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Tuberculosis Drug Development: History and Evolution of the Mechanism-Based Paradigm

Sumit Chakraborty1,2 and Kyu Y. Rhee1,2

1Division of Infectious Diseases, Department of Medicine, Weill Cornell Medical College, New York, New York 10021 2Department of Microbiology and Immunology, Weill Cornell Medical College, New York, New York 10021 Correspondence: [email protected]

Modern tuberculosis (TB) chemotherapy is widely viewed as a crowning triumph of anti- infectives research. However, only one new TB drug has entered clinical practice in the past 40 years while drug resistance threatens to further destabilize the pandemic. Here, we review a brief history of TB drug development, focusing on the evolution of mechanism(s)-of-action studies and key conceptual barriers to rational, mechanism-based drugs.

istory recounts streptomycin and para- 2014). However, mounting evidence has impli- Haminosalicylic acid (PAS) among the first cated a growing inadequacy of existing tools and clinical antibiotics developed. Both showed ac- approaches (Nathan 2004; Nathan et al. 2008). tivity against Mycobacterium tuberculosis (Mtb) Although technologically advanced, anti-infec- and were followed in rapid succession by iso- tives research remains rooted in the same empir- niazid, pyrazinamide, cycloserine, ethionamide, ical paradigm that first gave rise to penicillin. ethambutol, and rifampicin, among others While this paradigm was initially reinforced by (Barry 2011; Zumla et al. 2013). Together, these its delivery of all major classes of clinical antibi- advances transformed tuberculosis (TB) from a otics, its productivity has since waned. We be- predictably fatal disease to a curable disease. lieve that this early success fostered an unintend-

www.perspectivesinmedicine.org However, current TB chemotherapies remain ed dissociation between our ability to develop far from optimal. TB remains the leading bacte- antibiotics and understanding of their mecha- rial cause of deaths worldwide, and drug resis- nisms. Rational, or mechanism-based, para- tance has emerged as a problem of singular im- digms have thus lagged behind. portance (Nathan 2009; Russell et al. 2010; Here, we review existing knowledge of the Raviglione et al. 2012). mechanisms of current TB drugs and emerging Somewhat unexpectedly, efforts to restock experimental agents. Wefocus specificallyon (1) this once celebrated medicine chest have fal- the frontline agents isoniazid, pyrazinamide, tered. The causes for this are multifactorial ethambutol, and rifampicin, because of their (IDSA 2004; Payne et al. 2007; Zumla et al. clinical importance and legacy in the history of

Editors: Stefan H.E. Kaufmann, Eric J. Rubin, and Alimuddin Zumla Additional Perspectives on Tuberculosis available at www.perspectivesinmedicine.org Copyright # 2015 Cold Spring Harbor Laboratory Press; all rights reserved Advanced Online Article. Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a021147

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S. Chakraborty and K.Y. Rhee

anti-infectives development and (2) bedaqui- drug-resistant (MDR) and extensively drug-re- line because of its role as the first new clinically sistant (XDR) epidemic. Historical details of its approved TB drug developed in the past 40 years. discovery and studies around its antimycobac- However, rather than providing an exhaustive terial activities, however, are probably far less recitation of the literature, which has been ex- well appreciated. tensively cataloged elsewhere (Zhang 2005; Bar- INH was first synthesized in 1912. However, ry 2011; Zumla et al. 2013), we review key find- its antitubercular activity was only later discov- ingsthat informed ourcurrent understanding of ered en route to the synthesis of amithiozone or drug mechanisms and the historical contexts in thioacetazone (p-acetaminobenzaldehyde thio- which they were discovered. By doing so, we semicarbazone), a pyridine-based thiosemicar- highlight recurrent themes that have guided bazone that was being developed in an effort to past and current TB drug development efforts improve on the tuberculostatic activity of sulfa- and yet unaddressed barriers in the path toward thiazole, first reported by Domagk (Fox 1952; more rational mechanism-based approaches. Long 1958). Whereas amithiozone was found to be tuberculostatic in vitro and therapeutically abandoned due to untoward gastrointestinal, HISTORICAL PERSPECTIVES ON TB DRUG allergic, and hematologic toxicities in patients, DEVELOPMENT INH was found to be vastly superior to all other antitubercular compounds tested in vitro and in Isoniazid (INH) animals at the time, including closely related Isoniazid (INH) (isonicotinic acid hydrazide) nicotinic acid and hydrazine derivatives (Fig. (Fig. 1(1)) is arguably among the most clinically 1). In addition to its potency, INH was found successful and extensively studied TB drugs ever to exhibit remarkable selectivity for mycobacte- developed (Vilcheze and Jacobs 2007). Howev- ria, with activityagainst strains resistant to strep- er, although once synonymous with TB chemo- tomycin, PAS, and amithiozone (Long 1958). therapy and prophylaxis, INH has paradoxically Numerous in vitro studies soon showed that become a defining feature of the current multi- INH exhibited activity against Mtb only when

