The Mechanism of Action of Inhibitors of Bacterial Two-component Signal Transduction Systems

Abstract

Two-component signal transduction systems allow bacteria to sense and respond rapidly to changes in their environment leading to specific gene activation or repression. These two-component systems are integral in the ability of pathogenic bacteria to mount and establish a successful infection within the host and, consequently, have been recognized as targets for new anti-microbial agents. In this paper, we define the site and mechanism of action of several previously identified inhibitors of bacterial two-component systems. We show that the most potent inhibitors target the carboxyl-terminal catalytic domain of the sensor kinase and exert their affect by causing structural alterations of the kinase leading to aggregation. Recognition of this phenomenon has important implications for the development of novel inhibitors of two-component systems and should facilitate the rapid identification and elimination of compounds with nonspecific affects from medicinal chemistry drug discovery programs. Two-component signal transduction systems allow bacteria to sense and respond rapidly to changes in their environment leading to specific gene activation or repression. These two-component systems are integral in the ability of pathogenic bacteria to mount and establish a successful infection within the host and, consequently, have been recognized as targets for new anti-microbial agents. In this paper, we define the site and mechanism of action of several previously identified inhibitors of bacterial two-component systems. We show that the most potent inhibitors target the carboxyl-terminal catalytic domain of the sensor kinase and exert their affect by causing structural alterations of the kinase leading to aggregation. Recognition of this phenomenon has important implications for the development of novel inhibitors of two-component systems and should facilitate the rapid identification and elimination of compounds with nonspecific affects from medicinal chemistry drug discovery programs. structure activity relationship adenosine 5′-β,γ-imidotriphosphate 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid polyacrylamide gel electrophoresis N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine Bacteria are highly adaptive organisms capable of growth in an extreme variety of carbon and nitrogen sources and environmental conditions. The key to adaptivity is the large reservoir of genes and pathways encoded in the genome that can be turned on and off at will to reflect the composition of the environment and the available carbon and energy sources. Signals specific for each potential pathway must be sensed and converted to transcription activation or repression by signal transduction systems to facilitate responses to environmental change. One of the major means by which this is accomplished is through two-component signal transduction systems (1Hoch, J. A., Silhavy, T. J., and (eds) (1995) Two-Component Signal Transduction, American Society for Microbiology, Washington, D. C.Google Scholar). In general, such systems consist of a signal ligand responsive histidine kinase and a response regulator transcription factor. Specific signal ligand binding by the kinase activates an autophosphorylation reaction to a histidine residue. This phosphoryl group is transferred to an aspartate of a response regulator domain, which serves to activate its transcriptional properties. In some cases, notably NRII kinase of Escherichia coli, signal ligands work in the opposite manner and activate the phosphatase properties of the kinase (2Ninfa A.J. Atkinson M.R. Kamberov E.S. Feng J. Ninfa E.G. Hoch J.A. Silhavy T.J. Two-Component Signal Transduction. American Society for Microbiology, Washington, D. C.1995: 67-88Google Scholar). Regardless of their mechanism of action such systems serve to interpret the condition of the cellular environment and make adjustments to the repertoire