Ethics and inclusion statement
This research represents a collaborative effort between multiple research groups based in Belgium, the UK, South Africa, the USA, Canada, France and Spain. Authorship was determined solely based on substantial contributions to the conception, design, execution or interpretation of the work, and the roles and responsibilities of each author were defined according to their area of expertise. All studies involving animals or human participants received approval from the appropriate institutional ethics committees (see relevant sections) and were carried out in accordance with international and institutional ethical guidelines. All experiments involving M. tuberculosis or other pathogens were conducted in the appropriate biosafety-level facilities under strict containment protocols. No part of the research involved the use of identifiable human data or samples without consent.
Animal ethics statement
All the in vivo efficacy and lung nucleobase studies were performed at Janssen Pharmaceutica in Beerse, in a certified BSL3 facility in agreement with European Directive (2010/63/EU) and national directives for the protection of animals for experimental purposes. All procedures were approved by the Ethics Committee of Janssen Pharmaceutica (license number LA1100119), located in Beerse, Belgium, which is accredited by Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) since 2004 under the unit number 001131 (https://www.aaalac.org/). For pharmacokinetic studies at Pharmaron, all procedures were approved by the Institutional Animal Care and Use Committee and Pharmaron holds full AAALAC accreditation.
Human ethics statement
The collection of human biological samples for this study was approved by the University of KwaZulu-Natal Biomedical Research Ethics Committee (BREC reference number BE019/13). The protocol includes the acquisition of lung tissue from anonymized patients undergoing pneumonectomy or lobectomy procedures at two public hospitals in Durban, South Africa: Inkosi Albert Luthuli Central Hospital and King Dinizulu Hospital Complex. Written, informed consent was obtained from all study participants who were routinely assessed for extent of disease and fitness for surgery according to standard imaging and clinical parameters. Of note, the patients with TB in this study cohort completed 9–24 months of anti-TB treatment before surgery, largely determined by M. tuberculosis susceptibility profiles. Lung tissue specimens from 15 patients with TB who were HIV negative (n = 15) had visible tubercles with a variable combination of associated haemorrhage, cavitation, fibrosis and bronchiectasis. Control lung specimens (n = 15) were obtained from patients with cancer who were TB and HIV negative undergoing lung surgery. Upon resection, representative tissue samples were immediately placed in conical tubes and snap frozen on dry ice before transport. Samples were then stored at −80 °C until thawed for homogenization.
Mice
Six-to-eight-week-old female Balb/cBy mice were purchased from Charles-River or Janvier. Mice were allocated in the BLS3 facility in individually ventilated cages in HEPA-filtered racks and rested for at least 5 days to allow acclimatization. Access to water and food was ad libitum.
Bacterial strains and growth conditions
The M. tuberculosis H37Rv strain was kindly provided by R. Brosch (Institut Pasteur). The strain was regularly passaged in mice to maintain virulence and retain PDIM-related genes. To prepare frozen stocks, H37Rv was grown in Middlebrook 7H9 (Becton-Dickinson) culture medium supplemented with 10% oleic acid–albumin–dextrose–catalase (OADC) complex (Becton-Dickinson), 0.2% glycerol and 0.05% Tween 80 (Sigma-Aldrich). Upon reaching stationary phase, the H37Rv culture was harvested in glycerol (15%; Becton-Dickinson) containing Middlebrook 7H9 medium and frozen at −80 °C.