Compounds

O H O N NH2 O OH O HO OH O OH OH N H O www.perspectivesinmedicine.org NH2 N NH N H NN N O N O OH N O (1) (2) (3) (4)

Br

N

OH N

O

(5)

Figure 1. Antitubercular compounds.

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TB Drug Development

replicating and that this activity was preceded showed that most INH resistance was accompa- by a highly reproducible, although still unex- nied by defects in catalase activity (Middlebrook plained, delay of approximately a generation 1954). Saroja and Gopinathan subsequently and a half (1–4 d duration). During this period showed that INH sensitivity could be restored (the initial 16–24 h of exposure), it was noted on transfer of a genetic locus from an INH- that INH-susceptible bacilli accumulated 14C- sensitive strain of Mycobacterium smegmatis labeled INH that could not be washed away encoding a catalase-like activity (manifest, at while resistant or dead bacilli did not, and this the time, as resistance to hydrogen peroxide) fixation was accompanied by a loss of its acid (Saroja and Gopinathan 1973). Together, these fastness and morphologic changes such as sur- findings introduced the concept that INH might face wrinkling and bulging (vide supra). function as a prodrug. Work by Zhang and col- Concurrent studies by Chorine (1945) ser- leagues later extended these findings to Mtb, endipitously reported that the structurally re- with the discovery of the katG-encoded cata- lated pyridine, nicotinamide, exhibited activity lase-peroxidase, which was found to harbor against Mtb in vitro and in infected guinea pigs, point mutations and deletions in INH-resistant prompting a litany of studies examining the clinical isolates and shown to be sufficient to potential antimetabolic activity of INH (Cho- restore INH susceptibility in resistant strains. rine 1945; Fox 1952; Long 1958; Vilcheze and Follow-up biochemical studies showed that Jacobs 2007). These studies revealed reductions KatG activates INH by conversion to a range of in NAD levels in INH-treated bacilli but failed chemically reactive intermediates, including to deliver evidence of its direct misincorpora- an isonicotinoyl radical (Johnsson and Schultz tion into NAD, whereas the more closely related 1994; Wilming and Johnsson 1999; Lei et al. species nicotinic acid was found to exhibit only 2000), first hypothesized by Winder (1960). Of weak in vitro activity and no activity in guinea interest, INH was biochemically also shown to pigs. Notwithstanding, INH was found to in- be a potent inhibitor of KatG (Marcinkeviciene duce numerous changes in the biochemical et al. 1995). composition and activity of Mtb. These includ- Adopting a similar approach, Jacobs and ed selective effects on its extractable lipids, ac- colleagues isolated and genetically characterized cumulations of soluble disaccharide trehalose mutants of M. smegmatis resistant to INH and and hexose phosphates, and reductions in respi- ethionamide (a closely related analog) but ex- ratory activity. Pope further showed that the hibiting wild-type levels of catalase activity and activity of INH could be antagonized by the ad- susceptibility to other antitubercular com- dition of pyridoxine (a precursor of the pyri- pounds (Banerjee et al. 1994). These studies www.perspectivesinmedicine.org doxal phosphate cofactor required for many identified a gene named inhA, that showed decarboxylation and transamination reactions) .40% identity to proteins involved in fatty and the a-ketoacids, a-ketoglutarate and pyru- acid biosynthesis in Escherichia coli and Salmo- vate. Although still unexplained, this antago- nella typhimurium. These mutants were found nism was particularly remarkable as pyridoxine to harbor a missense mutation (S94A) that con- also appeared to paradoxically resensitize resis- ferred an order of magnitude increase in resis- tant strainsto INH (Boone and Woodward1953; tance but could be corrected on allelic exchange Pope 1953, 1956; Long 1958). with a wild-type copy of the inhA gene. High- Serendipity aside, progress toward a func- level resistance could conversely be achieved by tional understanding of the mechanism of ac- overexpressing the wild-type inhA gene on a tion of INH was catalyzed by INH-resistant iso- multicopy plasmid. Biochemical studies showed lates that were recovered in vitro and in vivo, that InhA was a NADH-dependent enoyl-ACP either following treatment of experimentally in- (acyl carrier protein) reductase of the fatty acid fected animals or from the sputum of TB pa- synthase type II (FASII) system (Dessen et al. tients, almost immediately after the discovery of 1995; Quemard et al. 1995; Marrakchi et al. INH itself. Work by Middlebrook specifically 2000), involved in mycolic acid biosynthesis.

