The Expanding Diversity of Mycobacterium tuberculosis Drug Targets

Subscriber access provided by MT ROYAL COLLEGE

Review

The expanding diversity of Mycobacterium tuberculosis drug targets Samantha Wellington, and Deborah T. Hung ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00255 • Publication Date (Web): 07 Feb 2018 Downloaded from http://pubs.acs.org on February 8, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Infectious Diseases is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Infectious Diseases

The expanding diversity of Mycobacterium tuberculosis drug targets Samantha Wellington1,2,3 and Deborah T. Hung1,2,3,* 1

Broad Institute of MIT and Harvard, 415 Main St., Cambridge, MA 02142 USA

2

Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115

USA 3

Department of Molecular Biology and Center for Computational and Integrative Biology,

Massachusetts General Hospital, 185 Cambridge St., Boston, MA 02114 USA *

email: [email protected]

After decades of relative inactivity, a large increase in efforts to discover new antitubercular therapeutics has brought new insights into the biology of Mycobacterium tuberculosis (Mtb) and promising new drugs such as bedaquiline, which inhibits ATP synthase, and the nitroimidazoles delamanid and pretomanid, which inhibit both mycolic acid synthesis and energy production. Despite these advances, the drug discovery pipeline remains underpopulated. The field desperately needs compounds with novel mechanisms of action capable of inhibiting multi- and extensively-drug resistant Mtb (M/XDR-TB) and, potentially, non-replicating Mtb with the hope of shortening the duration of required therapy. New knowledge about Mtb, along with new methods and technologies, has driven exploration into novel target areas, such as energy production and central metabolism, that diverge from the classical targets in macromolecular synthesis. Here, we review new small molecule drug candidates that act on these novel targets to highlight the methods and perspectives advancing the field. These new targets bring with them the aspiration of shortening treatment duration as well as a pipeline of effective regimens against XDR-TB, positioning Mtb drug discovery to become a model for anti-infective discovery. Key Words: high-throughput screening, target identification, drug development, Mycobacterium tuberculosis, antibiotics

ACS Paragon Plus Environment

1

ACS Infectious Diseases 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 52

Mycobacterium tuberculosis (Mtb) recently surpassed human immunodeficiency virus (HIV) as the leading cause of death due to an infectious disease.(1) Perhaps the most underfunded disease relative to global burden, spending on Mtb research and development hovers around US$ 0.7 billion, well short of the US$ 5.4 billion and US$ 1.7 billion spent on HIV/AIDs and malaria, respectively.(1) While this robust investment has steadily reduced the mortality of HIV and malaria, consistent underfunding has prevented Mtb from following the same course. Mtb kills 1.4 million people per year and an estimated 2 billion people are latently infected worldwide.(1) A particular challenge of Mtb therapy is that the front-line combination of drugs is slow, requiring 6-9 months of treatment to cure an infection, which fuels the development of multi- and extensively drug-resistant tuberculosis (M/XDR-TB).(2) In cases of drug resistance, patients currently undergo a 2-year regimen with cure rates of only 50% for MDR-TB and 20% for XDR-TB(1), though recent early bactericidal activity (EBA) clinical trials have provided hope that new combinations could improve survival and shorten treatment times.(3) It is against this lengthy, suboptimal regimen that new clinical candidates are compared. Overall, there is general consensus that ideal new therapeutics should [1] be effective against M/XDR-TB, [2] shorten treatment time, possibly by targeting a non-replicating state, and [3] be compatible with current antitubercular and HIV therapeutics, as Mtb/HIV co-infection is prevalent.(2, 4-5)

Targeting Replicating and Non-Replicating Bacteria The lengthy treatment required for Mtb infections is potentially attributable to multiple factors, including the effects of lung pathology on drug distribution and on the physiology of infecting bacilli. Mtb typically infects the lungs where it first occupies alveolar macrophages(6) and stimulates the formation of granulomas, or dense clusters of immune cells.(7) Within a single patient, granuloma structures are heterogeneous and may be cellular and well vascularized, necrotic with extracellular bacteria residing in a lipid-rich caseum core, or cavitary, in which the granuloma merges with an airway.(8) In many cases, the granuloma structure restricts blood flow and drug diffusion into its 2 ACS Paragon Plus Environment

Page 3 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


center. While some drugs, such as rifampin and pyrazinamide, are able to penetrate the granuloma, others, such as moxifloxacin, cannot and instead accumulate in the cellular periphery.(9) These differences in penetration likely account for the inability of some promising drugs, such as moxifloxacin, to significantly impact clinical cures. Also contributing to treatment time is the effect of the lung environment on bacterial physiology. The various niches Mtb occupies, including inside macrophages and in the caseum of granulomas(10), are characterized by hypoxia(11-13), nutrient deprivation(14), reactive nitrogen and oxygen species(13), and low pH.(6) In vitro, these environmental stresses have been shown to induce a non-replicating, phenotypically drug-resistant state in Mtb.(15-18) It is hypothesized that in vivo, subpopulations of non-replicating bacilli contribute to the lengthy time required for Mtb treatment and serve as a reservoir from which drug-resistant bacteria emerge.(5, 19) Non-replicating bacteria are also hypothesized to underlie latent infections, though the physiology of bacilli in this infection state is even less well understood.(20) Pyrazinamide is capable of killing non-replicating Mtb and its introduction to the front-line regimen shortened standard therapy by three months.(21) Further, clofazimine, which also has activity against non-replicating Mtb, reduces treatment time required in murine models of drug-sensitive disease(22) and was a component of a drug cocktail that reduced clinical treatment for MDR-TB by 18 months.(23) These precedents have motivated interest in targeting non-replicating bacteria in the hope of further shortening treatment time. However, both pyrazinamide and clofazimine function via very complex and poorly understood mechanisms(21), restricting the idea that targeting non-replicating bacteria will reduce treatment time to a hypothesis. More recently, it was shown that bedaquiline, which kills both replicating and non-replicating bacilli, in combination with other drugs can shorten the treatment duration necessary to prevent relapse in murine models(24), giving additional support to the idea that compounds active against nonreplicating bacteria could reduce treatment time. With an interest in targeting non-replicating bacteria, numerous screens have been conducted in conditions meant to simulate the in vivo environment(15, 18) and against Mtb inside 3 ACS Paragon Plus Environment


Page 4 of 52

macrophages.(25) Assessment of whether a process is essential in non-replicating bacteria has been complicated, however, by the observation that inhibitors have variable efficacy across different nonreplicating models.(15, 26) For example, rifampin, streptomycin, and moxifloxacin have low micromolar activity against hypoxic bacteria, but are inactive against nutrient-deprived bacteria.(26) It remains unclear which, if any, model is most predictive of the potential to shorten treatment time and though activity against non-replicating Mtb has become a goal in drug discovery efforts, it remains to be seen what impact this will have clinically. Another major challenge is the lack of robust models to predict clinical efficacy and treatmentshortening potential. Murine models and EBA trials, in which colony forming units (CFU) in a patient’s sputum are measured over the first 7-14 days of treatment, are proving insufficient to forecast the outcomes of long-term clinical trials. This is perhaps not surprising in that it has long been known that viable bacteria survive even after sputum smears become negative for tuberculous bacilli.(27) Conflicting data, such as with bedaquiline which tends to kill Mtb slowly in vitro and in EBA trials(28) but reduces treatment duration in murine models, suggest the development of new models and alternative biomarkers must accompany the development of inhibitors against new targets. Recent work analyzing patients’ transcriptional response to Mtb infection(29-30), for example, has promise for delivering sensitive and effective biomarkers. Despite these challenges, much progress had been made in recent years. Armed with new knowledge about in vivo environments and non-replicating Mtb along with novel techniques to define essential genes, validate targets, and discover active small molecules, the field is shifting from its historical narrow focus on macromolecular synthesis to consider an expanding candidacy of diverse functions. Encouragingly, there are more targets currently being explored than can be discussed in this review. Here we discuss new approaches to address active infection only, as the physiology of latent infections remains poorly understood. We focus on new developments against the most wellvalidated pathways in macromolecular synthesis, new candidates in the rapidly expanding area of


4

Page 5 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


energy production, and then finally, new targets that are emerging as a result of the development and application of novel methods and technologies.

Macromolecular Synthesis: New targets in well-validated pathways With few exceptions, current antibacterial drugs as well as compounds in clinical trials target macromolecular (i.e. cell wall, protein, and nucleic acid) synthesis. This is true even of the majority of newly discovered compounds in earlier stages of development as recent unbiased, whole-cell phenotypic screens have predominantly identified new leads against well-established functions in macromolecular synthesis. This is primarily a product of screening methods, which have historically aimed to identify compounds active against cells replicating in rich media(31) where only a subset of all potential targets, including macromolecular synthesis, are required. Notably, while inhibition of macromolecular synthesis has historically been very effective against replicating bacteria, it has little effect on non-replicating Mtb and no new drugs against these targets have resulted in a significant reduction in treatment length, though some remain to be evaluated for this potential. Nevertheless, there has been progress in this area. Below, we highlight notable targets against which new drugs and clinical candidates have recently been developed, particularly within cell wall and protein synthesis, though progress is not limited to these two processes, as exemplified by efforts on the natural product griselimycin (Table 1), which inhibits DNA replication and repair.(32) We focus on new molecules and targets that demonstrate the challenges and opportunities in these pathways.

Cell Wall Synthesis: The historical favorite For most bacteria, cell wall biosynthesis is a critical antibiotic target with beta-lactams being arguably the most important class of cell wall active antibiotics historically. Beta-lactams, however, have limited efficacy against Mtb due to constitutive expression of a highly effective beta-lactamase (BlaC)(33), a particularly impenetrable cell wall(34), and expression of multidrug efflux pumps.(35)


5


Page 6 of 52

Nevertheless, targeting the mycobacterial cell wall is an effective strategy as illustrated by two of the front-line antitubercular drugs, isoniazid and ethambutol, and several second-line drugs including ethionamide, thiacetazone, cycloserine, and terizidone.(2) Success in targeting these pathways has led to numerous efforts to identify cell-wall active compounds, as exemplified by current work to potentiate the activity ethionamide(36) and find new inhibitors of InhA(37), which is the target of both isoniazid and ethionamide. Cell wall synthesizing enzymes are particularly effective targets because they are unique to bacteria, thereby limiting toxicity to mammalian cells, and because many enzymes and transporters involved in the construction of the cell wall are found at or in the cell membrane or in the periplasm, making them more accessible to xenobiotics than cytoplasmic targets.(38) The mycobacterial cell wall, unique even among bacteria, is composed of a peptidoglycan layer covalently bound to arabinogalactan to which very long 60-90 carbon fatty acids called mycolic acids are attached (Figure 1).(39) Lipids intercalate into the long hydrophobic chains of the mycolic acids creating an outer membrane.(34) All three molecular entities, arabinogalactan, mycolic acids, and peptidoglycan, serve as targets for inhibition. In 2009, a class of benzothiazinone (BTZ) compounds (Table 1) was found to act by covalently binding the active site of decaprenylphosphoryl-β-D-ribose 2’ epimerase (DprE1).(40) DprE1, in concert with DprE2, epimerizes decaprenylphosphoryl ribose to decaprenylphosphoryl arabinose in an essential step of arabinogalactan and lipoarabinomannan biosynthesis (Figure 1).(41) Giving precedent to this particular cell wall target, one of the front-line drugs, ethambutol, is thought to act by inhibiting arabinogalactan synthesis.(42) Following the initial discovery of BTZs, several groups independently reported DprE1 inhibitors identified through unbiased, whole-cell phenotypic screens.(25, 31) The very diverse chemical structures of these inhibitors prompted investigation into the apparent promiscuity of DprE1. Unexpectedly, it was found that DprE1 localizes to the periplasmic space, making it more accessible to xenobiotics and at least in part explaining its vulnerability.(41) Given that target identification heavily relies on the isolation of resistant Mtb clones, it has also been


6

Page 7 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


speculated that proteins that easily retain function when mutated, such as DprE1, may bias drug discovery toward these redundant targets.(31) Reminiscent of the discovery of DprE1 inhibitors, in 2012, three groups independently published unique small molecules believed to inhibit the previously untargeted MmpL3.(39, 43-44) These studies implicated MmpL3 in the transport of the mycolic acid precursor trehalose monomycolate (TMM) from the cytoplasm to the periplasm. Quickly thereafter, several groups reported additional chemical scaffolds that seem to inhibit MmpL3 based on the identification of resistance mutations in mmpl3.(31, 45-46) As with DprE1, the location of MmpL3 in the cell membrane is thought to be a key factor in the large number of unrelated compounds that inhibit its function and MmpL3 also appears tolerant to mutations. MmpL3 inhibitors demonstrate antitubercular activity in mice(46), supporting MmpL3 as a valuable drug target and SQ109, a 1,2-diamine structurally related to ethambutol (Table 1), was already in clinical trials at the time it was proposed to inhibit MmpL3.(44) Trial results have not been overly promising for its efficacy; in EBA trials, SQ109 alone(47) or in combination with rifampin and moxifloxacin(48) has shown no significant efficacy. The lack of efficacy may be due to pharmacokinetics of the drug or may reflect the limited predictive power EBA studies, leaving hope yet for the target. It has also recently been shown that SQ109 does not inhibit the transport of TMM through MmpL3 in a spheroplast assay and likely instead acts through indirect mechanisms.(49) These findings serve as an important reminder that resistance mutations may point toward a target, but they are not confirmatory. The two scaffolds discovered in 2012, BM212(43) and AU1235(39), do, however, inhibit MmpL3 in this assay.(49) Further, depletion of MmpL3 is lethal for Mtb in vitro and in mice, demonstrating the essentiality of the target during infection.(50) With the recent availability of a direct assay of MmpL3 activity, optimization of MmpL3 inhibitors may more rapidly advance. Several other enzymes involved in mycolic acid synthesis are also being investigated as drug targets including FadD32 and Pks13, which directly interact with each other. FadD32 activates the meromycolic acid branch from the type-II fatty acid synthase (FAS-II) system; then Pks13 condenses 7 ACS Paragon Plus Environment


Page 8 of 52

it with the alpha-alkyl fatty acid branch from the type-I fatty acid synthase (FAS-I) system.(51) FadD32 inhibitors have been identified via unbiased, whole-cell screening in which a series of diarylcoumarins (Table 1) were shown to inhibit mycolic acid synthesis in whole cells.(52) The recently reported crystal structure of Mtb FadD32 in its active conformation will facilitate continued efforts to develop inhibitors of this essential enzyme.(53) Inhibitors have also been identified against Pks13. In a screen for cell wall biosynthesis inhibitors, the iniBAC operon promoter was used to control the expression of lacZ as a reporter of cell wall stress.(51) This screen identified a class of thiophene compounds (Table 1) that targets Pks13. A separate study to develop a scalable platform for target identification based on resistance generation identified a benzofuran that also targets Pks13 (Table 1).(45) The benzofuran has since been optimized through structure-guided medicinal chemistry; a lead compound from this series is effective in mice, making Pks13 a promising new target.(54) Finally, peptidoglycan synthesis has also been an area of interest. For example, two natural products, capuramycin(55) (Table 1) and caprazamycin(56), were found target MurX (translocase I), an essential enzyme required for peptidoglycan synthesis (Figure 1). While capuramycin and its analogs suffer from poor solubility, IV administration of a phospholipid-based nanoemulsion of the capuramycin analog SQ641 has proven effective in a murine model(57) and there are ongoing efforts to identify new chemical scaffolds that inhibit the enzyme and to explore aerosol delivery.(58) Additionally, despite historical limitations to beta-lactam efficacy, several groups have worked to alter their structures or develop combinations that circumvent the intrinsic beta-lactamase activity of Mtb. Compared with rapidly growing bacteria, effective treatment of Mtb in murine models requires a larger percentage of time during which beta-lactam concentration exceeds its MIC – likely a direct effect of Mtb’s slow growth.(59) With their activity tied to cell division and replication, beta-lactams are typically effective only against replicating bacteria. Surprisingly, and unlike other beta-lactams, carbapenems in combination with the beta-lactamase inhibitor clavulanate are able to reduce the viability of non-replicating hypoxic bacteria.(60) Mtb’s unique peptidoglycan structure may account for 8 ACS Paragon Plus Environment

