Source: http://www.asmscience.org/content/book/10.1128/9781555816827.ch05
Timestamp: 2019-04-21 08:21:32+00:00

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The chapter talks about selected cell-based assays, and the selection of the corresponding target(s), amenable for the discovery of novel antibacterial and antifungal agents from microbial products, screened as complex mixtures resulting from partial processing of fermentation broths. The authors also provide a framework to view target choice and assay design in the context of the microbial diversity available for screening, highlighting the importance of designing an overall strategy for an effective drug discovery program. In the anti-infective field, a drug discovery program based on microbial products has the ultimate goal of discovering a new, patentable chemical entity possessing desired properties, such as antimicrobial spectrum, molecular weight, solubility, and preferred route of administration. Cell-free assays directly measure the effect of a sample on the biological activity of one or more relevant targets. In general, these types of assays are more specific and sensitive than cell-based assays. The objective of the assay optimization phase is to ensure that the assay is more sensitive than growth inhibition of the same test strain. The ultimate goal of microbial product screening is to establish the chemical identity of the hits and their biological properties. In antibacterial and antifungal screening programs, hits are characterized for their activity on different microbial strains.
Scheme of a screening process.
From target to screens. Main conceptual passages required (top) and the experimental steps required at each stage (bottom).
Concept of reporter assays. In a bacterial cell, the antibiotics 1 and 2 act on the different pathways a and b, respectively (top panel), leading to pathway perturbation due to specific stress-response signals and upregulation of genes under the same stimulon, as exemplified by the promoters pa and pb, which responds to perturbations in pathways a and b, respectively (middle panel). Two reporter assays can be designed by engineering cells so that expression of the reporter gene rep is under the control of the inducible promoter pa or pb, resulting in increased activity of the reporter enzyme Enz (bottom panel).
1. Alksne, L. E.,, P. Burgio,, W. Hu,, B. Feld,, M.P. Singh,, M. Tuckman,, P.J. Petersen,, P. Labthavikul,, M. McGlynn,, L. Barbieri,, L. McDonald,, P. Bradford,, R. G. Dushin,, D. Rothstein, and, S. J. Projan. 2000. Identification and analysis of bacterial protein secretion inhibitors utilizing a SecA-LacZ reporter fusion system. Antimicrob. Agents Chemother. 43: 1418– 1427.
2. Baltz, R. H. 2005. Antibiotic discovery from actinomycetes: will a renaissance follow the decline and fall? SIM News 55: 186– 196.
3. Boucher, H. W.,, G. H. Talbot,, J. S. Bradley,, J. E. Edwards,, D. Gilbert,, L. B. Rice,, M. Scheld,, B. Spellberg, and, J. Bartlett. 2009. Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America. Clin. Infect. Dis. 48: 1– 12.
4. Brandi, L.,, A. Fabbretti,, A. La Teana,, M. Abbondi,, D. Losi,, S. Donadio, and, C. O. Gualerzi. 2006. Specific, efficient, and selective inhibition of prokaryotic translation initiation by a novel peptide antibiotic. Proc. Natl. Acad. Sci. USA 103: 39– 44.
5. Brötz-Oesterhelt, H.,, D. Beyer,, H. P. Kroll,, R. Endermann,, C. Ladel,, W. Schroeder,, B. Hinzen,, S. Raddatz,, H. Paulsen,, K. Henninger,, J. E. Bandow,, H. G. Sahl, and, H. Labischinski. 2005. Dysregulation of bacterial proteolytic machinery by a new class of antibiotics. Nat. Med. 11: 1082– 1087.
6. DeBacker, M. D.,, B. Nelissen,, M. Logghe,, J. Viaene,, I. Loonen,, S. Vandoninck,, R. de Hoogt,, S. Dewaele,, F. A. Simons,, P. Verhasselt,, G. Vanhoof,, R. Contreras, and, W. H. Luyten. 2001. An antisense-based functional genomics approach for identification of genes critical for growth of Candida albicans. Nat. Biotechnol. 19: 235– 241.
7. De Pascale, G.,, C. Grigoriadou,, D. Losi,, I. Ciciliato,, M. Sosio, and, S. Donadio. 2007. Validation for high-throughput screening of a VanRS-based reporter gene assay for bacterial cell wall inhibitors. J. Appl. Microbiol. 103: 133– 140.
8. DeVito, J. A.,, J. A. Mills,, V. G. Liu,, A. Agarwal,, C. F. Sizemore,, Z. Yao,, D. M. Stoughton,, M. G. Cappiello,, M. D. Barbosa,, L. A. Foster, and, D. L. Pompliano. 2002. An array of target-specific screening strains for antibacteri-als discovery. Nat. Biotechnol. 20: 478– 483.
