FEATURE

New Strategies for Antibacterial Drug Discovery Targeting Nonmultiplying Bacteria1

Executive summaryDuring the past two decades, the number of new antibacterial drugs entering the market each year has declined, while bacterialresistance to existing drugs has increased. Consequently, new antibacterials are urgently needed. Antibacterial drug candidates arenormally identified by screening libraries of natural, recombinant or chemically synthesized compounds against actively dividingbacteria. However, as well as replicating cells, clinically important bacterial infections also contain a population of latent, nonmultiplyingbacteria that are tolerant of existing antibacterial drugs. This means that long courses of antibacterial therapy are normally required,increasing the risk of resistant bacteria emerging. An emerging approach to antibacterial drug discovery is the identification of novelcompounds active against nonmultiplying bacteria, which have the potential to shorten the duration of therapy and reduce theemergence of antibacterial resistance.However, there is much to learn about nonmultiplying bacteria, particularly the mechanisms that lie behind their profound tolerance ofcurrent antibacterials. New terminology has been proposed for susceptibility tests for antibacterial agents against nonmultiplyingbacteria, namely: the minimum stationary-cidal concentration and the minimum dormicidal concentration, which are defined as theminimum concentrations of drug that will kill stationary-phase (nondividing) and dormant bacteria, respectively. The relationshipbetween the antibiotic susceptibility of stationary and rapidly dividing (logarithmic-phase) bacteria is the stationary/logarithmic ratio.This terminology is suitable for both planktonic and biofilm cultures. In the future, an increasing number of new compounds mayemerge from screening against nonmultiplying bacteria.

have become resistant to amoxicillin, and up to 60% ofAntibacterial drugs comprise a major segment of theclinically isolated strains of Staphylococcus aureus havepharmaceutical industry. The top four antibacterials inbecome resistant to methicillin, giving rise toterms of worldwide sales are Augmentin® (amoxicillin/methicillin-resistant S. aureus (MRSA). Streptococcusclavulanic acid; GlaxoSmithKline), Klacid®

pneumoniae is now often resistant to penicillin (18%) and(clarithromycin; Abbott Laboratories), Zithromax®

to macrolides (25%), and 30% of Pseudomonas(azithromycin; Pfizer) and Levaquin® (levofloxacin;aeruginosa isolates are resistant to quinolones in someJohnson & Johnson), all with sales in excess ofcountries.$US1 billion in the year to March 2005 (table I). The four

main classes of antibacterials are the penicillins, theBig Pharma bugs outcefalosporins, the macrolides and the fluoroquinolones.

Unfortunately, resistance to an antibacterial agent develops Because of the emergence of bacterial resistance, drugquickly after approval of the drug for clinical use. During treatment for many infections will become useless unlessthe past 15–20 years, up to 56% of Escherichia coli the pharmaceutical industry can produce a constant flowstrains isolated from patients with bloodstream infections of effective new agents. However, many large

Table I. Global top 10 antibacterial drugs in year ending March 2005Drug Brandname Company Sales ($US million)

Azithromycin Zithromax Pfizer 2002

Amoxicillin/clavulanic acid Augmentin GlaxoSmithKline 1972

Levofloxacin Levaquin Johnson & Johnson 1551

Clarithromycin Klacid Abbott Laboratories 1495

Ciprofloxacin Cipro, Ciproxin Bayer 878

Amoxicillin Amoxil GlaxoSmithKline 781

Cefdinir Cefzon, Omnicef Astellas Pharma 551

Cefuroxime axetil Zinnat GlaxoSmithKline 499

Moxifloxacin Avelox Bayer 382

Ceftriaxone Rocephin Roche 343

1 The full text of this article was published in Drugs in R&D 2006; 7 (3): 133-151. The original article was written by Anthony Coates andYanmin Hu from the Department of Cellular and Molecular Medicine, Medical Microbiology, Centre for Infection, St George’s University ofLondon, UK.