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Moreover, these studies showed that INH could and/or contribute to the remarkable whole-cell inhibit InhA activity in cell-free lysates but that potency of INH itself, however, remains to be this inhibition was reduced in inhA mutant elucidated. strains. Using methods of specialized genetic trans- Pyrazinamide (PZA) duction, Jacobs et al. finally showed that the S94A mutation was sufficient to confer coresist- As previously described, pyrazinamide (PZA) ance to both the phenotypic and mycolic acid (Fig. 1(2)) emerged as an outgrowth of research inhibitory activities of INH and ETH at clinical- conducted by Vital Chorine who discovered the ly relevant levels of INH (Vilcheze et al. 2006). ability of subcutaneous nicotinamide to pro- Together, these findings helped elucidate the long survival of Mtb-infected guinea pigs (Cho- significance of (1) decades-long observations rine 1945). Motivated by this finding, PZA reporting INH’s effects on acid-fastness (Barclay emerged from Lederle Laboratories of Ameri- et al. 1954; Koch-Weser et al. 1955), which was can Cynamid as the most active pyrazine analog later found to be caused by the unique dye-bind- of nicotinamide tested in mice (Malone et al. ing properties of the mycolic acids covalently 1952). Somewhat unexpectedly, however, PZA linked to the 50-hydroxyl groups of the arabino- was found to lack activity under typical in vitro galactan polymer of the mycobacterial cell wall culture conditions, instead requiring incuba- (McNeil et al. 1991); (2) the demonstration that tion at an acidic pH (e.g., 5.5) similar to that isoniazid inhibited the synthesis of mycolic acids associated with active inflammation (McDer- in Mtb (Winder and Collins 1970; Winder et al. mott and Tompsett1954). However, even under 1970) and its correlation with viability (Ta- such conditions, PZA showed only bacterio- kayama et al. 1972); and (3) the specific inhibi- static activity with minimum growth inhibitory tory effect of isoniazid on the synthesis of satu- concentrations (MIC) ranging from 6 to 50 mg rated fatty acids greater than 26 carbons (Wang mL21 and minimal bactericidal concentrations and Takayama 1972; Takayama et al. 1975; Da- (MBC) of .1000 mgmL21 (Zhang and Mitch- vidson and Takayama 1979). ison 2003). This dissociation was further em- Building on this work, structural studies of phasized by the subsequent discovery of PZA’s the orthologous enoyl reductase of E. coli re- unique treatment shortening (or sterilizing) ac- vealed the presence of a covalent NAD inhibitor tivity in animals and patients at achieved serum species tightly bound to the same proposed re- concentrations generally at or below its in vitro gion associated with INH resistance in InhA MICs (Ellard 1969). Studies of PZA’s mecha- (Baldock et al. 1996). These studies paved the nism(s) of action have thus been hindered by www.perspectivesinmedicine.org way for the unifying discovery by Rozwarski fundamental limitations in the ability to faith- et al. (1998) that INH did not bind directly to fully model its therapeutic activities in vitro. InhA but, instead, as a covalent adduct with Limitations notwithstanding, studies of NAD. This INH-NAD species was shown to PZA’s mechanism(s) of action, like INH, were act as a potent, long residence time inhibitor driven by the in vitro characterization of PZA- of InhA (Lei et al. 2000; Nguyen et al. 2002; resistant strains. McDermott and colleagues Rawat et al. 2003; Vilcheze et al. 2006). The showed that, like INH, PZA is a prodrug but current working model of the INH mechanism activated by an amidase, later found to be en- thus proposes that INH is a prodrug activated coded by the gene pncA, whose ortholog in the by the catalase-peroxidase KatG which gives rise closely related and naturally PZA-resistant vac- to a diverse array of INH-derived radicals and cine strain Mycobacterium bovis BCG (Bacillus adducts, some of which are capable of killing Calmette–Gue´rin) was found to harbor an in- Mtb by potently inhibiting its ability to synthe- activatingmissensemutation(Konnoetal.1967; size mycolic acids. The identities, quantities, Scorpio and Zhang 1996). Moreover, transfor- andextenttowhichadditionalINH-derivedspe- mation of BCG or pncA-defective Mtb strains cies produced by KatG may also synergize with was shown to restore or confer in vitro suscept-

