Balanced dual targeting of novel bacterial topoisomerase inhibitor-fluoroquinolone (NBTI-FQ) hybrids as an innovative approach in fighting bacterial resistance

The discovery of antibacterial agents and their remarkable success in the fight against bacterial infections have yielded security for the humanity. Yet, their excessive and uncontrolled use has led to an increase in bacterial resistance, which undermines the effectiveness of these drugs and questions their future. The extremely small number of new antibacterial agents approved in recent decades indicates difficulties in their development, which has led to the introduction of new therapeutic strategies to successfully overcome the bacterial resistance. One such strategy is a balanced antibacterial activity on two biological targets, which ensures that in the event of a development of bacterial resistance in one target, antibacterial efficacy is maintained through the other target. This can be achieved through so-called hybrid molecules, in which two biologically active antibacterial compounds are incorporated into a single molecule. The well-known fluoroquinolones (FQ) are an example of antibacterial compounds that owe their success to the simultaneous inhibition of both DNA gyrase and topoisomerase IV (Topo IV) enzymes, but their antibacterial effect is unfortunately not balanced. Consequently, FQs lose their antibacterial effect mainly by developing bacterial resistance in only one enzyme target. Despite all the challenges associated with resistance, DNA gyrase and Topo IV remain suitable targets for the development of new antibacterial agents. One such very promising new class are the novel bacterial topoisomerase inhibitors (NBTIs), which share mechanistical similarities with FQs, but differ enough to avoid the FQ acquired bacterial resistance. This is due to the fact that NBTIs bind to a distinct but very close binding pocket on both enzymatic targets. Like FQs, NBTIs exhibit unbalanced antibacterial activity, with a higher priority on one enzyme target. The primary target of NBTIs is just the opposite as of FQs. Namely, DNA gyrase is the primary target of FQs in Gram-negative organisms and Topo IV forms the primary target in Gram-positive organisms. In NBTIs, DNA gyrase is primarily targeted in Gram-positive bacteria, whereas NBTIs are more effective on Topo IV in Gram-negative organisms. Therefore, the proposed research project focuses on the hybridization of NBTI and FQ antibacterial agents into a single compound, to achieve a balanced targeting inhibition of both DNA gyrase and Topo IV, which will help to reduce the development of bacterial resistance. Since the binding of both NBTI and FQ is unbalanced with opposite primary targets in Gram-staining pathogens, the added value of NBTI-FQ hybrids would also be in a more balanced Gram-positive and Gram-negative activity. Hybrids will be formed in two ways, by fusing the NBTI and FQ fragments into a single compound and by connecting NBTI and FQ compounds with a linker. NBTI-FQ fused hybrids will be able to bind to the NBTI or FQ binding site, while the second approach is based on the spatial proximity of NBTI and FQ binding sites, allowing the simultaneous binding of such hybrids to both sites. Hybrids designed with both approaches will be subjected to intensive computer simulation methods to study the stability and to quantify their enzyme affinity. By the calculated binding free energy of such hybrid compounds, we will obtain the information about the ability of a balanced dual binding to both enzyme targets in the same bacteria, which consequently determines the enzyme inhibitory and further antibacterial activity. Moreover, the calculated binding free energy will also allow the prediction of Gram-positive/Gram-negative antibacterial activity. Finally, hybrids for which computer-assisted research will predict the strongest and most balanced simultaneous binding to DNA gyrase and Topo IV, will be further chemically synthesized, and their enzyme inhibition and antibacterial activity will be determined by in vitro assays to confirm the balanced dual activity.