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
Correspondence
Raymond Gilmour
Gilmour_Raymond{at}Lilly.com
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ABSTRACT |
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Present address: MDS Proteomics, 251 Attwell Dr., Toronto, ON, Canada M9W 7H4.
Present address: Quality Control Laboratories, Lilly Technology Center, Indianapolis, IN 46285, USA.
Present address: Jordan Hall 142, 1001 East 3rd Street, Bloomington, IN 47405, USA.
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INTRODUCTION |
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Besides their role in virulence, TCSs have been implicated in the antibiotic resistance of a number of clinically important pathogenic strains (Walsh, 2003; Wright et al., 1993
). Vancomycin is considered one of the last lines of defence in antibiotic therapy. However, vancomycin resistance has now become common in enterococci. The resistance to vancomycin in these strains is mediated by the VanRS TCS, which responds to cell wall damage by turning on expression of a set of genes that results in the synthesis of a peptidoglycan containing a modified pentapeptide chain (reviewed by Walsh, 2003
). Vancomycin-binding to strains carrying this modification is significantly reduced, rendering the antibiotic ineffective. A TCS designated temporarily as VncRS was reported to mediate vancomycin tolerance in Strep. pneumoniae, possibly through the action of a toxic peptide (Novak & Tuomanen, 2003
; Novak et al., 1999
). However, later work showed that this tolerance to vancomycin depended in part on reduced growth caused by erythromycin in growth media (Robertson et al., 2002
) and not on the VncRS TCS per se.
The roles of TCSs in viability, pathogenesis and antibiotic resistance have led to numerous efforts by industrial and academic laboratories to develop small molecules that inhibit these systems (Hilliard et al., 1999; Hubbard et al., 2003
; Matsushita & Janda, 2002
; Stephenson & Hoch, 2002
). Some approaches have targeted specific TCSs, such as AgrCA (Lyon et al., 2000
), whereas other approaches have been aimed at identifying inhibitors that target a broad collection of TCSs (reviewed by Matsushita & Janda, 2002
; Stephenson & Hoch, 2002
). In the latter case, the presence of conserved domains within histidine kinases and response regulators (Hoch & Varughese, 2001
; Stock et al., 2000
; West & Stock, 2001
) has provided the rationale that a single molecule could inhibit multiple TCSs. Most searches for small-molecule inhibitors have been based on assays of either autophosphorylation of histidine kinases or phosphotransfer from histidine kinases to response regulators (Matsushita & Janda, 2002
; Stephenson & Hoch, 2002
). Each of these assays has certain limitations, and kinetic analyses of inhibitors have not been widely reported, partly because of these limitations (e.g. see Stephenson & Hoch, 2002
). Here we report a new coupled assay using the HpkA histidine kinase and DrrA response regulator of Thermotoga maritima, first characterized by Stock and coworkers (Goudreau et al., 1998
; Lee & Stock, 1996
). The coupled assay, which allows analysis of the steady-state turnover of histidine kinase activity, was used to determine the kinetic properties of several new classes of compounds that inhibit autophosphorylation or phosphotransfer of TCSs.
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METHODS |
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Construction of plasmids expressing His-tagged HpkA and DrrA.
Expression vectors were constructed to allow His-tagged versions of HpkA and DrrA to be purified from Escherichia coli (Novagen technical literature, 2003; www.novagen.com). A DNA fragment corresponding to a truncated HpkA, lacking 77 N-terminal amino acids (HpkA77), was amplified from chromosomal DNA of Thermotoga maritima [American Type Culture Collection (ATCC); product no. 43589] by PCR with oligonucleotides primers hpkA5' (5'-AACTTTCTCACGCATATGAACAGCCTCAGCGAACC-3') and hpkA3' (5'-CCATCTGGTAGTCTGGATCCTCACCTTTCACGTATC-3') and cloned into the NdeI and BamHI restriction sites of pET-16B (Novagen), producing N-terminally His-tagged HpkA77. The same procedure was used to produce a full-length, N-terminally His-tagged DrrA using the following primers: drrA5' (5'-GGGATCGCATATGGCGAAAAAGAAGATTCTGGTGGTTG-3') and drrA3' (5'-GAGCAGGACGAATAGAACGGATCCTATGACGAACAGG-3'). Following PCR amplification and ligation into pET16B, the constructs were transformed into E. coli strain DH5, and positive clones were selected on LB plates containing 100 µg ampicillin ml1. The resulting constructs were confirmed by DNA sequencing.
Expression and purification of His-HpkA77.
