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Assays for Β-Lactamase Activity and Inhibition

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New Antibiotic Targets

Part of the book series: Methods In Molecular Medicine™ ((MIMM,volume 142))

Summary

The ability, either innate or acquired, to produce β-lactamases, enzymes capable of hydrolyzing the endocyclic peptide bond in β-lactam antibiotics, would appear to be a primary contributor to the ever-increasing incidences of resistance to this class of antibiotics. To date, four distinct classes, A, B, C, and D, of β-lactamases have been identified. Of these, enzymes in classes A, C, and D utilize a serine residue as a nucleophile in their catalytic mechanism while class B members are Zn+2-dependent for their function. Efforts have been and still continue to be made toward the development of potent inhibitors of these enzymes as a means to ensure the efficacy of β-lactam antibiotics in clinical medicine. This chapter concerns procedures for the evaluation of the catalytic activity of β-lactamases as a means to screen compounds for their inhibitory potency.

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References

  1. Knowles, J. R. (1985) Penicillin resistance–-The chemistry of β-lactamase inhibition. Acc. Chem. Res. 18, 97–104.

    Article  CAS  Google Scholar 

  2. Livermore, D. M., and Wooford, N. (2006) The β-lactamase threat in Enterobacteriacae, Pseudomonas and Acinetobacter. Trends Microbiol. 9, 413–420.

    Article  Google Scholar 

  3. Payne, D. J., Du, W., and Bateson, J. H. (2000) β-Lactamase epidemiology and the utility of established and novel β-lactamase inhibitors. Expert Opin. Invest. Drugs 9, 247–261.

    Article  CAS  Google Scholar 

  4. Fisher, J. F., Meroueh, S. O., and Mobashery, S. (2005) Bacterial resistance to β-lactam antibiotics: Compelling opportunism, compelling opportunity. Chem. Rev. 105, 395–424.

    Article  CAS  PubMed  Google Scholar 

  5. Walsh, C. (2000) Molecular mechanisms that confer antibacterial drug resistance. Nature 406, 775–781.

    Article  CAS  PubMed  Google Scholar 

  6. Martinez, J. L., and Baquero, F. (2000) Mutation frequencies and antibiotic resistance. Antimicrob. Agents Chemother. 44, 1771–1777.

    Article  CAS  PubMed  Google Scholar 

  7. Helfand, M. S., and Bonomo, R. A. (2003) β-lactamases: A survey of protein diversity. Curr. Drug Targets-Infect. Disord. 3, 9–23.

    Article  CAS  PubMed  Google Scholar 

  8. Matagne, A., Dubos, A., Galleni, M., and Frére, J. M. (1999) The β-lactamase cycle: A tale of selective pressure and bacterial ingenuity. Nat. Prod. Rep. 16, 1–19.

    Article  CAS  PubMed  Google Scholar 

  9. Page, M. I., and Laws, A. P. (1998) The mechanism of catalysis and the inhibition of β-lactamases. Chem. Comm. 16, 1609–1617.

    Article  Google Scholar 

  10. Rasmussen, B. A., and Bush, K. (1997) Carbapenem-hydrolyzing β-lactamases. Antimicrob. Agents Chemother. 41, 223–232.

    CAS  PubMed  Google Scholar 

  11. Sykes, R. B., Bonner, D. P., Bush, K., and Georgopapadakou, N. H. (1982) Aztreonam (SQ.26,776) a synthetic monobactam specifically active against Gram-negative bacteria, Antimicrob. Agents Chemother. 21, 85–92.

    CAS  PubMed  Google Scholar 

  12. Fisher, J., Charnas, R., and Knowles, J. R. (1978) Kinetic studies on the inactivation of Escherichia coli RTEM β-lactamase by clavulanic acid. Biochemistry 17, 2180–2184.

    Article  CAS  PubMed  Google Scholar 

  13. English, A. R., Retsema, J. A., Girard, A. E., Lynch, J. E., and Barth, W. E. (1978) CP-45,899, a β-lactamase inhibitor that extends the antibacterial spectrum of β-lactams: Initial bacteriological characterization. Antimicrob. Agents Chemother. 14, 414–419.

