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Identification of a chemically validated target in replicating and non-replicating Mycobacterium tuberculosis with the aid of a small molecule probe

, , , , , , , , , , , , , , , , , , , , , , and .

Author Information and Affiliations

Received: ; Last Update: January 16, 2015.

The problem of tuberculosis (TB) continues to take a tremendous toll on global health, accounting for almost 2 million deaths per year, despite the discovery of antitubercular chemotherapy more than half a century ago. In fact, the crisis is growing due to the alarming increase in multi-drug resistant and even totally-drug resistant strains, coupled with the little progress made in discovering new TB drugs. One of the major barriers to discovering new, potentially more effective agents has been the lack of a fundamental understanding of the physiology of the M. tuberculosis bacilli as they exist within the infected human host and the identification of biologically and chemically validated targets. Toward the goal of developing chemical tools to help understand the M. tuberculosis bacterial state during infection and to identify chemically validated targets that represent vulnerabilities in an in vivo relevant state, this report describes initial steps in the development of a benzimidazole probe that targets both replicating and dormant non-replicating M. tuberculosis bacilli using a model of starvation. It takes advantage of the ability to identify such chemical leads using a model for drug tolerance based on nutrient deprivation. As studies have suggested the relevance of the bacterial physiologic state adopted under nutrient deprivation during TB infection, this probe represents an invaluable tool both for identifying a chemically validated target that is critical for M. tuberculosis survival under both replicating and nutrient deprived, non-replicating M. tuberculosis bacilli and for studying the role of targeting both of these states in vivo during infection.

Assigned Assay Grant #: 1R03 MH877444-01

Screening Center Name & PI: Southern Research Specialized Biocontainment Screening Center, E. Lucile White; Broad institute Probe Development Center, Stuart L. Schreiber, PhD

Chemistry Center Name & PI: Broad institute Probe Development Center, Stuart L. Schreiber, PhD

Assay Submitter & Institution: Deborah T. Hung, PhD, Broad Institute of MIT and Harvard, Cambridge, MA.

PubChem Summary Bioassay Identifier (AID): 687033

Probe Structure & Characteristics

ML408.

ML408

CID/ML

No.
TargetsCFU non-replicating IC90: (μM)

[SID, AID]
CFU replicating IC90: (μM)

[SID, AID]
Anti-Target IC50: (μM)
CID 72725758

ML408
Replicating and Non-replicating

M. tuberculosis
32


[SID 172131671, AID 743450]
8


[SID 172131671, AID 743404]
HEK293

HepG2

A549

>84

1. Recommendations for Scientific Use of the Probe

While anti-tuberculosis drugs can kill Mycobacterium tuberculosis bacilli in in vitro, axenic culture in a few days, treatment of an infected human host requires at least six months. The difference between the in vitro and in vivo requirement is thought to be due to M. tuberculosis bacilli existing in a variety of different physiologic states in vivo that include an actively replicating state that is susceptible to current anti-tubercular chemotherapeutic agents but also additional, “drug tolerant” states due to the assumption of a metabolically dormant, non-replicating state (1). These drug tolerant cells are able to survive in the presence of chemotherapy that is effective under normal, replicating conditions. Understanding chemically validated in vivo targets essential to both states of M. tuberculosis bacilli as they adapt to the host microenvironment would provide invaluable insight for the development of more effective, potentially shorter course TB therapy.

Genome-wide methods such as Tn-seq and transposon site hybridization (TraSH) have been applied to M. tuberculosis and attempted to define essential genes required for survival in replicating bacilli, bacilli in macrophages, and in a mouse model of infection. However, such genetic studies have several limitations including the inability to examine genes that are required under replicating conditions under other growth conditions (as these mutants cannot be made) and the fact that genetic validation of a target does not necessarily equate with chemical validation. Thus, small molecule probes that modulate the physiology of replicating and non-replicating bacteria play an incredibly powerful role in the elucidation of this biology.