The His-HpkA77 domain was expressed in E. coli BL21(DE3)pLysS (Novagen). Cells were grown to mid-exponential phase (OD600 0·5) in LB medium containing 100 µg ampicillin ml1 and expression was induced by the addition of IPTG to a final concentration of 0·4 mM. Induction was continued at 25 °C for 4·5 h. For purification, cells were lysed by sonication on ice (6 min total, 30 s pulse on, 45 s off). All remaining steps were performed at 4 °C. Membranes and cell debris were removed by ultracentrifugation (Beckman Ti70 rotor, 40 000 r.p.m., 60 min). Supernates were applied to a 5 ml HiTrap chelating Sepharose column (previously charged with NiCl2 in water) equilibrated in 20 mM sodium phosphate, 0·5 M NaCl, pH 7·4, containing protease inhibitor cocktail III lacking EDTA (Calbiochem). After washing the 5 ml HiTrap chelating column with 10x the column volume of binding buffer, His-HpkA77 was eluted with a gradient of 00·5 M imidazole in the binding buffer. His-HpkA77 fractions were pooled, dialysed extensively against buffer (20 mM sodium phosphate, 0·2 M NaCl, pH 7·4), concentrated using an Ultra-15 centrifugal filter unit (Amicon), and stored at 80 °C. Protein concentrations were determined using a Coomassie Plus Protein Assay Kit (Pierce), and purity was assessed by Coomassie staining of SDS-PAGE gels (see Fig. S1, available as supplementary data with the online version of this paper at http://mic.sgmjournals.org). Approximately 510 mg of His-HpkA77 was obtained from 1 litre of cells. His-HpkA77 at a concentration of 2 µM was shown to exist primarily as a dimer by chemical cross-linking experiments (see below). Dimerization was also likely at concentrations of 80 nM and 20 nM, because His-HpkA77 was active in autophosphorylation and coupled reactions, respectively, at these lower concentrations (see below).
Expression and purification of His-DrrA.
His-DrrA was expressed in E. coli BL21(DE3)pLysS and purified as described above for His-HpkA, except that the following buffers were used. The binding buffer for DrrA was 50 mM Tris/HCl, 0·1 M KCl, pH 8·0, and the dialysis buffer was 20 mM Tris/HCl, pH 8·0, containing 0·5 M arginine to retard protein aggregation (see supplementary Fig. S1).
Initial velocity of autophosphorylation.
The rate of autophosphorylation was determined by incubating 80 nM His-HpkA77 in autophosphorylation buffer (50 mM Tris/HCl, pH 8·5, 50 mM KCl, 5 mM MgCl2, 0·5 mM EDTA and 0·1 mM DTT) (Goudreau et al., 1998) containing ATP concentrations ranging from 1 to 400 µM with 90180 µCi (3·36·6 MBq) [
-32P]ATP, in the presence of 10 % DMSO. Following equilibration at 37 °C, reactions were initiated by addition of ATP. For kinetic analyses, 6 µl aliquots were removed at various times over 5 min and quenched with 60 mM EDTA, pH 8·0, which was shown to stop the reaction in control experiments. SDS-PAGE sample buffer was added to the quenched reactions, and samples were applied without heating to 420 % (w/v) Tris-glycine SDS-polyacrylamide gels. After electrophoresis, the bottoms of the gels were removed to lower the background from the unincorporated radiolabelled ATP. Gels were dried without staining on a Bio-Rad GelAir drying system and exposed to a PhosphorImager screen, which was analysed using a PhosphorImager SF and ImageQuant software (Molecular Dynamics). The amount of 32P-His-HpkA77 in each lane was quantified by comparison with a standard curve of spotted [
-32P]ATP. Initial rates of autophosphorylation of His-HpkA77 were calculated from linear regression analysis of plots of phosphorylated His-HpkA77 (HpkA-P) formed versus time.
HpkA77-DrrA combined assay.
