Polyphosphate kinase from intracellular pathogens as a novel antibacterial target

Abstract

To circumvent the established mechanisms of bacterial resistance towards the available antibiotics, previously unexploited pathways critical for bacterial survival and pathogenicity must be targeted. Polyphosphate metabolism plays a crucial role in bacterial survival, in the expression of several virulence factors, in bacterial persistence and in the adaptation to stress conditions. The bacterial enzyme polyphosphate kinase (PPK) catalyses the reversible synthesis of polyphosphate by the transfer of a phosphate unit from ATP onto the growing polyphosphate chain. The presence of PPK in the genome of many pathogens and the attenuation of virulence observed in ppk deletion mutants make the PPK a potential target for the development of a new class of antibiotics.

The objective of this project has been the biochemical characterization of PPK2 from Francisella tularensis (FtPPK), the discovery of potential inhibitors and their evaluation as antibacterial agents. Using recombinant FtPPK and firefly luciferase, a coupled assay was developed to measure the FtPPK activity and further optimised to produce a robust assay in a format suitable for high throughput screening (384 well plate, Z'=0.70). The optimised FtPPK- luciferase coupled assay was used to characterize the kinetic properties of FtPPK and for the screening of two small libraries: a protein kinase inhibitor library (Protein Kinase Inhibitor Set, released by GSK), and a nucleotide analogue library (Reynolds library). The screening yielded 27 and 34 initial hits, respectively. The hits were validated by independent
luminescence assay, thermal shift assay and HPLC assay. The most potent hit was resynthesised for detailed characterization. IC50 and preliminary mode of inhibition have been investigated and, in conjunction with some preliminary SAR and with the modelling in the FtPPK active site, provide a starting point for later medicinal chemistry optimisation.

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Identifiers

ORCID for Peter L. Roach: orcid.org/0000-0001-9880-2877

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