Signal Transduction

Generate and apply methods for characterization and inhibition of the primary bacterial signal transduction pathways

Given the alarming rate of resistance to currently available antibacterial agents, it is clear that validation of ubiquitous new bacterial targets is vital. Histidine kinase proteins could provide such an advance. Histidine kinases (HKs) are mediators of bacterial cell signaling, ultimately enabling cells to respond to the outside world (Figure 1). HKs are paired with a cognate response regulator (RR) to compose a two-component system (TCS; Figure 2). When an extracellular signal is sensed, the HK catalyzes autophosphorylation of a conserved histidine (His) residue with the γ-phosphate of adenosine triphosphate (ATP). The phosphoryl group is subsequently transferred from the HK to a response regulator, which commonly facilitates transcription regulation. HKs have been implicated in acute and chronic infections caused by both Gram-negative and Gram-positive bacteria and have been correlated with antibiotic resistance. Despite this fact, global and efficient methods for the profiling and inhibition of TCS-associated proteins, HKs and RRs, are lacking (Sci. Transl. Med. 2013, 5, 203ps12; Kinomics: Approaches and Applications, WILEY-VCH2015, pp 233-254).

Studying HKs in TCS is difficult due to the labile nature of the phoshohistidine bond. Prior work in the Carlson lab resulted in the development of the first non-radioactive ATP-based probe to globally label HKs in vitro, BODIPY-FL-ATPγS (J. Am. Chem. Soc. 2012, 134, 9150–9153). This probe has great success in stabilizing the P-N bond through the thiophosphohistidine linkage to label HKs. Further studies demonstrated that the bulky BODIPY causes sluggish phosphorylation kinetics (Biochemistry 2018, 57, 4368-4373). As a result, we are currently pursuing the development of a new generation of γ-modified ATP-based probes (ACS Chem. Biol. 2020, doi.org/10.1021/acschembio.9b01024), which will enable us to  stabilize the γ-phosphate onto the HKs with the ultimate goal of globally profiling HKs, through proteomic studies.

The high degree of sequence conservation in the catalytic and ATP-binding (CA) domain of HKs, their absence in eukaryotic organisms, and their essential role in bacterial signal transduction make them an attractive target for broad-spectrum antibacterial activity. Inhibitors that prevent bacterial signal transduction could lead to a new mechanism for the treatment of infectious disease. To identify inhibitors of the HKs, we have combined molecular modeling, high-throughput screening, and structural biology to identify inhibitors that bind to the CA domain (Med. Chem. Commun. 20134, 269-277; ACS Chem. Biol201510, 328-335; J. Med. Chem.201760, 8170-8182; Bioorg. Med. Chem. 201826, 5322-5326). To date, we have identified several scaffolds that inhibit HKs in vitro and show activity reducing virulent phenotypes in several species of pathogenic bacteria (Chem. Sci. 2018, 10, 7332-7337).

What types of methods will you learn/use on this project?

Organic synthesis, chemical proteomics and mass spectrometry, medicinal chemistry, molecular modeling, protein expression/purification, microbiology, and molecular biology.

sensing bacteria

Figure 1. Bacteria possess many different two-component systems to enable them to sense and respond to their external environment.

tcs info

Figure 2. (A) A stimulus is received by the extracellular domain, initiating the autophosphorylation event. First, ATP binds to the catalytic domain (CA) in the ATP-binding pocket. Next, the γ-phosphate is transferred to the conserved histidine on the dimerization and histidine phosphotransfer (DHp) domain. The phosphoryl group is transferred to the response regulator (RR), initiating a cellular response. (B) The structures of ATP and the resulting phosphorylated histidine with the transferred phosphate boxed in pink. 

pseudmonas

Figure 3. Inhibition of histidine kinases in Pseudomonas aeruginosa resulted in massive decreases in multiple virulence phenotypes.