Antibacterial Gyrase Inhibitors

Discovery of novel gyrase B and gyrase B/topoisomerase IV (ParE) dual inhibitors with in vitro antibacterial activity (WP1/WP3)

Prof. Danijel Kikelj
University of Ljubljana

Gyrase plays an essential role in the cell by maintaining DNA in a negatively-supercoiled conformation. Gyrase is comprised of two subunits – GyrA and GyrB. The GyrA subunit is involved in nicking the DNA and is the target of the quinolone antibiotics. GyrB binds and hydrolyses ATP during the topoisomerase catalytic cycle and is the target of the aminocoumarin antibiotics, which compete with ATP for binding to the enzyme. We have recently identified a new structural class of compounds that target GyrB, and our project involves extending these findings and characterising the action of these compounds in considerable detail. This will involve in vitro screening of the compounds for inhibitory activity on bacterial gyrase B and topoisomerase IV (from E. coli) and determination of IC50 values using a newly-developed HTP assay (Brvar, et al. Bioorg. Med. Chem. Lett. 20, 958-962 (2010)). Hits will be validated using ITC and SPR to evaluate binding parameters, and the antibacterial activity of promising compounds against ESKAPE pathogens and other key bacterial species will be determined. Novel gyrase reporter gene-based assays and the transcriptional/proteomic profiling of key hits will be developed/assessed.

The increasing resistance to fluoroquinolones, the main class of gyrase inhibitors in clinical practice, has shifted the attention towards the discovery of new GyrB and/or ParE topoisomerase IV inhibitors. Based on our recent research towards the discovery of DNA gyrase B inhibitors possessing novel chemical scaffolds, and on our current promising results on marine natural products-inspired gyrase B inhibitors, for which a co-crystal structure with gyrase B from E. coli will be shortly available, we will focus our research on design, synthesis and evaluation of several novel structural types of gyrase B inhibitors and explore their potential for dual inhibition of gyrase B and topoisomerase IV. Additionally, we will study how physicochemical properties and modifications of our most promising Gyrase B and/or ParE inhibitors with known and innovative permeability-increasing moieties influence the compounds’ permeability into bacteria to ensure efficient discovery of potent, small-molecule compounds active against Gram-negative strains.

In vitro screening of compounds designed and synthesised for inhibitory activity on bacterial gyrase B and topoisomerase IV (from E. coli), determination of IC50 values, activity spectrum profiling, binding parameters (Kd, ΔH, ΔS) via ITC and SPR, MoA confirmation and transcriptional/proteomic profiling of hits, development of novel reporter gene-based gyrase assays (in collaboration with University of Cambridge).

Objectives: Design, synthesis and evaluation of structurally novel gyrase B inhibitors and gyrase B/topoisomerase IV (ParE) dual inhibitors and achieving their in vitro antibacterial activity by modifications which will improve entry of inhibitors into bacteria.

Expected Results: Structurally novel gyrase B inhibitors and balanced gyrase B/topoisomerase IV (ParE) dual inhibitors with in vitro antibacterial activity. Assessment of novel scaffolds and permeability enhancing moieties for achieving antibacterial activity in gyrase B inhibitors and gyrase B/topoisomerase IV (ParE) dual inhibitors.

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska Curie grant agreement No  642620
European Union