Chemical Biology Applied to the Study of Bacterial Pathogens

Rebecca Anthouard,* Victor J. DiRita

Laboratory of Genetics & Molecular Biology of Intestinal Pathogens, Department of Microbiology & Immunology, University of Michigan Medical School, AnnArbor, Michigan, USA

In recent years, chemical biology and chemical genomics have been increasingly applied to the field of microbiology to uncovernew potential therapeutics as well as to probe virulence mechanisms in pathogens. The approach offers some clear advantages, asidentified compounds (i) can serve as a proof of principle for the applicability of drugs to specific targets; (ii) can serve as condi-tional effectors to explore the function of their targets in vitro and in vivo; (iii) can be used to modulate gene expression in oth-erwise genetically intractable organisms; and (iv) can be tailored to a narrow or broad range of bacteria. This review highlightsrecent examples from the literature to illustrate how the use of small molecules has advanced discovery of novel potential treat-ments and has been applied to explore biological mechanisms underlying pathogenicity. We also use these examples to discusspractical considerations that are key to establishing a screening or discovery program. Finally, we discuss the advantages andchallenges of different approaches and the methods that are emerging to address these challenges.

Researchers have taken increasing interest in small-moleculescreening programs because of their dual use in discovering

new potential therapeutics and in generating molecular probesuseful for studying virulence pathways in vitro and in vivo. Thefields of drug discovery and microbial biology have both benefitedfrom findings made using small-molecule screening programs.

The Centers for Disease Control and Prevention recently esti-mated that over 2,000,000 illnesses are caused by antibiotic-resis-tant bacteria and fungi annually, resulting in over 23,000 deaths,though this is likely an underestimate (1). Despite the huge initialsuccess of antibiotic therapy, overuse and misuse have led to theirincreasingly limited effectiveness, and today, at least one resistantstrain of bacteria exists for every known antibiotic (2). Equallytroubling is that antibiotic therapy kills a large portion of the hostmicrobiota in addition to the targeted pathogen. The resultingdysbiosis can lead to acute and chronic intestinal problems (3, 4)and is a leading cause of hospital-acquired infections by Clostrid-ium difficile (5). As more and more pathogens become resistant toantibiotics, and with increasing awareness of the protective effectsof the microbiota, researchers have started to look for alternativetherapies for treating bacterial infections.

Anti-infective drugs, also known as antivirulence drugs, areattractive alternatives because they disarm pathogens rather thankilling them, providing significant advantages over antibiotictreatment. First, resistance developed against anti-infective drugsmay be driven by a weaker selective pressure; thus, resistancewould take longer to develop, if it develops at all. Second, by tar-geting a virulence trait, anti-infectives affect only the bacteria thatpossess that pathogenic trait—ideally leaving the microbiota rel-atively unaffected.

In addition to their use in drug discovery, small molecules haveplayed important roles in biomedical research because of their usein uncovering new virulence requirements. There are several ad-vantages to probing pathogenesis with small molecules at thebench: (i) they act quickly, (ii) they may act reversibly, (iii) they donot require manipulation of the genome, a quality that is espe-cially advantageous in studying genetically intractable organisms,(iv) the dose can be adjusted to fine-tune the effects, and (v) theycan be used across multiple bacterial species to determine howconserved a pathway is between different species or strains.

Pathogenic bacteria have evolved numerous strategies for es-tablishing infection and causing disease in various hosts. For anygiven pathogen, there are multiple points in the infection processthat can be inhibited to reduce virulence potential. Figure 1 de-picts small-molecule inhibitors (shown in red) discussed in thisreview in a generalized model of pathogenesis. Some of these vir-ulence mechanisms are restricted to only one or a few organisms(for example, actin-mediated cell-to-cell spread is used promi-nently by Listeria monocytogenes, Shigella spp., and Rickettsiaspp.), while others are more broadly conserved. Such an “Achilles’heel” of pathogenesis can be exploited to develop new treatmenttherapies. We begin this review with a brief introduction of thescreening process and then discuss examples of successful screens(summarized in Table 1) to demonstrate how they have influ-enced our understanding of pathogenesis and uncovered new po-tential drug targets. We explore the advantages and limitations ofsmall-molecule research and conclude with some ways in whichsmall-molecule screens could further our understanding ofpathogenesis.

THE SCREENING PROCESS

To understand the impact of using small molecules to study andtarget bacterial pathogens and pathogenicity, it is important toappreciate the process involved in their identification. A briefcomment is warranted regarding the sort of chemical libraries thatmay be used in the basic screening process outlined in Fig. 2. Adiverse library enables researchers to probe more chemical space,increasing the likelihood of finding a novel inhibitor, but less is

Accepted manuscript posted online 17 November 2014

Citation Anthouard R, DiRita VJ. 2015. Chemical biology applied to the study ofbacterial pathogens. Infect Immun 83:456 – 469. doi:10.1128/IAI.02021-14.

Editor: H. L. Andrews-Polymenis

Address correspondence to Victor J. DiRita, vdirita@umich.edu.

* Present address: Rebecca Anthouard, Department of Microbiology &Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NorthCarolina, USA.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.02021-14

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known regarding the molecules in these libraries. Some of theexamples we provide below arose through screening diverse li-braries. Probing more-defined libraries of molecules with knownbiological function, on the other hand, has the advantage that themechanism of action for these molecules has been previously de-scribed in at least some settings. However, such libraries lack thebroad chemical space of the diverse libraries. We also providesome examples where these more-defined libraries have beenscreened successfully.

