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 J. Estelle Foster, 1 3 Qin Sheng, 1 Jonathan R. McClain, 1 Mark Bures, 1 Thalia I. Nicas, 1 4 Kenneth Henry, 1 Malcolm E. Winkler 1,2 1 and Raymond Gilmour 1 Correspondence Raymond Gilmour Gilmour_Raymond@Lilly.com 1 Lilly Research Laboratories, Eli Lilly and Company, Drop Code 0428, Lilly Corporate Center, Indianapolis IN 46285, USA 2 Department of Biology, Indiana University, Bloomington, IN, 47405, USA Received 10 October 2003 Revised 8 December 2003 Accepted 8 December 2003 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. INTRODUCTION Bacteria have the ability to sense and respond to changes in their environment. This capability is necessary for growth and survival of many bacteria and for virulence of patho- gens. Much of this ability to sense and adapt to environ- mental stimuli is mediated through two-component signal transduction systems (TCSs) (reviewed by Hoch & Varughese, 2001; Inouye & Dutta, 2003; Stock et al., 2000; West & Stock, 2001). These TCSs typically consist of a sensor histidine kinase and a cognate response regulator (Fig. 1). The histidine kinase is often an integral membrane pro- tein in which the sensor domain is extracellular and the catalytic kinase domain is intracellular (Inouye & Dutta, 2003). Upon binding of a ligand or sensing a change in the 3Present address: MDS Proteomics, 251 Attwell Dr., Toronto, ON, Canada M9W 7H4. 4Present address: Quality Control Laboratories, Lilly Technology Center, Indianapolis, IN 46285, USA. 1Present address: Jordan Hall 142, 1001 East 3rd Street, Bloomington, IN 47405, USA. Abbreviations: AMP-PNP, adenosine 59-b,c-imidotriphosphate; CAA, cyanoacetoacetamide; DrrA-P, phosphorylated DrrA; EPPS, 4-(2-hydroxyethyl)-1- piperazinepropanesulfonic acid; SAR, structure–activity relationship; TCS, two-component signal transduction system. 0002-6824 G 2004 SGM Printed in Great Britain 885 Microbiology (2004), 150, 885–896 DOI 10.1099/mic.0.26824-0