Page 9 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


this activity. Mtb peptidoglycan contains a high percentage of 33 crosslinks synthesized by L,Dtranspeptidases compared with other organisms’ peptidoglycan which contains primarily 34 crosslinks synthesized by D,D-transpeptidases. 33 crosslinks are particularly abundant in stationary-phase Mtb, suggesting a role for cell wall remodeling in non-replicating survival.(61) In contrast to most beta-lactams which inhibit D,D transpeptidases, carbapenems target L,Dtranspeptidases(61), potentially explaining their efficacy against non-replicating bacilli. Using crystal structures of carbapenems bound to the L,D-transpeptidases LdtMt1 and LdtMt2, researchers have been able to design even more potent carbapenems.(62) Further adding to their potential, carbapenems are poor substrates for BlaC and, in fact, function as slow, tight-binding inhibitors of the enzyme.(60) More recently, novel cephalosporins were shown to have activity against only nonreplicating Mtb.(63) Given that inhibitors of peptidoglycan synthesis rely on cell division for their bactericidal effect and that beta-lactams are known to have noncanonical targets outside the transpeptidases, it has been suggested that additional targets could contribute to the observed activity of the carbapenems and the new cephalosporins against non-replicating Mtb.(63)

Protein Synthesis: An old target with new approaches Protein synthesis inhibitors are another mainstay in the treatment of many bacterial infections and serve as second-line therapeutics for Mtb. More than 70 years ago, streptomycin, an inhibitor of the bacterial ribosome, was the first antibiotic discovered to be effective against Mtb.(64) Due to its initial use as a monotherapy, streptomycin resistance rapidly emerged in the clinic.(65) Further preventing more wide-spread use, streptomycin must be administered by injection. Orally bioavailable ribosome inhibitors such as chloramphenicol and tetracycline inhibit the Mtb ribosome in cell-free assays, but are unable to permeate the mycobacterial cell well thereby rendering them inactive.(66) Streptomycin and chloramphenicol are also limited by their risk for severe side effects (ototoxcitiy(67) and aplastic anemia(68), respectively) – a common challenge for antitubercular drugs as they must be administered over extended periods of time. ACS Paragon Plus Environment

9


Page 10 of 52

Excitement in newer orally bioavailable ribosome inhibitors, the oxazolidinones (Table 1), has been fueled by the performance of linezolid in off-label, retrospective studies against MDR-TB.(69) More recently, prospective clinical trials in M/XDR-TB patients have shown linezolid to achieve sputum clearance with no relapse for a majority of patients.(70) Unexpectedly, its human efficacy was superior than that predicted from earlier murine models. Later murine studies showed better efficacy for linezolid(71), perhaps due to differences in the mouse model or in the strain of Mtb. This discrepancy between murine models and clinical efficacy is not unique to linezolid and supports the need for more effective animal models. A major challenge of linezolid treatment, however, is the high risk of severe adverse side effects, including myelosuppression and neuropathy, which are experienced by as many as 82% of patients.(70) Significant efforts are thus being invested in developing alternative oxazolidinones, including sutezolid (formerly PNU-100480). Sutezolid is 1-2 orders of magnitude more effective than linezolid in murine models(72-73), is less toxic, and had efficacy in EBA trials.(74) Altogether, these studies suggest sutezolid may be superior to linezolid for Mtb. Spectinamides (Table 1), a class of ribosome inhibitors derived from the natural product spectinomycin, have antitubercular activity resulting from inhibition of ribosome translocation – a different molecular mechanism than that of the oxazolidinones. While the efflux pump Rv1258c limits the potency of spectinomycin, researchers used structure based design to synthesize spectinamides, which are unaffected by this pump.(75) Despite bacteriostatic activity against replicating Mtb in vitro, both the oxazolidinones and spectinamides have modest activity in certain models of non-replicating Mtb(75), suggesting active protein synthesis is required to enter some non-replicating states. The spectinamides are not orally bioavailable, and so, resolving this property is a focus for optimization. Due to their high safety margin in mice, spectinamides have moved into pre-clinical development via aerosol delivery(75), which has been of great interest in the Mtb field for its potential to overcome solubility challenges and increase dosing while minimizing systemic side effects.(76)


10

Page 11 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


As an alternative to traditional ribosomal inhibitors, tRNA synthetase inhibitors have also progressed into preclinical development. Building on the success of antifungals targeting Leucyl-tRNA synthetase (LeuRS), a small library of benzoxaboroles was synthesized and screened against Mtb.(77) Optimization using crystal structures of an inhibitor bound to LeuRS resulted in orally bioavailable compounds that are effective in murine models (Table 1). With the exception of streptomycin which is bactericidal(78), protein synthesis inhibitors, including LeuRS inhibitors, are bacteriostatic in vitro and it remains to be seen whether they will shorten treatment time clinically. Finally, it is interesting to note that while natural products play a large role in ribosome inhibition, they have recently also had an impact at the opposite end of the biological spectrum; multiple distinct natural products have been discovered that act on the ClpP1P2 protein degradation machinery.(7981)

Energy Metabolism – A critical pathway for non-replicating Mtb As a complement to macromolecular synthesis, energy production has become an area of great interest and large advances. Importantly, ATP homeostasis and the maintenance of an energized membrane are particularly vulnerable targets in hypoxic and nutrient-deprived nonreplicating bacteria.(26, 82) In both aerobic and anaerobic conditions, the electron transport chain (ETC; Figure 2) is responsible for creating a proton gradient that is then used to generate ATP. NADH dehydrogenases (NDH-1 and NDH-2) and succinate dehydrogenase (SDH) serve as entry points to the ETC, transferring electrons from NADH or succinate to menaquinone (MK), a lipid-soluble electron carrier. MK then transfers electrons to nitrate reductase, fumarate reductase, the cytochrome bc1-aa3 complex, or cytochrome bd oxidase.(83-84) These steps are accompanied by the movement of protons from the intracellular to the extracellular space, creating the proton motive force that fuels ATP synthesis by ATP synthase. The recent discovery of bedaquiline, an inhibitor of ATP synthase,


11


Page 12 of 52

has intensified interest in targeting these pathways and there are now inhibitors of many components of the ETC including ATP synthase, cytochrome bc1-aa3, NDH-2 and MK biosynthesis.(83) Discovered in 2005, the diarylquinoline bedaquiline (also known as TMC207 or R207910; Table 2) has bactericidal activity against replicating and non-replicating Mtb in vitro and in mice.(85) Mutations in atpE, part of the F0 subunit of ATP synthase, confer resistance to bedaquiline.(85) Mycobacteria are able to survive ATP depletion, and so it is believed bedaquiline’s bactericidal effect may arise from the uncoupling of proton flow from ATP synthesis which collapses the proton motive force.(86) In 2012, bedaquiline became the first new TB therapeutic to be approved in the US in 40 years. Amongst several promising studies, in clinical trials against MDR-TB, bedaquiline reduces the time to sputum conversion when added to the standard five-drug background regimen.(87) In this particular trial, while a greater number of patients in the group receiving bedaquiline died than in the control group, the trial deaths were not attributed to side effects from the drug and the drug has proven critical for the treatment of M/XDR-TB.(88) Bedaquiline is currently part of multiple ongoing trials evaluating new regimens for their ability to shorten the treatment of XDR-TB as well as drugsensitive infections, with promising preliminary results.(24, 89) Because bedaquiline has a “black box” warning from the United States Food and Drug Administration (FDA) due to its potential effects on cardiac function, there are also ongoing efforts to identify safer analogs.(90) Inhibitors have also been developed against enzymes upstream of ATP synthase such as the cytochrome b subunit (QcrB, Rv2196) of cytochrome bc1, which is responsible for transferring electrons from MK to the aa3-type cytochrome c oxidase.(84, 91)

Currently in clinical trials, the

imidazopyridine amide Q203 (Table 2) was identified through a screen against Mtb infected macrophages.(91) Mutations in QcrB confer resistance to Q203 and the compound triggers a reduction in intracellular ATP, further supporting QcrB as the target.(91) Adding to growing interest in this target, similar compounds were identified through an independent whole-cell screening campaign, with genetic evidence again suggesting the compounds inhibits QcrB(92) and lansoprazole, an FDA-approved gastric proton-pump inhibitor, was also discovered to act against this 12 ACS Paragon Plus Environment

Page 13 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


same target.(93) Unlike bedaquiline, Q203 is only bacteriostatic in vitro and has no activity against non-replicating Mtb.(94) Genetic disruption of cytochrome bd oxidase, encoded by cydAB, is synthetically lethal with Q203, producing a bactericidal response in replicating and non-replicating Mtb.(94) This suggests cytochrome bd oxidase has overlapping function with cytochrome bc1-aa3 and can maintain membrane potential in cells treated with Q203. Several other enzymes in the ETC are under investigation, including NDH-2 (Figure 2).(84) In studying the mechanism of phenothiazine analogs (Table 2) that had been previously shown to disrupt ATP synthesis in Mycobacterium leprae, it was determined the compounds inhibit both Mtb NDH-2s (Ndh and NdhA).(95) The phenothiazines suppress Mtb growth in mice and they are highly effective against non-replicating Mtb.(96) Commercially available phenothiazines are psychotropic, with negative side effects at concentrations required for antitubercular activity. Chemical optimization has, therefore, been targeted at reducing neuroreceptor binding while maintaining anti-Mtb activity.(97) Separately, NdhA was implicated in the mechanism of a 2-mercapto-quinazolinone when it was found that mutations resulting in increased ndhA expression confer resistance to the compound.(45) Interestingly, NDH-2 also plays a role in the mechanism clofazimine by reducing clofazmine and producing toxic reactive oxygen species.(84) Finally, numerous MK biosynthetic enzymes are also being explored as targets. MenA, the penultimate enzyme of MK biosynthesis, and MenE, the fourth enzyme in the pathway, are both essential for growth in mice.(98) Aurachin RE (Table 2), a natural product isolated from Rhodococcus erythropolis, inhibits MenA resulting in potent killing of non-replicating Mtb.(98) Because Aurachin RE has therapeutic limitations due to interactions with anti-HIV drugs, current efforts are focused on modifying the natural product.(98) There have also been efforts to design inhibitors based on predicted reaction intermediates of MenE (Table 2)(99) and a biphenyl benzamide inhibitor of MenG (Table 2), the final enzyme of MK biosynthesis, was recently discovered using a pathway-specific screen in which the promoter of the cydAB operon was fused to a fluorescent reporter to identify inhibitors of respiration.(100) ACS Paragon Plus Environment

13


Page 14 of 52

An important class of drugs with a more complex mechanism of action, the nitroimidazoles, bridges inhibition of macromolecular (cell wall) synthesis and energy production. Delamanid (OPC67683; Table 2) has been approved for use in the European Union and pretomanid (PA-824) is in clinical trials as part of new combination therapies. Delamanid was discovered in a program in which compounds were assessed for their ability to inhibit mycolic acid biosynthesis via thin layer chromatograph of

14

C-labeled mycolic acids.(101) Though no specific enzyme in cell wall

biosynthesis has been identified as a target, delamanid inhibits the synthesis of methoxy- and ketomycolic acids, but not alpha-mycolic acids(101-102); this is in contrast to isoniazid, which inhibits the synthesis of all mycolic acid classes. Unlike isoniazid, the nitroimidazoles are active against non-replicating Mtb.(102) Interestingly, the structure activity relationships governing activity against non-replicating Mtb do not correlate with activity against replicating Mtb, suggesting a different mechanism of action against each state.(103) Through genetic techniques including the isolation of resistant mutants, it was found that both delamanid and pretomanid rely on functional copies of ddn, which encodes an F420-dependent nitroreductase.(102, 104) Ddn converts pretomanid into three metabolites in a process that generates nitric oxide (NO).(104) It is hypothesized that this NO reacts with cytochrome c oxidase, interfering with respiration and thereby conferring the compounds’ activity against non-replicating Mtb.(103-104) Along with pyrazinamide and SQ109, which are reported to affect multiple targets(21, 105-106), the nitroimidazoles thus demonstrate that multi-target activity may be highly desirable for conferring increased potency and activity against multiple physiological states of Mtb.(107) As prodrugs, singlestep high-level resistance to delamanid and pretomanid can occur at high rates(108), but this challenge should be minimized by the appropriate use of combination regimens. Pretomanid in combination with bedaquiline and/or oxazolidinones are superior to the current front-line regimen in mouse models(109) and positive preliminary clinical trial results of bedaquiline, pretomanid and linezolid against drug-resistant Mtb (Nix-TB)(110) bring the hope of a shortened treatment time.


14

Page 15 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Ongoing work aims at further understanding the activity of and optimizing these promising nitroimidazoles.(111)

New Strategies Yielding New Targets Historically, the front-line antitubercular were discovered and developed from the 1940s through the 1960s based on their in vitro or in vivo activity against intact Mtb cells. Isoniazid and pyrazinamide are synthetic drugs that are structurally similar to nicotinamide, which had been previously shown to be active against Mtb in a guinea pig model. Early murine testing of isoniazid showed it was more effective than any other known antitubercular, sparking deep interest in and rapid development of the compound.(112) Pyrazinamide was discovered through direct testing in mice infected with Mtb, and was only later discovered to have no in vitro activity against Mtb cells in standard broth culture.(113) Similarly, ethambutol was discovered through screening of randomly selected synthetic compounds in a murine model.(114) Finally, rifampin is a semisynthetic derivative of a natural product that was discovered via screening of culture extracts followed by structural elucidation, modification, and optimization.(115) Contrary to most modern efforts where target identification is considered crucial for hit optimization, it was not until the drugs had been in the clinic for multiple years that mechanisms of action were determined. For each front-line drug, resistance mutations helped to elucidate the primary target (116-117), though for pyrazinamide, this is an ongoing area of investigation.(21) Following this period of rapid success, Mtb drug discovery efforts lapsed, with little activity until the past decade. As it is not practical to screen all compounds in mice, modern drug discovery has shifted to whole-cell screening without bias toward a particular molecular target or to cell-free, targetbased methods (Figure 3). Though resulting in some success, there are limitations to both of these approaches. While unbiased whole-cell screening identifies compounds that penetrate the mycobacterial cell and produce a phenotype, it appears to favor the identification of inhibitors that


15


Page 16 of 52

repeatedly hit the same targets (e.g. MmpL3 and DprE1).(31, 38) Target-based methods, on the other hand, can identify potent inhibitors of novel targets, but often fail to deliver compounds with whole-cell activity.(118-119) More recently, the development of new technologies and advances in our understanding of Mtb biology are poised to transform this landscape by expanding it beyond classical thinking in several ways. First, the concept of targeting bacteria in different physiologic states, including the nonreplicating state, is not only changing how one prioritizes different targets, but is also changing the conditions in which whole-cell screening is performed, with a reorientation towards conditions which might better mimic the in vivo microenvironment.(15, 18) Secondly, with the desire for new, different targets but the continued desire for a whole-cell screening approach to minimize the challenges of cell permeation, whole-cell, pathway-directed screening combines the benefits of both approaches. In this approach, the screening metric is often something other than simply inhibition of cell growth. For example, driven by the finding that ATP homeostasis is particularly important for non-replicating Mtb, researchers screened for inhibitors of this process by measuring ATP levels in treated cells. These efforts identified, among other hits, imidazopyridines related to Q203.(120) Common approaches also include small molecule screens against strains with essential protein targets knocked-down(121) or against strains engineered to report the activity of a specific enzyme or pathway.(51, 100) Thirdly, as the ability to define novel essential targets under different environmental conditions has advanced with the latest genomic technologies, new genetic tools are allowing the validation of these targets in vivo. Tn-Seq, in which transposon insertion mutagenesis is combined with highthroughput sequencing to perform negative genetic selection studies, has identified about 625 genes necessary for Mtb growth in vitro(122) and another 194 genes that are required in mice, but not in vitro.(123) Strikingly, numerous genes involved in nutrient acquisition and biosynthesis are essential in vivo but not in vitro. Such data suggest the potential to target these “conditionally essential” metabolic processes for efficacy in vivo even when such functions are dispensable in vitro. Lastly, borrowing from a concept that has been increasingly explored in the context of other bacterial 16 ACS Paragon Plus Environment

Page 17 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


infections, the realization that infection outcome is affected by more than bacterial viability but is also related to pathogenicity is resulting in the development of small molecules that target virulence. Utilizing all of these different strategies, numerous novel targets are currently being explored – too many to comprehensively discuss within one review. Therefore, we highlight these novel concepts and strategies with selected examples of new inhibitors of the glyoxylate shunt, pantothenate biosynthesis, amino acid biosynthesis, and iron scavenging.