For screening and postscreening evaluation, the process (out-lined in Fig. 2) is essentially as follows. First, a robust assay isdeveloped. Second, a series of high-throughput screens are per-formed to identify small-molecule inhibitors. A primary screen isperformed with selected libraries of compounds to identify thosethat affect the assay. A secondary screen is performed to confirmgenuine hits and determine whether they behave in a dose-depen-dent manner. A counterscreen may also be performed, in parallelto or subsequent to the primary and secondary screens, to provideadditional information for triage of hit compounds. Counter-screens may, for example, report on the specificity or general tox-icity of a compound. After triage of hits from the screen, a smallmolecule is selected for further characterization. There are manyways to characterize a small molecule, and the choice largely de-pends on the aim of the study. If, for example, the principal goal isto develop a novel therapeutic, emphasis will be placed on opti-mizing in vivo efficacy. However, if the compound is primarilyintended as a biological probe, determining its target and mecha-

nism of action will be prioritized. Several approaches to com-pound characterization are often performed in parallel becauseinformation gleaned from one approach can impact the design orinterpretation of data obtained by other approaches. As we discusssmall molecules that target bacterial pathogenesis in this review,we also point out how different strategies used in the assay, screen-ing, and characterization steps affect the type of small moleculeidentified.

SMALL MOLECULES THAT AFFECT VIRULENCE FACTORSAND THEIR REGULATION

Many chemical screens have targeted bacterial virulence factors ortheir regulation, an approach that boasts numerous advantages.From a drug discovery standpoint, antivirulence compounds havethe advantages that (i) they presumably would have no (or per-haps a very limited) effect on the host microbiota in comparisonto traditional antibiotics, which act more indiscriminately, and(ii) they disarm the pathogen rather than kill it, theoretically im-posing a lower selective pressure with respect to resistance thantraditional antibiotics, reducing the risk for the emergence of re-sistant strains. Also, targeting virulence factors enables drugging arange of species, depending on how conserved the target is. Fi-nally, antivirulence molecules are valuable as research tools, asthey deepen our understanding of the molecular mechanisms re-quired for virulence gene activation.

Virstatin. One early example of such a molecule is virstatin(see Table 1). Virstatin targets Vibrio cholerae specifically by inhib-

FIG 1 Critical steps in the pathogenicity of microbes and the small molecules that inhibit each step. Inhibitors discussed in this review (shown in red) target manyaspects of pathogenesis (shown in black), including specific virulence factors and their regulation (steps 1 to 5, 10, and 13) and broader aspects of host-pathogeninteractions (steps 6 to 9, 11 to 12, and 14).

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iting its ability to produce cholera toxin (CT) (step 1 in Fig. 1)(17). Virstatin was identified using a live-cell-based reporter assayin which the promoter for the genes encoding cholera toxin(ctxAB) directed expression of the tetracycline resistance genetetA. In this screen, compounds that inhibited expression of thecholera toxin genes resulted in poor growth of the reporter strainin media containing tetracycline. Such cell-based assays are par-ticularly useful to basic biology because the identified compoundsmay inhibit a protein or pathway not yet known to play a role invirulence, thus allowing new discoveries about biological systemsto be made.

In contrast to screens against purified proteins, a cell-basedscreen requires postscreen target identification. This process canbe extremely challenging because the target (i) may have an un-specified role in virulence, (ii) may be expressed only under cer-tain conditions, and/or (iii) may not be a single protein but mayrather be a protein complex or (iv) not a protein at all but ratherDNA, RNA, lipids, or the redox state of the cell, to name a few ofthe possibilities.

In the case of virstatin, target identification was accomplishedby taking advantage of the fact that the virulence regulatory path-way in V. cholerae is well defined (20–26). Expression profiles of

genes expressed upstream of ctxAB transcription in the regulatorycascade were examined by quantitative reverse transcription-PCR(qRT-PCR), and none was affected by virstatin, suggesting that itstarget is late in the transcription cascade leading to ctxAB expres-sion. Virstatin could still inhibit ctxAB transcription even whentoxT, encoding the direct activator of ctxAB transcription, wasexpressed ectopically, indicating that virstatin inhibits ToxT ac-tivity (17).

To examine further whether ToxT is the likely target of virsta-tin, a library of toxT mutant alleles was generated and screened forresistance to the compound; one allele, toxTL113P, was identified(17). The protein expressed from this mutant allele resembledwild-type ToxT in that it was found predominantly in the multi-meric form; however, in the presence of virstatin, wild-type ToxTis largely in the monomeric state, while ToxTL113P remains pre-dominantly multimeric (18, 19). This supported a long-held hy-pothesis, based on the arrangement of the binding sites on ToxT-activated promoters known as toxboxes (27), that ToxT is active asa dimer. Virstatin was then used to probe ToxT activity at otherToxT-dependent promoters, which led to the discovery that somepromoters favor the monomeric form whereas others favordimerized ToxT (28).

FIG 2 Identifying and characterizing small-molecule inhibitors of pathogenesis require many considerations at each step of the process. The major strategies areoutlined here and are discussed further in the text.