    CAS  PubMed  Google Scholar 

  14. Micetich, R. G., Maiti, S. N., Spevak, P., Hall, T. W., Yamabe, S., Ishida, N., Tanaka, M., Yamazaki, T., Nakai, A., and Ogawa, K. (1987) Synthesis and β-lactamase inhibitory properties of 2β-[(1,2,3-triazol-1-yl)]-2a-methylpenam-3a-carboxylic acid 1,1-dioxide and related triazolyl derivatives. J. Med. Chem. 30, 1469–1474.

    Article  CAS  PubMed  Google Scholar 

  15. Ambler, R. P. (1980) The structure of β-lactamases. Philos. Trans. R. Soc. London, B, Biol. Sci. 289, 321–331.

    Article  CAS  Google Scholar 

  16. Ghuysen, J. M. (1991) Serine β-lactamases and penicillin-binding proteins. Ann. Rev. Microbiol. 45, 37–67.

    Article  CAS  Google Scholar 

  17. Galleni, M., Lamotte-Brasseur, J., Rossolini, G. M., Spencer, J., Dideberg, O., and Frére, J. M. (2001). Standard numbering scheme for class B β-lactamases. Antimicrob. Agents Chemother. 45, 660–663.

    Article  CAS  PubMed  Google Scholar 

  18. Hernandez-Valladares, M., Felici, A., Weber, G., Adolph, H. W., Zeppezauer, M., Rossilini, G. M., Amicosante, G., Frére, J. M., and Galleni, M. (1997) Zn(II) dependence of the Aeromonas hydrophila AE036 metallo-β-lactamase activity and stability. Biochemistry 36, 11534–11541.

    Article  CAS  PubMed  Google Scholar 

  19. Sharma, N. P., Hajdin, C., Chandrasekar, S., Bennett, B., Yang, K.-W., and Crowder, M. W. (2006) Mechanistic studies on the mononuclear ZnII-containing metallo-β-lactamase ImiS from Aeromonas sobria. Biochemistry 45, 10729–10738.

    Article  CAS  PubMed  Google Scholar 

  20. Crowder, M. W., Spencer, J., and Vila, A. J. (2006) Metallo-β-lactamases: Novel weaponry for antibiotic resistance in bacteria. Acc. Chem. Res. 39, 721–728.

    Article  CAS  PubMed  Google Scholar 

  21. Murphy, T. A., Catto, L. E., Halford, S. E., Hadfield, A. T., Minor, W., Walsh, T. R., and Spencer, J. (2006) Crystal structure of Pseudomonas aeruginosa SPM-1 provides insights into variable zinc affinity of metallo-β-lactamases. J. Mol. Biol. 357, 890–903.

    Article  CAS  PubMed  Google Scholar 

  22. Siemann, S., Badiei, H. R., Karanassios, V., Viswanatha, T., and Dmitrienko, G. I. (2006) 68Zn isotope exchange experiments reveal an unusual kinetic lability of the metal ions in the di-zinc form of IMP-1 metallo-β-lactamase. Chem. Comm., 532–534.

    Google Scholar 

  23. Badarau, A., Damblon, C., and Page, M. I. (2007) The activity of dinuclear cobalt-β-lactamase from Bacillus cereus in catalysing the hydrolysis of β-lactams. Biochem. J. 401, 197–203.

    Article  CAS  PubMed  Google Scholar 

  24. Wang, Z., Fast, W., Valentine, A. M., and Benkovic, S. J. (1999) Metallo-β-lactamase: Structure and mechanism. Curr. Opin. Chem. Biol. 3, 614–622.

    Article  CAS  PubMed  Google Scholar 

  25. Hou, J. P., and Poole, J. W. (1972) Measurement of β-lactamase activity and rate of inactivation of penicillins by a pH-stat alkalimetric titration method. J. Pharm. Sci. 61, 1594–1598.

    Article  CAS  PubMed  Google Scholar 

  26. Saz, A. K., Lowery, D. L., and Jackson, L. J. (1961) Staphyloccocal penicillinase: Inhibition and stimulation of activity. J. Bacteriol. 82, 298–304.

