Bacterial histidine kinase inhibitors as novel antibacterials with low potential for resistance development

Infections caused by multidrug-resistant (MDR) bacteria continue to be a global problem. There is a rapidly growing problem of resistance among Gram-positive (Gr+) and Gram-negative (Gr-) pathogens causing infections in the nosocomial environment and the general community(1, 2). Several MDR pathogens, especially Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. – collectively referred to by the acronym ESKAPE – cause the majority of nosocomial infections(3). Recently, more people die of infections caused by MDR ESKAPE pathogens than of HIV/AIDS and tuberculosis combined(3) highlighting the urgency for novel antibacterial medicines development. Tuberculosis (TB) causes around 1.5 mln deaths per year(4). Mainly due to improper use of antibiotics MDR-TB is on the raise, and it is estimated that around 0.5 mln people developed MDR-TB in 2014. Another emerging health problem is posed by zoonotic infections and MDR in zoonotic pathogens(5). Zoonotic infectious diseases are diseases transmitted from vertebrate animals to humans. Zoonoses account for an estimated 60% of all human infectious diseases(6). Pasteurella multocida or Mannheimia haemolytica are the etiologic agents of variety of diseases in domestic animals and the infections can be transmitted to humans(7,8). Streptococcus suis is considered an emerging zoonotic agent with increasing numbers of human cases over the last years. In the environment, both avirulent and virulent strains occur in pigs, and virulent strains appear to infect pigs and human with the same efficiency(9,10). The growing problem of MDR and the lack of new antibiotics entering the clinic presents a major societal problem and threat to human and animal health. Bringing new antibacterials with low potential of resistance development to the clinic is critical as currently antibacterial therapy is the only way to ensure the level of infection control required for many medical procedures, e.g. during invasive surgery, in cancer chemotherapy, and in treatment of elderly and immuno-compromised patients. The best solution to the MDR problem would be the discovery and development of several novel classes of antibacterials for which there is no pre-existing resistance among human and animal bacterial pathogens (11). Bacterial histidine kinases (HK), and particularly the conserved catalytic and ATP-binding (CA) domain, have been recognized and explored as promising targets for novel antibacterials (12-17). HK together with a cognate response regulator (RR) form two-component systems (TCS), the major signalling systems in bacteria (18). TCS are involved in the response and adaptation to environmental stimuli, regulation of processes such as virulence and antimicrobial resistance, making them attractive antibacterial and antivirulence medicine drug targets(12, 14, 19). Due to the presence of the conserved catalytic and ATP-binding (CA) domain in all HKs, HK inhibitors targeted at the CA domain (i) inhibit multiple HKs in vitro, likely leading to shut down of multiple signalling pathways simultaneously and ultimately to inability of the bacteria to survive, (ii) show broad-spectrum antibacterial effect (12,15,17,20), and (iii) possess low probability to develop resistance due to the inhibition of multiple HKs, which may be essential or contribute to fitness and virulence. Here, we propose the further development of putative HK inhibitors (HKIs) we previously discovered as novel human and veterinary antibacterial medicines (15, 20). Within 5 years upon finalizing the project we anticipate to have a drug candidate in clinical development. The HKIs we have discovered so far inhibit the autophosphorylation of multiple HKs in vitro with IC50s for some HKs, e.g. S. aureus PhoR below 10 µM (Figure 1), and have antibacterial effect against Gr+, susceptible Gr-, and Mycobacterium marinum with minimal inhibitory concentrations (MIC) equeal or higher than 1µg/ml, 25 µg/ml, and 63 µg/ml, respectively. In regard to developing a broad-spectrum antibiotic, the main setback of the latest HKIs is the lack of susceptibility observed with E. coli, P. aeruginosa and other Gr-. The main objectives of the research proposed here (4 years) are (i) to develop 3 lead-candidates for broad-spectrum antibacterial medicines against Gr+ pathogens belonging to ESKAPE and zoonotic Gr+ pathogens, susceptible Gr-, e.g. the zoonotic pathogen P. multocida, and Mycobacterium spp. via the inhibition of ATP binding to the CA domain of multiple bacterial HK (Objective 1 (O1), WP1, 3 and 4), (ii) to improve the activity of the HKIs and broaden the spectrum against Gr- by elucidating the molecular mechanisms underlying the lack of susceptibility observed with E. coli and P. aeruginosa (O2, WP2), and (iii) to confirm the targets and to study the potential for resistance development and possible mechanisms of resistance in target pathogens(O3, WP3).