We herein report the discovery and development of a benzimidazole probe that represents the first intentionally designed small molecule which targets both replicating and dormant, non-replicating M. tuberculosis bacilli in which a completely drug tolerant state has been adopted through nutrient deprivation. While there have been recent reports of molecules that target both replicating and non-replicating bacilli (i.e., bedaquiline, delaminid, clofazimine), this represents the first molecule to focus on nutrient deprivation for induction of drug tolerance in non-replicating bacilli. Evidence exists that nutrient deprivation is relevant to TB infection in animal models, playing an important role in inducing the bacilli to become dormant. Thus, the probe will be used to examine and identify essential functions required for both a replicating and non-replicating physiologic state. Further, the compound will be used to probe in vivo M. tuberculosis bacilli and to validate the relevance of targeting both the replicating and nutrient deprivation models and the essential functions identified as the target of the developed probe during infection. Thus, studies will not only have the potential to provide insight into new targets but also inform the application of relevant models for identifying more effective TB chemotherapy.

2. Materials and Methods

  1. SRSBSC Detailed Assay Protocol (AID 488890, AID 492952, AID 489018, AID 489025, AID 492998)
    1. MATERIALS.
      1. Strain.
        1. M. tuberculosis H37Rv.
      2. Reagents.
        1. 7H9 complete medium (1 L):
          1. Middlebrook 7H9 powder (BD, catalog # 271310) - 4.7 g
          2. 50% Glycerol (Fisher, catalog # G33) - 4 ml
          3. 20% Tween-80 (Sigma, catalog # P1754) - 2.5 ml
          4. Water up to 900 mL
          5. Autoclave, cool to 50 °C and add
          6. OADC (BD/Difco) – 100 mL
        2. 7H9/Tyloxapol (1L):
          1. Middlebrook 7H9 powder (BD, catalog # 271310) - 4.7 g
          2. 20% Tyloxapol – 2.5 mL
          3. Water up to 1 L
        3. Concentrated Supplemental Media (1 L):
          1. Middlebrook 7H9 powder (BD, catalog # 271310) - 4.78 g
          2. Glycerol (Fisher, catalog # G33) - 20 mL
          3. 20% Tyloxapol – 2.25 mL
          4. Water up to 500 mL
          5. Autoclave, cool to 50 °C and add
          6. OADC (BD/Difco) – 500 mL
        4. Alamar Blue (Life Technologies DAL1100)
    2. TOXICITY ASSAYS
      1. THP1 Cytotoxicity Assay
        1. REAGENTS:
          1. COMPLETE MEDIA:
            1. RPMI-1640 (ATCC #30-2001)
            2. 10% FBS (GIBCO #16000)
            3. 1% Penicillin/Streptomycin (GIBCO #15140)
            4. 0.05 mM Β-2-Mercaptoethanol (Sigma #M3148)
          2. CellTiter Glo (Promega #G7573) (CTG)
      2. VERO E6 Cytotoxicity Assay
        1. REAGENTS:
          1. COMPLETE MEDIA:
            1. EMEM (ATCC #30-2003)
            2. 10% Fetal bovine serum (GIBCO # 16000)
            3. 1% Penicillin/Streptomycin (GIBCO # 15140)
          2. CellTiter Glo (Promega #G7573)
      3. HEPG2 Cytotoxicity Assay
        1. REAGENTS:
          1. COMPLETE MEDIA:
            1. EMEM (ATCC #30-2003)
            2. 10% Fetal bovine serum (GIBCO # 16000)
            3. 1% Penicillin/Streptomycin (GIBCO # 15140)
          2. CellTiter Glo (Promega #G7573)
  2. c.

    MATERIALS.

    1. Strain.
      1. M. tuberculosis H37Rv pUV3583c:GFP. This strain contains an episomal plasmid with the constitutive promoter from gene Rv3583c driving expression of green fluorescent protein. This plasmid confers hygromycin resistance.
    2. Reagents.
      1. Difco Middlebrook 7H9 powder [Becton Dickinson Cat# 271310]
      2. Becton Dickinson Catalogue Number: 271310 OADC Enrichment
      3. VWR Catalog Number: 90000-614 Glycerol
      4. Sigma Catalogue Number: G5516-1L Tween-80
      5. VWR Catalogue Number: 95043-486 Hygromycin
      6. Invitrogen Catalogue Number: 10687-010 Tyloxapol
      7. Sigma-Aldrich Catalogue Number: T8761-50G
    3. Disposable Equipment.
      1. Corning roller bottle (Corning, 490cm2).
      2. Sigma-Aldrich Catalogue Number: CLS430195
      3. Corning 384 well black clear bottom plates Corning brand #3712
      4. VWR Catalogue Number: 29444-078
      5. Aerosol Barrier Tips
      6. VWR Catalogue Numbers: 53509-138, 16466-008, 53510-106
      7. Nalgene Reservoirs (Nalgene #1200-2301, 300mL convoluted bottom)
      8. Nalgene Catalogue Number: 1200-2301
      9. Tupperware (6-1/4″ × 8-5/8″ × 5-7/8″ h)
    4. Large Equipment.
      1. Wheaton roller apparatus (Wheaton Science Products Modular Cell Production Roller Culture Apparatus, www​.wheatonsci.com)
      2. Wheaton Science Products Catalogue Number: 348961
      3. Matrix well mate
      4. Thermo Scientific Catalogue Number: 201-10001
      5. CyBiWell
      6. No Catalogue Number
      7. 37 °C Incubator (non-humidified, VWR 14231-488)
      8. VWR Catalogue Number: 14231-488
      9. Bravo Velocity 11
      10. No Catalogue Number
      11. 5 Spectromax
      12. Molecular Devices Part Number: M5