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To assess its in vivo efficacy, virstatin was tested in an infantmouse model of V. cholerae colonization (17). Mice inoculatedwith wild-type V. cholerae and given virstatin had a 4-log decreasein colonization relative to mice given the dimethyl sulfoxide(DMSO) control, suggesting that virstatin may have therapeuticpotential. To determine whether the decreased colonization wasToxT dependent, an atypical strain of V. cholerae that colonizesthe mouse in a ToxT-independent manner was used. This straincolonized mice treated with the DMSO control and mice treatedwith virstatin equally well, indicating that virstatin reduces colo-nization of the typical endemic strains of V. cholerae by inhibitingToxT activity in vivo. Although it remains to be directly tested, thatcolonization by ToxT-independent strains of V. cholerae is notaffected by virstatin suggests that other bacteria lacking ToxT,including the host microbiota, would be unaffected by virstatintreatment, making virstatin an attractive therapeutic lead.

The cell-based screening approach leading to the discovery ofvirstatin and the subsequent work characterizing its mechanism ofaction advanced the field of V. cholerae research in several ways.First, this approach provided further strong evidence for the im-portance of the dimerization state of ToxT. Second, it proveduseful for determining whether a V. cholerae isolate colonizes in aToxT-dependent or -independent manner. Finally, as virstatinwas shown to be a compound that significantly reduces coloniza-tion of a pathogen by specifically inhibiting its virulence factors invivo, it provided a proof of principle for the concept of antiviru-lence drugs.

Toxtazins. The toxtazins (step 1 in Fig. 1) constitute anotherclass of antivirulence compounds that inhibit expression of viru-lence factors in V. cholerae (15). Toxtazins A and B were identifiedin a cell-based screen using a toxT-gfp reporter strain to probe theToxT pathway directly. As indicated in Fig. 2, assays can be de-signed to target a phenotype or a molecular pathway, and eachapproach has unique advantages. Targeting a pathway restrictshits to those that affect only that pathway, while targeting a phe-notype, CT production, for example, would include hits that tar-get the ToxT pathway as well as inhibitors of CT folding and se-cretion.

The toxtazins significantly reduce expression of both choleratoxin and the toxin-coregulated pilus (15). Furthermore, toxtazinB reduced the level of V. cholerae colonization of infant mice by 2logs, suggesting that it may have therapeutic potential in the treat-ment or prevention of cholera. Further analysis demonstrated thattoxtazin A does not affect expression of ToxR or TcpP, the tran-scriptional activators of toxT, while toxtazin B reduced expressionof both tcpP transcript and TcpP protein levels. TcpP is transcrip-tionally activated by AphA and AphB, neither of which is affectedby toxtazin B treatment. In fact, ectopic expression of ToxT in cellstreated with either toxtazin A or B restored expression of choleratoxin, confirming that both of these compounds act upstream ofToxT expression. From these results, it was determined that tox-tazin A targets toxT transcription whereas toxtazin B targets tcpPtranscription (15).

The exact mechanisms of action for these compounds havenot been identified, but preliminary evidence showing that cellstreated with toxtazin A mount a stress response indicates the pres-ence of a pathway which was not previously known to influenceregulation of toxT expression but which affects the virulence po-tential of V. cholerae.

FPSS. Unlike virstatin and the toxtazins, which target species-

specific virulence regulators, fluoro-phenyl-styrene-sulfonamide(FPSS; see Table 1) targets a general microbial regulatory path-way—the sigma B (�B) regulon (step 2 in Fig. 1) (9). Sigma factorsare dissociable subunits of RNA polymerase (RNAP) that associ-ate with RNAP in response to certain environmental signals anddirectly activate or repress subsets of genes, resulting in a rapidchange in global transcription that is appropriate for the givenenvironment. Different sigma factors respond to different sets ofenvironmental signals, allowing bacteria to quickly respond tospecific environments.

Different sigma factors can respond to the same signal, anddifferent sigma factors can regulate the same genes in response todistinct signals, creating a complex web of regulation (29). In ad-dition, some sigma factors are essential in some or all growthconditions, making them difficult to study using classical geneticapproaches. Small-molecule inhibitors of specific sigma factorsenable the study of individual sigma factors in isolation, so thecontributions of individual sigma factors and of different environ-mental signals can be explored. This class of inhibitor may alsohave therapeutic potential because sigma factors regulate viru-lence gene expression in many pathogens (30–32), as reviewed inreference 33.

In Listeria monocytogenes, �B responds to environmental stress(i.e., acidic conditions, ethanol, or high salt concentrations), en-ergy stress, and growth at low temperatures (34). In response tothese signals, �B activates or represses genes involved in centralmetabolism and activates PrfA, the master regulator of virulence(35). FPSS was identified as an inhibitor of �B by screening adiverse library of synthetic compounds using a cell-based reporterassay (9).

FPSS was discovered using a screening strain containing a �B-dependent promoter, opuCA, fused to the gene for glucuronidase,gus, such that glucuronidase activity could be used as a readout for�B activity. Inhibition of �B activity by FPSS was confirmed byqRT-PCR. Transcription of �B-dependent promoters was at thelevel of a �B mutant after FPSS treatment, indicating that FPSScompletely inhibits �B activity at these promoters. Microarrayanalysis showed that L. monocytogenes cells treated with FPSS phe-nocopy an L. monocytogenes �B mutant, affecting 91% of previ-ously defined �B-regulated genes (9). FPSS also affected 83 othergenes, which, excluding side effects, could potentially expand the�B regulon. Gene-set enrichment analysis (GSEA) determinedthat genes specifically regulated by �H or by �L are not signifi-cantly enriched among genes differentially transcribed in FPSS-treated cells, indicating that FPSS inhibits �B specifically.