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TB Drug Development

ibility to PZA (Scorpio et al. 1997; Zhang et al. mycobacteria but active only against replicat- 1999; Boshoff and Mizrahi 2000). Curiously, ing bacilli, where it was found to impair glycerol mutations associated with PZA resistance apart metabolism as well as RNA synthesis (Forbes from those mapping to pncA have been only et al. 1962, 1965; Kuck et al. 1963). Subsequent rarelydescribed, and attemptsto isolate mutants biochemical studies (isotopic-labeling studies resistant to POA have been unsuccessful. Me- and analysis of cellular sugar content) showed ticulous biochemical studies by Zhang and that EMB induced an almost immediate accu- colleagues nonetheless showed that in vitro sus- mulation of the major intermediate of arabino- ceptibility to PZA was associated with an in- galactan biosynthesis, b-D-arabinofuranosyl-1- tracellular accumulation of its activated form, monophosphodecaprenol, followed by the se- pyrazinoic acid (POA), as revealed by high rates quential accumulation of trehalose mono- and of POA efflux and the natural resistance of the dimycolates and inhibition of mycolic acid in- amidase-proficient mycobacterial species, M. corporation into the cell wall (Takayama et al. smegmatis (Zhang et al. 1999). This accumula- 1979; Kilburn et al. 1981; Takayama and Kil- tion was associated with a dissipation of myco- burn 1989; Mikusova et al. 1995; Blanchard bacterial membrane potential, although the 1996; Goude et al. 2009). These findings sug- causal significance of this effect remains unre- gested that the primary inhibitory effect of EMB solved (Zhang et al. 2003). More recent affinity- lay at the polymerization of the mycobacterial based studies of POA identified several addi- cell wall arabinan. tional high-affinity binding targets. Prominent As for the case with INH and PZA, this pre- among these was a subunit of the 30S ribosome, diction was genetically confirmed with the in named RpsA, which plays a critical role in a spe- vitro generation of EMB-resistant strains and cialized process termed trans-translation which characterization of EMB-resistant clinical iso- involves the rescue of stalled ribosomes and has lates, both of which were found to map to a been shown to be critical for stress survival and cluster of genes (embCAB) associated with ara- virulence of other pathogens (Shi et al. 2011b). binogalactan biosynthesis (Belanger et al. 1996; Reflecting the uncertain fidelity of current in Telentiet al. 1997). Curiously, however, evidence vitro models, however, it remains unclear which, of a specific molecular target remains lacking as if any, of the foregoing findings pertain to the even the most common mutations associated unique sterilizing activity of PZA. with EMB resistance (such as EmbB306) have been also found in clinically susceptible isolates, whereas both EmbB and EmbC encoded arabi- Ethambutol (EMB) nosyltransferases are predicted integral mem- www.perspectivesinmedicine.org Like PZA, ethambutol (2,20 ethylenediimino- brane proteins that have proven refractory to di-1-butanol) (Fig. 1(3)) was first discovered biochemical study (Safi et al. 2008; Srivastava at Lederle Laboratories of American Cynamid et al. 2009; Shi et al. 2011a; Guerrero et al. 2013). and tested immediately in animals following the Studies of EMB resistance have proven similarly discovery of its remarkable stereospecific activ- challenging to interpret in that high-level resis- ity (Thomas et al. 1961; Shepherd et al. 1966). tance, which had been traditionally used as a Comparison of all stereoisomers specifically marker of potential primary targets, was only showed that the dextro (S,S) form was 12 times observed in the setting of multiple mutations more active than the meso form, whereas the (Safi et al. 2013). levo form was entirely inactive, raising the pos- Primary target aside, early work by Peets sibility of a discrete macromolecular target and colleagues showed that the growth inhibi- (Thomas et al. 1961). However, its small size tory activity of, and recovery from, EMB treat- and simple structure provided few clues. ment could be specifically ameliorated (and Early biochemical studies nonetheless often accompanied by MIC shifts of as much showed that ethambutol (EMB) was rapidly as 16-fold) by addition of either the polyamine taken up by both replicating and nonreplicating spermidine or magnesium (but not amino ac-