9. Donadio, S.,, L. Brandi,, S. Serina,, M. Sosio, and, S. Stinchi. 2005. Discovering novel antibacterial agents by high throughput screening. Front. Drug Design Discov. 1: 3– 16.
10. Donadio, S.,, P. Monciardini, and, M. Sosio. 2009. Approaches to discovering novel antibacterial and anti-fungal agents. Methods Enzymol. 458: 3– 28.
11. Fischer, H. P.,, N. A. Brunner,, B. Wieland,, J. Paquette,, L. Macko,, L. Ziegelbauer, and, C. Freiberg. 2004. Identification of antibiotic stress-inducible promoters: a systematic approach to novel pathway-specific reporter assays for antibacterial drug discovery. Genome Res. 14: 90– 98.
12. Forsyth, R. A.,, R. J. Haselbeck,, K. L. Ohlsen,, R. T. Yamamoto,, H. Xu,, J. D. Trawick,, D. Wall,, L. Wang,, V. Brown-Driver,, J. M. Froelich,, G. C. Kedar,, P. King,, M. McCarthy,, C. Malone,, B. Misiner,, D. Robbins,, Z. Tan,, Z.-Y. Zhu,, G. Carr,, D. A. Mosca,, C. Zamudio,, J. G. Foulkes, and, J. W. Zyskind. 2002. A genome-wide strategy for the identification of essential genes in Staphylococcus aureus. Mol. Microbiol. 43: 1387– 1400.
13. Fossum, S.,, G. De Pascale,, C. Weigel,, W. Messer,, S. Donadio, and, K. Skarstad. 2008. A robust screen for novel antibiotics: specific knockout of the initiator of bacterial DNA replication. FEMS Microbiol. Lett. 281: 210– 214.
14. Gerdes, S. Y.,, M. D. Scholle,, J. W. Campbell,, G. Balázsi,, E. Ravasz,, M. D. Daugherty,, A. L. Somera,, N. C. Kyrpides,, I. Anderson,, M. S. Gelfand,, A. Bhattacharya,, V. Kapatral,, M. D’Souza,, M. V. Baev,, Y. Grechkin,, F. Mseeh,, M. Y. Fonstein,, R. Overbeek,, A. L. Barabási,, Z. N. Oltvai, and, A. L. Osterman. 2003. Experimental determination and system level analysis of essential genes in Escherichia coli MG1655. J. Bacteriol. 185: 5672– 5684.
15. Giaever, G.,, A. M. Chu,, L. Ni,, C. Connelly,, L. Riles, and, S. Véronneau. 2002. Functional profiling of the Saccharomyces cerevisiae genome. Nature 418: 387– 391.
16. Goh, E. B.,, G. Yim,, W. Tsui,, J. McClure,, M. G. Surette, and, J. Davies. 2002. Transcriptional modulation of bacterial gene expression by subinhibitory concentrations of antibiotics. Proc. Natl. Acad. Sci. USA 99: 17025– 17030.
17. Hu, W.,, S. Sillaots,, S. Lemieux,, J. Davison,, S. Kauffman,, A. Breton,, A. Linteau,, C. Xin,, J. Bowman,, J. Becker,, B. Jiang, and, T. Roemer. 2007. Essential gene identification and drug target prioritization in Aspergillus fumigatus. PLoS Pathog. 3: e24.
18. Huang, L.,, S. Stevens-Miles, and, R. B. Lingham. 1999. Screening for bioactivities, p. 21– 28. In A. L. Demain and, J. E. Davies (ed.), Manual of Industrial Microbiology and Biotechnology, 2nd ed. ASM Press, Washington, DC.
19. Hutter, B.,, C. Fischer,, A. Jacobi,, C. Schaab, and, H. Loferer. 2004. Panel of Bacillus subtilis reporter strains indicative of various modes of action. Antimicrob. Agents Chemother. 48: 2588– 2594.
20. Ji, Y.,, B. Zhang,, S. F. Van Horn,, P. Warren,, G. Woodnutt,, M. K. Burnham, and, M. Rosenberg. 2001. Identification of critical staphylococcal genes using conditional phenotypes generated by antisense RNA. Science 293: 2266– 2269.
21. Kavermann, H.,, B. P. Burns,, K. Angermüller,, S. Odenbreit,, W. Fischer,, K. Melchers, and, R. Haas. 2003. Identification and characterization of Helicobacter pylori genes essential for gastric colonization. J. Exp. Med. 197: 813– 822.
