Inactivation of Multiple Bacterial Histidine Kinases by Targeting the ATP-Binding Domain

Abstract

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.

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

“…Hence, we explored the SAR of adenine ring functionalization as well as the key components of ATP to facilitate the design of potent and selective HK inhibitors (Figure 2a). To test the importance of the α -, β -, and γ -phosphates, we measured the affinity of HK853 for ATP ( 5 ), ADP ( 6 ), and AMP ( 7 ) 22 using the activity-based fluorescence gel assay previously developed by our group (Figure 2b). 30 5 and 6 had similar IC 50 values (27 and 2.4 μ M, respectively), whereas 7 had marginal activity (1030 μ M), suggesting that one phosphate group is not enough for octahedral Mg 2+ coordination or interactions with Arg on the ATP-lid and Lys of the N-box for optimal binding.…”

“…22 Highly conserved across HKs, it contains the Bergerat fold that is characteristic of the GHKL ( G yrase, H eat shock proteins, histidine K inase, Mut L ) family but is absent in mammalian kinases. 23 We envisioned that targeting this conserved domain would inactivate several HKs simultaneously for a broad-spectrum-type antibiotic therapy.…”

“…Further analysis for dose-dependent activity against HK853, as well as VicK ( Streptococcus pneumoniae ) and CheA ( Escherichia coli ), yielded nine lead compounds capable of inhibiting multiple HKs. 22 …”

“…Interestingly, all of our adenine-based leads [6- N , N -dipropyl adenine ( 1 ), 6- N , N -morpholine adenine ( 2 ), 6- N- phenylethyladenine ( 3 ), 6- N , N -(3,3,5-trimethylcyclohexyl) adenine ( 4 )] were functionalized at the 6-position on the ring with various secondary or tertiary amines (Figure 1a). 22 To better understand the structural requirements for inhibition of these proteins, we conducted structure–activity relationship (SAR) studies with complementary adenine analogues. In addition, given the importance of other ATP-binding proteins in eukaryotic biology, we sought to identify compounds that selectively target HKs over eukaryotic kinases (EKs) and other GHKL proteins (i.e., heat shock proteins).…”

Sensor histidine kinase is a β-lactam receptor and induces resistance to β-lactam antibiotics

“…Defining the VbrK/ VbrR regulatory pathway that controls the expression of β-lactamase would provide a promising target to inhibit β-lactamase production and β-lactam resistance. Lead compounds that can inhibit the activity of histidine kinase have been identified for a number of TCSs (35)(36)(37)(38). There is future promise in rationally designing VbrK inhibitors to enhance the efficacy of β-lactam antibiotics in treating infections caused by Vibrio species.…”