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ids, nucleotides, Zn, or Mn) (Forbes et al. 1965). unit of RNA polymerase responsible for rifam- More precise understanding of both its primary picin binding (Levin and Hatfull 1993; Telenti and downstream effects thus awaits further in- et al. 1993). vestigation. Bedaquiline (BDQ) Rifampicin Apart from being the only clinical drug to be Rifampicin (Fig. 1(4)) is a semisynthetic deriv- approved for the treatment of TB in .40 yr, ative of a natural product ansamycin, which was bedaquiline (BDQ) (previously known as originally discovered as an antimicrobial activ- TMC207 and R207910) (Fig. 1(5)) is also the ity in the conditioned media of the soil bacte- first FDA-approved drug to have been devel- rium Nocardia mediterranei termed rifamycins oped in the modern era of molecular sciences (named after a popular French movie about (Cohen 2013). BDQ was discovered in a high- jewel heists). Remarkably, the only component throughput phenotypic screen for compounds of this extract (termed rifamycin B) that could active against the saprophytic mycobacteria, be isolated in pure crystalline form was a minor M. smegmatis, and subsequently shown to dem- (5%210%) species with comparatively weak onstrate activity against M. bovis BCG and Mtb activity that was serendipitously noted to gain (Andries et al. 2005). Early SAR studies of close- activity on incubation in oxygenated aqueous ly related congeners revealed its activity was solution. Detailed chemical studies soonshowed stereoselective, such that the (R,S) stereoisomer that this activation was due to a reversible oxi- was the most potent with MICs against Mtb dation of a “quinonoid-like” core followed by ranging from 4 to 70 ng/mL, whereas the hydrolytic loss of a glycolic acid moiety, giving (S,R) isomer exhibited MICs of 0.35–8.8 mg/ rise to the far more active rifamycins S and SV mL (Andries et al. 2005; Koul et al. 2007). This (the latter of which was found to show potent stereoselectivity suggested the existence of a activity against Mtb). The discovery of this specific binding target. Remarkably, BDQ was structure-activity relationship (SAR) provided also discovered to also show activity against the first evidence for a potentially specific bio- hypoxic, nonreplicating Mtb (Rao et al. 2008). logic mode of action that, in conjunction with Apart from its potency, BDQ was also found to the poor bioavailability of rifamycin SV, moti- show an unusually slow time-dependent killing vated a heroic chemical campaign to develop a such that only bacteriostatic activity was ob- more potent and orally bioavailable compound. served during the first 3–4 d but was then fol- These efforts culminated in the introduction lowed by a bactericidal phase with a 4 log10 re- www.perspectivesinmedicine.org of rifampicin in the 1960s with detailed knowl- duction in measurable colony-forming units by edge of key molecular determinants of its ac- day 14 (Koul et al. 2014). tivity (e.g., ansa chain C21 and C23 hydroxyl Insight into the functional target of BDQ groups and C1 and C8 phenols among others) first emerged with the generation of resistant (Sensi 1983; Marriner et al. 2011). mutants followed by whole-genome resequenc- Armed with this knowledge, concurrent ing. These studies identified mutations in the studies of protein and nucleic acid biosynthesis atpE gene encoding the membrane bound c revealed that rifampicin specifically inhibited subunit of F0ATP synthase, which were inde- RNA synthesis and, with the identification of pendently confirmed through comparative se- RNA polymerase, its molecular mode of inhi- quence analyses of naturally BDQ-susceptible bition (Wehrli and Staehelin 1971). The study and -resistant mycobacteria, and BDQ-selected of rifampicin-resistant mutants later confirmed resistant Mtb strains (Andries et al. 2005; Hui- that this inhibition, in fact, accounted for its tric et al. 2007, 2010). Interestingly, sequence antitubercular activity as .96% of all resistance comparisons to the orthologous mitochondrial mutations could be mapped to an 81-nucleo- ATP synthase revealed an alanine-to-methio- tide region in the coding sequence of the b sub- nine substitution at position 63 that was hy-

