availability of structural data for this enzyme . In addition to PqsD, several attempts to develop small‐molecule inhibitors of PqsA were undertaken despite the absence of structural insight …”