Mammalian Cytotoxicity Assay Panel

A mammalian cell panel was subjected to incubation with compounds of interest for 72 hours. Subsequent cell viability was determined via measuring luminescence using Cell Titer Glo reagent. Toxicity at less than 20 μM was considered to be below the acceptable threshold and resulted in exclusion of the associated compound from further consideration.

Materials and Reagents

White 384-well plates (Corning 8867BC; Lot 35808016); Geldanamycin (AG Scientific, G-1047) 15 mM stock solution in DMSO; CellTiter Glo (Promega G7573); PBS without Calcium and Magnesium (Cellgro 21-040-CV)

Assay Medium

Optimem medium (Invitrogen, Catalog No. 31985-070; Lot No. 548536); 2.5% (v/v) Fetal Bovine Serum (Hyclone, Catalog No.30071.03; Lot No. ARF26748); 1% (v/v) Pen/Strep solution (Invitrogen, Catalog No.15140-122; Lot No. 529891)

Cell

  1. HEK293 (ATCC CLR-1573) human embryonic kidney cells
  2. HepG2 (ATCC HB-8065) human hepatocellular carcinoma cells
  3. A549 (ATCC CCL-185) human lung carcinoma cells

2.1. Assays

Bacterial strains and growth conditions at SRSBSC. M. tuberculosis H37Rv, wild-type strain, was used for all experiments. Mtb H37Rv was grown at 37 °C in Middlebrook 7H9 broth supplemented with 10% OADC (oleic acid-albumin-dextrose complex), 0.2% glycerol and .05% Tween-80.

Bacterial strains and growth conditions at the Broad Institute. The strain M. tuberculosis H37Rv was used for all experiments. GFP was expressed using a constitutive episomal plasmid driven by the Rv3583c promoter. An inducible firefly luciferase expression plasmid was constructed using an anhydrotetracycline inducible system, as described previously (31). Mtb H37Rv was grown at 37 °C in Middlebrook 7H9 broth supplemented with 10% OADC (oleic acid-albumin-dextrose complex), 0.2% glycerol and .05% Tween-80 or on Middlebrook 7H10 plates supplemented with 10% OADC enrichment.

Carbon Starvation at SRSBSC. Freezer stocks of H37Rv wildtype were diluted 1:20 in fresh 7H9 OADC media and cultured until OD600 between 0.3 and 0.9. The bacteria were centrifuged at 2800 × g for five minutes and resuspended in 50 mL of starvation media (7H9 and 0.05% Tyloxapol without any supplementation) to an OD600 of 0.2 and 50 mL of culture aliquoted into a sterile 250mL flask. The starvation culture was incubated standing at 37 °C for 5 weeks.

Carbon Starvation at the Broad Institute. Freezer stocks of H37Rv were diluted 1:50 in fresh 7H9 OADC media and cultured until late log phase, OD600 between 0.6 and 1.0. The bacteria were centrifuged at 2800 × g for five minutes and resuspended in 50 mL of starvation media (7H9 and .05% Tyloxapol without any supplementation). The cells were then washed an additional two times with starvation media. After the final wash the cells were resuspended in starvation media to an OD600 of 0.2 and 50 mL of culture aliquoted into a sterile roller bottle. The starvation culture was incubated standing at 37 °C for 5 weeks.