Assays were performed at 37 °C, either in 96-well microtitre plates or in microcentrifuge tubes. Two assay formats were employed for determination of enzyme activity: SDS-PAGE gels and Immobilon-P 96 PVDF filters contained in a 96-well multiscreen vacuum manifold (Millipore). In each well or tube, 20 nM His-HpkA77, 4 µM His-DrrA and 5 % DMSO in reaction buffer (50 mM Tris/HCl, pH 8·5, 50 mM KCl, 5 mM MgCl2 and 1 mM DTT) were pre-incubated at 37 °C for 15 min. Reactions were initiated by the addition of ATP, with concentrations ranging from 1 to 512 µM [-32P]ATP [4245 Ci mmol1 (1489065 GBq mmol1), gel format] or from 1 to 512 µM [
-33P]ATP [3121 Ci mmol1 (1104480 GBq mmol1), filter format]. Reactions were stopped at various times (see figure legends) by the addition of either equal volumes of SDS sample buffer (gel format) or 60 mM EDTA, pH 8·0 (filter format). For the gel format, samples were applied directly to SDS-PAGE gels without heating and analysed as described above. For the filter format, samples were transferred to a pre-wetted 96-well filter plate connected to a vacuum line at room temperature and washed with buffer (50 mM Tris/HCl, pH 8·5, 500 mM NaCl), and then water. Filters were dried for 2 h at 37 °C. Microscint 20 scintillation fluid (Packard) was dispensed into each well and the radioactivity was determined using a Packard Top Count liquid scintillation counter.
Temperature dependence of HpkA77-DrrA combined assay.
The HpkA77-DrrA combined assay was performed as described above except that 100 µM [-32P]ATP (45 Ci mmol1, 1665 GBq mmol1) was used at a range of temperatures. The samples were pre-incubated at temperatures from 27 to 82 °C for 15 min before initiation of the reaction with ATP. At the indicated times, 30 µl aliquots were removed and added to 10 µl SDS-PAGE sample buffer. The reaction products were applied without heating to polyacrylamide gels as described above.
Inhibitor studies.
Unless otherwise indicated, inhibitors were dissolved in 100 % DMSO. When testing inhibitors, the final DMSO concentration in the assays was either 5 % or 10 % (v/v), as indicated in the figure legends. Controls lacking inhibitors contained an equal concentration of DMSO. Inhibitorresponse curves were analysed by three-parameter fits using GraphPad Prism 3.0 software to determine IC50 values, defined as the concentration of compound giving 50 % inhibition of enzyme activity (Copeland, 2000).
Aggregation studies.
The effect of enzyme inhibitors on the oligomeric state of His-HpkA77 was investigated by a modification of the method reported by Stephenson et al. (2000). Inhibitors at a range of concentrations were incubated with 2 µM His-HpkA77 in a pH 8·5 assay buffer (5 mM K-EPPS, pH 8·5, 20 mM MgCl2, 0·1 mM EDTA, 5 %, v/v, glycerol) (Stephenson et al., 2000
). After a 2 min pre-incubation at 42 °C, autophosphorylation reactions were initiated by the addition of 45 µCi [
-32P]ATP diluted to a final concentration of 20 µM with unlabelled ATP. After 30 min the sample was split into two equal parts. One fraction was cross-linked for 30 min by the addition of glutaraldehyde to a final concentration of 0·22 % (v/v). The autophosphorylation and cross-linking reactions were terminated by addition of SDS-PAGE sample buffer containing glycine to a final concentration of 400 mM. The reaction products were applied to denaturing polyacrylamide gels. Activity gels (non-cross-linked fractions) were analysed as described above. Gels of cross-linked fractions were silver-stained (Invitrogen), dried, and scanned using ImageQuant software. Backgrounds of approximately equal areas were subtracted from the intensities of the His-HpkA bands, and percentage activity or percentage dimer were calculated relative to control samples that had not been treated with inhibitor. DC50 values, defined as the concentration of an inhibitor at which only 50 % of His-HpkA was present as a dimer, were determined by three-parameter fits using GraphPad Prism 3.0 software.
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RESULTS |
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The combined assay was temperature dependent between 27 and 82 °C. The initial rate increased sharply from 42 °C, reached an optimum at 67 °C, and declined at temperatures greater than 67 °C (data not shown). The high temperature for optimum activity is consistent with the source of the enzyme from a thermophilic organism and with the maximum activities of the individual autophosphorylation and phosphotransfer reactions described previously (Goudreau et al., 1998). The Vmax(ATP) at 67 °C was calculated to be 2·19 nM DrrA-P formed min1, which was 10-fold greater than that at 37 °C (Table 1
). The Km(ATP) also increased significantly with temperature, from 26·0 µM at 37 °C to 216 µM at 67 °C (Table 1
). Although the enzymes were more active at 67 °C, the DrrA-P product was less stable. The His-DrrA-P was stable for up to 2 h at 37 °C, but only for 30 min at 60 °C (data not shown). The decreasing stability of DrrA-P at increasing temperatures confirmed the results of Goudreau et al. (1998)
.