The Glyoxylate Shunt: A target-based approach against non-replicating Mtb Though there has been interest in expanding target space to include metabolic enzymes, validating these enzymes as in vivo targets has been particularly challenging. Metabolic pathways are necessarily robust, required to function stably over many environmental conditions. As such, there is often redundancy built into the pathways, with bacteria capable of rewiring to bypass certain enzymes or entire pathways.(124) Further, some metabolic enzymes are essential in one condition, but dispensable in others. This challenge is exemplified by Novartis’s pyrimidine-imidazole inhibitors. The compounds progressed through lead optimization only to fail in murine efficacy studies. It was later found that the inhibitors function by causing an accumulation of glycerol phosphate in glycerol metabolizing cells, leading to ATP depletion and cell death.(125) Though glycerol is often used as a carbon source in in vitro axenic culture, it is irrelevant in vivo where fatty acids are the primary carbon source of Mtb.(126-127) Glucose metabolism, which is connected to glycerol metabolism via dihydroxyacetone phosphate, is however, required for Mtb survival in mice.(128) Mtb in the context of an infection is thus not susceptible to inhibitors that rely on glycerol metabolism, but may be susceptible to inhibitors of other carbon metabolism pathways. The increased recognition that carbon source is often critical for inhibitor activity in Mtb has resulted in interest in isocitrate lyase (ICL) as a target. In the absence of carbohydrates, most microorganisms use the glyoxylate cycle for growth on fatty acids or acetate(126, 129), with ICL first converting isocitrate to succinate and glyoxylate followed by malate synthase (GlcB) producing 17 ACS Paragon Plus Environment


Page 18 of 52

malate from glyoxylate and acetyl-CoA (Figure 4). In Mtb, ICL is bifunctional, playing a role in both the glyoxylate pathway and the methylcitrate cycle. In cholesterol- and propionate-containing growth environments, toxic metabolites from the methylcitrate cycle accumulate in ICL knockouts, accounting for at least one aspect of the enzyme’s essentiality.(130) Consistent with this hypothesis, recently identified inhibitors of the cholesterol-catabolizing enzyme HsaAB and of the methylcitrate cycle enzyme PrpC restore the growth of ICL mutants in the presence cholesterol.(131) Further supporting the glyoxylate shunt as a target, non-replicating Mtb relies on the glyoxylate shunt and ATP synthase to maintain continuous respiration.(26), With the notable exception of H37Rv, which has a frameshift mutation in icl2, Mtb has two functional ICLs(126) and both are upregulated in bacilli isolated from mouse lungs.(129) While deletion of icl1 or icl2 individually has little effect on Mtb fitness, deletion of both genes results in severe attenuation in mice.(126) Thus, compounds likely must inhibit both enzymes to be effective. As humans do not possess ICL homologs, numerous groups have attempted to identify ICL inhibitors, though it has been suggested that ICL’s small, polar active site has made finding potent inhibitors challenging. At the time Mtb’s ICL was identified, 3-nitropropionate (3-NP; Table 3), a structural analog of succinate, was already known to inhibit ICL from other organisms. 3-NP is indeed active against recombinant Mtb ICL(129) and eliminates Mtb growth on fatty acids and in macrophages, with little activity against Mtb grown on glycerol or glucose.(126) Conditional ICL knock-down strains are hypersensitive to 3-NP, confirming ICL’s role in the activity of the compound within whole Mtb cells.(121) 3-NP is, however, bactericidal against not only wild-type Mtb, but also ICL knockouts under both replicating and hypoxic conditions(132), demonstrating ICL cannot be the only target of 3-NP. The compound is also known to inhibit SDH, a TCA cycle enzyme that also functions in the ETC (Figure 2), which may account for 3-NPs activity against ICL knockouts. Other groups have tested whether compounds active against non-replicating Mtb may function via ICL inhibition(133-135) and target-based high-throughput screening has been used to directly search for inhibitors of recombinant ICL.(136) Each of these efforts has resulted in compounds that partially 18 ACS Paragon Plus Environment

Page 19 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


inhibit ICL in vitro, but it is not yet clear if ICL is the relevant target of any of these compounds in whole cells. In contrast to the shallow active site of ICL, GlcB has a much larger active site. Recent fragment-based screening against GlcB, in which very small molecules were assessed for their ability to bind GlcB and then co-crystallized with the protein, revealed a dynamic enzyme with multiple conformations, providing ample opportunity to design unique inhibitors of the enzyme.(137) Researchers have used target-based methods with considerably more success against GlcB than ICL. Phenyl-diketo acid derivatives (Table 3) designed to inhibit GlcB in vitro have activity against whole Mtb cells grown on acetate and in low-oxygen conditions, and reduce Mtb CFU in an acute mouse model of infection(138), chemically validating this target in vivo. Further supporting this novel drug target, recent work has shown that GlcB is required for Mtb survival during the chronic phase of a mouse infection and that GlcB is required for glyoxylate detoxification and for resistance to fattyacid associated toxicity.(139)

Pantothenate and Coenzyme A Biosynthesis: Hypomorphs pave the way Antibacterial drug discovery has been greatly advanced by the development of new genetic tools, namely conditional knockdown (hypomorph) technology in which essential proteins are expressed or degraded in an inducible and tunable fashion.(140) Knock-down strategies may be employed at the transcriptional level, e.g. replacing the native promoter with a weak promoter or an inducible promoter, or at the post-translational level, i.e. targeted degradation of a protein of interest.(140) By using transcriptional regulation, the final protein is unchanged whereas proteolytic methods require the addition of a tag to the protein, but result in more rapid and efficient depletion. Applied to antibiotic discovery in a method pioneered in Staphylococcus aureus with the discovery of platensimycin(141), the regulated depletion of essential proteins enables screens that exploit the fact that this depletion can make the strain hypersusceptible to small molecule inhibitors that target the


19


Page 20 of 52

depleted protein, thereby combining the advantages of whole-cell screening with focus on a particular target or pathway. These methods have been applied to pantothenate, an essential precursor for the synthesis of coenzyme A (CoA) and acyl carrier protein (ACP) which is itself synthesized from pantoate and betaalanine by pantothenate synthetase (PanC). While humans lack panC, it is essential for Mtb survival in mice.(121, 127) Additionally, pantothenate biosynthesis was recently implicated as part of the mechanism of pyrazinamide via the observation of panD mutations amongst pyrazinamide-resistant Mtb strains(106), fueling interest in targeting this pathway. Prior to the application of conditional knockdown strategies, PanC inhibitors were primarily been identified through target-based methods using fragment-based screening(142), rational design based on reaction intermediates(143), and unbiased screening of small compound libraries(127, 144), resulting in inhibitors of PanC with only weak activity against whole Mtb cells. With the advent of the conditional knockdown strategy, whole-cell, target-based screening against PanC was carried out by Abrahams et al.(121) A PanC hypomorph was constructed using a tet-OFF system in which the promoter of the native gene was replaced with a tet operator and mycobacterial promoter thereby allowing the expression level of panC to be controlled by the addition of anhydrotetracycline. A screen identified compounds with preferential activity against the hypomorph over wild-type Mtb, with the hypothesis that such compounds would function by inhibiting pantothenate biosynthesis. Though the compounds identified in the screen did not directly inhibit PanC in cell-free assays, addition of pantothenate rescued whole Mtb cells from the growth inhibitory effect of the compounds, suggesting they do indeed function via a mechanism related to pantothenate and validating this targeted whole-cell screening approach against the pantothenate pathway. Interestingly, while panB, panC and coaE knockdowns are bacteriostatic in vitro, knockdown of coaBC, which is further downstream in the CoA pathway, is bactericidal in vitro and in vivo(145), perhaps making CoaBC the best target in this pathway. Though researchers do not have a good 20 ACS Paragon Plus Environment

Page 21 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


understanding of the biology underlying this phenomenon, these differences highlight that not all targets within a single pathway are equal in terms of effect on the bacterial cell. Tools such as conditional knockdowns of essential genes greatly facilitate our ability to study and understand such pathways and better define essentiality. Importantly, these conditional knockdown strains are being used both in vitro and in vivo, transforming our ability to assess the essentiality of genes at different times during infection and to perform in vivo target validation of less classical targets, which has been important in the push beyond macromolecular synthesis. It is important to proceed with caution when relying on genetic target validation, however, as it can be difficult to identify small molecules potent enough to achieve the level of growth inhibition or cell killing achieved through genetic disruption of a target.

Amino Acid Biosynthesis: Metabolic targets with variable essentiality Historically, there has been reluctance to target other metabolic pathways, such as amino acid biosynthesis, because of concern over the ability of Mtb to scavenge nutrients from the host, thereby rendering their biosynthesis nonessential during infection. Recently however, the concept of conditional essentiality and the ability to genetically validate targets (even in the absence of chemical validation) has provided a path forward to explore targets that might be conditionally essential, i.e., are required in one condition – in vivo – even when they are dispensable in another – in vitro. For example, not all amino acid biosynthetic pathways are equivalent in Mtb and targeting some may prove to be a better strategy than others. While deletion or inhibition of certain amino-acidsynthesizing enzymes results in Mtb cell death in vitro (e.g. enzymes in tryptophan, histidine, and methionine biosynthesis), other amino acid starvations (e.g. proline and leucine) are better tolerated, producing only a static response.(146-147) This is even more complicated in vivo, where Mtb can scavenge nutrients from host cells(148) and where nutrient availability varies between tissues(149) and likely also between various parts of the granuloma. Despite some level of amino acid availability in vivo, both tryptophan and branched-chain amino acid auxotrophs are highly attenuated in 21 ACS Paragon Plus Environment


Page 22 of 52

mice.(148, 150) Further there are no human homologs of the genes in these pathways, suggesting they could be promising therapeutic targets. Tryptophan biosynthesis has long been recognized as essential for Mtb in vitro and in vivo.(146) Recent reports have shown that upon stimulation by CD-4 T cell-produced interferon-γ (IFNγ), the host enzyme indoleamine 2,3-dioxygenase (IDO) degrades host tryptophan, thereby increasing Mtb’s reliance on its endogenous biosynthetic machinery.(148) Building on the knowledge that tryptophan biosynthesis is essential in vivo, Zhang et al. tested halogenated anthranilates (Table 3), which are structural mimetics of tryptophan intermediates, and found them to be effective against Mtb in vitro and in mice.(148) Though the precise target of the anthranilates remains unclear, these findings supported increased interest in inhibiting the pathway. Other groups have used in silico screening to identify inhibitors of indole-3-glycerol-phosphate synthase (TrpC) resulting in a competitive inhibitor, ATB107 (Table 3), with activity against Mtb in axenic culture as well as inside macrophages.(151) The effect of ATB107 is not reversed by the addition of tryptophan however, suggesting it likely has targets outside tryptophan biosynthesis and highlighting one of the challenges of target-based drug discovery methods, and even of whole-cell methods, as hit compounds often are found to have multiple targets. More recently, using unbiased, whole-cell screening, we found an allosteric inhibitor of tryptophan synthase (TrpAB; Table 3), the final enzyme of tryptophan biosynthesis.(152) Shortly after, a collaborative team at the University of Birmingham and GlaxoSmithKline (GSK) reported two series of compounds (Table 3), also discovered through whole-cell screening, that inhibit recombinant Mtb TrpAB in vitro and appear to target TrpAB in whole cells.(153) It should be noted that mutations in both TrpAB and an asparagine permease confer resistance to the GSK compounds in M. bovis BCG, suggesting the potential for multiple mechanisms of action. One of the compounds is effective against Mtb in an acute murine model in which adaptive immune responses play a limited role and we also found, through genetic techniques, that TrpAB may be required in vivo even before CD-4 T cell-


22

Page 23 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


mediated adaptive immune responses deplete host tryptophan, which would be important for targeting TrpAB in the HIV-positive population. As exemplified by efforts against the glyoxylate shunt and other tryptophan biosynthetic enzymes, a predominant strategy for inhibiting metabolic enzymes has been the design of synthetic mimetics of substrates or reaction intermediates. Such inhibitors, in contrast to allosteric inhibitors, often suffer from suboptimal potency because substrate binding pockets tend to be small, shallow, and hydrophilic(124) and inhibitors face competition from natural substrates in whole-cell environments. Further, substrate mimetics may have off-target effects on enzymes with similar substrates due to the fact that there are a limited number of natural substrates and co-factors used in metabolic processes, e.g. ATP, sugars, amino acids, resulting in overlapping recognition sites across enzymes. Critically, the overlapping substrates create the potential for off-target inhibition of human enzymes.(124) At the same time, metabolic enzymes provide an opportunity for an alternate strategy due to their tendency to be highly dynamic, adopting several conformations throughout the course of a reaction, with activity strongly linked to the ability to progress through these conformations.(154) Many of these properties emerge because the enzymes are naturally allosterically modulated as part of the cellular regulatory program to control their activities in fluctuating nutritional microenvironments. Thus, metabolic enzymes may be primed for targeting by synthetic allosteric inhibitors that affect this conformational equilibrium. Indeed, our recently reported allosteric inhibitor of TrpAB demonstrated several of these principles. Mtb drug discovery against metabolic enzymes has also benefited from discoveries in other fields. Antimicrobial agents have been developed based on the herbicide sulphometuron methyl (SMM; Table 3)(155), which inhibits branched-chain amino acid synthesis in plants as well as in Mtb by targeting acetohydroxyacid synthase (IlvB1), the first dedicated enzyme of branched-chain amino acid biosynthesis. IlvB1 is essential in the absence of supplemented amino acids; when ilvB1 is deleted, Mtb dies in vitro and is attenuated in mice.(150) Chemically validating this target, SMM is potent against Mtb in vitro and restricts the growth of Mtb in the lungs of infected mice.(155) 23 ACS Paragon Plus Environment


Page 24 of 52

Motivated by these initial examples, the future development of amino acid biosynthesis inhibitors progresses, enabled by the ability to validate pathways in vivo in animal models and in vitro under conditions mimicking human lungs and granulomas.