22. Knuth, K.,, H. Niesalla,, C. J. Hueck, and, T. M. Fuchs. 2004. Large-scale identification of essential Salmonella genes by trapping lethal insertions. Mol. Microbiol. 51: 1729– 1744.
23. Kobayashi, K.,, S. D. Ehrlich,, A. Albertini,, G. Amati,, K. K. Andersen,, M. Arnaud,, K. Asai,, S. Ashikaga,, S. Aymerich,, P. Bessieres,, F. Boland,, S. C. Brignell,, S. Bron,, K. Bunai,, J. Chapuis,, L. C. Christiansen,, A. Danchin,, M. Débarbouille,, E. Dervyn,, E. Deuerling,, K. Devine,, S. K. Devine,, O. Dreesen,, J. Errington,, S. Fillinger,, S. J. Foster,, Y. Fujita,, A. Galizzi,, R. Gardan,, C. Eschevins,, T. Fukushima,, K. Haga,, C. R. Harwood,, M. Hecker,, D. Hosoya,, M. F. Hullo,, H. Kakeshita,, D. Karamata,, Y. Kasahara,, F. Kawamura,, K. Koga,, P. Koski,, R. Kuwana,, D. Imamura,, M. Ishimaru,, S. Ishikawa,, I. Ishio,, D. Le Coq,, A. Masson,, C. Mauël,, R. Meima,, R. P. Mellado,, A. Moir,, S. Moriya,, E. Nagakawa,, H. Nanamiya,, S. Nakai,, P. Nygaard,, M. Ogura,, T. Ohanan,, M. O’Reilly,, M. O’Rourke,, Z. Pragai,, H. M. Pooley,, G. Rapoport,, J. P. Rawlins,, L. A. Rivas,, C. Rivolta,, A. Sadaie,, Y. Sadaie,, M. Sarvas,, T. Sato,, H. H. Saxild,, E. Scanlan,, W. Schumann,, J. F. Seegers,, J. Sekiguchi,, A. Sekowska,, S. J. Séror,, M. Simon,, P. Stragier,, R. Studer,, H. Takamatsu,, T. Tanaka,, M. Takeuchi,, H. B. Thomaides,, V. Vagner,, J. M. van Dijl,, K. Watabe,, A. Wipat,, H. Yamamoto,, M. Yamamoto,, Y. Yamamoto,, K. Yamane,, K. Yata,, K. Yoshida,, H. Yoshikawa,, U. Zuber, and, N. Ogasawara. 2003. Essential Bacillus subtilis genes. Proc. Natl. Acad. Sci. USA 100: 4678– 4683.
24. Lancini, G. 2006. Forty years of antibiotic discovery at Lepetit: a personal journey. SIM News 56: 192– 212.
25. Mascher, T.,, S. L. Zimmer,, T. A. Smith, and, J. D. Helmann. 2004. Antibiotic-inducible promoter regulated by the cell envelope stress-sensing two-component system LiaRS of Bacillus subtilis. Antimicrob. Agents Chemother. 48: 2888– 2896.
26. Miller, J. R.,, S. Dunham,, I. Mochalkin,, C. Banotai,, M. Bowman,, S. Buist,, B. Dunkle,, D. Hanna,, H. J. Harwood,, M. D. Huband,, A. Karnovsky,, M. Kuhn,, C. Limberakis,, J. Y. Liu,, S. Mehrens,, W. T. Mueller,, L. Narasimhan,, A. Ogden,, J. Ohren,, J. V. Prasad,, J. A. Shelly,, L. Skerlos,, M. Sulavik,, V. H. Thomas,, S. Vanderroest,, L. Wang,, Z. Wang,, A. Whitton,, T. Zhu, and, C. K. Stover. 2009 A class of selective antibacterials derived from a protein kinase inhibitor pharmacophore. Proc. Natl. Acad. Sci. USA 106: 1737– 1742.
27. Nikaido, H. 2003. Molecular basis of bacterial outer membrane permeability revisited. Microbiol. Mol. Biol. Rev. 67: 593– 656.
28. Payne, D. J.,, M. N. Gwynn,, D. J. Holmes, and, D. L. Pompliano. 2007. Drugs for bad bugs: confronting the challenges of antibacterial discovery. Nat. Rev. Drug Discov. 6: 29– 40.
29. Peláez, F. 2006. The historical delivery of antibiotics from microbial natural products—can history repeat? Biochem. Pharmacol. 71: 981– 990.
30. Rake, J. B.,, R. Gerber,, R. J. Mehta,, D. J. Newman,, Y. K. Oh,, C. Phelen,, M. C. Shearer,, R. D. Sitrin, and, L. J. Nisbet. 1986. Glycopeptide antibiotics: a mechanism-based screen employing a bacterial cell wall receptor mimetic. J. Antibiot. 39: 58– 67.
