Kinetic and mechanistic analyses of new classes of inhibitors of two-component signal transduction systems using a coupled assay containing HpkA–DrrA from Thermotoga maritima

Abstract

Two-component signal transduction systems (TCSs) play fundamental roles in bacterial survival and pathogenesis and have been proposed as targets for the development of novel classes of antibiotics. A new coupled assay was developed and applied to analyse the kinetic mechanisms of three new kinds of inhibitors of TCS function. The assay exploits the biochemical properties of the cognate HpkA-DrrA histidine kinase-response regulator pair from Thermotoga maritima and allows multiple turnovers of HpkA, linear formation of phosphorylated DrrA, and Michaelis-Menten analysis of inhibitors. The assay was validated in several ways, including confirmation of competitive inhibition by adenosine 59-b,c-imidotriphosphate (AMP-PNP). The coupled assay, autophosphorylation and chemical cross-linking were used to determine the mechanisms by which several compounds inhibit TCS function. A cyanoacetoacetamide showed non-competitive inhibition with respect to ATP concentration in the coupled assay. The cyanoacetoacetamide also inhibited autophosphorylation of histidine kinases from other bacteria, indicating that the coupled assay could detect general inhibitors of histidine kinase function. Inhibition of HpkA autophosphorylation by this compound was probably caused by aggregation of HpkA, consistent with a previous model for other hydrophobic compounds. In contrast, ethodin was a potent inhibitor of the combined assay, did not inhibit HpkA autophosphorylation, but still led to aggregation of HpkA. These data suggest that ethodin bound to the HpkA kinase and inhibited transfer of the phosphoryl group to DrrA. A peptide corresponding to the phosphorylation site of DrrA appeared to inhibit TCS function by a mechanism similar to that of ethodin, except that autophosphorylation was inhibited at high peptide concentrations. The latter mechanism of inhibition of TCS function is unusual and its analysis demonstrates the utility of these approaches to the kinetic analyses of additional new classes of inhibitors of TCS function.

The histidine kinase inhibitor Sda binds near the site of autophosphorylation and may sterically hinder autophosphorylation and phosphotransfer to Spo0F

“…6A), Histidine kinases have long been considered attractive targets for the development of new antimicrobial drugs (Macielag and Goldschmidt, 2000;Matsushita and Janda, 2002;Hubbard et al, 2003;Lyon and Muir, 2003;Stephenson and Hoch, 2004;Rowland and King, 2007). Specific inhibitors of histidine kinases have recently been reported based on lead compounds that were identified by high-throughput screening of chemical libraries or from the rational design of compounds (Lyon et al, 2000;Besant et al, 2002;Foster et al, 2004;Gilmour et al, 2005;Qin et al, 2006;Okada et al, 2007). The surface that we have identified as being important for Sda and KipI to inhibit KinA may prove useful as a new target for the rational design of compounds that bind and inhibit histidine kinases.…”

New Class of Competitive Inhibitor of Bacterial Histidine Kinases

“…Fifty-percent inhibitory concentration (IC 50 ) determinations performed by methods described previously (12) showed that TEP competes with ATP in the coupled assay (Fig. 2).…”

“…The coupled assay measures the formation of phosphorylated DrrA (Drr-P) in reaction mixtures containing catalytic amounts of HpkA and excess amounts of ATP and DrrA, which are treated as substrates in the kinetic analyses ( Fig. 2 and 3) (12). In the high-throughput screens, we used a filter format of the coupled assay, as described previously (12), which employs 96-well Immobilon P filter plates (Millipore, Inc.).…”

“…2 and 3) (12). In the high-throughput screens, we used a filter format of the coupled assay, as described previously (12), which employs 96-well Immobilon P filter plates (Millipore, Inc.).…”

“…We identified TEP as an inhibitor of TCS function in a high-throughput screen of compound libraries. In this screen, we used a coupled assay containing the HpkA HK and DrrA RR of Thermotoga maritima that allows multiple turnovers of HpkA, linear formation of phosphorylated DrrA, and Michaelis-Menten analysis of inhibitors of HpkA autokinase activity or phosphoryl group transfer from HpkA to DrrA (12). The coupled assay measures the formation of phosphorylated DrrA (Drr-P) in reaction mixtures containing catalytic amounts of HpkA and excess amounts of ATP and DrrA, which are treated as substrates in the kinetic analyses ( Fig.…”