Bacillus subtilis was used to determine the mechanism by whichFPSS inhibits �B activity, because its �B activity is also inhibitedby FPSS (9) and the components of its well-characterized �B regu-lon are highly conserved with the components of the insufficientlystudied �B regulon of L. monocytogenes. In B. subtilis, �B activity isregulated by three distinct branches (shown in Fig. 3 and reviewedin reference 36) that convey conditions of environmental stress(i.e., acidic conditions, ethanol, or high salt concentrations), en-ergy stress (i.e., limitation of glucose, ATP, GTP, or phosphate),and growth at low temperatures.

In B. subtilis, �B is kept in the off state by an anti-sigma factor,RsbW. The ability of RsbW to sequester �B is controlled by thephosphorylation state of another protein, RsbV; phosphorylatedRsbV cannot bind RsbW, leaving RsbW free to sequester �B,thereby keeping the regulon from being expressed. In contrast,

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unphosphorylated RsbV binds RsbW, liberating �B and leading toactivation of the �B regulon (37). RsbV phosphorylation is con-trolled by energy and environmental stresses (see Fig. 3) (38), andFPSS inhibited �B activation by several such stresses, indicatingthat it works on a factor common to these pathways (9). Thepossibility that FPSS could bind or interact with �B was ruled outby in vitro and in vivo experiments (10). Taken together, theseresults support a model whereby FPSS targets the partner-switch-ing mechanism between RNA polymerase, �B, and its anti-sigmafactor, RsbW.

FPSS provides a unique tool to study �B activity in multipleorganisms. Microarray analysis of FPSS-treated cultures (9) sug-gests that the �B regulon is broader than currently understood,showing another value of using this compound to study �B. Whileanimal studies with FPSS have not been reported, �B is an attrac-tive therapeutic target because it is conserved and important in theactivation of virulence genes in several pathogenic bacteria (39,40). One potential drawback to the therapeutic potential of thismolecule, however, is that FPSS may also inhibit �B in commensalstrains of the microbiota, which could lead to dysbiosis, althoughthis has not been experimentally assessed.

TSS29. Secretion mechanisms have been targeted in small-molecule screens for antivirulence drugs. In particular, the type 3secretion system (T3SS) injectosome has been attractive becauseof its broad conservation in Gram-negative pathogens (step 3 inFig. 1). Inhibitors of the Salmonella enterica serovar TyphimuriumT3SS were identified using a reporter strain that secretes phospho-lipase in a T3SS-dependent manner (16). This phenotypic ap-proach to cell-based screening can result in hits that affect any of

several steps in production of a functional phospholipase, includ-ing transcription, translation, protein folding, protein-protein in-teraction, and translocation. Eighty-nine compounds remainedafter the primary and secondary screens. Tertiary screens aresometimes performed to select compounds with particular char-acteristics (Fig. 2). In that study, the authors performed two ter-tiary screens to eliminate compounds that affect bacterial growthand those that are not specific (i.e., that inhibit bacterial transla-tion, sec-dependent secretion, or disulfide bond isomerization).Seven compounds passed the tertiary screens, and one of these, a2-imino-5-arylidene thiazolidinone termed TTS29, was investi-gated further (16). Cultures grown with TTS29 displayed an over-all reduction in the levels of type 3 secretion system needle com-plexes. Levels of needle complex protein constituents were notreduced in whole-cell lysates, indicating that the proteins werebeing produced but that their assembly into the needle complexwas inhibited by TTS29 (16).

Because components of the T3SS are conserved in other bac-teria, TTS29 has the potential to work against other T3S-encodingbacteria. Yersinia species express two types of T3SS: (i) the plas-mid-encoded Ysc system in Yersinia pestis, Yersinia enterocolitica,and Yersinia pseudotuberculosis, which secretes Yop (Yersiniaouter protein) into the cytosol of target cells, and (ii) the chromo-somally encoded Ysa system in Y. enterocolitica, which secrets Ysp(Yersinia secreted protein) (41, 42). TSS29 inhibited secretion ofboth Yop and Ysp into Y. enterocolitica culture supernatants, in-dicating its potential utility as a broad inhibitor of T3SS (16). Incontrast, TSS29 did not alter flagellar motility or decrease the lev-els of flagellar components in either S. Typhimurium or Pseu-

FIG 3 FPSS targets sigma B. Multiple stresses activate �B activity in B. subtilis. Energy stress is relayed via RsbPQ, environmental stress is relayed via the“stressosome” and RsbU, and low-temperature stress is relayed via an as-yet-unknown mechanism. Sensing of stress results in dephosphorylation of the RsbVanti-anti-sigma factor, allowing it to bind the RsbW anti-sigma factor, which in turn releases �B, to interact with RNAP and activate the �B regulon. Phosphor-ylated RsbV interacts poorly with RsbW, which is then free to bind �B, leading to low-level expression of the regulon. FPSS inhibits �B activity by drivingequilibrium toward the state in which �B is bound to RsbW. SNP, single nucleotide polymorphism.