    CAS  PubMed  Google Scholar 

  27. Henry, R. J., and Housewright, R. D. (1947) Studies on penicillinase: II. Manometric method of assaying penicillinase and penicillin, kinetics of the penicillin-penicillinase reaction, and the effects of inhibitors on penicillinase. J. Biol. Chem. 167, 559–571.

    CAS  PubMed  Google Scholar 

  28. Grove, D. C., and Randall, W. A. (1955) Assay methods of antibiotics. A laboratory manual Medical Encyclopedia, New York, p. 16.

    Google Scholar 

  29. Perret, C. J. (1954) Iodometric assay of penicillinase. Nature, 174, 1012–1013.

    Article  CAS  PubMed  Google Scholar 

  30. Masuda, G. (1976) Studies on bactericidal activities of β-lactam antibiotics on agar plates: The correlation with the antibacterial activities determined by the conventional methods. J. Antibiot. 29, 1237–1240.

    CAS  PubMed  Google Scholar 

  31. McGhie, D., Clarke, P. D., Johnson, T., and Hutchison, J. G. P. (1977) Detection of β-lactamase activity of Haemophilus influenzae. J. Clin. Path. 30, 585–587.

    Article  CAS  PubMed  Google Scholar 

  32. Sykes, R. B., and Mathew, M. (1979) Detection and immunology of β-lactamases. In β-Lactamases (J. M. T. Hamilton-Smith and J. T. Smith, eds.). Academic Press Ltd., London, pp. 17–49.

    Google Scholar 

  33. Lucas, T. J. (1979) An evaluation of 12 methods for the demonstration of penicillinase. J. Clin. Pathol. 32, 1061–1065.

    Article  CAS  PubMed  Google Scholar 

  34. O’Callaghan, C. H., Muggleton, P. W., and Ross, G. W. (1968) Effects of β-lactamase from gram-negative organisms on cephalosporins and penicillins. Antimicrob. Agents Chemother. 8, 57–63.

    PubMed  Google Scholar 

  35. Samuni, A. (1975) A direct spectrophotometric assay and determination of Michaelis constants for the β-lactamase reaction. Anal. Biochem. 63, 17–26.

    Article  CAS  PubMed  Google Scholar 

  36. Waley, S. G. (1974) A spectrophotometric assay of β-lactamase action on penicillins. Biochem. J. 139, 789–790.

    CAS  PubMed  Google Scholar 

  37. O’Callaghan, C. H., Morris, A., Kirby, S., and Shingler, A. H. (1972) Novel method for detection of β-lactamases by using a chromogenic cephalosporin substrate. Antimicrob. Agents Chemother. 1, 283–288.

    PubMed  Google Scholar 

  38. Bebrone, C., Moali, C. Mahy, F., Rival, S., Docquier, J. D., Rossolini, G. M., Fastrez, J., Pratt, R. F., Frére, J. M., and Galleni, M. (2001) CENTA as a chromogenic substrate for studying β-lactamases. Antimicrob. Agents Chemother. 45, 1868–1871.

    Article  CAS  PubMed  Google Scholar 

  39. Perumal, S. K., and Pratt, R. F. (2006) Synthesis and evaluation of ketophosph (on)ates as β-lactamase inhibitors. J. Org. Chem. 71, 4778–4785.

    Article  CAS  PubMed  Google Scholar 

  40. Jones, R. N., Wilson, H. W., and Novick, W. J. Jr. (1982) In vitro evaluation of pyridine-2-azo-p-dimethylaniline cephalosporin, a new diagnostic chromogenic reagent, and comparison with nitrocefin, cephacetrile, and other β-lactam compounds. J. Clin. Microbiol. 15, 677–683.

    CAS  PubMed  Google Scholar 

  41. Durkin, J. P., Dmitrienko, G. I., and Viswanatha, T. (1977) N-(2-Furyl) acryloyl penicillin: A novel compound for the spectrophotometric assay of β-lactamase I. J. Antibiot. 30, 883–885.

