Combinatorial Chemistry & High Throughput Screening, 2006, 9, 501-514 501 1386-2073/06 $50.00+.00 © 2006 Bentham Science Publishers Ltd. Whole Cell Strategies Based on lux Genes for High Throughput Applica- tions Toward New Antimicrobials Lorenzo Galluzzi 1,2 and Matti Karp *,2 1 CNRS-FRE2939, Institut Gustave Roussy, 39, rue Camille Desmoulins, F-94805 Villejuif, France 2 Institute of Environmental Engineering and Biotechnology, Tampere University of Technology, P.O. Box 541, FIN- 33101 Tampere, Finland Abstract: The discovery/development of novel drug candidates has witnessed dramatic changes over the last two dec- ades. Old methods to identify lead compounds are not suitable to screen wide libraries generated by combinatorial chem- istry techniques. High throughput screening (HTS) has become irreplaceable and hundreds of different approaches have been described. Assays based on purified components are flanked by whole cell-based assays, in which reporter genes are used to monitor, directly or indirectly, the influence of a chemical over the metabolism of living cells. The most conven- ient and widely used reporters for real-time measurements are luciferases, light emitting enzymes from evolutionarily dis- tant organisms. Autofluorescent proteins have been also extensively employed, but proved to be more suitable for end- point measurements, in situ applications – such as the localization of fusion proteins in specific subcellular compartments – or environmental studies on microbial populations. The trend toward miniaturization and the technical advances in de- tection and liquid handling systems will allow to reach an ultra high throughput screening (uHTS), with 100,000 of com- pounds routinely screened each day. Here we show how similar approaches may be applied also to the search for new and potent antimicrobial agents. Keywords: Antibiotics, bacterial luciferase, biosensors, high density plates, HTS, light emission, luxCDABE, bioluminescence. INTRODUCTION Resistance to antimicrobial agents commonly used in therapeutic protocols is spreading at fast rates among bacte- rial pathogens [1, 2]. Prescriptions based on empirical obser- vations rather than on the actual identification of the patho- gen, inappropriate compliance to the therapy, transfer in the food chain and exchange of mobile genetic elements among commensal and pathogenic bacteria are main contributors to the emergence of drug resistance. This has measurable con- sequences for public health, among which (1) the reduced efficacy of previously established therapies, (2) increased rates of infection transmission and (3) the possible co- selection of virulence traits. Ineffective regimens are substi- tuted with more aggressive combinations, resulting in more frequent and severe side-effects [3, 4]. The wide scale eco- nomical costs of drug resistance are enormous, as can be eas- ily envisioned [5]. This has raised the need for antibiotics with unexploited mechanisms of action. Novel molecules are being developed which target very selectively well-known and characterized biochemical pathways or recently identified prokaryotic me- tabolisms [6-8]. In the first case, enzyme structures have been characterized and crystallographic data is available for computationally assisted drug design. However, new ap- proaches based upon functional genomics have been sug- gested to identify novel drug-target pairs. Such approaches aim to identify essential proteins with significant dissimilar- *Address correspondence to this author at the Tampere University of Tech- nology, Institute of Environmental Engineering and Biotechnology, P.O. Box 541, FIN–33101 Tampere, Finland; Tel: +358-3-31153522; Fax: +358- 3-31152869; E-mail: matti.karp@tut.fi ity to possible human counterparts and broad conservation among bacteria [9]. Combinatorial chemistry has provided the drug develop- ers with huge chemical libraries, by allowing the simultane- ous synthesis of a wide array of compounds through an itera- tive process of linking component “building blocks” [10, 11]. Two different approaches have been described. Accord- ing to a first one, monomeric building blocks such as pep- tides or nucleotides are assembled into linear or branched oligomers. As an alternative, a basic scaffold structure is substituted with different functional groups. Hundreds of thousands of related but distinct molecules are available and efficient screening procedures have become of the utmost importance to select compounds with defined properties for further development [8]. The ideal high throughput screening (HTS) assay should be as much cheap, fast, simple, homogeneous and reliable as possible. Homo- geneous systems, in which there is no need of dispensing ex- tra reagents (e.g. substrates or cofactors), are preferable be- cause the number of steps is kept to a minimum, resulting in improved simplicity and possibility for automation. The reli- ability of an assay gives an idea of its performance in identi- fying candidate molecules among huge numbers of dis- cardable compounds. It can be quantified by the rate of false negative and false positive results and by intra-assay and in- ter-assay coefficients of variation. These parameters are usu- ally evaluated in test procedures with well-known model molecules. Low values characterize reliable and performing assays, which can be safely applied to the screening of chemical libraries. Finally, the ideal assay should avoid the involvement of radioactivity, and more generally of harmful reagents or end products difficult to dispose of.