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domonas aeruginosa, bacteria that depend on a flagellum-specificT3SS for motility (16). This suggests that TSS29 targets a compo-nent of the T3SS that is not conserved with the evolutionarilyrelated flagellum-specific T3SS (42).

Because one component of the T3SS, secretin, is conserved inthe type 2 secretion system (T2SS) that delivers enzymes and otherproteins across the Gram-negative envelope (43), TSS29 wastested for its ability to inhibit such systems. Secretion of elastase(44) by the T2SS in P. aeruginosa was inhibited by TSS29, as wastwitching motility, which is controlled by a pilus whose assemblyincludes components similar to those of the T2SS (16). Theseresults demonstrate that TSS29 is a broad inhibitor of secretionaffecting different secretion systems in multiple bacterial patho-gens.

The assessment of the in vivo effectiveness of TSS29 was sup-ported by demonstrating its ability to reduce killing of bone mar-row-derived macrophages (BMMs) in a tissue culture model ofinfection (16). It also inhibited Pseudomonas syringae pv. tomatoDC3000 from inducing a hypersensitivity response in tobaccoplants (16). Thus, TSS29 has broad therapeutic potential.

Pilicides. Pili are recognized as important virulence factorsbecause of their role in colonization of many pathogens in their

respective hosts (step 4 in Fig. 1). While some microbes secretepilus adherence structures through type 2-like secretion systemsthat can be inhibited by TSS29 as described above, others assemblepili using pathways that rely on a periplasmic chaperone (45–47)and an outer membrane usher (48, 49) (see references 50 and 51for reviews). The chaperone mediates the folding, stabilization,and transport of pilus subunits, while the usher aids in incorpo-rating subunits into the growing pilus (51).

Because the chaperone-usher pathway of pilus assembly is con-served in a wide range of pathogens (51), inhibitors of these sys-tems would theoretically be effective against a broad range of bac-terial species, making them attractive targets for therapeuticdevelopment. Substituted bicyclic 2-pyridones, called pilicides(pharmacophore shown in Table 1), are a well-studied group ofsynthetic small-molecule inhibitors that prevent formation of piliin uropathogenic Escherichia coli (UPEC) (51).

UPEC bacteria produce different pathogenicity-associated pili,the most representative of which are P pili and type I pili; theseadhesion structures and others like them are generally termedchaperone-usher pili for the key proteins associated with theirassembly (Fig. 4) (51). P pili are made up of subunits called PapA(the major subunit), PapE, PapF, PapG, PapH, and PapK. These

FIG 4 Pilicides affect both P pili and the type 1 pili, which have similar structures. Pili consist of several repeating subunits arranged in a helical structure.Subunits are translocated from the cytoplasm to the periplasm, where a chaperone (PapD or FimC) folds the protein, stabilizes it, and transfers it to the usherprotein (PapC or FimD), which secretes the protein and incorporates it into the pilus structure. Pilicides inhibit formation of pili by preventing the chaperonefrom passing the subunit to the usher protein.

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are assembled by the PapD chaperone and the usher PapC. Type 1pili are made up of FimA (the major subunit), FimG, and FimHand are assembled by the chaperone FimC and the usher FimD(51).

In contrast to inhibitors discovered by screening compoundlibraries, pilicides were developed using a rational design ap-proach (12). Pilus biogenesis requires the chaperone protein tobind its natural ligands (52), so the solved crystal structure of thePapD-PapG complex was used to chemically design molecularmimetics that would bind within the active site of periplasmicchaperones PapD and FimC (12). A library of pilicides was syn-thesized and demonstrated to be effective at inhibiting pilus for-mation (13).

PiIlicides do not affect cell growth or viability but inhibit man-nose-sensitive hemagglutination (MSHA) and hemagglutination(HA), traits of type 1 pili and P pili, respectively (13). Pilicides alsoreduce biofilm formation and bacterial attachment to host cells,both of which require type 1 pili (13). Pilicide 2c in particularreduced the ability of E. coli strains to adhere to cultured bladdercells by 90% (13).

Cocrystallization of pilicide 2c with the PapD chaperone (13)demonstrated close contact with a hydrophobic patch that runsacross the back of the F1-C1-D1 beta sheet on PapD, a regionhighly conserved in all pilus periplasmic chaperones, which maymediate interactions between the chaperone and the N-terminaldomain of the usher protein (13, 53). Surface plasmon resonanceshowed that pilicide 2c also prevents FimC-FimH (chaperone-adhesin) from binding to FimD (usher) (13), suggesting that pili-cide 2c inhibits the biogenesis of pili by preventing the chaperonefrom passing the subunit to the usher.

Studies of pilicides have uncovered knowledge about molecu-lar mechanisms involved in the biogenesis of pili, and their dis-covery and characterization demonstrate the power of crystallog-raphy to guide chemists to create a small molecule with apredictable activity. This technique, called structure-based drugdesign (SBDD), can be extended by cocrystallizing the compoundand target to provide information useful for improving com-pound activity.

SMALL MOLECULES THAT AFFECT HOST-PATHOGENINTERACTIONS

Small molecules can provide valuable insight into underlying bi-ological mechanisms in pathogenicity and a relevant system forstudying pathogens in the context of their hosts. While the previ-ously discussed compounds targeted the pathogen, the followingexamples are of small molecules that work by disrupting impor-tant host-pathogen interactions and demonstrate that this is alsoan effective strategy for generating compounds with therapeuticpotential.