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pharmaceutical companies have discontinued their Wyeth, a member of the new class of glycylcyclines,antibacterial drug-discovery and development programs. entered the US market. In addition, there are no classes ofEli Lilly, Bristol-Myers Squibb and Wyeth have all new agents in clinical development for multidrug-resistantwithdrawn from antibacterial research and development. Gram-negative organisms, especially P. aeruginosa andBayer plans to spin off its anti-infective research and Acinetobacter spp.Roche has already spun off its dihydrofolate pipeline intoArpida and its cefalosporin pipeline into Basilea

With the emergence of bacterialPharmaceutica. Sanofi-Aventis has spun off itsresistance, drug treatment for manyantimicrobial pipeline into Novexel. A major reason for

infections will become useless unless thethe departure of large companies is the perception thatpharma industry can produce a constantprofits from antibacterials are not worth the effort. Other

flow of effective new agents; however,factors include:many large pharma companies have• the failure of the genomics approach to lead to adiscontinued their antibacterial R&Dplethora of new marketed antibacterials;

programs.• increasing regulatory requirements leading to largerdrug trials;

Current methods of antibacterial• a decrease in the discovery rate of new broad-spectrumdrug discoveryantibacterials with novel mechanisms of action;

• the clinical preference for narrow- rather than broad- The sources of compounds for antibacterial drug-spectrum compounds; discovery screening programs are diverse. Almost all

• the risk of restrictions on the use of new antimicrobials existing antibacterials are natural compounds or are(for example, pressure to ‘keep it in reserve’, and derived from such agents. Pharmaceutical companiesquestions about the efficacy of antibacterials in create derivatives of natural compounds, screen them forcommon clinical diseases such as otitis media and antibacterial activity, and develop a family of newbronchitis); antibacterials. Libraries of natural compounds from

• and the limited effective life of an antibacterial because bacteria, fungi, plants and even higher animals are used toof the emergence of antibacterial resistance. select early leads from which analogs are derived.

Chemical synthesis, recombinant DNA technology andFortunately, several large pharmaceutical companies arefermentation are used to produce new compounds.still active in the field. GlaxoSmithKline has at least twoLibraries of synthetic molecules are also available assystemic antibacterials from different new classes in latestarting material. Combinatorial chemistry can be used topreclinical/early clinical development. Johnson & Johnsonproduce libraries containing large numbers of derivativehas entered into a global collaboration with Basilea forcompounds.ceftobiprole, and added doripenem to its pipeline via the

acquisition of Peninsula Pharma in June 2005. Pfizer Genomics is a recent advance in which a mixture ofsuccessfully bid for Vicuron Pharmaceuticals and gained methodologies is used to select molecular targets such asthe late-stage compound dalbavancin, a glycopeptide bacterial enzymes, determine their structure, and createlincosamide in late preclinical development, and new- compounds that will inhibit the enzymatic function of thegeneration oxazolidinones from Pharmacia. Merck & target. The active compounds are then screened againstCo. and AstraZeneca also remain in the antibacterial whole bacterial cells in order to determine theirarena. However, Novartis is the only large pharmaceutical antibacterial activity. So far, no genomics-derivedcompany to have entered the antibacterial research and antibacterial compounds have reached the market, and thedevelopment field in recent years. technology has not proved as productive as initially

predicted.Overall, however, not enough new antibacterials arebeing produced to stem losses arising from the tide of

Targeting nonmultiplying bacteria – aantibacterial resistance. The number of new antibacterialsnew paradigmreaching the market has fallen by over 50% in the last 20

years, while levels of antibacterial resistance among Regardless of the source of compound, all existingcommon pathogens are increasing. In fact, no new antibacterials have been selected by screening againstantibacterials were launched in 2004, and only one – bacteria that are actively growing (logarithmic-phasedaptomycin from Cubist Pharmaceuticals – was bacterial cultures). However, nonmultiplying bacteria arelaunched in 2003. In 2005, Tygacil® (tigecycline) from not killed effectively by the majority of current