Screening. An initial assay was developed and utilized at the Broad Institute using a GFP-expressing M. tuberculosis reporter strain that measured fluorescence as a marker for growth and survival after an outgrowth period in rich 7H9 media. This assay was modified at the Southern Research Institute to accommodate the scale of the screen involving the MLPCN library. These modifications include using wild type H37Rv, outgrowth in 7H12 media, and using lamarBlue to measure surviving bacteria rather than the fluorescence.

Carbon starvation assay at the Broad Institute. For M. tuberculosis starvation screening assays, carbon-starved bacteria expressing GFP were diluted and plated into 384 well plates into which compounds had previously been pinned for a final OD600 of 0.05, a final volume of 40 μL and a final compound concentration of 30 μM. Plates were incubated for a period of 120h, at which time 10 μL 5× concentrated media was added to each well of the plate (7H9 media with 50% OADC, 1% glycerol, .05 % Tyloxapol). Plates were then incubated for an additional 96 hours, at which time fluorescence was read using an M5 Spectramax. Each compound was screened in duplicate, and composite z-scores were calculated using DMSO controls as reference. Hits from the M. tuberculosis screen were defined as compounds with a composite z-score of less than -6. This z-score cutoff was selected as the z-score of the concentration of the control antibiotic rifampicin that gave a Z'-factor of 0.

The HTS carbon starvation assay adapted at Southern Research Institute. Carbon starved bacteria were diluted and plated into 384 well plates into which compounds had already been added for a final OD600 of 0.005 and final volume of 50 μL. The plates were incubated for 120 h, at which time 12 μL of concentrated media was added. Plates were incubated for an additional 72 hours. For alamar Blue detection, a solution of 3 parts 18.2% Tween-80 to 4 parts alamarBlue (3/7th Tween-80 to 4/7th alamarBlue) was made and 9 μL added to each well in the plate. The plates were incubated (stacked 2-3 high) overnight at 37 °C in humidified incubator. The plates were then removed from the incubator, sealed with aluminum seals and fluorescence read using PerkinElmer's Envision plate reader (bottom read) with an excitation wavelength of 531nm and an emission wavelength of 595nm.

Replicating, logarithmic assay conducted at the Broad Institute. For M. tuberculosis screening assays for logarithmically growing, actively replicating activity, bacteria expressing GFP was grown to mid-log phase (OD600=0.6–0.8), diluted, and plated into 384 well plates into which compounds had previously been pinned for a final OD600 of 0.025. Plates were incubated for a period of 72 h, at which time GFP fluorescence was read. Each compound was screened in duplicate, and composite z-scores were calculated using DMSO controls as reference. Hits from the M. tuberculosis screen were defined as compounds with a composite z-score of less than −4. This z-score cutoff was selected using average of the z-scores of the concentrations of the control antibiotics clofazimine and rifampicin that gave a Z'-factor of 0.

Replicating, logarithmic assay conducted at SRSBSC. A comparative M. tuberculosis screening assay for actively logarithmically replicating H37Rv was assessed at Southern Research Institute. H37Rv was grown in 7H9 complete medium for 120 h, diluted, and plated into 384 well plates into which compounds had previously been added for a final OD600 of 0.001. Plates were incubated for seven days, alamarBlue was added and fluorescence was determined as previously detailed.

Replicating IC90 determination by OD600. For dose response curves and IC90 determinations by OD600, bacteria were grown to mid-log phase and plated in 96 well plates at OD600=0.05 in the presence of small molecule inhibitors for 7 days unless otherwise indicated, and growth was assessed by reading OD600. The IC90 was defined as the minimum concentration that inhibited growth by 90% relative to the DMSO control (32).

Non-replicating IC90 determination by luciferase. For the luciferase secondary screen that tests for activity of small molecules directly on non-replicating cells without an outgrowth phase, carbon-starved M. tuberculosis H37Rv containing an inducible firefly luciferase plasmid was dispersed into 96-well plates containing the small molecules and anhydotetracycline 50 nM (to induce luciferase expression). After 5 days the cells were lysed, luciferase reagent added and luminescence measured (Promega Corporation, Madison WI) in a Spectramax M5 (Molecular Devices). The antibiotic rifampicin (at 80× the MIC) was used as a positive control for the assay. Hits were defined as small molecules that resulted in ≥95% inhibition of luciferase signal.