LED209. Virulence gene expression is often induced by signalsfrom the host environment, making sensory transduction mech-anisms potential therapeutic targets (step 5 in Fig. 1). For exam-ple, E. coli, S. Typhimurium, and Francisella tularensis express asensor histidine kinase called QseC that detects host-derived ad-renergic signals (epinephrine and norepinephrine) as well as quo-rum-sensing autoinducer-3 (AI-3) (54, 55). In response to eitherof these signals, QseC autophosphorylates and then transfers thephosphate to transcription factor QseB, leading to transcription ofkey virulence genes, including the LEE1 operon in enterohemor-rhagic E. coli (EHEC) (56).

A diverse chemical library of 150,000 compounds was screenedfor those that block expression of a LEE1-lacZ reporter strain ofEHEC (11). Compounds were screened in an assay using spentmedium (which contains AI-3) to activate QseC and induce re-porter gene expression. The most potent inhibitor identified wasimproved by structure-activity relationship (SAR) studies andnamed LED209 (see Table 1 for structure) (11).

LED209 did not inhibit bacterial growth but rather selectivelyinhibited virulence gene expression by inhibiting QseC autophos-phorylation (11). While LED209 was ineffective at reducing colo-nization loads in an infant rabbit model of EHEC infection (per-haps due to rapid absorption from the gastrointestinal tract), itsignificantly reduced the mouse pathogenicity of both S. Typhi-murium and F. tularensis, which express QseC homologs of 87%and 57% similarity to EHEC QseC, respectively (11). QseC is im-portant for motility in S. Typhimurium (41) and for systemicinfection in F. tularensis (57).

Similar to the ToxT inhibitors virstatin and the toxtazins,LED209 inhibits virulence by targeting a specific virulence regu-lator without affecting growth. Its target, QseC, is broader, how-ever, being conserved in over 25 pathogenic bacteria but absent inmammals (11), giving this molecule a bacterium-specific broadspectrum of activity and making it an attractive lead compound.

CCG-2979. In addition to targeting sensory transduction path-ways, screens can target the ability of pathogens to manipulate thehost. Such a strategy identified CCG-2979 (see Table 1 for struc-ture), which reduces production of streptokinase (SK) by Strepto-coccus pyogenes (see step 6 in Fig. 1). CCG-2979 targets the pro-moter of SK (7), which is secreted by S. pyogenes and other groupA streptococci (GAS) and activates the host zymogen plasmino-gen to form plasmin, the central protease of the fibrinolytic systemcritical for regulating blood clots (58, 59). CCG-2979 was identi-fied using a cell-based assay in which ska, the SK gene promoter,controlled expression of the kanamycin resistance gene; positivehits were those that decreased the growth of the reporter strain butnot growth of a constitutive kanamycin-resistant strain (7).

CCG-2979 reduces SK activity in a dose-dependent mannerwithout inhibiting bacterial growth. Furthermore, GAS treatedwith 5 or 50 �M CCG-2979 were more susceptible to phagocytosisby host cells. A series of structural analogs of CCG-2979 was gen-erated, and structure-activity relationship (SAR) studies were per-formed (8, 60). The effect of one chemical analog, CCG-102487,on global gene expression was examined by microarray analysis,which demonstrated that expression of 29% of GAS genes in ad-dition to that of ska was altered, with the vast majority of thembeing reduced in expression. Included among these were othervirulence-associated genes coding for adhesins and toxins, alongwith genes encoding some metabolic functions (7).

Transgenic mice expressing the human plasminogen gene wereused to determine the therapeutic potential of CCG-2979. Usingthis well-established model (59), mice were subcutaneously in-jected with GAS, given a day to establish an infection, and thenintraperitoneally treated with compound daily for 5 days. Micetreated with CCG-2979 showed a statistically significant improve-ment in survival, while those treated with the CCG-102487 analogwere not protected from GAS-induced mortality (7).

Another analog, CCG-203592, inhibited SK at a level 35-foldgreater than that seen with CCG-2979 (8). Subsequent work led tothe observation that this and other analogs are also effectiveagainst another Gram-positive pathogen, Staphylococcus aureus.

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S. aureus is a major public health problem due in part to its abilityto form biofilms on implantable devices (61). Biofilm formationof three different biofilm-producing S. aureus strains was reducedin the presence of 50 �M CCG-203592, both in laboratory micro-titer plates and on silicone, the most common surface used forimplantable medical devices (8). Thus, this class of compoundsmay have therapeutic applications against different traits ex-pressed by two classes of important human pathogens, GAS and S.aureus, making them interesting therapeutic leads.

Pimozide. In contrast to screens using large, diverse chemicallibraries whose functions are completely unknown, screens usingcompounds with known biological activities result in more readilyidentified mechanisms of action in pathogenic processes. Pimoz-ide (see Table 1 for structure) was identified as an inhibitor ofListeria monocytogenes infection by screening a library of FDA-approved drugs (14).

L. monocytogenes infects macrophages (step 7 in Fig. 1) (62)and escapes the phagocytic vacuole by producing a hemolysin,listeriolysin O (LLO) (step 8 in Fig. 1). It replicates in the cytosolbefore spreading to neighboring cells by polymerizing host cellactin to propel itself into adjacent cells (step 9 in Fig. 1) (13, 63).While genetic and cellular biological studies have uncoveredmuch about L. monocytogenes pathogenicity, the mechanisms reg-ulating infection remain incompletely defined.