of genes being expressed.Virulence and pathogenicity are properties of certain bacteria that find themselves in a suitable host environment. Genes and pathways specific for invasion and growth in the host need to be activated in response to signals emanating from that environment. Two-component signal transduction systems play a key role in controlling virulence responses of a wide variety of bacterial pathogens (3Barrett J.F. Isaacson R.E. Bristol J.A. Annual Reports in Medicinal Chemistry. Academic Press, San Diego, CA1995: 111-118Google Scholar). For example, many of the two-component systems of Streptococcus pneumoniae when mutated affect the ability of this organism to mount a successful infection in mouse models without having deleterious effects on growth in laboratory media (4Throup J.P. Koretke K.K. Bryant A.P. Ingraham K.A. Chalker A.F. Ge Y. Marra A. Wallis N.G. Brown J.R. Holmes D.J. Rosenberg M. Burnham M.K. Mol. Microbiol. 2000; 35: 566-576Crossref PubMed Scopus (274) Google Scholar). Two-component signal transduction systems are woven in the fabric of cellular regulation and play important roles in the complex cellular processes required for virulence and pathogenicity (5Barrett J.F. Hoch J.A. Antimicrob. Agents Chemother. 1998; 42: 1529-1536Crossref PubMed Google Scholar). Recognition of this role has led the pharmaceutical industry to spend untold millions of dollars on the search for suitable inhibitors of such systems as potential anti-infective agents. The results of several inhibitor screens and structure activity relationship (SAR)1 programs of medicinal chemistry have been published (6Barrett J.F. Goldschmidt R.M. Lawrence L.E. Foleno B. Chen R. Demers J.P. Johnson S. Kanojia R. Fernandez J. Bernstein J. Licata L. Donetz A. Huang S. Hlasta D.J. Macielag M.J. Ohemeng K. Frechette R. Frosco M.B. Klaubert D.H. Whiteley J.M. Wang L. Hoch J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5317-5322Crossref PubMed Scopus (109) Google Scholar, 7Domagala J.M. Alessi D. Cummings M. Gracheck S. Huang L. Huband M. Johnson G. Olson E. Shapiro M. Singh R. Song Y. Van Bogelen R. Vo D. Wold S. Adv. Exp. Med. Biol. 1998; 456: 263-286Google Scholar, 8Macielag M. Demers J.P. Fraga-Spano S.A. Hlasta D.J. Johnson S.G. Kanojia R.M. Russell R. Sui Z. Weidner-Wells M.A. Werblood H. Foleno B.D. Goldschmidt R.M. Loeloff M. Webb G.C. Barrett J.F. J. Med. Chem. 1998; 41: 2939-2945Crossref PubMed Scopus (128) Google Scholar, 9Sui Z. Guan J. Hlasta D.J. Macielag M.J. Foleno B.D. Goldschmidt R.M. Loeloff M.J. Webb G.C. Barrett J.F. Bioorg. Med. Chem. Lett. 1998; 8: 1929-1934Crossref PubMed Scopus (46) Google Scholar, 10Hlasta D.J. Demers J.P. Foleno B.D. Fraga-Spano S.A. Guan J. Hilliard J.J. Macielag M.J. Ohemeng K. Sheppard C.M. Sui Z. Webb G.C. Weidner-Wells M.A. Werblood H. Barrett J.F. Bioorg. Med. Chem. Lett. 1998; 8: 1923-1928Crossref PubMed Scopus (72) Google Scholar). While many different compounds have been described with inhibitory activity on these systems, the mechanism(s) of inhibition is largely unknown. In this paper studies to define the means by which several of such inhibitors function are presented. Bacteria are highly adaptive organisms capable of growth in an extreme variety of carbon and nitrogen sources and environmental conditions. The key to adaptivity is the large reservoir of genes and pathways encoded in the genome that can be turned on and off at will to reflect the composition of the environment and the available carbon and energy sources. Signals specific for each potential pathway must be sensed and converted to transcription activation or repression by signal transduction systems to facilitate responses to environmental change. One of the major means by which this is accomplished is through two-component signal transduction systems (1Hoch, J. A., Silhavy, T. J., and (eds) (1995) Two-Component Signal Transduction, American Society for Microbiology, Washington, D. C.Google Scholar). In general, such systems consist