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TB Drug Development

pothesized to explain the apparent 20,000-fold functions whose dysregulation affected activity. selectivity of binding to the Mtb over mamma- Although functionally powerful for its genome- lian enzyme (Haagsma et al. 2009). Biochemical wide scope, this shift had the unintended con- studies nonetheless showed accompanying re- sequence of conceptually constraining drug ac- ductions in bacterial ATP levels, lending or- tivity to a single best target and mechanism of thogonal evidence for the role of ATP synthase action because of its experimental proclivity to in its mechanism (Andries et al. 2005; Koul et al. select for and focus on isolated genes and/or 2007, 2014; Rao et al. 2008). Affinity purifica- proteins. tion-based efforts to identify binding partners Defined by their ability to suppress or kill (using an amine analog of BDQ immobilized to bacteria, most antibiotics act through the inhi- a sepharose resin) resulted in the identification bition of biochemical networks, and elicit mal- of a and b subunits of ATP synthase from the adaptive phenotypes that, although experimen- total membrane fraction of M. smegmatis, al- tally simple to assay, are no less complex than though the c subunit was not recovered, perhaps their adaptive physiologic counterparts. Their because of its high hydrophobicity (Koul et al. mechanisms of action are thus likely, if not cer- 2007). Interestingly, however, a recent study of tain, to be mediated through an equally com- resistant mutants from clinical isolates showed plex array of factors, whose identities, quantita- that 38% of mutations were unrelated to the tive contributions, and interactions have only ATPsynthase operon (Huitric et al. 2010). Thus, been partially elucidated. although the available data suggests that BDQ Looking ahead, it is encouraging to see targets ATP synthase, it is possible that addi- growing recognition of the complexity of anti- tional targets remain to be identified. biotic action and the advent of new level tech- nologies and disciplines to help respond to this challenge. One current area of potential prom- RETROSPECTIVE VIEWS ON ise is the rapidly emerging field of metabolo- MECHANISM-BASED STUDIES mics. Metabolomics is the youngest of systems- AND CONCLUSIONS level disciplines, defined as the global study of Looking back, it is interesting to note that as the metabolites in a biological system under a given tools used to develop and study antibiotics have set of conditions (Saghatelian and Cravatt 2005; steadily increased in scale and precision, our van der Werf et al. 2005, 2007; Patti et al. 2012). understanding of their mechanisms has some- Because metabolites are the biochemical fuel what paradoxically shrunken in scope. Indeed, of all physiologic processes, metabolomic ap- early studies of antibiotic mechanisms largely proaches offera global biochemicalwindow into www.perspectivesinmedicine.org consisted in broad phenotypic and biochemical the physiologic composition and state of a given characterizations of antimicrobial activity. The cell. From a pharmacologic point of view, me- advent of genetic tools and approaches, how- tabolomic readouts offer direct biochemical in- ever, fostered a systematic shift toward organ- sights into the intrabacterial “pharmacokinetic” ism-wide, but gene-specific, views of drug ac- fates and “pharmacodynamic” actions of a giv- tivity. This shift was largely driven by the en compound within Mtb (de Carvalho et al. successful use of drug resistance as a functional 2010, 2011; Pethe et al. 2010; Chakraborty et al. “loss-of-function” window into the targets and 2013). Although technologically advanced, cur- mechanisms of INH (InhA), rifampicin (RNA rent antibiotic development efforts rest heavily, polymerase b subunit), and BDQ (ATP syn- if not exclusively, on the use of indirect bacter- thase). This same experimental shift, however, iologic or genetic readouts or in vitro measure- was also accompanied by a limited appreciation ments of enzyme activity as the primary mea- of the only partial intersection between drug sures of compound activity. Although valuable, activity and resistance, and operational shift to- these same tools and readouts have left critical ward more empirical definitions of drug targets ambiguities. For example, in elucidating the and mechanisms based on the genes and/or structure-activity relationship of a given com-