A novel chemical genetics approach was taken in which bothbacterial cells and host cells were exposed to a library of com-pounds (14). Primary bone marrow-derived macrophages(BMMs) were infected with L. monocytogenes constitutively ex-pressing green fluorescent protein (GFP) to enable visualizationof internalized bacteria. A total of 480 compounds with diverse,known biological activities were screened; 21 of these altered L.monocytogenes infection in one of three ways (14). The majority ofinhibitory compounds inhibited BMM infection, as observed by adecrease in GFP fluorescence within BMMs (i.e., internalized bac-teria) relative to the DMSO control results. Others enhanced bac-terial uptake or intracellular replication, causing an increase inGFP fluorescence per BMM cell. Lastly, some compounds inhib-ited cell-to-cell spread, observed as an increase in GFP fluores-cence per BMM, but only in very few cells.

Compounds that answered the screen fell into four categoriesbased on their previously known activities, and each category en-genders testable hypotheses about the conditions required for L.monocytogenes infection. The first category of inhibitors includescompounds that disrupt actin, which is required for phagocytosisby BMMs and for cell-to-cell spread of L. monocytogenes (64). Asecond group targets kinases and phosphatases, important for ac-tin rearrangement. These may provide new insights into the roleof specific kinases and phosphatases in L. monocytogenes infectionbut were not further analyzed. The members of a third group ofcompounds were categorized together on the basis of lackingshared activities with other compounds from the screen; furtherstudies on these may also yield new insights into L. monocytogenesinfection. The fourth category, composed of nine compounds(43% of the hits), includes molecules that affect calcium pathways.Calcium is not only important in many cellular signaling path-ways such as phagocytosis (65), it is also released in response toprotein kinase C (PKC) activation, which occurs during L. mono-cytogenes infection and modulates bacterial uptake and escapefrom the vacuole (66). Pimozide falls into this fourth category andwas chosen for further study; it should be emphasized, however,

that whether the action of pimozide on calcium pathways is alsowhat disrupts any given aspect of L. monocytogenes infection hasyet to be resolved. This raises an issue that confounds design of anysmall-molecule inhibitor, whether used as a chemical probe forbiological effects or as an actual therapeutic: inhibitors effective inspecific screens may have other off-target effects.

Pimozide inhibited intracellular infection of BMMs by L.monocytogenes by an order of magnitude after a 10-h treatment(14). While the exact mechanism for this is unclear, pimozide wasfound to inhibit infection at three distinct steps. The most potenteffect of pimozide was inhibition of macrophage phagocytosis,which was not limited to phagocytosis of L. monocytogenes but wasseen with three other bacteria as well: B. subtilis, S. Typhimurium,and E. coli K-12. Pimozide inhibited internalization of bacteria bymacrophages by 99% in a calcium-independent manner (14).Pimozide also reduced vacuolar escape of L. monocytogenes by26%, and this was not due to inhibition of LLO. Finally, pimozidetreatment decreased cell-to-cell spread by approximately 50%(14).

This screen generated many testable hypotheses regarding thecellular requirements of L. monocytogenes infection. Additionally,pimozide can be used to probe the molecular mechanisms under-lying BMM phagocytosis of bacteria in general and of L. monocy-togenes specifically. Pimozide blocks postsynaptic dopamine re-ceptors and is an FDA-approved antipsychotic molecule used totreat severe Tourette’s syndrome and schizophrenia (67). The mo-lecular mechanism by which pimozide inhibits macrophage func-tion leading to alterations in Listeria infection is not clear, but,given the class of compound into which it fell in this screen, itsmechanism may involve calcium signaling. In addition, this com-pound was shown to affect this particular host-pathogen system inmultiple and yet synergistic ways and highlights the connectionbetween what otherwise appear to be different pathogenic mech-anisms. Molecules such as pimozide have increased therapeuticappeal because of the lowered probability of bacteria overcomingtheir multiple effects by mutation. Additionally, an added benefitof an FDA-approved compound such as pimozide is that there is awealth of information regarding safety, pharmacokinetics, andpharmacodynamics.

Type 4 secretion inhibitors. Compounds with known biolog-ical activities have also been used to probe the type 4 secretionsystem (T4SS; also called the Icm/Dot type IVB system) (step 10 inFig. 1) in Legionella pneumophila, which infects and replicates inlung alveolar macrophages and causes Legionnaires’ disease (68,69). L. pneumophila avoids phagosome-lysosome fusion by usingits T4SS to secrete effectors that interfere with vesicular traffick-ing, the host innate immune response, phosphoinositide metab-olism, and ubiquitination (reviewed in reference 70). Given itsimportance in intracellular survival and replication (71), the T4SSis an attractive target for drug development. The T4SS can beactivated by contact with the host cell (72), but the signals thattrigger secretion of effectors are not well understood.

Compounds with known biological targets were screened toprobe the mechanisms of type 4 secretion in L. pneumophila (6). AT4SS-secreted effector protein (LepA) was fused to the TEM-1�-lactamase (BlaM), which cleaves a green substrate, CCF4, toproduce a blue product. Host cells were incubated with com-pounds for 24 h and infected with the L. pneumophila lepA-blaMreporter strain, and host-bacterial cell contact was initiated by alow-speed centrifugation step. CCF4 was added 1 h later, and flu-

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orescence was measured 2 h later to quantify the ratio of cleavedCCF4 to uncleaved CCF4 (6).