Iron Scavenging – Targeting virulence As an alternative to inhibiting core essential processes, anti-virulence strategies against processes essential for pathology or virulence in vivo have gained attention in academic and industrial antibiotic discovery.(156-157) Inhibition of mycobactin biosynthesis highlights this strategy in Mtb. Because human serum and tissue have little free iron available, pathogens must synthesize iron-chelating siderophores to scavenge sufficient iron for survival and virulence.(158-159) Mtb produces two siderophores: mycobactin, which is cell wall-associated, and carboxymycobactin, which is secreted.(160) In this pathway (Figure 5), MbtI synthesizes salicylate from chorismate and then MbtA, the first module in an assembly chain, activates so that it can covalently bind the carrier protein domain of MbtB.(161) Next, MbtB condenses the salicylate molecule with L-threonine and cyclizes the intermediate. As the growing molecule continues down the synthetic line, MbtE condenses it with a modified L-lysine and then MbtD attaches a beta-keto group. Finally, MbtF condenses the molecule with another modified lysine and cyclization of this terminal moiety releases the mycobactin.(161) Supporting this pathway as a drug target, an mbtE deletion strain was found to be growth attenuated in vitro, a property that was reversed by the addition of iron and exogenous siderophores, and was unable to survive in a guinea pig model of infection.(160) Building on the mechanistic understanding of siderophore biosynthesis, several groups have used rational design to synthesize inhibitors of mycobactin biosynthetic enzymes. The first of these compounds, 5’-O-[N-salicyl-sulfamoyl adenosine] (salicyl-AMS; Table 3), was designed to mimic a reaction intermediate of MbtA.(162) Salicyl-AMS potently inhibits MbtA in vitro and Mtb growth in irondepleted media; it was also recently shown to have modest activity in a murine model of Mtb infection, providing in vivo chemical proof of concept for this new antitubercular target.(158) The 24 ACS Paragon Plus Environment

Page 25 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


clinical potential of this candidate is limited by toxicity which is potentially due to off-target effects or drug metabolites.(158) Rational design based on salicyl-AMS is ongoing to improve the specificity and the pharmacokinetic properties of this original compound.(163-164) MbtI has also been explored as a target with some inhibitors rationally designed based on substrate or proposed transition state structures (Table 3) (165-166), while others have been discovered through in vitro high-throughput screening against purified enzyme.(167) To date, inhibitors active against the enzyme in vitro have not translated to inhibitors with potent whole-cell activity. Though inhibitors of this target have yet to be reported, an alternate strategy is to inhibit siderophore export. It was recently shown that genetically disrupting export via deletion of mmpS4/S5 is significantly more toxic to Mtb than inhibiting the biosynthesis of siderophores, likely due to the buildup of siderophores inside the Mtb cell(168), again demonstrating that inhibition of various steps within the same pathway does not always result in the same phenotype. As the export systems is comprised of membrane proteins, it may also be more accessible to xenobiotics than the cytoplasmic siderophore synthesizing enzymes. Though more accessible, membrane targets pose their own challenges such as those of developing an assay to report on target activity and of identifying inhibitors with drug-like properties.(38, 169)

Lessons Learned and Prospects for Moving Forward Despite significant progress in the last 10-15 years, the number of compounds in clinical development for the treatment of Tuberculosis remains low and drugs with novel mechanisms of action are particularly underrepresented.(4) Encouragingly however, new methods are beginning to shift the field toward a deeper understanding of Mtb biology. This is resulting in progress in uncovering new drug targets for Mtb, including the identification of vulnerabilities in energy production and central carbon metabolism and the consideration of new areas such as amino acid biosynthesis, pantothenate biosynthesis, and iron acquisition. Notably, some of these processes have been of


25


Page 26 of 52

particular interest because of their importance for Mtb survival in non-replicating conditions. There is still much to learn, however, about which targets are the best. As shown in examples described in this review, not all targets within a pathway are equally amenable to inhibition and inhibition of different targets in the same pathway can result in different phenotypes. At the same time that consideration must be given to the biological vulnerabilities of each target, biochemical consideration must also be made in identifying the most chemically vulnerable, tractable targets. Classic kinetics tells us that drugs should be designed against the rate-determining enzyme of a pathway, as inhibiting what is already the slowest step will have the greatest effect on overall flux through the pathway.(170) And yet, we often do not know which enzymes are rate determining, if these enzymes have shallow binding pockets that will hamper attempts to identify potent inhibitors, or if inhibiting these ratedetermining steps will result in bacterial death or stasis. Chemical optimization must also take into account drug-target residence time. Given the difference between a closed in vitro and an open in vivo system, the correlation between in vitro and in vivo activity should be better for drugs with longer residence times.(171) In light of these varied considerations, a structural and biochemical understanding of drugs and their targets becomes crucial. Compounding this challenge of selecting biologically robust and chemically tractable targets is a lack of understanding about which physiological state(s) could be targeted to shorten treatment duration. There is a large focus on killing non-replicating bacteria, but definitive evidence that this strategy will work is lacking. Perhaps, success in shortening treatment time will come from identifying drugs that are able to penetrate particular granuloma compartments(8) or able to penetrate the thickened cell wall of dormant bacilli.(15) Maybe the ability to clear an infection is so intimately linked to the slow replication rate of Mtb, we need to resuscitate dormant bacteria(20) or to engage the host in more effectively clearing the infection.(172) With new methods and shifting focuses, the hope is that the collective advancement of tools to study Mtb infection will contribute to a greater understanding of this challenging bacterium and its greatest vulnerabilities during infection. It is imperative that we not only understand what essential 26 ACS Paragon Plus Environment

Page 27 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


genes and functions should be targeted, but at the same time, we must identify those enzymes or proteins that are most chemically tractable. Closing the many gaps in knowledge will require a combination of chemical and biological perspectives, marrying an understanding of drug-target engagement, cell penetration, and pharmacokinetics with one of host and bacterial physiology. If we succeed, ironically, Mtb may be poised to leap-frog from perhaps one of the most poorly understood to one of the best studied and understood bacterial infectious processes, leading the way in developing and implementing novel, innovative approaches to antibiotic discovery.

Author Information Corresponding Author * E-mail: [email protected] Notes The authors declare no competing financial interests. Acknowledgements We would like to thank A. Clatworthy for discussions and editing. This work was supported by the Broad Institute Tuberculosis donor group and the Pershing Square Foundation. Abbreviations Mtb Mycobacterium tuberculosis M/XDR-TB Multi- and extensively-drug resistant Mycobacterium tuberculosis HIV Human immunodeficiency virus AIDS Aquired immune deficiency syndrome EBA Early bactericidal activity CFU Colony forming units BTZ Benzothiazinone compounds TMM Trehalose monomycolate ETC Electron transport chain NDH Nitrogen dehydrogenase SDH Succinate dehydrogenase MK Menaquinone FDA United States Food and Drug Administration NO Nitric oxide ICL Isocitrate lyase 3-NP 3-nitropropionate CoA Coenzyme A ACP Acyl carrier protein IDO Indoleamine 2,3-dioxygenase GSK GlaxoSmithKline SMM Sulphometuron methyl ACS Paragon Plus Environment

27


Page 28 of 52

References 1.

WHO Global tuberculosis report World Health Organization: Geneva, Switzerland, 2016.

2. Zumla, A., Nahid, P., and Cole, S. T. (2013) Advances in the development of new tuberculosis drugs and treatment regimens. Nat. Rev. Drug Discov. 12, 388-404. DOI: 10.1038/nrd4001. 3. Dawson, R., Diacon, A. H., Everitt, D., van Niekerk, C., Donald, P. R., Burger, D. A., Schall, R., Spigelman, M., Conradie, A., Eisenach, K., Venter, A., Ive, P., Page-Shipp, L., Variava, E., Reither, K., Ntinginya, N. E., Pym, A., von Groote-Bidlingmaier, F., and Mendel, C. M. (2015) Efficiency and safety of the combination of moxifloxacin, pretomanid (PA-824), and pyrazinamide during the first 8 weeks of antituberculosis treatment: A phase 2b, open-label, partly randomised trial in patients with drug-susceptible or drug-resistant pulmonary tuberculosis. Lancet 385, 1738-1747. DOI: 10.1016/S0140-6736(14)62002-X. 4. Mdluli, K., Kaneko, T., and Upton, A. (2015) The tuberculosis drug discovery and development pipeline and emerging drug targets. Cold Spring Harb. Perspect. Med. 5, a021154. DOI: 10.1101/cshperspect.a021154. 5. Katsuno, K., Burrows, J. N., Duncan, K., van Huijsduijnen, R. H., Kaneko, T., Kita, K., Mowbray, C. E., Schmatz, D., Warner, P., and Slingsby, B. T. (2015) Hit and lead criteria in drug discovery for infectious diseases of the developing world. Nat. Rev. Drug Discov. 14, 751-758. DOI: 10.1038/nrd4683. 6. Cosma, C. L., Sherman, David R., and Ramkrishnan, L. (2003) The secret lives of pathogenic mycobacteria. Annu. Rev. Microbiol. 57, 641-676. DOI: 10.1146/annurev.micro.57.030502.091033. 7. Im, J. G., Itoh, H., Shim, Y. S., Lee, J. H., Ahn, J., Han, M. C., and Noma, S. (1993) Pulmonary tuberculosis: CT findings--early active disease and sequential change with antituberculous therapy. Radiology 186, 653-660. DOI: 10.1148/radiology.186.3.8430169. 8. Dartois, V. (2014) The path of anti-tuberculosis drugs: From blood to lesions to mycobacterial cells. Nat. Rev. Micro. 12, 159-167. DOI: 10.1038/nrmicro3200. 9. Prideaux, B., Via, L. E., Zimmerman, M. D., Eum, S., Sarathy, J., O'Brien, P., Chen, C., Kaya, F., Weiner, D. M., Chen, P.-Y., Song, T., Lee, M., Shim, T. S., Cho, J. S., Kim, W., Cho, S. N., Olivier, K. N., Barry Iii, C. E., and Dartois, V. (2015) The association between sterilizing activity and drug distribution into tuberculosis lesions. Nat. Med. 21, 1223-1227. DOI: 10.1038/nm.3937. 10. Kaplan, G., Post, F. A., Moreira, A. L., Wainwright, H., Kreiswirth, B. N., Tanverdi, M., Mathema, B., Ramaswamy, S. V., Walther, G., Steyn, L. M., Barry, C. E., and Bekker, L.-G. (2003) Mycobacterium tuberculosis growth at the cavity surface: A microenvironment with failed immunity. Infect. Immun. 71, 7099-7108. DOI: 10.1128/IAI.71.12.7099-7108.2003. 11. Via, L. E., Lin, P. L., Ray, S. M., Carrillo, J., Allen, S. S., Eum, S. Y., Taylor, K., Klein, E., Manjunatha, U., Gonzales, J., Lee, E. G., Park, S. K., Raleigh, J. A., Cho, S. N., McMurray, D. N., Flynn, J. L., and Barry, C. E. (2008) Tuberculous granulomas are hypoxic in guinea pigs, rabbits, and nonhuman primates. Infect. Immun. 76, 2333-2340. DOI: 10.1128/IAI.01515-07. 12. Schnappinger, D., Ehrt, S., Voskuil, M. I., Liu, Y., Mangan, J. A., Monahan, I. M., Dolganov, G., Efron, B., Butcher, P. D., Nathan, C., and Schoolnik, G. K. (2003) Transcriptional adaptation of ACS Paragon Plus Environment

28

Page 29 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Mycobacterium tuberculosis within macrophages: Insights into the phagosomal environment. J. Exp. Med. 198, 693-704. DOI: 10.1084/jem.20030846. 13. Voskuil, M. I., Schnappinger, D., Visconti, K. C., Harrell, M. I., Dolganov, G. M., Sherman, D. R., and Schoolnik, G. K. (2003) Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program. J. Exp. Med. 198, 705. DOI: 10.1084/jem.20030205. 14. 850.

Nyka, W. (1974) Studies on the effect of starvation on mycobacteria. Infect. Immun. 9, 843-

15. Grant, S. S., Kawate, T., Nag, P. P., Silvis, M. R., Gordon, K., Stanley, S. A., Kazyanskaya, E., Nietupski, R., Golas, A., Fitzgerald, M., Cho, S., Franzblau, S. G., and Hung, D. T. (2013) Identification of novel inhibitors of nonreplicating Mycobacterium tuberculosis using a carbon starvation model. ACS Chem. Biol. 8, 2224-2234. DOI: 10.1021/cb4004817. 16. Wayne, L. G., and Hayes, L. G. (1996) An in vitro model for sequential study of shiftdown of Mycobacterium tuberculosis through two stages of nonreplicating persistence. Infect. Immun. 64, 2062-2069. 17. Xie, Z., Siddiqi, N., and Rubin, E. J. (2005) Differential antibiotic susceptibilities of starved Mycobacterium tuberculosis isolates. Antimicrob. Agents and Chemother. 49, 4778-4780. DOI: 10.1128/AAC.49.11.4778-4780.2005. 18. Gold, B., Warrier, T., and Nathan, C., A multi-stress model for high throughput screening against non-replicating Mycobacterium tuberculosis. In Mycobacteria protocols, Parish, T.; Roberts, M. D., Eds. Springer New York: New York, NY, 2015; pp 293-315. 19. Keren, I., Minami, S., Rubin, Eric J., and Lewis, K. (2011) Characterization and transcriptome analysis of Mycobacterium tuberculosis persisters. mBio. 2, e00100-11. DOI: 10.1128/mBio.0010011. 20. Chao, M. C., Rubin E.J. (2010) Letting sleeping dos lie: Does dormancy play a role in tuberculosis? Annu. Rev. Microbiol. 64, 293-311. DOI: 10.1146/annurev.micro.112408.134043. 21. Dillon, N. A., Peterson, N. D., Feaga, H. A., Keiler, K. C., and Baughn, A. D. (2017) Antitubercular activity of pyrazinamide is independent of trans-translation and rpsa. Sci. Rep. 7, 6135. DOI: 10.1038/s41598-017-06415-5. 22. Tyagi, S., Ammerman, N. C., Li, S.-Y., Adamson, J., Converse, P. J., Swanson, R. V., Almeida, D. V., and Grosset, J. H. (2015) Clofazimine shortens the duration of the first-line treatment regimen for experimental chemotherapy of tuberculosis. Proc. Natl Acad. Sci. 112, 869-874. DOI: 10.1073/pnas.1416951112. 23. Van Deun, A., Maug, A. K. J., Salim, M. A. H., Das, P. K., Sarker, M. R., Daru, P., and Rieder, H. L. (2010) Short, highly effective, and inexpensive standardized treatment of multidrug-resistant tuberculosis. Am. J. Respir. Crit. Care Med. 182, 684-692. DOI: 10.1164/rccm.201001-0077OC. 24. Li, S.-Y., Tasneen, R., Tyagi, S., Soni, H., Converse, P. J., Mdluli, K., and Nuermberger, E. L. (2017) Bactericidal and sterilizing activity of a novel regimen with bedaquiline, pretomanid, moxifloxacin and pyrazinamide in a murine model of tuberculosis. Antimicrob. Agents and Chemother. 61, e00913-17. DOI: 10.1128/AAC.00913-17.


29


Page 30 of 52

25. Christophe, T., Jackson, M., Jeon, H. K., Fenistein, D., Contreras-Dominguez, M., Kim, J., Genovesio, A., Carralot, J.-P., Ewann, F., Kim, E. H., Lee, S. Y., Kang, S., Seo, M. J., Park, E. J., Škovierová, H., Pham, H., Riccardi, G., Nam, J. Y., Marsollier, L., Kempf, M., Joly-Guillou, M.-L., Oh, T., Shin, W. K., No, Z., Nehrbass, U., Brosch, R., Cole, S. T., and Brodin, P. (2009) High content screening identifies decaprenyl-phosphoribose 2′ epimerase as a target for intracellular antimycobacterial inhibitors. PLoS Pathog. 5, e1000645. DOI: 10.1371/journal.ppat.1000645. 26. Gengenbacher, M., Rao, S. P. S., Pethe, K., and Dick, T. (2010) Nutrient-starved, nonreplicating Mycobacterium tuberculosis requires respiration, atp synthase and isocitrate lyase for maintenance of atp homeostasis and viability. Microbiol. 156, 81-87. DOI: 10.1099/mic.0.033084-0. 27. Behr, M. A., Warren, S. A., Salamon, H., Hopewell, P. C., de Leon, A. P., Daley, C. L., and Small, P. M. (1999) Transmission of Mycobacterium tuberculosis from patients smear-negative for acid-fast bacilli. Lancet 353, 444-449. DOI: 10.1016/S0140-6736(98)03406-0. 28. Diacon, A. H., Dawson, R., von Groote-Bidlingmaier, F., Symons, G., Venter, A., Donald, P. R., van Niekerk, C., Everitt, D., Winter, H., Becker, P., Mendel, C. M., and Spigelman, M. K. (2012) 14day bactericidal activity of PA-824, bedaquiline, pyrazinamide, and moxifloxacin combinations: A randomised trial. Lancet. 380, 986-993. DOI: 10.1016/S0140-6736(12)61080-0. 29. Blankley, S., Graham, C. M., Turner, J., Berry, M. P. R., Bloom, C. I., Xu, Z., Pascual, V., Banchereau, J., Chaussabel, D., Breen, R., Santis, G., Blankenship, D. M., Lipman, M., and O’Garra, A. (2016) The transcriptional signature of active tuberculosis reflects symptom status in extrapulmonary and pulmonary tuberculosis. PLOS ONE. 11, e0162220. DOI: 10.1371/journal.pone.0162220. 30. Sweeney, T. E., Braviak, L., Tato, C. M., and Khatri, P. (2016) Genome-wide expression for diagnosis of pulmonary tuberculosis: A multicohort analysis. Lancet Respir. Med. 4, 213-224. DOI: 10.1016/S2213-2600(16)00048-5. 31. Stanley, S. A., Grant, S. S., Kawate, T., Iwase, N., Shimizu, M., Wivagg, C., Silvis, M., Kazyanskaya, E., Aquadro, J., Golas, A., Fitzgerald, M., Dai, H., Zhang, L., and Hung, D. T. (2012) Identification of novel inhibitors of M. tuberculosis growth using whole cell based high-throughput screening. ACS Chem. Biol. 7, 1377-1384. DOI: 10.1021/cb300151m. 32. Kling, A., Lukat, P., Almeida, D. V., Bauer, A., Fontaine, E., Sordello, S., Zaburannyi, N., Herrmann, J., Wenzel, S. C., König, C., Ammerman, N. C., Barrio, M. B., Borchers, K., BordonPallier, F., Brönstrup, M., Courtemanche, G., Gerlitz, M., Geslin, M., Hammann, P., Heinz, D. W., Hoffmann, H., Klieber, S., Kohlmann, M., Kurz, M., Lair, C., Matter, H., Nuermberger, E., Tyagi, S., Fraisse, L., Grosset, J. H., Lagrange, S., and Müller, R. (2015) Targeting DnaN for tuberculosis therapy using novel griselimycins. Science. 348, 1106. DOI: 10.1126/science.aaa4690. 33. Hugonnet, J.-E., and Blanchard, J. S. (2007) Irreversible inhibition of the Mycobacterium tuberculosis β-lactamase by clavulanate. Biochemistry 46, 11998-12004. DOI: 10.1021/bi701506h. 34. Bansal-Mutalik, R., and Nikaido, H. (2014) Mycobacterial outer membrane is a lipid bilayer and the inner membrane is unusually rich in diacyl phosphatidylinositol dimannosides. Proc. Natl Acad. Sci. 111, 4958. DOI: 10.1073/pnas.1403078111. 35. Danilchanka, O., Mailaender, C., and Niederweis, M. (2008) Identification of a novel multidrug efflux pump of Mycobacterium tuberculosis. Antimicrob. Agents and Chemother. 52, 2503-2511. DOI: 10.1128/aac.00298-08. ACS Paragon Plus Environment

