New leads for antimicrobial drug discovery: Omics-guided identification of new bacterial folate biosynthesis inhibitors.

Njanje, I.*1, Gurmessa, G. T.1,2, Swart, T.1, Tsele, T.1, Kalinski, J.-C. J.1,3, Isaacs, M.1, Veale, C.4, Dorrington, R. A.1,5

1 Department of Biochemistry, Microbiology and Bioinformatics Rhodes University, Makhanda 6140, South Africa.
2 Department of Chemistry, College of Natural and Computational Sciences, Wollega University, Nekemte P.O. Box 395, Ethiopia.
3 Department of Biochemistry, University of California Riverside, Riverside, USA.
4 Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
5 South African Institute for Aquatic Biodiversity, Makhanda 6140, South Africa

Folates are cofactors for the biosynthesis of macromolecules essential for life. Bacterial pathogens, unlike their mammalian hosts, synthesize these cofactors de novo, making the inhibition of folate biosynthesis an appealing target for antimicrobial development. The highly conserved enzyme, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), which catalyzes the first reaction in the folate biosynthetic pathway, is present only in microorganisms, and the aim of this study was to isolate natural product (NPs) inhibitors of HPPK as potential lead compounds for antimicrobial development. Approximately 1500 marine and microbial NPs extracts were screened for inhibition of Escherichia coli HPPK in vitro as well as in vivo antibacterial and cytotoxicity activities, resulting in the prioritization of Streptomyces sp. AMRU046. AMRU046 was selected for further study. Bioassay guided fractionation, metabolomics and molecular docking analyses resulted in the identification of HPPK inhibitors. The NPs produced by Streptomyces sp. AMRU046 demonstrated promising in vitro inhibition of HPPK, in vivo antibacterial activity, without significant cytotoxicity. Furthermore, whole genome sequencing and bioinformatic analysis uncovered 33 putative Biosynthetic Gene Clusters (BGCs), the majority of which lack homology with known BGCs, indicating that this strain has the capacity to produce potentially novel secondary metabolites. Some of these metabolites were detected in the active fractions during metabolomic profiling. This study underscores the value of integrating modern ‘omics’ approaches, cheminformatics and classical microbiology in the discovery of novel anti-HPPK natural products.

Keywords: biosynthetic gene cluster, HPPK, metabolomics, cheminformatics