of a signal ligand responsive histidine kinase and a response regulator transcription factor. Specific signal ligand binding by the kinase activates an autophosphorylation reaction to a histidine residue. This phosphoryl group is transferred to an aspartate of a response regulator domain, which serves to activate its transcriptional properties. In some cases, notably NRII kinase of Escherichia coli, signal ligands work in the opposite manner and activate the phosphatase properties of the kinase (2Ninfa A.J. Atkinson M.R. Kamberov E.S. Feng J. Ninfa E.G. Hoch J.A. Silhavy T.J. Two-Component Signal Transduction. American Society for Microbiology, Washington, D. C.1995: 67-88Google Scholar). Regardless of their mechanism of action such systems serve to interpret the condition of the cellular environment and make adjustments to the repertoire of genes being expressed. Virulence and pathogenicity are properties of certain bacteria that find themselves in a suitable host environment. Genes and pathways specific for invasion and growth in the host need to be activated in response to signals emanating from that environment. Two-component signal transduction systems play a key role in controlling virulence responses of a wide variety of bacterial pathogens (3Barrett J.F. Isaacson R.E. Bristol J.A. Annual Reports in Medicinal Chemistry. Academic Press, San Diego, CA1995: 111-118Google Scholar). For example, many of the two-component systems of Streptococcus pneumoniae when mutated affect the ability of this organism to mount a successful infection in mouse models without having deleterious effects on growth in laboratory media (4Throup J.P. Koretke K.K. Bryant A.P. Ingraham K.A. Chalker A.F. Ge Y. Marra A. Wallis N.G. Brown J.R. Holmes D.J. Rosenberg M. Burnham M.K. Mol. Microbiol. 2000; 35: 566-576Crossref PubMed Scopus (274) Google Scholar). Two-component signal transduction systems are woven in the fabric of cellular regulation and play important roles in the complex cellular processes required for virulence and pathogenicity (5Barrett J.F. Hoch J.A. Antimicrob. Agents Chemother. 1998; 42: 1529-1536Crossref PubMed Google Scholar). Recognition of this role has led the pharmaceutical industry to spend untold millions of dollars on the search for suitable inhibitors of such systems as potential anti-infective agents. The results of several inhibitor screens and structure activity relationship (SAR)1 programs of medicinal chemistry have been published (6Barrett J.F. Goldschmidt R.M. Lawrence L.E. Foleno B. Chen R. Demers J.P. Johnson S. Kanojia R. Fernandez J. Bernstein J. Licata L. Donetz A. Huang S. Hlasta D.J. Macielag M.J. Ohemeng K. Frechette R. Frosco M.B. Klaubert D.H. Whiteley J.M. Wang L. Hoch J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5317-5322Crossref PubMed Scopus (109) Google Scholar, 7Domagala J.M. Alessi D. Cummings M. Gracheck S. Huang L. Huband M. Johnson G. Olson E. Shapiro M. Singh R. Song Y. Van Bogelen R. Vo D. Wold S. Adv. Exp. Med. Biol. 1998; 456: 263-286Google Scholar, 8Macielag M. Demers J.P. Fraga-Spano S.A. Hlasta D.J. Johnson S.G. Kanojia R.M. Russell R. Sui Z. Weidner-Wells M.A. Werblood H. Foleno B.D. Goldschmidt R.M. Loeloff M. Webb G.C. Barrett J.F. J. Med. Chem. 1998; 41: 2939-2945Crossref PubMed Scopus (128) Google Scholar, 9Sui Z. Guan J. Hlasta D.J. Macielag M.J. Foleno B.D. Goldschmidt R.M. Loeloff M.J. Webb G.C. Barrett J.F. Bioorg. Med. Chem. Lett. 1998; 8: 1929-1934Crossref PubMed Scopus (46) Google Scholar, 10Hlasta D.J. Demers J.P. Foleno B.D. Fraga-Spano S.A. Guan J. Hilliard J.J. Macielag M.J. Ohemeng K. Sheppard C.M. Sui Z. Webb G.C. Weidner-Wells M.A. Werblood H. Barrett J.F. Bioorg. Med. Chem. Lett. 1998; 8: 1923-1928Crossref PubMed Scopus (72) Google Scholar). While many different compounds have been described with inhibitory activity on these systems, the mechanism(s) of inhibition is largely unknown. In this paper studies to define the means by which several of such inhibitors function are presented.