Replicating IC90 determinations by CFU. To confirm the replicating IC90 values determined using OD600, the activity of some small molecules was tested by plating for colony forming units (CFU). M. tuberculosis H37Rv was grown to mid-log phase and plated in 96 well plates at OD600 = 0.025 in the presence of small molecule inhibitors for specified time periods. The number of surviving bacteria was then determined by plating a dilution series of the culture for colony forming units (CFU). The IC90 was defined as the concentration tested that inhibited growth by at least 90% relative to the DMSO control.

Non-replicating IC90 determinations by CFU. To confirm the non-replicating IC90 values determined using the luciferase reporter, the activity of some small molecules was tested by plating for CFU. Carbon-starved bacteria were diluted to OD600 = .05 in starvation media and plated in 96 well plates in the presence of small molecule inhibitors for indicated time periods. The number of surviving bacteria was then determined by plating a dilution series of the culture for colony forming units (CFU). The IC90 was defined as the concentration tested that inhibited survival by at least 90% relative to the DMSO control.

Vero toxicity assay. To determine toxicity of selected hit compounds, Vero E6 cells were analyzed at Southern Research Institute. Compounds were added in a stacked plate dose-response method to 384-well microtiter plates in 5 μL at 6× concentration. VeroE6 cells were plated at 1.5 × 105 cells/ml in 20 μL and incubated for 72 h. The end-point was determined with CellTiter Glo (Promega Corporation) and read on the Perkin Elmer Envision using LUM settings.

HepG2 toxicity assay. To determine the toxicity of selected hit compounds in dose response, the biologically relevant liver hepatocyte cell line, HepG2, was selected for toxicity screening. Compounds were added in a stacked plate dose-response method to 384-well microtiter plates in 5 μL at 6× concentration. HepG2 cells were plated at 1.5 × 105 cells/ml in 20 μL and incubated for 72 h. The end-point was determined with CellTiter Glo (Promega Corporation) and read on the Perkin Elmer Envision using LUM settings.

THP-1 toxicity assay. To determine the toxicity of selected hit compounds in dose response, the biologically relevant blood monocyte cell line, THP-1, was selected for toxicity screening. Compounds were added in a stacked plate dose-response method to 384-well microtiter plates in 5 μL at 6× concentration. THP-1 cells were plated at 2.5 × 105 cells/mL in 20 μL and incubated for 72 h. The end-point was determined with CellTiter Glo (Promega Corporation) and read on the Perkin Elmer Envision using LUM settings.

2.2. Probe Chemical Characterization

The probe (ML408) was synthesized as described in Section 2.3 and was subsequently characterized by UPLC, 1H NMR, 13C NMR and high-resolution mass spectrometry. The data obtained from NMR and mass spectroscopy were consistent with the structure of the probe, and UPLC indicates an isolated purity of greater than 95%. The physical properties of the probe ML408 are summarized in Table 1. The solubility of ML408 was determined to be 12.0 μM in PBS with 1% (v/v) DMSO (PBS; pH 7.4, 23 °C).

Table 1. Summary of Probe Properties Computed from Structure ML408.

Table 1

Summary of Probe Properties Computed from Structure ML408.

The chemical stability of the probe ML408 was measured in the presence of PBS pH 7.4 with 1% DMSO. The probe ML408 was added (in triplicate at 1 μM) on six separate plates and allowed to equilibrate at room temperature for 48 hours. At each time point (0, 2, 4, 8, 24, and 48 hours), one plate was removed and an aliquot was taken out from each well and analyzed by UPLC-MS. After 48 h, 100% of ML408 remained (Figure 1).

Figure 1. Stability of the Probe (ML408, CID 72725758) in PBS Buffer (pH 7.4, 23 °C).

Figure 1

Stability of the Probe (ML408, CID 72725758) in PBS Buffer (pH 7.4, 23 °C).

The probe ML408 is stable in human and murine plasma. The stability of the probe was determined by measuring stability in human plasma (100% remaining) and murine plasma (>91% remaining) after a 5-hour incubation period at 37 °C (Table 2). ML408 showed that it was 91.2% bound in human plasma and 95.7% bound in mouse plasma (Table 2). This broad spectrum of stability experiments demonstrates ML408 as a valuable probe molecule.

Table 2. Plasma Stability and Plasma Protein Binding of ML408 (CID 72725758) and CID 780674.

Table 2

Plasma Stability and Plasma Protein Binding of ML408 (CID 72725758) and CID 780674.