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antibacterials. In other words, although existing Why are nonmultiplying bacteria so resistantto current antibacterials?antibacterials can kill logarithmic-phase bacteria, they only

prevent the growth of nonmultiplying bacteria. TheWhile existing antibacterials kill multiplying bacteria,identification of compounds that can kill nonmultiplying

persisters tolerant to antibacterials survive in abacteria is a fundamentally new approach to antibacterialnonmultiplying state. Persisters appear to be heterogenousdrug discovery and development that is still in thein their rates of multiplication and metabolism, may bepreclinical stage.nutritionally starved, and may be at a high cell density.Biofilms and stationary-phase planktonic cultures often

The rationale for targetinghave one or more of these characteristics. There appear to

nonmultiplying bacteria be many different mechanisms by which nonmultiplyingbacteria can tolerate antibacterials. Although an impaired

Most clinically symptomatic bacterial infections contain entry of antibacterials into biofilms has occasionally beenboth multiplying and nonmultiplying bacteria. In fact, observed, this phenomenon is relatively uncommon. β-nonmultiplying bacteria that live in a dispersed Lactamase enzymes that destroy β-lactam antibacterialsenvironment or in biofilms are predominant in over 60% are thought to accumulate in biofilms, and extracellularof clinically important infections requiring treatment in slime produced by S. epidermidis counteracts glycopeptidedeveloped countries. Nonmultiplying and slowly growing antibacterials. However, overall, the mechanisms ofbacteria exist in many forms in nature, including biofilms, antibacterial tolerance in nonmultiplying bacteria are stillplanktonic phase, latent and dormant states. The poorly understood.importance of nonmultiplying bacteria, regardless ofwhether they are dispersed or concentrated in a biofilm, isthat they are very tolerant of current antibacterials. Hence,

The identification of compounds that canlong-term therapy is required and treatment failures arekill nonmultiplying bacteria is acommon. For example, in a patient with a sore throat,fundamentally new approach toantibacterial treatment must be given for 7–10 days inantibacterial drug discovery andorder to kill fast-growing bacteria (which are rapidly

development and still in the preclinicalkilled), but also to slowly reduce the number ofstage.nonmultiplying bacteria until clinical remission is

achieved.

If the duration of antibacterial therapy is too short,Is it possible to develop new antibacterials

although fast-growing bacteria are killed, the slow- that kill nonmultiplying bacteria?growing ones remain and can spin off fast-growingorganisms, resulting in an increase in the number of

Since existing antibacterials are relatively inactivebacteria and a return of clinical symptoms. However,

against nonmultiplying bacteria, it could be argued that itlong-term treatment can result in the emergence of is impossible to kill nonmultiplying bacteria. However,antibacterial resistance. Consequently, rapid eradication of this may be a misconception, since all currentall bacteria – not just multiplying cells – is considered an antibacterials were developed against actively growingimportant aim of antibacterial therapy, to not only enhance bacteria and none was specifically selected for activityclinical outcome but also to minimize the emergence of against nonmultiplying bacteria. Therefore, we really doresistant strains. Unfortunately, tolerance of not know yet whether it is feasible to create newnonmultiplying cells to current antibacterials is seen in antibacterials that effectively kill nonmultiplying bacteria.most bacterial species. For instance, carbenicillin, The available data suggest that some existingofloxacin and tobramycin kill multiplying P. aeruginosa, antibacterials are better than others at killingbut are relatively inactive against nonmultiplying bacteria. nonmultiplying bacteria. Furthermore, slowly multiplyingNonmultiplying Haemophilus influenzae is killed less or nonmultiplying bacteria are metabolically active, albeitrapidly by antibacterials than multiplying bacteria. at a lower level than logarithmic-phase organisms, whichDaptomycin and vancomycin have higher kill rates against suggests that it may be possible to develop novelexponentially growing S. aureus than stationary-phase compounds that will interfere with the metabolism of

these nonmultipliers.organisms.