    CAS  PubMed  Google Scholar 

  42. Ellis, K. J., and Morrison, J. F. (1982) Buffers of constant ionic strength for studying pH dependent processes. Methods Enzymol. 87, 405–426.

    Article  CAS  PubMed  Google Scholar 

  43. Laraki, N., Franceschini, N., Rossolini, G. M., Santucci, P., Meunier, C., de Pauw, E., Amicosante, G., Frére, J. M., and Galleni, M. (1999) Biochemical characterization of the Pseudomonas aeruginosa 101/1477 metallo-ss-lactamase IMP-1 produced by Escherichia coli. Antimicrob. Agents Chemother. 43(4), 902–906.

    CAS  PubMed  Google Scholar 

  44. Kuwabara, S. (1970) Purification and properties of two extracellular β-lactamases from Bacillus cereus 569/H. Biochem. J. 118, 457–465.

    CAS  PubMed  Google Scholar 

  45. Mathonet, P., Deherve, J., Soumillion, P., and Fastrez, J. (2006) Active TEM-1 mutants with random peptides inserted in three contiguous surface loops. Protein Sci. 15, 2323–2334.

    Article  CAS  PubMed  Google Scholar 

  46. Toney, J. H., Wu, J. K., Overbye, K. M., Thompson, C. M., and Pompliano, D. L. (1997) High-yield expression, purification, and characterization of active, soluble Bacteroides fragilis metallo-beta-lactamase, CcrA. Protein Expr. Purification 9(3), 355–362.

    Article  CAS  Google Scholar 

  47. Poirel, L., Naas, T., Nicolas, D., Collet, L., Bellais, S., Cavallo, J.-D., and Nordmann, P. (2000) Characterization of VIM-2, a carbapenem-hydrolyzing metallo-beta-lactamase and its plasmid- and integron-borne gene from a Pseudomonas aeruginosa clinical isolate in France. Antimicrob. Agents Chemother. 44(4), 891–897.

    Article  CAS  PubMed  Google Scholar 

  48. Crowder, M. W., Walsh, T. R., Banovic, L., Pettit, M., and Spencer, J. (1998) Overexpression, purification, and characterization of the cloned metallo-β-lactamase L1 from Stenotrophomonas maltophilia. Antimicrob. Agents Chemother. 42(4), 921–926.

    CAS  PubMed  Google Scholar 

  49. Goward, C. R., Stevens, G. B., Hammond, P. M., and Scawen, M. D. (1988) Large-scale purification of the chromosomal β-lactamase from Enterobacter cloacae P99. J. Chromat. 457, 317–324.

    Article  CAS  Google Scholar 

  50. Maveyraud, L., Golemi-Kotra, D., Ishiwata, A., Meroueh, O., Mobashery, S., and Samama, J.-P. (2002) High-resolution X-ray structure of an acyl-enzyme species for the class D OXA-10 β-lactamase. J. Amer. Chem. Soc. 124(11), 2461–2465.

    Article  CAS  Google Scholar 

  51. Bush, K., and Sykes, R. B. (1986) Methodology for the study of β-lactamases. Antimicrob. Agents Chemother. 30, 6–10.

    CAS  PubMed  Google Scholar 

  52. Dixon, M. (1953) The determination of enzyme inhibitor constants. Biochem. J. 55, 170–171.

    CAS  PubMed  Google Scholar 

  53. Cornish-Bowden, A. (1974) A simple graphical method for determining the inhibition constants of mixed, uncompetitive and non-competitive inhibitors. Biochem. J. 137, 143–144.

    CAS  PubMed  Google Scholar 

  54. Siemann, S., Clarke, A. J., Viswanatha, T., and Dmitrienko, G. I. (2003) Thiols as classical and slow-binding inhibitors of IMP-1 and other binuclear metallo-β-lactamases. Biochemistry, 42, 1673–1683.

    Article  CAS  PubMed  Google Scholar 

  55. Doucet, N., DeWals, P. Y., and Pelletier, J. N. (2004) Site saturation mutagenesis of Tyr 105 reveals its importance in substrate stabilization and discrimination in TEM-1 β-lactamase. J. Biol. Chem. 279, 46295–46303.

