Using Microarray Gene Signatures to Elucidate Mechanisms of Antibiotic
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DDT • Volume 10, Number 18 • September 2005 REVIEWS Using microarray gene signatures to elucidate mechanisms of antibiotic TODAY:TARGETS DRUG DISCOVERY action and resistance Reviews • Michelle D.Brazas and Robert E.W.Hancock Microarray analyses reveal global changes in gene expression in response to environmental changes and, thus, are well suited to providing a detailed picture of bacterial responses to antibiotic treatment. These responses are represented by patterns of gene expression, termed expression signatures, which provide insight into the mechanism of action of antibiotics as well as the general physiological responses of bacteria to antibiotic-related stresses. The complexity of such signatures is challenging the notion that antibiotics act on single targets and this is consistent with the concept that there are multiple targets coupled with common stress responses. A more detailed knowledge of how known antibiotics act should reveal new strategies for antimicrobial drug discovery. Antimicrobial drug discovery and screening resulted in new candidates for clinical development, approaches albeit with seemingly decreased frequency [2], these Since Alexander Fleming’s discovery of the antimi- are only short-term solutions to the fundamental crobial activity of penicillin [1], the field of antimicro- problem of bacterial resistance because resistance to bial drug discovery has been largely dominated by the parent molecule foreshadows resistance devel- whole-cell screening assays, wherein new antimi- opment in the derivative, in that the same basic crobial compounds are chosen for their ability to resistance mechanisms can give rise to cross resistance inhibit the growth of actively multiplying bacteria. in both the parent and derivative. Moreover, our Although the mechanism of action of such com- limited understanding of the target or the mecha- pounds is not always clear, this approach was nism of action of the parent compound often hinders successful in the early days of antibiotic development. rational improvement of antibiotic structures. Whereas this approach still holds potential for the In the past decade, antimicrobial drug discovery screening of large synthetic chemical libraries with research has incorporated a complementary strategy, novel chemistries or naturally occurring antimicro- led by the identification of prospective novel targets bials, including peptides, no major novel leads have important for bacterial growth or survival. An improved Michelle D. Brazas Robert E.W. Hancock* resulted in the past 40 years [2,3]. understanding in bacterial biology and metabolism Centre for Microbial Diseases To combat the emergence of bacterial resistance, and the sequencing of many genes involved in these and Immunity Research, researchers have taken to modifying the chemical processes [4] has facilitated the identification of 2259 Lower Mall Research Station, structure of existing antibiotics to yield derivatives novel targets. Screening systems designed specifically University of British Columbia, that are more potent, have broader spectrum or are to target individual proteins known to be essential Vancouver, B.C., Canada,V6T 1Z4 more effective in vivo (e.g. better oral bioavailability, for cell survival are then used to identify inhibitors *e-mail: [email protected] longer half-life). Although such modifications have in a high-throughput target-specific fashion. Such 1359-6446/05/$ – see front matter ©2005 Elsevier Ltd. All rights reserved. PII: S1359-6446(05)03566-X www.drugdiscoverytoday.com 1245 REVIEWS DDT • Volume 10, Number 18 • September 2005 target-based screening approaches have some advantages attributed to interaction with a predominant PBP, β-lactams over random screening methods, particularly with respect can interact with multiple PBPs. Indeed, the majority of Reviews • to their ability to facilitate lead compound optimization, β-lactams bind to several PBPs with similar affinities [13] as the target is already understood. and it has been suggested that inhibition of more than DRUG DISCOVERY TODAY:TARGETS DRUG DISCOVERY The popularity of such approaches has grown tremen- one PBP could account for the action of these antibiotics dously with the advent of bacterial genome sequencing. [15]. Aminoglycoside antimicrobials have exceptionally The initial sequencing of the Haemophilus influenzae complicated modes of action too, which can be best genome in 1995 [5], followed in rapid succession by the explained by multiple targets [16,17]. Likewise, cationic elucidation of the genomic sequences of more than 200 antimicrobial peptides can act on the permeability barrier organisms, including many medically important pathogens, of bacterial