Exploration of the Effects of γ-Phosphate-Modified ATP Analogues on Histidine Kinase Autophosphorylation.

While two-component systems (TCSs), composed of a sensor histidine kinase (HK) and a response regulator, are the main signaling pathways in bacteria, global TCS activity remains poorly described. Here, we report the kinetic parameters of the HK autophosphorylation reaction using previously uncharacterized γ-phosphate-modified ATP analogues to further elucidate their utility as activity-based probes for global TCS analysis. Given the increased stability of thiophosphorylated histidine in comparison to that of the native phosphoryl modification, which is attributed to the decreased electrophilicity of this moiety, we anticipated that ATPγS may be turned over much more slowly by the HKs. Surprisingly, we found this not to be the case, with the turnover numbers decreasing <1 order of magnitude. Instead, we found that alkylation of the thiophosphate had a much more dramatic effect on turnover and, in one case, the binding affinity of this substrate analogue (BODIPY-FL-ATPγS).

Screening serine/threonine and tyrosine kinase inhibitors for histidine kinase inhibition.

Histidine kinases of bacterial two-component systems are promising antibacterial targets. Despite their varied, numerous roles, enzymes in the histidine kinase superfamily share a catalytic core that may be exploited to inhibit multiple histidine kinases simultaneously. Characterized by the Bergerat fold, the features of the histidine kinase ATP-binding domain are not found in serine/threonine and tyrosine kinases. However, because each kinase family binds the same ATP substrate, we sought to determine if published serine/threonine and tyrosine kinase inhibitors contained scaffolds that would also inhibit histidine kinases. Using select assays, 222 inhibitors from the Roche Published Kinase Set were screened for binding, deactivation, and aggregation of histidine kinases. Not only do the results of our screen support the distinctions between ATP-binding domains of different kinase families, but the lead molecule identified also presents inspiration for further histidine kinase inhibitor development.

Activity-based probe for histidine kinase signaling.

Bacterial two-component systems (TCSs) are signaling pathways composed of two proteins: a histidine kinase (HK) and a response regulator (RR). Upon stimulation, the HK autophosphorylates at a conserved histidine. The phosphoryl group is subsequently transferred to an aspartate on an RR, eliciting an adaptive response, often up- or downregulation of gene expression. TCS signaling controls many functions in bacteria, including development, virulence, and antibiotic resistance, making the proteins involved in these systems potential therapeutic targets. Efficient methods for the profiling of HKs are currently lacking. For direct readout of HK activity, we sought to design a probe that enables detection of the phosphotransfer event; however, analysis of the phosphohistidine species is made difficult by the instability of the P-N bond. We anticipated that use of a γ-thiophosphorylated ATP analogue, which would yield a thiophosphorylated histidine intermediate, could overcome this challenge. We determined that the fluorophore-conjugated probe, BODIPY-FL-ATPγS, labels active HK proteins and is competitive for the ATP binding site. This activity-based probe provides a new strategy for analysis of TCSs and other HK-mediated processes and will facilitate both functional studies and inhibitor identification.

Rational Design of Selective Adenine-Based Scaffolds for Inactivation of Bacterial Histidine Kinases.

Bacterial histidine kinases (HKs) are quintessential regulatory enzymes found ubiquitously in bacteria. Apart from their regulatory roles, they are also involved in the production of virulence factors and conferring resistance to various antibiotics in pathogenic microbes. We have previously reported compounds that inhibit multiple HKs by targeting the conserved catalytic and ATP-binding (CA) domain. Herein, we conduct a detailed structure-activity relationship assessment of adenine-based inhibitors using biochemical and docking methods. These studies have resulted in several observations. First, interaction of an inhibitor's amine group with the conserved active-site Asp is essential for activity and likely dictates its orientation in the binding pocket. Second, a N-NH-N triad in the inhibitor scaffold is highly preferred for binding to conserved Gly:Asp:Asn residues. Lastly, hydrophobic electron-withdrawing groups at several positions in the adenine core enhance potency. The selectivity of these inhibitors was tested against heat shock protein 90 (HSP90), which possesses a similar ATP-binding fold. We found that groups that target the ATP-lid portion of the catalytic domain, such as a six-membered ring, confer selectivity for HKs.

Thiol-ene-Enabled Detection of Thiophosphorylation as a Labeling Strategy for Phosphoproteins.

The adenosine triphosphate (ATP) analogue adenosine 5'-O-(3-thiotriphosphate) (ATPγS) has been applied as a tool to study kinase-substrate phosphorylation. Not only does the transfer of a thiophosphate group represent a unique modification amid the phosphoproteome, but it can also be stable to phosphatase activity. However, detection of this species is complicated due to the similar chemical reactivity of thiophosphate and proteinaceous thiols. Here, we describe a novel method for detection of protein thiophosphorylation utilizing the thiol-ene reaction. By first chemoselectively capping protein thiols through radical chemistry, kinase-catalyzed thiophosphorylation can be visualized specifically.

Inactivation of multiple bacterial histidine kinases by targeting the ATP-binding domain.

Antibacterial agents that exploit new targets will be required to combat the perpetual rise of bacterial resistance to current antibiotics. We are exploring the inhibition of histidine kinases, constituents of two-component systems. Two-component systems are the primary signaling pathways that bacteria utilize to respond to their environment. They are ubiquitous in bacteria and trigger various pathogenic mechanisms. To attenuate these signaling pathways, we sought to broadly target the histidine kinase family by focusing on their highly conserved ATP-binding domain. Development of a fluorescence polarization displacement assay facilitated high-throughput screening of ∼53 000 diverse small molecules for binding to the ATP-binding pocket. Of these compounds, nine inhibited the catalytic activity of two or more histidine kinases. These scaffolds could provide valuable starting points for the design of broadly effective HK inhibitors, global reduction of bacterial signaling, and ultimately, a class of antibiotics that function by a new mechanism of action.

All signals lost.

Inhibitors of bacterial histidine kinases that globally deactivate bacterial signaling may offer a new offensive against antibiotic resistance.

Mechanistic insight into inhibition of two-component system signaling.

Two-component signal transduction systems (TCSs) are commonly used by bacteria to couple environmental stimuli to adaptive responses. Targeting the highly conserved kinase domain in these systems represents a promising strategy for the design of a broad-spectrum antibiotic; however, development of such compounds has been marred by an incomplete understanding of the conserved binding features within the active site that could be exploited in molecule design. Consequently, a large percentage of the available TCS inhibitors demonstrate poor target specificity and act via multiple mechanisms, with aggregation of the kinase being the most notable. In order to elucidate the mode of action of some of these compounds, molecular modeling was employed to dock a suite of molecules into the ATP-binding domain of several histidine kinases. This effort revealed a key structural feature of the domain that is likely interacting with several known inhibitors and is also highly conserved. Furthermore, generation of several simplified scaffolds derived from a reported inhibitor and characterization of these compounds using activity assays, protein aggregation studies and saturation transfer differential (STD) NMR suggests that targeting of this protein feature may provide a basis for the design of ATP-competitive compounds.