Two-component systems (TCSs) are the primary mechanisms through which bacteria respond to their environment. Comprised of histidine kinases (HKs) and response regulators (RRs), these phosphorylation cascades result in myriad intracellular responses, often alterations in gene expression. HKs are excellent antibiotic targets: they are primarily found in bacteria, possess an ATP-binding domain distinct from Ser/Thr/Tyr kinases, participate in several pathogenic roles, contain a conserved catalytic core within the HK superfamily, and present a mechanism of inhibition not yet exploited medicinally. For the elucidation of the cellular roles, activity, and inhibition of these proteins, we are developing assorted chemical probes. BODIPY-ATP?S is an activity-based probe that features a modified ?-phosphate group. We have demonstrated that this molecule facilitates the detection of the phosphohistidine species formed by catalytically active HKs. Moreover, we have shown the subsequent transfer of the modified phosphate group to the cognate RR. At present, we are exploring the use of the identified probe in whole proteomes to illuminate HK regulation in bacteria and its role in virulence. In another effort, we have studied the conservation of the ATP-binding domain of HKs using molecular modeling. In silico screening of previously reported but failed HK inhibitors directed us to the generation of guanidine-bearing fragments as scaffolds that bind deep in the pocket by a salt bridge with a conserved aspartate. In combination with the analysis of ATP binding in HKs versus other proteins, this study provided insights into the rational design of HK inhibitors for potentially broad-spectrum use in bacteria. Lastly, we have developed an assay with an HK fluorescence polarization probe that has enabled the high-throughput screening of small molecule libraries for HK inhibitory activity. Together, these endeavors will facilitate a better understanding of TCS regulation in bacteria.

Histidine kinase profiling and inhibitor discovery with small-molecule probes

246th National Meeting (2013)

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