membranes, cell division or macromolecular has provided fuel to the notion that novel antibacterial synthesis and it has been proposed that peptides affect targets are abundant. Armed with a set of criteria that multiple anionic targets [18,19]. Even antibiotics such define an ideal antibiotic target, namely conservation as tetracycline and chloramphenicol are known to have across pathogens, target absence in the host, essentiality secondary effects on bacterial membrane permeability and accessible location in the microbe, a multitude of at concentrations above the minimum inhibitory concen- tools has been developed to mine this wealth of bacterial tration (supra-MIC) [20]. Thus, it appears unlikely that genomic information. For example, advanced sequence most antibiotics have single, easily definable targets. homology programs, such as genomic BLAST (NCBI, www. Rather, such complicated mechanisms of action are indica- ncbi.nlm.nih.gov/sutils/genom_table.cgi), have facilitated tive of multiple molecular targets contributing to bacterial the identification of genes ubiquitously present across a inhibition. These could be primary targets, leading range of bacterial genomes. Similarly, genomic knockout directly to inhibition of bacterial growth, or secondary libraries are defining core sets of genes present in all targets contributing to the overall inhibitory effect. bacterial genomes that encode proteins essential to bac- Despite their limited impact on antimicrobial discovery terial growth and survival; these sets range in number to date, genomic methods have shed light on the com- from 50 to 300, depending on the number of genomes plexities of antibiotic action, and in so doing have high- compared [6–9]. Such approaches have led to the discovery lighted possible reasons for the void in antimicrobial drug of inhibitors of targets essential for microbial survival, discovery. The value of genomics was illustrated for yeast, such as polypeptide deformylase [10] or the fatty-acid for which target validation studies by Marton et al. [21] biosynthesis pathway [11]. were based on the assumption that some inhibitors act Although bacterial genomics and its associated tech- on single targets. Using whole genome yeast microarrays, nologies were believed to be the launching pad for a whole gene-expression profiles were determined following new era in antimicrobial drug discovery [4], there are still addition of the inhibitor FK506 to yeast cultures. At low no new antimicrobial agents in late clinical development inhibitor concentrations, expression patterns were shown that have originated solely from genomics-based approaches to correlate with the deletion of the gene encoding [4,12]. Attempts by several pharmaceutical companies to calcineurin, the FK506-specific target. However, when utilize such strategies in antimicrobial drug discovery have higher concentrations of FK506 were used, changes in the been seemingly without a major return on investment, expression of genes outside the target pathway were also although a few antimicrobials have recently entered into observed, indicating that FK506 had additional secondary early clinical development [12]. This lack of success in an targets [21]. Intriguingly, some recent reviews on tran- area of research with so much potential, leads one to spec- scriptional profiling in antibiotic research have advocated ulate that there might be few new practical antibacterial the use of sub-inhibitory doses of antibiotics and the targets and modes of action left to be discovered [4]. analysis of early time points following antimicrobial expo- sure [22,23] so to rule out complicating components (e.g. Complex modes of action inhibition of secondary targets and downstream effects) A perusal of medical and microbiology textbooks leads from an inhibitor’s transcriptional profile. Whereas the one to believe that all antibiotics work by simple mech- use of sub-inhibitory concentrations might simplify the anisms, involving single targets. This, however, does not experimental output, such concentrations might restrict appear to be correct, at least for the bactericidal antibiotics. the discovery of secondary and downstream effects that For example, β-lactams, the class of antibiotics utilized contribute to the mechanism of action. Thus, it seems most often in clinical setting, are known to have several prudent to examine simultaneously the transcriptional molecular targets [13]. β-lactams inhibit the activity of profiles of inhibitors administered at different physio- penicillin-binding proteins (PBPs), a group of enzymes logically relevant concentrations, so as to learn how important for cell-wall synthesis, and activate murein ‘off-target’ effects [21] contribute to the activity of the hydrolases, which are active in bacterial cell-wall degra- inhibitor [9].