Validation of the combined assay with AMP-PNP
We validated the combined assay format for the determination of the kinetic mechanisms of action of inhibitor compounds by using AMP-PNP, which is a non-hydrolysable analogue of ATP (Fig. 3A). AMP-PNP often inhibits ATP-dependent reactions by competing for the active site of enzymes (e.g. Stephenson et al., 2000
). The experiments were done at 37 °C. Besides the inherent stability of DrrA-P, another advantage of performing the combined assay at 37 °C was preservation of potential inhibitor compounds, which potentially would degrade at higher temperatures. AMP-PNP inhibited the combined reaction (Fig. 4
). The degree of inhibition was dependent on ATP concentration, and a mechanistic analysis indicated that the AMP-PNP was competitive with ATP (Fig. 4
). The Ki for AMP-PNP was calculated as 100 µM. The concentration of DrrA in the reaction had no effect on the inhibition by AMP-PNP (data not shown), suggesting that this compound did not affect the phosphotransfer reaction. Thus the combined assay was a valid format to study mechanisms of action of inhibitor compounds.
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New inhibitors
We carried out a programme to target TCSs for antibiotic drug discovery. In the course of these studies, we identified a number of novel inhibitor compounds from high-throughput screening of compound libraries and molecular modelling, including a cyanoacetoacetamide compound (CAA), ethodin and a DrrA peptide (Fig. 3). The assays described above were applied to characterize the mechanisms of action of these inhibitors.
Cyanoacetoacetamide (CAA).
The compound designated CAA (Fig. 3C) is the parent compound of a structureactivity relationship (SAR) chemistry effort that was developed around the cyanoacetoacetamide group. The cyanoacetoacetamide series inhibited the autophosphorylation activities of several bacterial histidine kinases with IC50 values as low as
30 µM (see below), did not strongly inhibit other classes of kinases, such as the mammalian Raf serine/threonine kinase, showed MICs against Gram-positive and Gram-negative bacteria as low as
1 µg ml1, did not exhibit acute cytotoxicity of mammalian cell lines in 24 h assays, and did not disrupt cellular membranes at 4x MIC (W. Luo, D. Mullen, A. Riley, J. Zhao, T. I. Nicas, R. Gilmour & M. E. Winkler, unpublished data). Unfortunately, the MICs and inhibition of autophosphorylation were abolished by the addition of serum and purified serum albumin (W. Luo, D. Mullen, A. Riley, J. Zhao, T. I. Nicas, R. Gilmour & M. E. Winkler, unpublished data), and the SAR was not able to separate serum binding from inhibition of autophosphorylation.
CAA inhibited both the combined autophosphorylationphosphotransfer and the autophosphorylation assays (Figs 6 and 7a). In the combined assay, the CAA compound acted as a non-competitive inhibitor with ATP (Fig. 6
). For CAA, there appeared to be a direct relationship between inhibition of autophosphorylation and the oligomeric state of the enzyme. Activity gels revealed that the autophosphorylation activity of His-HpkA77 decreased with increasing amount of CAA (Fig. 7a
). Silver-stained protein gels of His-HpkA77 cross-linked in the presence of CAA clearly demonstrated that the dimer disappeared as the compound concentration increased (Fig. 7b
). The disappearance of His-HpkA77 in lanes 69 of Fig. 7(b)
was attributed to formation of cross-linked aggregates that were unable to enter these PAGE gels. Control experiments demonstrated that samples that were not cross-linked contained the same amount of protein in each lane (data not shown). Similar to the case for closantel (above), the data displayed a strong correlation between the loss of autophosphorylation activity (IC50 129 µM) and the loss of protein dimer through aggregation (DC50 102 µM) with increasing CAA concentrations (Fig. 7c
). Finally, CAA inhibited autophosphorylation of His-HpkA77 less strongly (IC50 129 µM; Fig. 7c
) than it inhibited the autophosphorylation of truncated versions of the Strep. pneumoniae VicK (YycG) and E. faecium VanS histidine kinases (IC50 50 µM and 76 µM, respectively; data not shown). This result indicates that the coupled assay can detect general histidine kinase inhibitors that are not specific for the HpkA histidine kinase (see Discussion).