    Article  CAS  PubMed  Google Scholar 

  56. Abeles, R. H., and Maycock, A. L. (1976) Suicide enzyme inactivators. Acc. Chem. Res. 9, 313–319.

    Article  CAS  Google Scholar 

  57. Auld, D. S. (1988) Use of chelating agents to inhibit enzymes. Methods Enzymol. 158, 110–114.

    Article  CAS  PubMed  Google Scholar 

  58. Kitz, R., and Wilson, I. B. (1962) Esters of methanesufonic acid as irreversible inhibitors of acetylcholinesterase. J. Biol. Chem. 237, 3245–3249.

    CAS  PubMed  Google Scholar 

  59. Silverman, R. B. (2000) The organic chemistry of enzyme catalyzed reactions. Appendix 1 Academic Press, New York, pp. 584–587.

    Google Scholar 

  60. Siemann, S., Brewer, D., Clarke, A. J., Dmitrienko, G. I., Lajoie, G., and Viswanatha, T. (2002) IMP-1 metallo-β-lactamase: Effect of chelators and assessment of metal requirement by electrospray mass spectrometry. Biochim. Biophys. Acta 1571, 190–200.

    CAS  PubMed  Google Scholar 

  61. Payne, D. J., Coleman, K., and Cramp, R. (1991) The automated in-vitro assessment of β-lactamase inhibitors. J. Antimicrob. Chemother. 28, 775–776.

    Article  CAS  PubMed  Google Scholar 

  62. Yang, Z., Ho, P.-L., Liang, G., Chow, K. H., Wang, W., Cao, Y., Guo, Z., and Xu, B. (2006) Using β-lactamase to trigger supramolecular hydrogelation. J. Amer. Chem. Soc. ASAP Article; DOI: 10.1021/ja0675604.

    Google Scholar 

  63. Fitzgerald, F. M. D., Wu, J. K., and Toney, J. H. Unanticipated inhibition of metallo- β-lactamase from Bacterioides fragilis by 4-morpholinoethane sulfonic acid (MES): A crystallographic study at 1.85 ‘A resolution. Biochemistry 37, 6791–6800.

    Google Scholar 

  64. Oliver, R. W. A., and Viswanatha, T. (1968) Reaction of tris(hydroxymethyl)- aminomethane with cinnamoylimidazole and cinnamoyltrypsin. Biochem. Biophys. Acta 156, 422–425.

    CAS  PubMed  Google Scholar 

  65. Jones, R. N., Wilson, H. W., Novick, W. J. Jr., Barry, A. L., and Thornsberry, C. (1982) In vitro evaluation of CENTA, a new β-lactamase-susceptible chromogenic cephalosporin reagent. J. Clin. Microbiol. 15, 954–958.

    CAS  PubMed  Google Scholar 

  66. Ellman, G. L. (1959) Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70–77.

    Article  CAS  PubMed  Google Scholar 

  67. Riddles, P. W., Blakely, R. L., and Zerner, B. (1979) Ellman’s reagent: 5,5′- dithiobis-92-nitrobenzoic acid: a reexamination. Anal. Biochem. 94, 75–81.

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Chemistry, UniversityofWaterloo, W,Waterloo, Ontario, Canada

    Thammaiah Viswanatha, Laura Marrone, Valerie Goodfellow & Gary I. Dmitrienko

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  1. Thammaiah Viswanatha
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  2. Laura Marrone
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  4. Gary I. Dmitrienko
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  1. Department of Biochemistry and Molecular Biology, East Tennessee State University, Johnson, Tennessee

    W. Scott Champney

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Viswanatha, T., Marrone, L., Goodfellow, V., Dmitrienko, G.I. (2008). Assays for Β-Lactamase Activity and Inhibition. In: Champney, W.S. (eds) New Antibiotic Targets. Methods In Molecular Medicine™, vol 142. Humana Press. https://doi.org/10.1007/978-1-59745-246-5_19

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  • DOI: https://doi.org/10.1007/978-1-59745-246-5_19

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-915-4

  • Online ISBN: 978-1-59745-246-5

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