Developing Small Molecules to Study and Inhibit Histidine Kinases, Key Regulators of Antibiotic Resistance and Bacterial Virulence

Abstract

Antibiotic resistance currently is one of the biggest global health threats according to the World Health Organization (WHO). This situation results from antibiotic misuse and lack of new antibacterial agents and targets. Therefore, innovative methods to fight antibiotic resistance need to be developed. Two-component systems, composed of a histidine kinase (HK) and a response regulator, are the main signal transduction pathways in bacteria. They are involved in growth and cell maintenance, but also bacterial virulence and antibiotic resistance, which make them an ideal target for the development of antibacterial drugs. The transmembrane HK recognizes stimuli outside of the cell. Once a signal is received by this protein, an autophosphorylation event is initiated resulting in the transfer of the γ-phosphate of adenosine triphosphate (ATP) to a conserved histidine within the HK, followed by translocation to the response regulator. The latter protein typically acts as a DNA transcriptional factor, triggering a cellular response. Histidine phosphorylation occurs in both eukaryotes and prokaryotes but has been shown to dominate signaling networks in the latter due to its role in microbial TCSs. Methods to investigate histidine phosphorylation have lagged behind those to study serine, threonine and tyrosine modifications due to its inherent instability and the historical view that this protein modification was rare. An important strategy to overcome the reactivity of phosphohistidine is the development of substrate-based probes with altered chemical properties that improve modification longevity, but that do not suffer from poor recognition or transfer by the protein. Our research group has developed the first non-radioactive HK probe, BODIPY-FL-ATPγS, which enabled the visualization of HK activity. Nevertheless, efforts to enrich HKs with biotin-ATPγS were unsuccessful. In this thesis, we sought to understand the molecular basis for the development of efficient HK probes based on the ATP scaffold by comparison of the native kinase substrate, ATP, and alkylated ATP derivatives. Additionally, novel diazirine-based ATP probes were developed by adding a photoreactive crosslinker that facilitates the formation of a nonhydrolyzable bond between the HK and the phosphate group, ultimately enabling HK enrichment via biotin labeling. Due to the negatively charged phosphates, ATP cannot cross the bacterial cell envelope. Methods utilizing cationic polymers were explored to deliver the devised probes into bacteria for live cell studies. Finally, efforts towards the development of HK inhibitors are being presented. A class of HK inhibitors that diminish numerous virulence factors of methicillin-resistant Staphylococcus aureus, one of the antibiotic-resistant "priority pathogens" listed by the WHO, was developed. These compounds do not kill bacteria like most antibiotics but act as anti-virulence agents. Therefore, we anticipate that evolution of resistance to these molecules would be dramatically slower than for traditional bactericidal agents. We foresee their use as adjuvants with current antibiotics or as a stand-alone therapy, decreasing pathogenesis, which provides and advantage to the immune system in the fight against bacterial infectivity.