30

Page 31 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


36. Willand, N., Dirie, B., Carette, X., Bifani, P., Singhal, A., Desroses, M., Leroux, F., Willery, E., Mathys, V., Deprez-Poulain, R., Delcroix, G., Frenois, F., Aumercier, M., Locht, C., Villeret, V., Deprez, B., and Baulard, A. R. (2009) Synthetic EthR inhibitors boost antituberculous activity of ethionamide. Nat. Med. 15, 537-544. DOI: 10.1038/nm.1950. 37. Martínez-Hoyos, M., Perez-Herran, E., Gulten, G., Encinas, L., Álvarez-Gómez, D., Alvarez, E., Ferrer-Bazaga, S., García-Pérez, A., Ortega, F., Angulo-Barturen, I., Rullas-Trincado, J., Blanco Ruano, D., Torres, P., Castañeda, P., Huss, S., Fernández Menéndez, R., González del Valle, S., Ballell, L., Barros, D., Modha, S., Dhar, N., Signorino-Gelo, F., McKinney, J. D., García-Bustos, J. F., Lavandera, J. L., Sacchettini, J. C., Jimenez, M. S., Martín-Casabona, N., Castro-Pichel, J., and Mendoza-Losana, A. (2016) Antitubercular drugs for an old target: Gsk693 as a promising InhA direct inhibitor. EBioMedicine 8, 291-301. DOI: 10.1016/j.ebiom.2016.05.006. 38. Goldman, R. C. (2013) Why are membrane targets discovered by phenotypic screens and genome sequencing in Mycobacterium tuberculosis? Tuberculosis 93, 569-588. DOI: 10.1016/j.tube.2013.09.003. 39. Grzegorzewicz, A. E., Pham, H., Gundi, V. A. K. B., Scherman, M. S., Elton J North, T. H., Jones, V., Gruppo, V., Born, S. E. M., Kordulakova, J., Chavadi, S. S., Morisseau, C., Lenaerts, A. J., Lee, R. E., McNeil, M. R., and Jackson, M. (2012) Inhibition of mycolic acid transport across the Mycobacterium tuberculosis plasma membrane. Nat. Chem. Biol. 8, 334-341. DOI: 10.1038/nchembio.794. 40. Makarov, V., Manina, G., Mikusova, K., Mollmann, U., Ryabova, O., Saint-Joanis, B., Dhar, N., Pasca, M. R., Buroni, S., Lucarelli, A. P., Milano, A., De Rossi, E., Belanova, M., Bobovska, A., Dianiskova, P., Kordulakova, J., Sala, C., Fullam, E., Schneider, P., McKinney, J. D., Brodin, P., Christophe, T., Waddell, S., Butcher, P., Albrethsen, J., Rosenkrands, I., Brosch, R., Nandi, V., Bharath, S., Gaonkar, S., Shandil, R. K., Balasubramanian, V., Balganesh, T., Tyagi, S., Grosset, J., Riccardi, G., and Cole, S. T. (2009) Benzothiazinones kill Mycobacterium tuberculosis by blocking arabinan synthesis. Science. 324, 801-4. DOI: 10.1126/science.1171583. 41. Brecik, M., Centárová, I., Mukherjee, R., Kolly, G. S., Huszár, S., Bobovská, A., Kilacsková, E., Mokošová, V., Svetlíková, Z., Šarkan, M., Neres, J., Korduláková, J., Cole, S. T., and Mikušová, K. (2015) DprE1 is a vulnerable tuberculosis drug target due to its cell wall localization. ACS Chem. Biol. 10, 1631-1636. DOI: 10.1021/acschembio.5b00237. 42. Takayama, K., and Kilburn, J. O. (1989) Inhibition of synthesis of arabinogalactan by ethambutol in Mycobacterium smegmatis. Antimicrob. Agents and Chemother. 33, 1493-1499. DOI: 10.1128/AAC.33.9.1493. 43. La Rosa, V., Poce, G., Canseco, J. O., Buroni, S., Pasca, M. R., Biava, M., Raju, R. M., Porretta, G. C., Alfonso, S., Battilocchio, C., Javid, B., Sorrentino, F., Ioerger, T. R., Sacchettini, J. C., Manetti, F., Botta, M., De Logu, A., Rubin, E. J., and De Rossi, E. (2012) MmpL3 is the cellular target of the antitubercular pyrrole derivative BM212. Antimicrob. Agents Chemother. 56, 324-331. DOI: 10.1128/AAC.05270-11. 44. Tahlan, K., Wilson, R., Kastrinsky, D. B., Arora, K., Nair, V., Fischer, E., Barnes, S. W., Walker, J. R., Alland, D., Barry, C. E., and Boshoff, H. I. (2012) SQ109 targets MmpL3, a membrane transporter of trehalose monomycolate involved in mycolic acid donation to the cell wall core of Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 56, 1797-1809. DOI: 10.1128/AAC.05708-11. ACS Paragon Plus Environment

31


Page 32 of 52

45. Ioerger, T. R., O’Malley, T., Liao, R., Guinn, K. M., Hickey, M. J., Mohaideen, N., Murphy, K. C., Boshoff, H. I. M., Mizrahi, V., Rubin, E. J., Sassetti, C. M., Barry, C. E., III, Sherman, D. R., Parish, T., and Sacchettini, J. C. (2013) Identification of new drug targets and resistance mechanisms in Mycobacterium tuberculosis. PLoS ONE. 8, e75245. DOI: 10.1371/journal.pone.0075245. 46. Rao, S. P. S., Lakshminarayana, S. B., Kondreddi, R. R., Herve, M., Camacho, L. R., Bifani, P., Kalapala, S. K., Jiricek, J., Ma, N. L., Tan, B. H., Ng, S. H., Nanjundappa, M., Ravindran, S., Seah, P. G., Thayalan, P., Lim, S. H., Lee, B. H., Goh, A., Barnes, W. S., Chen, Z., Gagaring, K., Chatterjee, A. K., Pethe, K., Kuhen, K., Walker, J., Feng, G., Babu, S., Zhang, L., Blasco, F., Beer, D., Weaver, M., Dartois, V., Glynne, R., Dick, T., Smith, P. W., Diagana, T. T., and Manjunatha, U. H. (2013) Indolcarboxamide is a preclinical candidate for treating multidrug-resistant tuberculosis. Sci. Transl. Med. 5, 214ra168-214ra168. DOI: 10.1126/scitranslmed.3007355. 47. Heinrich, N., Dawson, R., du Bois, J., Narunsky, K., Horwith, G., Phipps, A. J., Nacy, C. A., Aarnoutse, R. E., Boeree, M. J., Gillespie, S. H., Venter, A., Henne, S., Rachow, A., Phillips, P. P. J., Hoelscher, M., Diacon, A. H., Mekota, A. M., Heinrich, N., Rachow, A., Saathoff, E., Hoelscher, M., Gillespie, S., Colbers, A., van Balen, G. P., Aarnoutse, R., Boeree, M., Bateson, A., McHugh, T., Singh, K., Hunt, R., Zumla, A., Nunn, A., Phillips, P., Rehal, S., Dawson, R., Narunsky, K., Diacon, A., du Bois, J., Venter, A., Friedrich, S., Sanne, I., Mellet, K., Churchyard, G., Charalambous, S., Mwaba, P., Elias, N., Mangu, C., Rojas-Ponce, G., Mtafya, B., Maboko, L., Minja, L. T., Sasamalo, M., Reither, K., Jugheli, L., Sam, N., Kibiki, G., Semvua, H., Mpagama, S., Alabi, A., Adegnika, A. A., Amukoye, E., and Okwera, A. (2015) Early phase evaluation of SQ109 alone and in combination with rifampicin in pulmonary tb patients. J. Antimicrob. Chemother. 70, 1558-1566. DOI: 10.1093/jac/dku553. 48. Boeree, M. J., Heinrich, N., Aarnoutse, R., Diacon, A. H., Dawson, R., Rehal, S., Kibiki, G. S., Churchyard, G., Sanne, I., Ntinginya, N. E., Minja, L. T., Hunt, R. D., Charalambous, S., Hanekom, M., Semvua, H. H., Mpagama, S. G., Manyama, C., Mtafya, B., Reither, K., Wallis, R. S., Venter, A., Narunsky, K., Mekota, A., Henne, S., Colbers, A., van Balen, G. P., Gillespie, S. H., Phillips, P. P. J., and Hoelscher, M. (2017) High-dose rifampicin, moxifloxacin, and SQ109 for treating tuberculosis: A multi-arm, multi-stage randomised controlled trial. Lancet Infect. Dis. 17, 39-49. DOI: 10.1016/S14733099(16)30274-2. 49. Xu, Z., Meshcheryakov, V. A., Poce, G., and Chng, S.-S. (2017) MmpL3 is the flippase for mycolic acids in mycobacteria. Proc. Natl Acad. Sci. 114, 7993-7998. DOI: 10.1073/pnas.1700062114. 50. Li, W., Obregón-Henao, A., Wallach, J. B., North, E. J., Lee, R. E., Gonzalez-Juarrero, M., Schnappinger, D., and Jackson, M. (2016) Therapeutic potential of the Mycobacterium tuberculosis mycolic acid transporter, MmpL3. Antimicrob. Agents and Chemother. 60, 5198-5207. DOI: 10.1128/AAC.00826-16. 51. Wilson, R., Kumar, P., Parashar, V., Vilchèze, C., Veyron-Churlet, R., Freundlich, J. S., Barnes, S. W., Walker, J. R., Szymonifka, M. J., Marchiano, E., Shenai, S., Colangeli, R., Jacobs Jr, W. R., Neiditch, M. B., Kremer, L., and Alland, D. (2013) Antituberculosis thiophenes define a requirement for Pks13 in mycolic acid biosynthesis. Nat. Chem. Biol. 9, 499-506. DOI: 10.1038/nchembio.1277. 52. Stanley, S. A., Kawate, T., Iwase, N., Shimizu, M., Clatworthy, A. E., Kazyanskaya, E., Sacchettini, J. C., Ioerger, T. R., Siddiqi, N. A., Minami, S., Aquadro, J. A., Grant, S. S., Rubin, E. J., and Hung, D. T. (2013) Diarylcoumarins inhibit mycolic acid biosynthesis and kill Mycobacterium ACS Paragon Plus Environment

32

Page 33 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


tuberculosis by targeting FadD32. Proc. Natl Acad. Sci. 110, 11565-11570. DOI: 10.1073/pnas.1302114110. 53. Kuhn, M. L., Alexander, E., Minasov, G., Page, H. J., Warwrzak, Z., Shuvalova, L., Flores, K. J., Wilson, D. J., Shi, C., Aldrich, C. C., and Anderson, W. F. (2016) Structure of the essential mtb FadD32 enzyme: A promising drug target for treating tuberculosis. ACS Infect. Dis. 2, 579-591. DOI: 10.1021/acsinfecdis.6b00082. 54. Aggarwal, A., Parai, M. K., Shetty, N., Wallis, D., Woolhiser, L., Hastings, C., Dutta, N. K., Galaviz, S., Dhakal, R. C., Shrestha, R., Wakabayashi, S., Walpole, C., Matthews, D., Floyd, D., Scullion, P., Riley, J., Epemolu, O., Norval, S., Snavely, T., Robertson, G. T., Rubin, E. J., Ioerger, T. R., Sirgel, F. A., van der Merwe, R., van Helden, P. D., Keller, P., Böttger, E. C., Karakousis, P. C., Lenaerts, A. J., and Sacchettini, J. C. (2017) Development of a novel lead that targets m. Tuberculosis polyketide synthase 13. Cell. DOI: 10.1016/j.cell.2017.06.025. 55. Yamaguchi, H., Sato, S., Toshida, S., Takada, K., Itoh, M., Seto, H., and Otake, N. (1986) Capuramycin, a new nucleoside antibiotic. Taxonomy, fermentation, isolation and characterization. J. Antibiot. 39, 1047-53. DOI: 10.7164/antibiotics.39.1047. 56. Salomon, J. J., Galeron, P., Schulte, N., Morow, P. R., Severynse-Stevens, D., Huwer, H., Daum, N., Lehr, C.-M., Hickey, A. J., and Ehrhardt, C. (2013) Biopharmaceutical in vitro characterization of CPZEN-45, a drug candidate for inhalation therapy of tuberculosis. Ther. Deliv. 4, 915-923. DOI: 10.4155/tde.13.62. 57. Nikonenko, B., Reddy, V. M., Bogatcheva, E., Protopopova, M., Einck, L., and Nacy, C. A. (2014) Therapeutic efficacy of SQ641-ne against Mycobacterium tuberculosis. Antimicrob. Agents and Chemother. 58, 587-589. DOI: 10.1128/AAC.01254-13. 58. Siricilla, S., Mitachi, K., Skorupinska-Tudek, K., Swiezewska, E., and Kurosu, M. (2014) Biosynthesis of a water-soluble lipid I analogue and a convenient assay for translocase I. Anal. Biochem. 461, 36-45. DOI: 10.1016/j.ab.2014.05.018. 59. Solapure, S., Dinesh, N., Shandil, R., Ramachandran, V., Sharma, S., Bhattacharjee, D., Ganguly, S., Reddy, J., Ahuja, V., Panduga, V., Parab, M., Vishwas, K. G., Kumar, N., Balganesh, M., and Balasubramanian, V. (2013) In vitro and in vivo efficacy of β-lactams against replicating and slowly growing/nonreplicating Mycobacterium tuberculosis. Antimicrob. Agents and Chemother. 57, 2506-2510. DOI: 10.1128/AAC.00023-13. 60. Hugonnet, J.-E., Tremblay, L. W., Boshoff, H. I., Barry, C. E., and Blanchard, J. S. (2009) Meropenem-clavulanate is effective against extensively drug-resistant Mycobacterium tuberculosis. Science. 323, 1215. DOI: 10.1126/science.1167498. 61. Dubée, V., Triboulet, S., Mainardi, J.-L., Ethève-Quelquejeu, M., Gutmann, L., Marie, A., Dubost, L., Hugonnet, J.-E., and Arthur, M. (2012) Inactivation of Mycobacterium tuberculosis l,dtranspeptidase LdtMt1 by carbapenems and cephalosporins. Antimicrob. Agents and Chemother. 56, 4189-4195. DOI: 10.1128/AAC.00665-12. 62. Kumar, P., Kaushik, A., Lloyd, E. P., Li, S.-G., Mattoo, R., Ammerman, N. C., Bell, D. T., Perryman, A. L., Zandi, T. A., Ekins, S., Ginell, S. L., Townsend, C. A., Freundlich, J. S., and Lamichhane, G. (2016) Non-classical transpeptidases yield insight into new antibacterials. Nat. Chem. Biol. 13, 54. DOI: 10.1038/nchembio.2237. ACS Paragon Plus Environment

