Iron is an essential mineral for the growth and proliferation of most microorganisms, and the efficiency with which pathogenic bacteria obtain iron generally parallels their virulence. Petrobactin is a non-ribosomal peptide synthase (NRPS) independent siderophore synthesized by pathogenic members of the Bacillus cereus sensu lato group, which includes the organism responsible for anthrax, B. anthracis. Petrobactin has shown to be absolutely necessary for the establishment of full virulence of B. anthracis within mammalian hosts. The biosynthetic pathway of petrobactin involves six enzymes (AsbA-F), and within these, the asbF gene encodes a dehydroshikimate (DHS) dehydratase enzyme responsible for the synthesis of the 3,4-dihydroxybenzoic acid groups that ultimately serve to coordinate ferric iron within the siderophore itself. In this work, inhibitors of AsbF were pursued as a new means to impede petrobactin production by pathogenic Bacillus strains. A library of diverse small molecules was screened against recombinant AsbF from B. thuringiensis 97-27 in vitro using an ultraviolet spectroscopic kinetic assay. Our results indicate that the presence of a carboxylate is requisite for efficient binding, and the presence of hydrogen bond acceptors near this functionality is also influential. Furthermore, the effectiveness of these inhibitors against petrobactin-producing Bacillus strains was examined using an in vivo assay. Computational docking models (AutoDockVina) were correlated with the experimental results, and the data from these screens are being used to inform the synthesis of second-generation enzyme inhibitors.

Development of inhibitors of petrobactin biosynthesis: Dehydroshikimate dehydratase as a target for anti-anthrax therapeutics

249th National Meeting (2015)

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