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pound, it is difficult, if not impossible, to deter- Accelerator Program and National Institutes of mine the extent to which the activity of a given Health for funding. compound is mediated by its ability to accu- mulate within a bacterial cell, its biochemical affinity for a specific target, and/or its biotrans- REFERENCES formation into one or more bioactive spe- Andries K, Verhasselt P, Guillemont J, Gohlmann HW, cies. Direct biochemical readouts of a com- Neefs JM, Winkler H, Van Gestel J, Timmerman P, Zhu pound’s intrabacterial “pharmacokinetic” fates M, Lee E, et al. 2005. A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis. Science and “pharmacodynamic” actions thus represent 307: 223–227. a major unmet scientific need of direct phar- Baldock C, Rafferty JB, Sedelnikova SE, Baker PJ, Stuitje AR, macologic relevance. In a recent study, Chakra- Slabas AR, Hawkes TR, Rice DW. 1996. A mechanism of drug action revealed by structural studies of enoyl reduc- borty and colleagues reported one such appli- tase. Science 274: 2107–2110. cation of metabolomic technologies using PAS, Banerjee A, Dubnau E, Quemard A, Balasubramanian V, a close structural analog and competitive in Um KS, Wilson T, Collins D, de Lisle G, Jacobs WR Jr. vitro inhibitor of the folate biosynthetic en- 1994. inhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis. Science 263: zyme dihydropteroate synthase (DHPS) (Chak- 227–230. raborty et al. 2013). Using this platform, the Barclay WR, Koch-WeserD, Ebert RH. 1954. Mode of action investigators showed that, contrary to long- of isoniazid. II. Am Rev Tuberc 70: 784–792. standing inferences, PAS inhibited Mtb by func- Barry CE. 2011. Lessons from seven decades of antitubercu- tioning as a replacement substrate, rather than losis drug discovery. Curr Top Med Chem 11: 1216–1225. Belanger AE, Besra GS, Ford ME, Mikusova K, Belisle JT, inhibitor, that, in turn, gave rise to dysfunc- Brennan PJ, Inamine JM. 1996. The embAB genes of My- tional folate analogs. In contrast, far more po- cobacterium avium encode an arabinosyl transferase in- tent sulfonamide-based competitive inhibitors volved in cell wall arabinan biosynthesis that is the target for the antimycobacterial drug ethambutol. Proc Natl of Mtb’s DHPS (sulfamethoxazole, sulfanilia- Acad Sci 93: 11919–11924. mide,anddiaminodiphenylsulfone)wereshown Blanchard JS. 1996. Molecular mechanisms of drug resis- to lack growth-inhibitory activity because of tance in Mycobacterium tuberculosis. Annu Rev Biochem their rapid intrabacterial inactivation, rather 65: 215–239. Boone IU, Woodward KT. 1953. Relationship of pyridoxine than failure to penetrate its notoriously thick and its derivatives to the mechanism of action of isonia- and hydrophobic envelope. These studies thus zid. Proc Soc Exp Biol Med 84: 292–296. not only showed the highly unpredictable Boshoff HI, Mizrahi V. 2000. Expression of Mycobacterium intrabacterial fates of two well-studied anti- smegmatis pyrazinamidase in Mycobacterium tuberculosis confers hypersensitivity to pyrazinamide and related am- biotics, but more importantly revised our fun- ides. J Bacteriol 182: 5479–5485. damental understanding of their accompanying Chakraborty S, Gruber T,Barry CE III, Boshoff HI, Rhee KY. www.perspectivesinmedicine.org activities (or lack thereof ). Metabolomics not 2013. Para-aminosalicylic acid acts as an alternative sub- withstanding, it remains important to recognize strate of folate metabolism in Mycobacterium tuberculosis. Science 339: 88–91. that no one approach is likely to prove sufficient Chorine V. 1945. Action de l’amide nicotinique sur les ba- to predictably determine the target and/or cilles du genre mycobcaterium. C R Hebd Seances Acad mechanism of any antibiotic. It is thus likely Sci 220: 150–151. that rational mechanism-based drug devel- Cohen J. 2013. Infectious disease. Approval of novel TB drug celebrated—With restraint. Science 339: 130. opment will emerge only with a continued ex- Davidson LA, Takayama K. 1979. Isoniazid inhibition of pansion and integration of systems-level ap- the synthesis of monounsaturated long-chain fatty acids proaches. in Mycobacterium tuberculosis H37Ra. Antimicrob Agents Chemother 16: 104–105. de Carvalho LP, Fischer SM, Marrero J, Nathan C, Ehrt S, ACKNOWLEDGMENTS Rhee KY.2010. Metabolomics of Mycobacterium tubercu- losis reveals compartmentalized co-catabolism of carbon We apologize that many papers could not be substrates. Chem Biol 17: 1122–1131. discussed owing to the lack of space. The au- de Carvalho LP, Darby CM, Rhee KY, Nathan C. 2011. Ni- tazoxanide disrupts membrane potential and intrabacte- thors gratefully acknowledge the support of the rial pH homeostasis of Mycobacterium tuberculosis. ACS Bill and Melinda Gates Foundation TB Drug Med Chem Lett 2: 849–854.