Of 2,640 compounds screened, 22 inhibited translocation withefficiencies ranging from 63% to 100% and were categorized intogroups based on their previously known targets: ionophore orprotonophores, calmodulin, cytoskeleton dynamics, NF-�B, ser-ine proteases, kinases or phosphatases, and others (6). The major-ity of the inhibitors are molecules previously known to affect hostcytoskeleton dynamics, including those affecting tubulin (step 11in Fig. 1), actin (step 12 in Fig. 1), and phosphoinositol 3-kinase(PI3K). Ionophores and protonophores discharge membraneelectric potential (�) and collapse the proton gradient (�pH),both components of the proton motive force (PMF) (step 13 inFig. 1) (73, 74). All three identified ionophores and protono-phores inhibited Icm/Dot-dependent lysis of red blood cells by L.pneumophila (6), indicating that translocation of effectors is atleast partially dependent on the PMF. Furthermore, one iono-phore, carbonyl cyanide m-chlorophenylhydrazone (CCCP)(shown in Table 1), inhibited LepA translocation in L. pneumo-phila in a dose-dependent and reversible manner (6). The PMFwas not previously known to play a role in T4SS-mediated trans-location.

Nineteen of the 22 identified inhibitors significantly affectedthe ability of macrophages to phagocytose other bacteria (step 7 inFig. 1) (6). To test whether phagocytosis is required for transloca-tion of L. pneumophila Icm/Dot effector proteins, cytoskeletoninhibitors were used to inhibit coiling phagocytosis, the type ofphagocytosis used by L. pneumophila to enter macrophages (75).Host-bacterium contact was instead initiated by opsonizationwith L. pneumophila-specific antibodies (76). Opsonization re-stored effector translocation in the absence of phagocytosis (6),indicating that host-cell binding, and not phagocytosis, is re-quired for translocation by the Icm/Dot system. These results sup-port a model where the T4SS in L. pneumophila is in a “locked andloaded” state, ready to inject effector proteins upon contact with ahost cell (step 10 in Fig. 1).

Another important finding from this study came by furtheranalyzing the RWJ-60475 translocation inhibitor (shown in Table1), previously known to inhibit a receptor tyrosine phosphatephosphatase (step 14 in Fig. 1) (77). Work with this compoundrevealed that CD45 and CD148 are required to phagocytose L.pneumophila (6).

In this Legionella work, screening just 2,500 small moleculeseventually generated many new testable hypotheses from the hitsthat arose. Additionally, new information was uncovered relatingphagocytosis and effector translocation to the PMF, host cell con-tact, and CD45 or CD148, providing a great example of howchemical genetics deepens our understanding of pathogenicitymechanisms.

ADVANCES IN TARGET IDENTIFICATION

Several thorough reviews (78–82) have been published discussingadvances made in improved target identification, and some ofthese approaches are briefly noted here. Target identification canbe the most challenging, time-consuming aspect of small-mole-cule discovery programs. As noted in the discussion of pimocide,one of the major challenges is that small molecules may alter tar-gets other than the one of interest. The potential for this must beconsidered during target identification.

In this review, we have noted several approaches that have been

used to characterize the molecules discussed. Generally, these canbe classified into three categories: genetic, proteomic, and chem-ical. Genetic approaches include comparing transcriptomes oftreated and untreated samples using microarray analysis (83, 84)or transcriptome sequencing (RNA-seq) (85), sequencing a resis-tant mutant (86–88), and error-prone PCR-based target identifi-cation (89). Proteomic methods include comparing the pro-teomes of treated and untreated samples using affinity pulldownanalysis (90), stable isotope labeling by amino acids in cell culture(SILAC) (91, 92), or isobaric tags for relative and absolute quan-tification (iTRAQ) (93, 94). Finally, chemical approaches, includ-ing click chemistry (95–97) and synergy (98–100), are also becom-ing fruitful for identifying molecular targets, as is in silico chemicalmodeling (101, 102).

CONCLUSIONS

Chemical genetics can uncover small-molecule inhibitors affect-ing a range of targets important for pathogenesis, including fac-tors involved in virulence regulation and host-pathogen interac-tions (Fig. 1). This review has covered molecules that are excellentchemical probes for studying molecular mechanisms in living cellsand that have potential as therapeutic leads, with various degreesof pathogen specificity and minimal effects on the microbiota. Wehave also tried to illustrate common strategies used in small-mol-ecule screening (outlined in Fig. 2), for example, the use of large,diverse chemical libraries or of smaller libraries of compoundswhose biological targets and mechanisms of action are unknown.Each approach can lead to important new discoveries. For thera-peutic lead development, however, the use of screens of previouslystudied compounds (sometimes termed “repurposing” [103]) hasthe advantage of reducing the costs, risks, and time associated withdrug development. We have covered how secondary and tertiaryscreens can help eliminate false hits and improve the specificity,efficacy, and toxicity of a given compound.

The power and appeal of using chemical genetics—such as inthe examples we have covered—are in how it supports both basicand translational research, answering questions about host-pathogen biology and providing potential therapeutic lead com-pounds to combat the increasing threat of pathogens resistant totraditional antibiotics.

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