The probe ML408 is stable in human liver microsomes but not in murine liver microsomes after 1 hour (Table 3). In contrast, HTS hit CID 780674 was found to be unstable to both human and murine liver microsomes.

Table 3. Liver Microsome Stability of the Probe ML408 (CID 72725758) and CID 780674.

Table 3

Liver Microsome Stability of the Probe ML408 (CID 72725758) and CID 780674.

Table A3Probe and Analog Information

BRDSIDCIDP/AMLSIDML
BRD-K31455027-001-01-117213167172725758PMLS005925240ML408
BRD-K95786485-001-01-717213169772725774AMLS005925243NA
BRD-K08883440-001-01-217213168814174644AMLS005925244NA
BRD-K12840275-001-01-616433637535280384AMLS005925245NA
BRD-K45227969-001-01-817213170172725763AMLS005925246NA
BRD-K32593642-001-10-4161005184780674AMLS005925247NA

A = analog; NA= not applicable; P = probe

2.3. Probe Preparation

Probe ML408 was synthesized by the one-step protocol outlined in Scheme 1. Coupling reaction between 3-methyl-3H-imidazo[4,5-h]isoquinolin-2-amine and benzoyl chloride in presence triethylamine in dichloromethane resulted the probe N-(3-methyl-3H-imidazo[4,5-h]isoquinolin-2-yl)benzamide (ML408).

Scheme 1. Synthesis of the Probe ML408.

Scheme 1

Synthesis of the Probe ML408.

3. Results

3.1. Dose Response Curves for Probe

Image ml408f4
Figure 2. Dose response curves for ML408.

Figure 2Dose response curves for ML408

A. Dose response against replicating, logarithmically growing bacteria measured by CFU/mL. (AID 743485, AID 743404), MIC90 = 8 μM B. Dose response against non-replicating, starved cells by CFU/mL. (AID 743486, AID 743450), MIC90 = 32 μM.

3.2. Cellular Activity

All the assays described in this report are cell-based experiments that measure the activity of compounds against intact, wild type M. tuberculosis strain H37Rv (unless otherwise indicated). Studies across several eukaryotic host cells including HEK 293, HepG2 and A549 cell lines were also performed to determine small molecule toxicity, which determine the ability to perform cellular studies on M. tuberculosis infection of intact macrophage cells. A summary of the assays is described in Section 2.1.

3.3. Profiling Assays

No profiling assays were conducted.

4. Discussion

4.1. Comparison to Existing Art and How the New Probe is an Improvement

Investigation into relevant prior art entailed searching the following databases: SciFinder, Reaxys, PubChem, PubMed, US Patent and Trademark Office (USPTO) PatFT and AppFT, and World Intellectual Property Organization (WIPO) databases. Abstracts were obtained for all references returned and were analyzed for relevance to the current project. The searches were performed on December 6, and are current as of December 6, 2013. The literature and patent searches uncovered no small molecules which had shown bactericidal effects against non-replicating M. tuberculosis (under carbon starvation condition) and also against the replicating variant. Few small molecules like TMC207 (Bedaquiline), clofazimine (24, 32) with bactericidal effects against replicating M. tuberculosis were reported to be active against non-replicating M. tuberculosis albeit in different stress condition (Figure 3).

Figure 3. Small molecules targeting non-replicating Mtb in different stress condition.

Figure 3

Small molecules targeting non-replicating Mtb in different stress condition.

Recently, a small molecule TCA1 was identified to target non-replicating Mtb in modified nutrient deprivation assay (30) and we were able to test TCA1 side by side with CID 780674 (HTS hit). TCA1 showed poor bactericidal effects in our 14-day model using carbon-starved M. tuberculosis in basal M7H9 medium (Figure 4), in contrast to its reported activity after 3 weeks of exposure to PBS-starved M. tuberculosis. Thus, ML408 has more rapid activity our model, which restricts growth exclusively through carbon limitation while still providing some minimal essential nutrients. At this point, ML408 is a suitable candidate for development as it shows activity against both replicating and carbon starved non-replicating M. tuberculosis adopting a completely drug tolerant state. ML408 represents an invaluable small molecule probe both for identifying essential functions and vulnerabilities of the M. tuberculosis bacilli in nutrient deprivation state and for proving the relevance of this state in vivo during infection.

Figure 4. Activity profile of TCA1 in carbon starvation assay.

Figure 4

Activity profile of TCA1 in carbon starvation assay.

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