33


Page 34 of 52

63. Gold, B., Smith, R., Nguyen, Q., Roberts, J., Ling, Y., Lopez Quezada, L., Somersan, S., Warrier, T., Little, D., Pingle, M., Zhang, D., Ballinger, E., Zimmerman, M., Dartois, V., Hanson, P., Mitscher, L. A., Porubsky, P., Rogers, S., Schoenen, F. J., Nathan, C., and Aubé, J. (2016) Novel cephalosporins selectively active on nonreplicating Mycobacterium tuberculosis. J. Med. Chem. 59, 6027-6044. DOI: 10.1021/acs.jmedchem.5b01833. 64. Schatz, A., Bugle, E., and Waksman, S. A. (1944) Streptomycin, a substance exhibiting antibiotic activity against gram-positive and gram-negative bacteria. Exp. Biol. Med. 55, 66-69. DOI: 10.3181/00379727-55-14461. 65. Pfuetze, K. H., and Pyle, M. M. (1949) Streptomycin in the treatment of tuberculosis. JAMA 139, 634-639. DOI: 10.1001/jama.1949.02900270018005. 66. Shaila, M. S., Gopinathan, K. P., and Ramakrishnan, T. (1973) Protein synthesis in Mycobacterium tuberculosis H37Rv and the effect of streptomycin in streptomycin-susceptible and resistant strains. Antimicrob. Agents and Chemother. 4, 205-213. DOI: 10.1128/IAI.73.6.34923501.2005. 67. McDermott, W. (1947) Toxicity of streptomycin. Am. J. Med. 2, 491-500. DOI: 10.1016/00029343(47)90095-8. 68. Rheingold, J. J., and Spurling, C. L. (1952) Chloramphenicol and aplastic anemia. JAMA 149, 1301-1304. DOI: 10.1001/jama.1952.02930310037008. 69. Fortún, J., Martín-Dávila, P., Navas, E., Pérez-Elías, M. J., Cobo, J., Tato, M., Pedrosa, E. G.G. D. l., Gómez-Mampaso, E., and Moreno, S. (2005) Linezolid for the treatment of multidrugresistant tuberculosis. J. Antimicrob. Chemother. 56, 180-185. DOI: 10.1093/jac/dki148. 70. Lee, M., Lee, J., Carroll, M. W., Choi, H., Min, S., Song, T., Via, L. E., Goldfeder, L. C., Kang, E., Jin, B., Park, H., Kwak, H., Kim, H., Jeon, H.-S., Jeong, I., Joh, J. S., Chen, R. Y., Olivier, K. N., Shaw, P. A., Follmann, D., Song, S. D., Lee, J.-K., Lee, D., Kim, C. T., Dartois, V., Park, S.-K., Cho, S.-N., and Barry, C. E. (2012) Linezolid for treatment of chronic extensively drug-resistant tuberculosis. N. Engl. J. Med. 367, 1508-1518. DOI: 10.1056/NEJMoa1201964. 71. Driver, E. R., Ryan, G. J., Hoff, D. R., Irwin, S. M., Basaraba, R. J., Kramnik, I., and Lenaerts, A. J. (2012) Evaluation of a mouse model of necrotic granuloma formation using C3HeB/FeJ mice for testing of drugs against Mycobacterium tuberculosis. Antimicrob. Agents and Chemother. 56, 31813195. DOI: 10.1128/AAC.00217-12. 72. Cynamon, M. H., Klemens, S. P., Sharpe, C. A., and Chase, S. (1999) Activities of several novel oxazolidinones against Mycobacterium tuberculosis in a murine model. Antimicrob. Agents Chemother. 43, 1189-1191. 73. Williams, K. N., Stover, C. K., Zhu, T., Tasneen, R., Tyagi, S., Grosset, J. H., and Nuermberger, E. (2009) Promising antituberculosis activity of the oxazolidinone PNU-100480 relative to that of linezolid in a murine model. Antimicrob. Agents Chemother. 53, 1314-1319. DOI: 10.1128/AAC.01182-08. 74. Wallis, R. S., Dawson, R., Friedrich, S. O., Venter, A., Paige, D., Zhu, T., Silvia, A., Gobey, J., Ellery, C., Zhang, Y., Eisenach, K., Miller, P., and Diacon, A. H. (2014) Mycobactericidal activity of sutezolid (PNU-100480) in sputum (EBA) and blood (WBA) of patients with pulmonary tuberculosis. PLoS ONE. 9, e94462. DOI: 10.1371/journal.pone.0094462. ACS Paragon Plus Environment

34

Page 35 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


75. Lee, R. E., Hurdle, J. G., Liu, J., Bruhn, D. F., Matt, T., Scherman, M. S., Vaddady, P. K., Zheng, Z., Qi, J., Akbergenov, R., Das, S., Madhura, D. B., Rathi, C., Trivedi, A., Villellas, C., Lee, R. B., Rakesh, Waidyarachchi, S. L., Sun, D., McNeil, M. R., Ainsa, J. A., Boshoff, H. I., GonzalezJuarrero, M., Meibohm, B., Böttger, E. C., and Lenaerts, A. J. (2014) Spectinamides: A new class of semisynthetic anti-tuberculosis agents that overcome native drug efflux. Nat. Med. 20, 152-158. DOI: 10.1038/nm.3458. 76. Misra, A., Hickey, A. J., Rossi, C., Borchard, G., Terada, H., Makino, K., Fourie, P. B., and Colombo, P. (2011) Inhaled drug therapy for treatment of tuberculosis. Tuberculosis 91, 71-81. DOI: 10.1016/j.tube.2010.08.009. 77. Palencia, A., Li, X., Bu, W., Choi, W., Ding, C. Z., Easom, E. E., Feng, L., Hernandez, V., Houston, P., Liu, L., Meewan, M., Mohan, M., Rock, F. L., Sexton, H., Zhang, S., Zhou, Y., Wan, B., Wang, Y., Franzblau, S. G., Woolhiser, L., Gruppo, V., Lenaerts, A. J., O'Malley, T., Parish, T., Cooper, C. B., Waters, M. G., Ma, Z., Ioerger, T. R., Sacchettini, J. C., Rullas, J., Angulo-Barturen, I., Pérez-Herrán, E., Mendoza, A., Barros, D., Cusack, S., Plattner, J. J., and Alley, M. R. K. (2016) Discovery of novel oral protein synthesis inhibitors of Mycobacterium tuberculosis that target leucyltRNA synthetase. Antimicrob. Agents Chemother. 60, 6271-6280. DOI: 10.1128/AAC.01339-16. 78. Dickinson, J. M., Aber, V. R., and Mitchison, D. A. (1977) Bactericidal activity of streptomycin, isoniazid, rifampin, ethambutol, and pyrazinamide alone and in combination against Mycobacterium tuberculosis. Am. Rev. Respir. Dis. 116, 627-635. DOI: 10.1164/arrd.1977.116.4.627. 79. Gavrish, E., Sit, C. S., Cao, S., Kandror, O., Spoering, A., Peoples, A., Ling, L., Fetterman, A., Hughes, D., Bissell, A., Torrey, H., Akopian, T., Mueller, A., Epstein, S., Goldberg, A., Clardy, J., and Lewis, K. (2014) Lassomycin, a ribosomally synthesized cyclic peptide, kills Mycobacterium tuberculosis by targeting the atp-dependent protease ClpC1P1P2. Chem. Biol. 21, 509-518. DOI: 10.1016/j.chembiol.2014.01.014. 80. Schmitt, E. K., Riwanto, M., Sambandamurthy, V., Roggo, S., Miault, C., Zwingelstein, C., Krastel, P., Noble, C., Beer, D., Rao, S. P. S., Au, M., Niyomrattanakit, P., Lim, V., Zheng, J., Jeffery, D., Pethe, K., and Camacho, L. R. (2011) The natural product cyclomarin kills Mycobacterium tuberculosis by targeting the ClpC1 subunit of the caseinolytic protease. Angew. Chem. Int. Ed. 50, 5889-5891. DOI: 10.1002/anie.201101740. 81. Gao, W., Kim, J.-Y., Anderson, J. R., Akopian, T., Hong, S., Jin, Y.-Y., Kandror, O., Kim, J.-W., Lee, I.-A., Lee, S.-Y., McAlpine, J. B., Mulugeta, S., Sunoqrot, S., Wang, Y., Yang, S.-H., Yoon, T.M., Goldberg, A. L., Pauli, G. F., Suh, J.-W., Franzblau, S. G., and Cho, S. (2015) The cyclic peptide ecumicin targeting ClpC1 is active against Mycobacterium tuberculosis in vivo. Antimicrob. Agents and Chemother. 59, 880-889. DOI: 10.1128/AAC.04054-14. 82. Rao, S. P. S., Alonso, S., Rand, L., Dick, T., and Pethe, K. (2008) The protonmotive force is required for maintaining ATP homeostasis and viability of hypoxic, nonreplicating Mycobacterium tuberculosis. Proc. Natl Acad. Sci. 105, 11945-11950. DOI: 10.1073/pnas.0711697105. 83. Cook, G. M., Hards, K., Dunn, E., Heikal, A., Nakatani, Y., Greening, C., Crick, D. C., Fontes, F. L., Pethe, K., Hasenoehrl, E., and Berney, M. (2017) Drug discovery & development: State-of-theart and future directions” on the topic of “targets”: Oxphos as a target space for tuberculosis: Success, caution, and future directions. Microbiol Spectr. 5, DOI: 10.1128/microbiolspec.TBTB20014-2016.


35


Page 36 of 52

84. Black, P. A., Warren, R. M., Louw, G. E., Helden, P. D. v., Victor, T. C., and Kana, B. D. (2014) Energy metabolism and drug efflux in Mycobacterium tuberculosis. Antimicrob. Agents and Chemother. 58, 2491-2503. DOI: 10.1128/AAC.02293-13. 85. Andries, K., Verhasselt, P., Guillemont, J., Gohlmann, H., Neefs, J., Winkler, H., Van Gestel, J., Timmerman, P., Zhu, M., Lee, E., Williams, P., de Chaffoy, D., Hultric, E., Hoffner, S., Cambau, E., Truffot-Pernot, C., Lounis, N., and Jarlier, V. (2005) A diarylquinoline drug active on the atp synthase of Mycobacterium tuberculosis. Science. 307, 223-7. DOI: 10.1126/science.1106753. 86. Hards, K., Robson, J. R., Berney, M., Shaw, L., Bald, D., Koul, A., Andries, K., and Cook, G. M. (2015) Bactericidal mode of action of bedaquiline. J. Antimicrob. Chemother. 70, 2028-2037. DOI: 10.1093/jac/dkv054. 87. Diacon, A. H., Pym, A., Grobusch, M. P., de los Rios, J. M., Gotuzzo, E., Vasilyeva, I., Leimane, V., Andries, K., Bakare, N., De Marez, T., Haxaire-Theeuwes, M., Lounis, N., Meyvisch, P., De Paepe, E., van Heeswijk, R. P. G., and Dannemann, B. (2014) Multidrug-resistant tuberculosis and culture conversion with bedaquiline. N. Engl. J. Med. 371, 723-732. DOI: 10.1056/NEJMoa1313865. 88. Brigden, G., Hewison, C., and Varaine, F. (2015) New developments in the treatment of drugresistant tuebrculosis: Clinical utiltiy of bedaquiline and delamanid. Infect. Drug. Resist. 8, 367-378. DOI: 10.2147/IDR.S68351. 89. Cohen, J. (2017) Simpler, safer treatment hailed as ‘breakthrough’ against drug-resistant tb. Science DOI: 10.1126/science.aal0769. 90. Tong, A. S. T., Choi, P. J., Blaser, A., Sutherland, H. S., Tsang, S. K. Y., Guillemont, J., Motte, M., Cooper, C. B., Andries, K., Van den Broeck, W., Franzblau, S. G., Upton, A. M., Denny, W. A., Palmer, B. D., and Conole, D. (2017) 6-cyano analogues of bedaquiline as less lipophilic and potentially safer diarylquinolines for tuberculosis. ACS Med. Chem Lett. 8, 1019-1024. DOI: 10.1021/acsmedchemlett.7b00196. 91. Pethe, K., Bifani, P., Jang, J., Kang, S., Park, S., Ahn, S., Jiricek, J., Jung, J., Jeon, H. K., Cechetto, J., Christophe, T., Lee, H., Kempf, M., Jackson, M., Lenaerts, A. J., Pham, H., Jones, V., Seo, M. J., Kim, Y. M., Seo, M., Seo, J. J., Park, D., Ko, Y., Choi, I., Kim, R., Kim, S. Y., Lim, S., Yim, S.-A., Nam, J., Kang, H., Kwon, H., Oh, C.-T., Cho, Y., Jang, Y., Kim, J., Chua, A., Tan, B. H., Nanjundappa, M. B., Rao, S. P. S., Barnes, W. S., Wintjens, R., Walker, J. R., Alonso, S., Lee, S., Kim, J., Oh, S., Oh, T., Nehrbass, U., Han, S.-J., No, Z., Lee, J., Brodin, P., Cho, S.-N., Nam, K., and Kim, J. (2013) Discovery of Q203, a potent clinical candidate for the treatment of tuberculosis. Nat. Med. 19, 1157-1160. DOI: 10.1038/nm.3262. 92. Abrahams, K. A., Cox, J. A. G., Spivey, V. L., Loman, N. J., Pallen, M. J., Constantinidou, C., Fernndez, R., Alemparte, C., RemuiÒn, M. J., Barros, D., Ballell, L., and Besra, G. S. (2013) Identification of novel imidazo[1,2-a]pyridine inhibitors targeting M. tuberculosis qcrb. PLoS ONE 7, e52951. DOI: 10.1371/journal.pone.0052951. 93. Rybniker, J., Vocat, A., Sala, C., Busso, P., Pojer, F., Benjak, A., and Cole, S. T. (2015) Lansoprazole is an antituberculous prodrug targeting cytochrome bc1. Nat. Commun. 6, 7659. DOI: 10.1038/ncomms8659. 94. Kalia, N. P., Hasenoehrl, E. J., Ab Rahman, N. B., Koh, V. H., Ang, M. L. T., Sajorda, D. R., Hards, K., Grüber, G., Alonso, S., Cook, G. M., Berney, M., and Pethe, K. (2017) Exploiting the ACS Paragon Plus Environment

36

Page 37 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


synthetic lethality between terminal respiratory oxidases to kill Mycobacterium tuberculosis and clear host infection. Proc. Natl Acad. Sci. 114, 7426-7431. DOI: 10.1073/pnas.1706139114. 95. Weinstein, E. A., Yano, T., Li, L.-S., Avarbock, D., Avarbock, A., Helm, D., McColm, A. A., Duncan, K., Lonsdale, J. T., and Rubin, H. (2005) Inhibitors of type II NADH:Menaquinone oxidoreductase represent a class of antitubercular drugs. Proc. Natl Acad. Sci. 102, 4548-4553. DOI: 10.1073/pnas.0500469102. 96. Advani, M. J., Siddiqui, I., Sharma, P., and Reddy, H. (2012) Activity of trifluoperazine against replicating, non-replicating and drug resistant m. Tuberculosis. PLoS ONE. 7, e44245. DOI: 10.1371/journal.pone.0044245. 97. Salie, S., Hsu, N.-J., Semenya, D., Jardine, A., and Jacobs, M. (2014) Novel non-neuroleptic phenothiazines inhibit Mycobacterium tuberculosis replication. J. Antimicrob. Chemother. 69, 15511558. DOI: 10.1093/jac/dku036. 98. Debnath, J., Siricilla, S., Wan, B., Crick, D. C., Lenaerts, A. J., Franzblau, S. G., and Kurosu, M. (2012) Discovery of selective menaquinone biosynthesis inhibitors against Mycobacterium tuberculosis. J. Med. Chem. 55, 3739-3755. DOI: 10.1021/jm201608g. 99. Lu, X., Zhang, H., Tonge, P. J., and Tan, D. S. (2008) Mechanism-based inhibitors of mene, an acyl-CoA synthetase involved in bacterial menaquinone biosynthesis. Bioorg. Med. Chem. Lett 18, 5963-5966. DOI: 10.1016/j.bmcl.2008.07.130. 100. Sukheja, P., Kumar, P., Mittal, N., Li, S.-G., Singleton, E., Russo, R., Perryman, A. L., Shrestha, R., Awasthi, D., Husain, S., Soteropoulos, P., Brukh, R., Connell, N., Freundlich, J. S., and Alland, D. (2017) A novel small-molecule inhibitor of the Mycobacterium tuberculosis demethylmenaquinone methyltransferase meng is bactericidal to both growing and nutritionally deprived persister cells. mBio. 8, DOI: 10.1128/mBio.02022-16. 101. Matsumoto, M., Hashizume, H., Tomishige, T., Kawasaki, M., Tsubouchi, H., Sasaki, H., Shimokawa, Y., and Komatsu, M. (2006) OPC-67683, a nitro-dihydro-imidazooxazole derivative with promising action against tuberculosis in vitro and in mice. PLoS Med. 3, e466. DOI: 10.1371/journal.pmed.0030466. 102. Stover, C. K., Warrener, P., VanDevanter, D. R., Sherman, D. R., Arain, T. M., Langhorne, M. H., Anderson, S. W., Towell, J. A., Yuan, Y., McMurray, D. N., Kreiswirth, B. N., Barry, C. E., and Baker, W. R. (2000) A small-molecule nitroimidazopyran drug candidate for the treatment of tuberculosis. Nature. 405, 962-966. DOI: 10.1038/35016103. 103. Manjunatha, U., Boshoff, H. I. M., and Barry, C. E. (2009) The mechanism of action of PA-824: Novel insights from transcriptional profiling. Commun. Integr. Biol. 2, 215-218. 104. Singh, R., Manjunatha, U., Boshoff, H. I. M., Ha, Y. H., Niyomrattanakit, P., Ledwidge, R., Dowd, C. S., Lee, I. Y., Kim, P., Zhang, L., Kang, S., Keller, T. H., Jiricek, J., and Barry, C. E. (2008) PA-824 kills nonreplicating Mycobacterium tuberculosis by intracellular NO release. Science. 322, 1392-1395. DOI: 10.1126/science.1164571. 105. Li, K., Schurig-Briccio, L. A., Feng, X., Upadhyay, A., Pujari, V., Lechartier, B., Fontes, F. L., Yang, H., Rao, G., Zhu, W., Gulati, A., No, J. H., Cintra, G., Bogue, S., Liu, Y.-L., Molohon, K., Orlean, P., Mitchell, D. A., Freitas-Junior, L., Ren, F., Sun, H., Jiang, T., Li, Y., Guo, R.-T., Cole, S.