Guided compound screening and MIC assay
Compounds screened in dose–response were tested in fourfold dilutions from 5 mM to 0.005 µM in black, clear bottom, 384-well microtitre plates (Greiner). Using an Echo liquid handler, a low volume dilution range in 100% DMSO was dispensed in the plates (150 nl per well). Reference plates were included as well as positive and negative control wells in each plate. M. tuberculosis H37Rv (5 × 105 CFU ml−1) diluted in Middlebrook 7H9 medium supplemented with 10% OADC, 0.2–0.5% glycerol and 0.05% Tween 80 was added to the compound plates (30 µl). Plates were incubated for 7 days at 37 °C. Before absorbance measurements (optical density at 620 nm (OD620)) using an Envision multimode plate reader (Perkin Elmer), the plates were shaken (4 min at 1,000 rpm). MIC50 values were determined as the lowest drug concentration, inhibiting at least 50% growth observed in the positive control wells using Microsoft Excel and presented as pMIC50 (Supplementary Fig. 1). For dose–response curves with JNJ-6640 or the CRISPRi-mediated transcript knockdown strain, plates were generated as above, with compounds tested in twofold dilutions, with the presence or absence of 100 ng ml−1 anhydrotetracycline (ATc). MIC90 values were determined as the lowest drug concentration inhibiting at least 90% growth observed in the positive control wells.
MBC99.9 assay
M. tuberculosis H37Rv (1 × 105 CFU ml−1) diluted in Middlebrook 7H9 medium supplemented with 10% OADC, 0.5% glycerol and 0.05% Tween 80 was added to 96-well plates containing compounds in dose–response. In each compound plate, two reference compounds in dose–response and positive control wells were included. After incubation of the plates for 17 days, 5 µl of each condition was stamped on 7H10 plates supplemented with 0.4% charcoal (Sigma-Aldrich), 0.5% glycerol and 10% OADC. In brief, a tenfold serial dilution was prepared of each concentration in PBS, plated on 7H10 six-well plates (plus 0.4% charcoal, 0.5% glycerol and 10% OADC) and incubated for 21 days before counting CFU. The MBC99.9 was defined as the concentration giving at least 3-log10 reduction compared with the initial CFU. For nucleotide rescue experiments, M. tuberculosis (5 × 105 CFU ml−1) was treated for 17 days in the presence of 1 µM JNJ-6640 supplemented with either various concentrations of hypoxanthine or adenosine.
Resistance generation
To isolate resistant colonies, agar plates containing approximately 25× MIC90 of either compound JNJ-7310, JNJ-6627 or JNJ-6640 were inoculated with WT M. tuberculosis (1–5 × 108 CFU) to select resistant colonies. Individual colonies were re-plated in the presence of compound for approximately 3 weeks to confirm resistance. Genomic DNA was isolated from resistant colonies using the Quick-DNA fungal and bacterial miniprep kit (Zymo Research). Following whole-genome sequencing, reads were aligned to the M. tuberculosis H37Rv genome (release 4, 2021-03-23; mycobrowser.epfl.ch). Comparison of the sequences of PurF homologues from different species, with the location of resistance mutations, is shown in Supplementary Fig. 2.
Molecular modelling
A model of MtPurF was generated using an AlphaFold model (AF-P9WHQ7-F1) and compared with crystal structures of other PRPP amindotransferases9,35,36,37,38. The SiteMap detection tool was used to predict putative binding pockets39,40 (D-score of 1.03 was detected at the PRTase active site; Supplementary Fig. 3), and JNJ-6640 was docked in the putative binding site using IFD-MD (Schrödinger)41,42,43. All SiteMap and IFD-MD calculations were performed using Schrödinger software (release 2024-2, Schrödinger).