31. Reich, K. A.,, L. Chovan, and, P. Hessler. 1999. Genome scanning in Haemophilus influenzae for identification of essential genes. J. Bacteriol. 181: 4961– 4968.
32. Riedlinger, J.,, A. Reicke,, H. Zähner,, B. Krismer,, A. T. Bull,, L. A. Maldonado,, A. C. Ward,, M. Goodfellow,, B. Bister,, D. Bischoff,, R. D. Süssmuth, and, H. P. Fiedler. 2004. Abyssomicins, inhibitors of the para-aminobenzoic acid pathway produced by the marine Verrucosispora strain AB-18–032. J. Antibiot. 57: 271– 279.
33. Roemer, T.,, B. Jiang,, J. Davison,, T. Ketela,, K. Veillette,, A. Breton,, F. Tandia,, A. Linteau,, S. Sillaots,, C. Marta,, N. Martel,, S. Veronneau,, S. Lemieux,, S. Kauffman,, J. Becker,, R. Storms,, C. Boone, and, H. Bussey, 2003. Large-scale essential gene identification in Candida albi-cans and applications to antifungal drug discovery. Mol. Microbiol. 50: 167– 181.
34. Scott, J. J.,, D. C. Oh,, M. C. Yuceer,, K. D. Klepzig,, J. Clardy, and, C. R. Currie. 2008. Bacterial protection of beetle-fungus mutualism. Science 322: 63.
35. Selva, E.,, N. Montanini,, S. Stella,, A. Soffientini,, L. Gastaldo, and, M. Denaro. 1997. Targeted screening for elongation factor Tu binding antibiotics. J. Antibiot. 50: 22– 26.
36. Shapiro, E., and, F. Baneyx. 2002. Stress-based identification and classification of antibacterial agents: second-generation Escherichia coli reporter strains and optimization of detection. Antimicrob. Agents Chemother. 46: 2490– 2497.
37. Urban, A.,, S. Eckermann,, B. Fast,, S. Metzger,, M. Gehling,, K. Ziegelbauer,, H. Rübsamen-Waigmann, and, C. Freiberg. 2007. Novel whole-cell antibiotic biosensors for compound discovery. Appl. Environ. Microbiol. 73: 6436– 6443.
38. Wang, J.,, S. M. Soisson,, K. Young,, W. Shoop,, S. Kodali,, A. Galgoci,, R. Painter,, G. Parthasarathy,, Y. S. Tang,, R. Cummings,, S. Ha,, K. Dorso,, M. Motyl,, H. Jayasuriya,, J. Ondeyka,, K. Herath,, C. Zhang,, L. Hernandez,, J. Allocco,, A. Basilio,, J. R. Tormo,, O. Genilloud,, F. Vicente,, F. Pelaez,, L. Colwell,, S. H. Lee,, B. Michael,, T. Felcetto,, C. Gill,, L. L. Silver,, J. D. Hermes,, K. Bartizal,, J. Barrett,, D. Schmatz,, J. W. Becker,, D. Cully, and, S. B. Singh. 2006. Platensimycin is a selective FabF inhibitor with potent antibiotic properties. Nature 441: 358– 361.
39. Washburn, R. S.,, A. Marra,, A. P. Bryant,, M. Rosenberg, and, D. R. Gentry. 2001. Rho is not essential for viability or virulence in Staphylococcus aureus. Antimicrob. Agents Chemother. 45: 1099– 1103.
40. Webber, M. A., and, L. J. Piddock. 2003. The importance of efflux pumps in bacterial antibiotic resistance. J. Anti-microb. Chemother. 51: 9– 11.
41. Weinstein, M. P. 2004. Micromonospora antibiotic discovery at Schering-Plough (1961–1973): a personal reminiscence. SIM News 54: 56– 66.
42. Young, K.,, H. Jayasuriya,, J. G. Ondeyka,, K. Herath,, C. Zhang,, S. Kodali,, A. Galgoci,, R. Painter,, V. Brown-Driver,, R. Yamamoto,, L. L. Silver,, Y. Zheng,, J. I. Ventura,, J. Sigmund,, S. Ha,, A. Basilio,, F. Vicente,, J. R. Tormo,, F. Pelaez,, P. Youngman,, D. Cully,, J. F. Barrett,, D. Schmatz,, S. B. Singh, and, J. Wang. 2006. Discovery of FabH/FabF inhibitors from natural products. Antimicrob. Agents Chemother. 50: 519– 526.
43. Zhang, J. H.,, T. D. Chung, and, K. R. Oldenburg. 1999. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen. 4: 67– 73.

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