37


Page 38 of 52

T., Gennis, R. B., Crick, D. C., and Oldfield, E. (2014) Multitarget drug discovery for tuberculosis and other infectious diseases. J. Med. Chem. 57, 3126-3139. DOI: 10.1021/jm500131s. 106. Zhang, Y., Shi, W., Zhang, W., and Mitchison, D. (2013) Mechanisms of pyrazinamide action and resistance. Microbiol. Spectr. 2, 1-12. DOI: 10.1128/microbiolspec.MGM2-0023-2013. 107. Silver, L. L. (2007) Multi-targeting by monotherapeutic antibacterials. Nat. Rev. Drug Discov. 6, 41-55. DOI: 10.1038/nrd2202. 108. Haver, H. L., Chua, A., Ghode, P., Lakshminarayana, S. B., Singhal, A., Mathema, B., Wintjens, R., and Bifani, P. (2015) Mutations in genes for the F(420) biosynthetic pathway and a nitroreductase enzyme are the primary resistance determinants in spontaneous in vitro-selected PA824-resistant mutants of Mycobacterium tuberculosis. Antimicrob. Agents and Chemother. 59, 53165323. DOI: 10.1128/AAC.00308-15. 109. Tasneen, R., Betoudji, F., Tyagi, S., Li, S.-Y., Williams, K., Converse, P. J., Dartois, V., Yang, T., Mendel, C. M., Mdluli, K. E., and Nuermberger, E. L. (2016) Contribution of oxazolidinones to the efficacy of novel regimens containing bedaquiline and pretomanid in a mouse model of tuberculosis. Antimicrob. Agents and Chemother. 60, 270-277. DOI: 10.1128/AAC.01691-15. 110. Conradie, F., Diacon, A. H., Everitt, D., Mendel, C., Niekerk, C. v., Howell, P., Comins, K., and Spigelman, M. The Nix-TB trial of pretomanid, bedaquiline, and linezolid to treat XDR-TB, Conference on Retroviruses and Opportunistic Infections, Seattle, WA, USA, Feb 13-16, 2017; Abstract 80LB. 111. Thomspon, A. M., Bonnet, M., Lee, H. H., Franzblau, S. G., Wan, B., Wong, G. S., Cooper, C. B., and Denny, W. A. (2017) Antitubercular nitroimidazoles revisited: Synthesis and activity of the authentic 3-nitro isomer of pretomanid. ACS Med. Chem Lett. 8, 1275-1280. DOI: 10.1021/acsmedchemlett.7b00356. 112. Fox, H. H. (1952) The chemical approach to the control of tuberculosis. Science 116, 129-124. DOI: 10.1126/science.116.3006.129. 113. Malone, L., Schurr, A., Lindh, H., Mckenzie, D., Kiser, J. S., and Williams, J. H. (1952) The effect of pyrazinamide (aldinamide) on experimental tuberuclosis in mice. Am. Rev. Tuberc. 65, 511518. 114. Thomas, J., Baughn, C., Wilkinson, R., and Shepherd, R. (1961) A new synthetic compound with antituberculous activity in mice: Ethambutol (dextro-2, 2′-(ethylenediimino)-di-1-butanol). Am. Rev. Respir. Dis. 83, 891-893. DOI: 10.1164/arrd.1961.83.6.891. 115. Verbist, L., and Gyselen, A. (1968) Antituberculous activity of rifampin in vitro and in vivo and the concentrations attained in human blood. Am. Rev. Respir. Dis. 98, 923-932. DOI: 10.1164/arrd.1968.98.6.923. 116. Banerjee, A., Dubnau, E., Quemard, A., Balasubramanian, V., Um, K. S., Wilson, T., Collins, D., de Lisle, G., and Jacobs, W. R. (1994) InhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis. Science 263, 227. DOI: 10.1126/science.8284673. 117. Telenti, A., Imboden, P., Marchesi, F., Matter, L., Schopfer, K., Bodmer, T., Lowrie, D., Colston, M. J., and Cole, S. (1993) Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis. Lancet 341, 647-651. DOI: 10.1016/0140-6736(93)90417-F.


38

Page 39 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


118. Payne, D. J., Gwynn, M. N., Holmes, D. J., and Pompliano, D. L. (2007) Drugs for bad bugs: Confronting the challenges of antibacterial discovery. Nat. Rev. Drug Discov. 6, 29-40. DOI: 10.1038/nrd2201. 119. Tommasi, R., Brown, D. G., Walkup, G. K., Manchester, J. I., and Miller, A. A. (2015) Eskapeing the labyrinth of antibacterial discovery. Nat. Rev. Drug Discov. 14, 529-542. DOI: 10.1038/nrd4572. 120. Mak, P. A., Rao, S. P. S., Ping Tan, M., Lin, X., Chyba, J., Tay, J., Ng, S. H., Tan, B. H., Cherian, J., Duraiswamy, J., Bifani, P., Lim, V., Lee, B. H., Ling Ma, N., Beer, D., Thayalan, P., Kuhen, K., Chatterjee, A., Supek, F., Glynne, R., Zheng, J., Boshoff, H. I., Barry, C. E., Dick, T., Pethe, K., and Camacho, L. R. (2012) A high-throughput screen to identify inhibitors of ATP homeostasis in non-replicating Mycobacterium tuberculosis. ACS Chemical Biology 7, 1190-1197. DOI: 10.1021/cb2004884. 121. Abrahams, G. L., Kumar, A., Savvi, S., Hung, Alvin W., Wen, S., Abell, C., Barry Iii, Clifton E., Sherman, David R., Boshoff, Helena I. M., and Mizrahi, V. (2012) Pathway-selective sensitization of Mycobacterium tuberculosis for target-based whole-cell screening. Chem. Biol. 19, 844-854. DOI: 10.1016/j.chembiol.2012.05.020. 122. DeJesus, M. A., Gerrick, E. R., Xu, W., Park, S. W., Long, J. E., Boutte, C. C., Rubin, E. J., Schnappinger, D., Ehrt, S., Fortune, S. M., Sassetti, C. M., and Ioerger, T. R. (2017) Comprehensive essentiality analysis of the Mycobacterium tuberculosis genome via saturating transposon mutagenesis. mBio. 8, DOI: 10.1128/mBio.02133-16. 123. Sassetti, C. M., and Rubin, E. J. (2003) Genetic requirements for mycobacterial survival during infection. Proc. Natl Acad. Sci. 100, 12989-12994. DOI: 10.1073/pnas.2134250100. 124. Murima, P., McKinney, J. D., and Pethe, K. (2014) Targeting bacterial central metabolism for drug development. Chem. Biol. 21, 1423-1432. DOI: 10.1016/j.chembiol.2014.08.020. 125. Pethe, K., Sequeira, P. C., Agarwalla, S., Rhee, K., Kuhen, K., Phong, W. Y., Patel, V., Beer, D., Walker, J. R., Duraiswamy, J., Jiricek, J., Keller, T. H., Chatterjee, A., Tan, M. P., Ujjini, M., Rao, S. P. S., Camacho, L., Bifani, P., Mak, P. A., Ma, I., Barnes, S. W., Chen, Z., Plouffe, D., Thayalan, P., Ng, S. H., Au, M., Lee, B. H., Tan, B. H., Ravindran, S., Nanjundappa, M., Lin, X., Goh, A., Lakshminarayana, S. B., Shoen, C., Cynamon, M., Kreiswirth, B., Dartois, V., Peters, E. C., Glynne, R., Brenner, S., and Dick, T. (2010) A chemical genetic screen in Mycobacterium tuberculosis identifies carbon-source-dependent growth inhibitors devoid of in vivo efficacy. Nat. Commun. 1, 57. DOI: 10.1038/ncomms1060. 126. Munoz-Elias, E. J., and McKinney, J. D. (2005) Mycobacterium tuberculosis isocitrate lyases 1 and 2 are jointly required for in vivo growth and virulence. Nat. Med. 11, 638-644. DOI: 10.1038/nm1252. 127. Kumar, A., Casey, A., Odingo, J., Kesicki, E. A., Abrahams, G., Vieth, M., Masquelin, T., Mizrahi, V., Hipskind, P. A., Sherman, D. R., and Parish, T. (2013) A high-throughput screen against pantothenate synthetase (PanC) identifies 3-biphenyl-4-cyanopyrrole-2-carboxylic acids as a new class of inhibitor with activity against Mycobacterium tuberculosis. PLoS ONE. 8, e72786. DOI: 10.1371/journal.pone.0072786.


39


Page 40 of 52

128. Marrero, J., Trujillo, C., Rhee, K. Y., and Ehrt, S. (2013) Glucose phosphorylation is required for Mycobacterium tuberculosis persistence in mice. PLOS Pathog. 9, e1003116. DOI: 10.1371/journal.ppat.1003116. 129. Höner Zu Bentrup, K., Miczak, A., Swenson, D. L., and Russell, D. G. (1999) Characterization of activity and expression of isocitrate lyase in Mycobacterium avium and Mycobacterium tuberculosis. J. Bacteriol. 181, 7161-7167. 130. Eoh, H., and Rhee, K. Y. (2014) Methylcitrate cycle defines the bactericidal essentiality of isocitrate lyase for survival of Mycobacterium tuberculosis on fatty acids. Proc. Natl Acad. Sci. 111, 4976-4981. DOI: 10.1073/pnas.1400390111. 131. VanderVen, B. C., Fahey, R. J., Lee, W., Liu, Y., Abramovitch, R. B., Memmott, C., Crowe, A. M., Eltis, L. D., Perola, E., Deininger, D. D., Wang, T., Locher, C. P., and Russell, D. G. (2015) Novel inhibitors of cholesterol degradation in Mycobacterium tuberculosis reveal how the bacterium’s metabolism is constrained by the intracellular environment. PLoS Pathog. 11, e1004679. DOI: 10.1371/journal.ppat.1004679. 132. Eoh, H., and Rhee, K. Y. (2013) Multifunctional essentiality of succinate metabolism in adaptation to hypoxia in Mycobacterium tuberculosis. Proc. Natl Acad. Sci. 110, 6554-6559. DOI: 10.1073/pnas.1219375110. 133. Krátký, M., Vinšová, J., Novotná, E., Mandíková, J., Wsól, V., Trejtnar, F., Ulmann, V., Stolaříková, J., Fernandes, S., Bhat, S., and Liu, J. O. (2012) Salicylanilide derivatives block Mycobacterium tuberculosis through inhibition of isocitrate lyase and methionine aminopeptidase. Tuberculosis 92, 434-439. DOI: 10.1016/j.tube.2012.06.001. 134. Sriram, D., Yogeeswari, P., Vyas, D. R. K., Senthilkumar, P., Bhat, P., and Srividya, M. (2010) 5-nitro-2-furoic acid hydrazones: Design, synthesis and in vitro antimycobacterial evaluation against log and starved phase cultures. Bioorg. Med. Chem. Lett 20, 4313-4316. DOI: 10.1016/j.bmcl.2010.06.096. 135. Sriram, D., Yogeeswari, P., Senthilkumar, P., Sangaraju, D., Nelli, R., Banerjee, D., Bhat, P., and Manjashetty, T. H. (2010) Synthesis and antimycobacterial evaluation of novel phthalazin-4ylacetamides against log- and starved phase cultures. Chem. Biol. Drug Des. 75, 381-391. DOI: 10.1111/j.1747-0285.2010.00947.x. 136. Liu, Y., Zhou, S., Deng, Q., Li, X., Meng, J., Guan, Y., Li, C., and Xiao, C. (2016) Identification of a novel inhibitor of isocitrate lyase as a potent antitubercular agent against both active and nonreplicating Mycobacterium tuberculosis. Tuberculosis 97, 38-46. DOI: 10.1016/j.tube.2015.12.003. 137. Huang, H.-L., Krieger, I. V., Parai, M. K., Gawandi, V. B., and Sacchettini, J. C. (2016) Mycobacterium tuberculosis malate synthase structures with fragments reveal a portal for substrate/product exchange. J. Biol. Chem. 291, 27421-27432. DOI: 10.1074/jbc.M116.750877. 138. Krieger, Inna V., Freundlich, Joel S., Gawandi, Vijay B., Roberts, Justin P., Gawandi, Vidyadhar B., Sun, Q., Owen, Joshua L., Fraile, Maria T., Huss, Sofia I., Lavandera, J.-L., Ioerger, Thomas R., and Sacchettini, James C. (2012) Structure-guided discovery of phenyl-diketo acids as potent inhibitors of M. tuberculosis malate synthase. Chem. Biol. 19, 1556-1567. DOI: 10.1016/j.chembiol.2012.09.018.