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Approaches to developing compounds their remaining colony-forming units can be assessed.Assessment of antibacterial activity against dormantagainst nonmultiplying bacteriabacteria can be achieved by measuring the minimumdormicidal concentration (MDC), which is defined as theThe current lack of detailed knowledge about theminimum concentration that results in complete kill of themechanisms of antibacterial tolerance in nonmultiplyingdormant bacteria. Since most antibacterials fail to killbacteria means a genomic approach to drug discovery is100% of dormant bacteria, the concentrations of drug thatnot feasible. In addition, structure-activity relationships arewill kill 50% (MDC50) or 99% (MDC99) are useful.not available for any drug against nonmultipliers, mainly

because the majority of existing drugs have poor activityThe stationary/logarithmic ratioagainst them. Thus, whole cell assays are currently the

way forward for drug discovery against nonmultiplyingSome antibacterials, such as quinolones, kill bothbacteria. Assays for M. tuberculosis stationary-phase

stationary-phase and logarithmic-phase bacteria while(Wayne model), for M. tuberculosis and pyogenicothers, such as penicillins, kill logarithmic-phase bacteriabacterial persisters (Hu-Coates model), and for pyogenicbut have no effect against stationary-phase organisms.bacterial biofilms have been developed. These models areCuriously, pyrazinamide is relatively inactive againstwhole bacterial assays that start with the organisms inlogarithmic cells, but kills slowly growing/nonmultiplyingdifferent growth phases. Bacteria may be converted intobacteria. So, in addition to measuring MSC or MDC for anonmultipliers by a reduced oxygen environment,particular antibacterial agent, it would be useful to knowstarvation, or the presence of antibacterials.the relationship between the kills of stationary-phase to

Second, methods for measuring the antibacterial logarithmic-phase. This is called the stationary/logarithmicactivity of a compound against nonmultiplying bacteria ratio (SLR). Clearly, the SLR must be measured atmust be developed. The gold-standard terminology for specific concentrations of antibacterial agent, such as thebacterial susceptibility testing is the minimum inhibitory MSC99 or MDC99, or at lower concentrations such as theconcentration (MIC), which is the lowest concentration of MSC50 or MDC50, which are achievable in serum. Thisa particular compound that inhibits multiplication of the terminology is suitable for both planktonic and biofilmtest organism. Clearly, MIC is unsuitable for cultures. The conditions in which each test is performednonmultiplying bacteria (since it is not possible to must be carefully controlled and equivalence betweenmeasure the inhibition of multiplication of a culture that is laboratories can only be achieved if the same conditionsnot growing). A less widely used measure is the minimum are used in each laboratory. The concentration of bacteria,bactericidal concentration (MBC), defined as the lowest the growth phase of the culture, the methodology of theconcentration of a compound that kills 99.9% of a test and the medium in which the test is performed are alllogarithmic-phase test organism overnight. In practice, critically important.neither MIC nor MBC can be used to assay the activity ofa compound against nonmultiplying bacteria. Therefore, Conclusionnew terminology for susceptibility testing of antimicrobialagents against nonmultiplying organisms is required.

The decline in number of new antibacterials enteringFor stationary-phase organisms, the test should be the market during the past 20 years is worrying in light of

capable of measuring small anti-nonmultiplier activity. For the rise in bacterial resistance against many current drugs.this purpose, the minimum stationary-cidal concentration New antibacterials are urgently required but although(MSC) has been proposed; this is defined as the minimum some sectors of the pharmaceutical industry areconcentration of compound that kills 100% of a culture of responding to this need, many large pharmaceuticalstationary-phase bacteria. Given that most current companies have departed the field. The genomic approachantibacterials will not kill 100% of the culture, the to discovering novel antibacterials has been disappointingconcentrations of compound that will kill 50% (MSC50) or so far. Targeting nonmultiplying bacteria is a new strategy99% (MSC99) are useful. Some dormant bacteria such as for drug discovery and development that could produceMicrococcus luteus and M. tuberculosis cannot be new drugs that can be administered over shorter periods ofcultivated on solid media and need resuscitation before time and thus may reduce the development of resistance.

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