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Tuberculosis Drug Development: History and Evolution of the Mechanism-Based Paradigm

Sumit Chakraborty and Kyu Y. Rhee

Cold Spring Harb Perspect Med published online April 15, 2015

Subject Collection Tuberculosis

Transmission and Institutional Infection Control Clinical Aspects of Adult Tuberculosis of Tuberculosis Robert Loddenkemper, Marc Lipman and Alimuddin Edward A. Nardell Zumla Innate and Adaptive Cellular Immune Responses Advances in Diagnostic Assays for Tuberculosis to Mycobacterium tuberculosis Infection Stephen D. Lawn Katrin D. Mayer-Barber and Daniel L. Barber Tuberculosis Comorbidity with Communicable Diagnosis and Management of Latent and Noncommunicable Diseases Tuberculosis Infection Matthew Bates, Ben J. Marais and Alimuddin Zumla Laura Muñoz, Helen R. Stagg and Ibrahim Abubakar Host-Directed Therapies for Tuberculosis Mycobacterial Growth David M. Tobin Iria Uhía, Kerstin J. Williams, Vahid Shahrezaei, et al. Immunity and Immunopathology in the Multidrug-Resistant Tuberculosis and Extensively Tuberculous Granuloma Drug-Resistant Tuberculosis Antonio J. Pagán and Lalita Ramakrishnan Kwonjune J. Seung, Salmaan Keshavjee and Michael L. Rich Tuberculosis Drug Development: History and The Mycobacterial Cell Wall−−Peptidoglycan and Evolution of the Mechanism-Based Paradigm? Arabinogalactan Sumit Chakraborty and Kyu Y. Rhee Luke J. Alderwick, James Harrison, Georgina S. Lloyd, et al. Genetic Approaches to Facilitate Antibacterial Tuberculosis and HIV Coinfection Drug Development Judith Bruchfeld, Margarida Correia-Neves and Dirk Schnappinger Gunilla Källenius The Tuberculosis Drug Discovery and Imaging in Tuberculosis Development Pipeline and Emerging Drug Targets Jamshed B. Bomanji, Narainder Gupta, Parveen Khisimuzi Mdluli, Takushi Kaneko and Anna Upton Gulati, et al.

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