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DISCUSSION |
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One of the challenges in determining the kinetic mechanism of action of histidine kinase inhibitors using autophosphorylation is that the reaction kinetics are nonlinear (see Results). Once phosphorylated, the histidine kinase forms a dead-end complex and cannot turn over unless the phosphoryl group is removed by the response regulator, a phosphatase, spontaneous dissociation, or the back reaction. Moreover, the autophosphorylation reaction consumes a considerable amount of enzyme, which reduces the sensitivity of detecting moderate inhibitors in high-throughput screens. Using the DrrA response regulator as a substrate in the assay, we developed a coupled assay format that allows analysis of multiple turnover events of the HpkA histidine kinase and linear formation of the DrrA-P product (supplementary Figs S3 and S4). This format depended on the stability at 37 °C of DrrA-P from the thermophile T. maritima (Results; Goudreau et al., 1998; Lee & Stock, 1996
). Coupled assay formats linking receptor function to phosphorylation of CheY protein have previously been used extensively to study the signal transduction pathway of bacterial chemotaxis (e.g. see Borkovich et al., 1989
; Bornhorst & Falke, 2000
; Ninfa et al., 1991
).
The HpkADrrA coupled assay was validated in several ways. The Km(ATP) for the coupled reaction (26·0 µM) was comparable to that determined for the HpkA autophosphorylation reaction (25·4 µM) (Results; Table 1). The coupled format allowed variation of either the ATP or DrrA substrate. Although not identical, the Vmax(ATP) and Vmax(DrrA) for the coupled reaction (0·287 nM min1 and 0·165 nM min1, respectively) were comparable (Table 1
). The Km(ATP) of the HpkADrrA coupled reaction increased significantly with temperature compared to that of the HpkA autophosphorylation reaction (Table 1
). This unexpected result suggests that the presence of DrrA may somehow destabilize the HpkAATP complex at higher temperatures (e.g. see Copeland, 2000
).
The linear rates of product formation and multiple turnover events allowed us to characterize inhibitors using traditional MichaelisMenten analysis. As validation for the assay, we demonstrated that AMP-PNP was an ATP-competitive inhibitor in the coupled assay, with a Ki of 100 µM (Fig. 4). Previously, Stephenson et al. (2000)
used a cross-linking approach to demonstrate that AMP-PNP did not cause aggregation of histidine kinase dimers. We confirmed this result using a protocol in which ATP and the AMP-PNP inhibitor were added to reactions simultaneously (Methods; Fig. 5
). In contrast, the compound closantel, which is a known toxin that inhibits histidine kinases (Sachsenmaier & Schachtele, 2002
; Stephenson et al., 2000
), inhibited HpkA autophosphorylation and caused concomitant aggregation of HpkA, as expected from previous results (Stephenson et al., 2000
).
As part of drug discovery efforts, we identified three new kinds of compounds (Fig. 3) that inhibited TCS function and determined their mechanisms by the HpkADrrA coupled assay, HpkA autophosphorylation, and cross-linking of HpkA. Each compound showed differences in its mechanism of inhibition of TCS function. The CAA compound, which had promising properties as an antibiotic but bound strongly to serum, showed non-competitive inhibition with respect to ATP concentration in the HpkADrrA coupled assay (Fig. 6
). Similar to closantel and other hydrophobic inhibitors of histidine kinases analysed before (Stephenson et al., 2000
), CAA led to aggregation of the HpkA kinase (Fig. 7
). Furthermore, the inhibition of HpkA kinase activity followed the same curve as aggregation of the HpkA dimer, consistent with inhibition being caused by aggregation. The inhibition patterns of CAA fit the model proposed by Stephenson and coworkers in which some hydrophobic inhibitors intercalate into the hydrophobic core of the four-helix bundle near the C-terminus of histidine kinases and thereby drive the bundle apart, which leads to aggregation and inhibition of kinase activity (Stephenson & Hoch, 2002
; Stephenson et al., 2000
).
One concern about the coupled assay might be whether it would detect only compounds that specifically inhibit autophosphorylation of the HpkA histidine kinase or phosphoryl group transfer from HpkA-P to DrrA. This concern can be dispelled for the autophosphorylation reaction. In the coupled assay, AMP-PNP and CAA showed different inhibition patterns (Figs 4 and 6) that were consistent with different mechanisms of inhibition of autophosphorylation (Figs 5 and 7
). Moreover, CAA inhibited the autophosphorylation of the E. faecium VanS and Strep. pneumoniae VicK histidine kinases even more strongly than it inhibited T. maritima HpkA (see Results). Finally, other classes of compounds that were competitive inhibitors of autophosphorylation of several different bacterial kinases showed competitive inhibition in the coupled HpkADrrA assay, similar to that of AMP-PNP (Fig. 4
; data not shown). Kinetic analysis of inhibition of phosphoryl group transfer has generally been hampered by the fast rates of these reactions and instability of the phosphorylated response regulators. The HpkADrrA coupled assay allows a starting point for the detection and detailed kinetic analysis of putative inhibitors of phosphoryl group transfer. The generality of putative inhibitors in the coupled assay can then be tested in other more challenging assay formats.