40

Page 41 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


139. Puckett, S., Trujillo, C., Wang, Z., Eoh, H., Ioerger, T. R., Krieger, I., Sacchettini, J., Schnappinger, D., Rhee, K. Y., and Ehrt, S. (2017) Glyoxylate detoxification is an essential function of malate synthase required for carbon assimilation in Mycobacterium tuberculosis. Proc. Natl Acad. Sci. 114, E2225-E2232. 140. Schnappinger, D., O’Brien, K. M., and Ehrt, S., Construction of conditional knockdown mutants in mycobacteria. In Mycobacteria protocols, Parish, T.; Roberts, D. M., Eds. Springer New York: New York, NY, 2015; pp 151-175. 141. Wang, J., Soisson, S. M., Young, K., Shoop, W., Kodali, S., Galgoci, A., Painter, R., Parthasarathy, G., Tang, Y. S., Cummings, R., Ha, S., Dorso, K., Motyl, M., Jayasuriya, H., Ondeyka, J., Herath, K., Zhang, C., Hernandez, L., Allocco, J., Basilio, Á., Tormo, J. R., Genilloud, O., Vicente, F., Pelaez, F., Colwell, L., Lee, S. H., Michael, B., Felcetto, T., Gill, C., Silver, L. L., Hermes, J. D., Bartizal, K., Barrett, J., Schmatz, D., Becker, J. W., Cully, D., and Singh, S. B. (2006) Platensimycin is a selective FabF inhibitor with potent antibiotic properties. Nature. 441, 358-361. DOI: 10.1038/nature04784. 142. Hung, A. W., Silvestre, H. L., Wen, S., Ciulli, A., Blundell, T. L., and Abell, C. (2009) Application of fragment growing and fragment linking to the discovery of inhibitors of Mycobacterium tuberculosis pantothenate synthetase. Angew. Chem. Int. Ed. 48, 8452-8456. DOI: 10.1002/anie.200903821. 143. Devi, P. B., Samala, G., Sridevi, J. P., Saxena, S., Alvala, M., Salina, E. G., Sriram, D., and Yogeeswari, P. (2014) Structure-guided design of thiazolidine derivatives as Mycobacterium tuberculosis pantothenate synthetase inhibitors. ChemMedChem 9, 2538-2547. DOI: 10.1002/cmdc.201402171. 144. White, E., Southworth, K., Ross, L., Cooley, S., Gill, R., Sosa, M., Manouvakhova, A., Rasmussen, L., Goulding, C., Eisenberg, D., and Fletcher, T. r. (2007) A novel inhibitor of Mycobacterium tuberculosis pantothenate synthetase. J. Biomol. Screen 12, 100-105. DOI: 10.1177/1087057106296484. 145. Evans, J. C., Trujillo, C., Wang, Z., Eoh, H., Ehrt, S., Schnappinger, D., Boshoff, H. I. M., Rhee, K. Y., Barry, C. E., and Mizrahi, V. (2016) Validation of coabc as a bactericidal target in the coenzyme a pathway of Mycobacterium tuberculosis. ACS Infect. Dis. 2, 958-968. DOI: 10.1021/acsinfecdis.6b00150. 146. Parish, T. (2003) Starvation survival response of Mycobacterium tuberculosis. J. Bacteriol. 185, 6702-6706. DOI: 10.1128/JB.185.22.6702–6706.2003. 147. Berney, M., Berney-Meyer, L., Wong, K.-W., Chen, B., Chen, M., Kim, J., Wang, J., Harris, D., Parkhill, J., Chan, J., Wang, F., and Jacobs, W. R. (2015) Essential roles of methionine and Sadenosylmethionine in the autarkic lifestyle of Mycobacterium tuberculosis. Proc. Natl Acad. Sci. 112, 10008-10013. DOI: 10.1073/pnas.1513033112. 148. Zhang, Y. J., Reddy, Manchi C., Ioerger, Thomas R., Rothchild, Alissa C., Dartois, V., Schuster, Brian M., Trauner, A., Wallis, D., Galaviz, S., Huttenhower, C., Sacchettini, James C., Behar, Samuel M., and Rubin, Eric J. (2013) Tryptophan biosynthesis protects mycobacteria from CD4 T-cell-mediated killing. Cell. 155, 1296-1308. DOI: 10.1016/j.cell.2013.10.045. 149. Silva, N. M., Rodrigues, C. V., Santoro, M. M., Reis, L. F. L., Alvarez-Leite, J. I., and Gazzinelli, R. T. (2002) Expression of indoleamine 2,3-dioxygenase, tryptophan degradation, and ACS Paragon Plus Environment

41


Page 42 of 52

kynurenine formation during in vivo infection with Toxoplasma gondii: Induction by endogenous gamma interferon and requirement of interferon regulatory factor 1. Infect. Immun. 70, 859-868. DOI: 10.1128/iai.70.2.859-868.2002. 150. Awasthy, D., Gaonkar, S., Shandil, R. K., Yadav, R., Bharath, S., Marcel, N., Subbulakshmi, V., and Sharma, U. (2009) Inactivation of the ilvb1 gene in Mycobacterium tuberculosis leads to branched-chain amino acid auxotrophy and attenuation of virulence in mice. Microbiol. 155, 2978– 2987. DOI: 10.1099/mic.0.029884-0. 151. Hongbo Shen, F. W., Ying Zhang, Qiang Huang, Shengfeng Xu, Hairong Hu, Jun Yue, Honghai Wang. (2009) A novel inhibitor of indole-3-glycerol phosphate synthase with activity against multidrug resistant Mycobacterium tuberculosis. FEBS J. 276, 144-154. DOI: 10.1111/j.17424658.2008.06763.x. 152. Wellington, S., Nag, P. P., Michalska, K., Johnston, S. E., Jedrzejczak, R. P., Kaushik, V. K., Clatworthy, A. E., Siddiqi, N., McCarren, P., Bajrami, B., Maltseva, N. I., Combs, S., Fisher, S. L., Joachimiak, A., Schreiber, S. L., and Hung, D. T. (2017) A small-molecule allosteric inhibitor of Mycobacterium tuberculosis tryptophan synthase. Nat. Chem. Biol. 13, 943-950. DOI: 10.1038/mchembio.2420. 153. Abrahams, K. A., Cox, J. A. G., Fütterer, K., Rullas, J., Ortega-Muro, F., Loman, N. J., Moynihan, P. J., Pérez-Herrán, E., Jiménez, E., Esquivias, J., Barros, D., Ballell, L., Alemparte, C., and Besra, G. S. (2017) Inhibiting mycobacterial tryptophan synthase by targeting the inter-subunit interface. Sci. Rep. 7, 9430. DOI: 10.1038/s41598-017-09642-y. 154. Peracchi, A., and Mozzarelli, A. (2011) Exploring and exploiting allostery: Models, evolution, and drug targeting. Biochim. Biophys. Acta. 1814, 922-933. DOI: 10.1016j.bbapap.2010.10.008. 155. Patil, V., Kale, M., Raichurkar, A., Bhaskar, B., Prahlad, D., Balganesh, M., Nandan, S., and Shahul Hameed, P. (2014) Design and synthesis of triazolopyrimidine acylsulfonamides as novel antimycobacterial leads acting through inhibition of acetohydroxyacid synthase. Bioorg. Med. Chem. Lett. 24, 2222-2225. DOI: 10.1016/j.bmcl.2014.02.054. 156. Clatworthy, A. E., Pierson, E., and Hung, D. T. (2007) Targeting virulence: A new paradigm for antimicrobial therapy. Nat. Chem. Biol. 3, 541-548. DOI: 10.1038/nchembio.2007.24. 157. Cegelski, L., Marshall, G. R., Eldridge, G. R., and Hultgren, S. J. (2008) The biology and future prospects of antivirulence therapies. Nat. Rev. Micro. 6, 17-27. DOI: 10.1038/nrmicro1818. 158. Lun, S., Guo, H., Adamson, J., Cisar, J. S., Davis, T. D., Chavadi, S. S., Warren, J. D., Quadri, L. E. N., Tan, D. S., and Bishai, W. R. (2013) Pharmacokinetic and in vivo efficacy studies of the mycobactin biosynthesis inhibitor Salicyl-AMS in mice. Antimicrob. Agents Chemother. 57, 51385140. DOI: 10.1128/AAC.00918-13. 159. Olakanmi, O., Schlesinger, L. S., Ahmed, A., and Britigan, B. E. (2002) Intraphagosomal Mycobacterium tuberculosis acquires iron from both extracellular transferrin and intracellular iron pools: Impact of interferon-γ and hemochromatosis. J. Biol. Chem. 277, 49727-49734. DOI: 10.1074/jbc.M209768200. 160. Reddy, P. V., Puri, R. V., Chauhan, P., Kar, R., Rohilla, A., Khera, A., and Tyagi, A. K. (2013) Disruption of mycobactin biosynthesis leads to attenution of Mycobacterium tuberculosis for growth and virulence. J. Infect. Dis. 208, 1255-1265. DOI: 10.1093/infdis/jit250. ACS Paragon Plus Environment

42

Page 43 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


161. McMahon, M. D., Rush, J. S., and Thomas, M. G. (2012) Analyses of MbtB, MbtE, and MbtF suggest revisions to the mycobactin biosynthesis pathway in Mycobacterium tuberculosis. J. Bacteriol. 194, 2809-2818. DOI: 10.1128/JB.00088-12. 162. Ferreras, J. A., Ryu, J.-S., Di Lello, F., Tan, D. S., and Quadri, L. E. N. (2005) Small-molecule inhibition of siderophore biosynthesis in Mycobacterium tuberculosis and Yersinia pestis. Nat. Chem. Biol. 1, 29-32. DOI: 10.1038/nchembio706. 163. Neres, J., Labello, N. P., Somu, R. V., Boshoff, H. I., Wilson, D. J., Vannada, J., Chen, L., Barry, C. E., Bennett, E. M., and Aldrich, C. C. (2008) Inhibition of siderophore biosynthesis in Mycobacterium tuberculosis with nucleoside bisubstrate analogues: Structure−activity relationships of the nucleobase domain of 5′-O-[N-(salicyl)sulfamoyl]adenosine. J. Med. Chem. 51, 5349-5370. DOI: 10.1021/jm800567v. 164. Dawadi, S., Kawamura, S., Rubenstein, A., Remmel, R., and Aldrich, C. C. (2016) Synthesis and pharmacological evaluation of nucleoside prodrugs designed to target siderophore biosynthesis in Mycobacterium tuberculosis. Bioorg. Med. Chem. 24, 1314-1321. DOI: 10.1016/j.bmc.2016.02.002. 165. Liu, Z., Liu, F., and Aldrich, C. C. (2015) Stereocontrolled synthesis of a potential transitionstate inhibitor of the salicylate synthase mbti from Mycobacterium tuberculosis. J. Org. Chem. 80, 6545-6552. DOI: 10.1021/acs.joc.5b00455. 166. Manos-Turvey, A., Bulloch, E. M. M., Rutledge, P. J., Baker, E. N., Lott, J. S., and Payne, R. J. (2010) Inhibition studies of Mycobacterium tuberculosis salicylate synthase (MbtI). ChemMedChem. 5, 1067-1079. DOI: 10.1002/cmdc.201000137. 167. Vasan, M., Neres, J., Williams, J., Wilson, D. J., Teitelbaum, A. M., Remmel, R. P., and Aldrich, C. C. (2010) Inhibitors of the salicylate synthase (MbtI) from Mycobacterium tuberculosis discovered by high-throughput screening. ChemMedChem. 5, 2079-2087. DOI: 10.1002/cmdc.201000275. 168. Jones, C. M., Wells, R. M., Madduri, A. V. R., Renfrow, M. B., Ratledge, C., Moody, D. B., and Niederweis, M. (2014) Self-poisoning of Mycobacterium tuberculosis by interrupting siderophore recycling. Proc. Natl Acad. Sci. 111, 1945-1950. DOI: 10.1073/pnas.1311402111. 169. Hurdle, J. G., O'Neill, A. J., Chopra, I., and Lee, R. E. (2010) Targeting bacterial membrane function: An underexploited mechanism for treating persistent infections. Nat. Rev. Micro. 9, 62. DOI: 10.1038/nrmicro2474. 170. Moreno-Sánchez, R., Saavedra, E., Rodríguez-Enríquez, S., and Olín-Sandoval, V. (2008) Metabolic control analysis: A tool for designing strategies to manipulate metabolic pathways. J. Biomed. Biotechnol. 2008, 30. DOI: 10.1155/2008/597913. 171. Lu, H., and Tonge, P. J. (2010) Drug-target residence time: Critical information for lead optimization. Curr. Opin. Chem. Biol. 14, 467-474. DOI: 10.1016/j.cbpa.2010.06.176. 172. Stanley, S. A., Barczak, A. K., Silvis, M. R., Luo, S. S., Sogi, K., Vokes, M., Bray, M.-A., Carpenter, A. E., Moore, C. B., Siddiqi, N., Rubin, E. J., and Hung, D. T. (2014) Identification of hosttargeted small molecules that restrict intracellular Mycobacterium tuberculosis growth. PLoS Pathog. 10, e1003946. DOI: 10.1371/journal.ppat.1003946.


43


Page 44 of 52

Figure 1. Mtb cell wall structure and targets as well as examples of compounds that inhibit them. Front-line drugs (isoniazid, ethambutol) are shown in purple.


44

Page 45 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Mtb electron transport chain and targets in energy metabolism. Several processes in energy generation are targeted by current drugs (bedaquiline and delamanid) as well as clinical and preclinical candidates. Inhibition of energy metabolism has proven to be a particularly vulnerable target in non-replicating Mtb.


45


Page 46 of 52

Figure 3: Drug discovery strategies in Mtb. Researchers employ numerous strategies for drug discovery. Target-based approaches frequently fail to produce cell-permeable clinical candidates while whole-cell screening suffers from the identification of redundant targets. Recently, researchers have bridged the two methods, creating target-specific, whole-cell screening methods.


46

Page 47 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Glyoxylate Shunt in which isocitrate lyase (ICL) converts isocitrate to succinate and glyoxylate and malate synthase (GlcB) converts glyoxylate and acetyl-CoA to malate. Mtb uses the glyoxylate shunt for growth on fatty acids and both ICL and GlcB are demonstrated to be essential in vivo.


47


Page 48 of 52

Figure 5. Model of Mycobactin Biosynthesis. The synthetic pathway consists of a combined nonribosomal peptide, polyketide synthase assembly chain. Various components of the siderophore (salicylate, modified lysine residues) are synthesized by enzymes outside the assembly chain. These building blocks are covalently tethered to members of the assembly chain and are added to the growing molecule as it moves down the line. This figure depicts building blocks and where they enter the chain, but not intermediates.


48

Page 49 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1. Inhibitors of macromolecular synthesis Target

Example Inhibitor(s)

Representative Structure

Method of Discovery

O N

N O

O

DnaN

N

N H

O

O O

O

N

Griselimycin

Unbiased whole-cell screen of natural product extracts

HN

O O

NH N

O

O N N

O

Cell Wall Synthesis DprE1

Benzothiazinones

Unbiased whole-cell methods N N

Cl

MmpL3

N

Unbiased whole-cell screen against replicating Mtb

BM212 Cl NH2

FadD32

O

O

O

O

F

O

F H N

F

Pks13


N

Diarylcoumarins

Thiophenes F

F

Pathway-specific whole-cell screen for activators of iniBAC operon

S

O

NH2 O

N

Benzofurans

O


NH

HO OH O HO

OH

OH O

O

NH

O

MurX

Capuramycin

N

O O

HN

H

O

O

O

NH2

O


HN

Protein Synthesis O

Ribosome

Oxazolidinones (Linezolid)

O N

N

O

H N

O

F H N

Spectinamides

OH

HO NH

H

H

O

O

H

Chemical optimization of synthetic compounds identified in screen against plant pathogens

O

Modifications to natural product

OH HN O

O

LeuRS

OH

Screen of analogs of compound known to inhibit fungal LeuRS

B

Benzoxaboroles

O

Br

NH2


49


Page 50 of 52

Table 2. Inhibitors of energy production Target

Example Inhibitor(s)


Method of Discovery

Br

ATP Synthase

Bedaquiline

N OH

O

Whole-cell screening against M. smegmatis

N

N

QcrB

Q203

F

N

F F

NH

Cl

O

N

O

High-content screening of macrophages infected with Mtb

N

NDH-2

Phenothiazine

Unbiased whole-cell methods

Cl

N

S

O

MenA

OH


Aurachin RE N OH

O

H2N OMe O

MenE

S

N

O N H

N HO

MenG

Mycolic acid synthesis & Cyt C oxidase

Biphenyl benzamide (DG70)

Designed based on chemical structure of reaction intermediates

N

O N

OH

O

O

Pathway-specific, whole-cell screen for activators of cydAB operon

N H

F

O

Cl

O

Nitroimidazoles (Delamanid)

N O

N

N+ -O

N

F O

O

F F

Program to identify cell wall synthesis inhibitors in whole Mtb cells

O


50

Page 51 of 52 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 3. Inhibitors of emerging targets Example Inhibitor(s)

Target


Method of Discovery

Glyoxylate shunt O

ICL & SDH

O

3-NP

Identified as toxin produced by plants

O

N O

Br

GlcB

O

Phenyl-diketo acids

Rational design (substrate analogs)

OH O

OH

Amino Acid Biosynthesis NH 2

Tryptophan synthesis

O

Fluorinated anthranilates

Rational design (substrate analogs)

OH

F N

TrpC & others

H N

N

ATB107

N

N N

in silico screening against TrpC structure

N HN

N

NH

TrpAB

BRD4592

F OH

HO

Cl

Sulfolanes

Unbiased whole-cell screening against replicating Mtb

N

N

S

O

Unbiased whole-cell screening against replicating Mtb

O

O

IlvB1

O O O

SMM S O

N H

Known herbicide

N N H

N

Mycobactin Synthesis

MbtA

Salicyl-AMS

Rational design (substrate analog)

OH

MbtI

Dihydroxybenzoate

HO

O

O O-

O

Benzimidazole-2thione

Rational design (substrate and intermediate analogs)

H N S N

in vitro target-based screen against MbtI

O


51


Page 52 of 52

For Table of Contents Use Only


52

The Expanding Diversity of Mycobacterium tuberculosis Drug Targets

Recommend Documents