Our studies identified two compounds that are putative inhibitors of phosphoryl group transfer. Ethodin is a small hydrophobic intercalator-like molecule that inhibited TCS function by a different mechanism from that of CAA or closantel. Ethodin was a potent inhibitor of the HpkADrrA combined assay (Fig. 8) and was shown to have antibacterial activity (data not shown). Due to its known toxic properties, ethodin was not pursued further as a possible antibiotic. Unlike CAA and closantel, ethodin appeared to compete with DrrA for binding to HpkA, and addition of excess DrrA eliminated inhibition of the coupled assay by ethodin (Fig. 8
). Interestingly, ethodin did not inhibit the HpkA autokinase activity, suggesting that the compound binds to DrrA or binds to HpkA, but only inhibits phosphoryl group transfer. Evidence for the binding of ethodin directly to the HpkA histidine kinase came from aggregation studies. Even though ethodin failed to inhibit HpkA autophosphorylation, it still caused loss of the HpkA dimer and presumably aggregation in cross-linking reactions (Fig. 8
). This result is consistent with ethodin acting by binding to the HpkA histidine kinase and inhibiting phosphoryl group transfer from the kinase to the DrrA response regulator. The ability of this compound to cause a histidine kinase to aggregate without inhibition is unusual and indicates a different mechanism of inhibition from CAA, closantel and the compounds studied by Stephenson et al. (2000)
.
A hydrophobic peptide corresponding to the phosphorylation site in DrrA (Fig. 3) inhibited the HpkADrrA coupled assay by a mechanism that was similar to that of ethodin, with one important difference (Fig. 9
). Like ethodin, the DrrA peptide appeared to compete with DrrA for binding to HpkA in the HpkADrrA coupled assay (Fig. 9
). Similar to ethodin, the DrrA peptide bound to HpkA and led to loss of dimer and presumed aggregation in the cross-linking assay without significant loss of HpkA autophosphorylation activity. However, unlike ethodin, higher concentrations of the DrrA peptide inhibited the HpkA autophosphorylation activity. Given the similarity of the inhibition patterns of ethodin and the DrrA peptide, the peptide may also act by binding to the HpkA kinase and inhibiting phosphoryl group transfer from the kinase to DrrA. However, we do not have evidence for folding of this peptide or for specific interactions with HpkA, analogous to those of DrrA.
Together, these results demonstrate the utility of the coupled assay reported here to elucidate rapidly the kinetic mechanisms of inhibition of TCSs by new compounds. Using this assay and approaches developed previously by other laboratories, we identified inhibitors, such as ethodin and DrrA peptide, with an unusual mechanism of inhibition of TCS function. To date, no TCS inhibitor has progressed to a clinical candidate antibiotic, despite considerable efforts by several companies and academic laboratories (e.g. see Barrett et al., 1998; Hilliard et al., 1999
; Matsushita & Janda, 2002
; Stephenson & Hoch, 2002
). Some inhibitors, such as the CAA reported here, probably cause aggregation of histidine kinases that leads to inhibition. Despite some favourable biological properties, CAA failed to progress as a drug candidate because of strong serum binding. Other histidine kinase inhibitors that cause aggregation kill bacterial cells by mechanisms other than TCS inhibition, such as RWJ-49815, which disrupts membranes (Hilliard et al., 1999
; Stephenson & Hoch, 2002
). The inability to develop high-quality drug candidates that target a broad spectrum of histidine kinases might relate to the fact that all of the compounds identified so far appear to act outside of the ATP-binding site. An effective, less toxic broad-spectrum histidine kinase inhibitor might come from identification of a potent, specific ATP-competitive inhibitor. Such a compound would have a novel mechanism and would probably not cause protein aggregation on the basis of the precedent set by our results with the analogue AMP-PNP. On the other hand, structure-based design of compounds targeted to the interaction between histidine kinases and response regulators may lead to new classes of TCS inhibitors (Hubbard et al., 2003
; Matsushita & Janda, 2002
; Stephenson & Hoch, 2002
). The coupled assay reported herein could be used to distinguish rapidly between these two different kinds of inhibitors.
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ACKNOWLEDGEMENTS |
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Received 10 October 2003;
revised 8 December 2003;
accepted 8 December 2003.
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