Stable isotope labelling
For the 15N stable isotope labelling experiment, 10 ml  M. tuberculosis H37Rv cultures (OD600 = 0.8 in 7H9 complete media) were treated with either DMSO (to 0.1% final concentration) or JNJ-6640 (100 nM final concentration) in DMSO (0.1% final concentration) and simultaneously supplemented with 15N-amide-glutamine (2 mM final concentration; Cambridge Isotope Laboratories). After 4 h incubation at 37 °C with shaking, cultures were centrifuged at 3,000g for 10 min at 4 °C. The supernatant was discarded and the pellet washed by resuspending in 150 mM ammonium acetate solution (pH 6.6) in ultra-pure water before centrifuging again as previously. Supernatant was discarded and pellet resuspended in 1 ml metabolite extraction solution (2:2:1 acetonitrile:methanol:water; all liquid chromatography–mass spectrometry (LC–MS) grade) and placed on dry ice for 10–20 min with frequent vortexing. Samples were then transferred to tubes containing 0.1-mm glass beads and the cells lysed using a Precellys 24 homogenizer (Bertin Technologies; 3 × 20 s at 6.0 ms−1). Samples were frozen at −80 °C overnight before being thawed, vortexed and centrifuged at 15,000g for 10 min at 4 °C. The supernatants were taken and filtered using prewashed Spin-X 0.22-μm centrifuge filters (Corning) by centrifuging for 15,000g for 10 min at 4 °C. Filtrates were stored at −70 °C until LC–MS analysis. For quality control purposes, additional bacterial samples were taken as above but using unlabelled glutamine, and background controls were taken by performing the same method on the media without bacteria and on the extraction solution only. Equal aliquots from each experimental sample were combined into a pooled sample. This pooled sample was used to create a dilution series and identical run control samples that were placed regularly throughout the LC–MS sample run.
LC–MS was performed using an Agilent 1290 Infinity II Bio LC system connected to an Agilent Accurate Mass 6546 QTOF mass spectrometer. Chromatography was performed using an InfinityLab Poroshell 120 HILIC-Z column (Agilent; 2.1 × 150 mm, 2.7 µm) at 15 °C. A binary gradient was used with 20 mM ammonium acetate in water (pH 9.3) containing 5 µM InfinityLab Deactivator Additive (Agilent) for mobile phase A and pure acetonitrile for mobile phase B. Flow rate was 400 μl min−1 with the solvent gradient changing linearly between the following times and holds: 0–1 min for 90% mobile phase B; 8 min for 78% phase B; 12 min for 60% phase B; and 15–18 min for 10% phase B. The solvent ratio was then returned to 90% phase B before re-equilibrating for 4.5 5 min. For MS, ionization was performed using ESI in negative mode with nebulizer pressure of 30 psig and a nitrogen drying gas flow rate of 9 l min−1 at a temperature of 225 °C. The capillary, nozzle and fragmentor voltages set to 3,000 V, 500 V and 80 V, respectively. The MS acquisition rate was 1 spectra per second. Metabolites were identified by their m/z values, MS/MS fragmentation, and comparison of fragmentation patterns and retention times to standards. Chromatogram alignment, targeted feature extraction, quantification and isotopologue analysis were performed using the Agilent MassHunter software suite. Stable isotope-labelled abundances were corrected for the natural abundance of 15N and confirmed by checking for lack of corresponding signal in the samples incubated with unlabelled glutamine. For quality control purposes, extracted metabolites with an r-squared value of less than 0.85 in the pooled sample dilution series or a relative standard deviation in the run control samples of more than 25% were removed from analysis. Metabolite abundances were normalized to the soluble protein content in each sample as measured by bichinchonic acid assay (Pierce BCA Protein assay kit, Thermo Scientific) performed as per the manufacturer’s instructions.
Measurement of glutamine levels
Ten millilitres of M. tuberculosis H37Rv cultures (OD600 = 0.8 in 7H9 complete media) was treated with either DMSO (to 0.1% final concentration) or JNJ-6640 (1,000 nM final concentration) in DMSO (0.1% final concentration). After 24 h of incubation at 37 °C with shaking, samples were processed for LC–MS analysis as described above. The LC–MS was performed using an Agilent 1290 Infinity II LC system connected to an Agilent Accurate Mass 6545 QTOF mass spectrometer. Chromatography was performed using an Cogent Diamond Hydride Type C silica HPLC column (MicroSolv) at 25 °C. A binary gradient was used with 0.2% acetic acid in water for mobile phase A and 0.2% acetic acid in acetonitrile for mobile phase B. The flow rate was 400 μl min−1 with the solvent gradient changing linearly between the following times and holds: 0–2 min for 85% mobile phase B; 3–5 min for 80% mobile phase B; 6–7 min for 75% mobile phase B; 8–9 min for 70% mobile phase B; 10–11 min for 50% mobile phase B; and 11–25 min for 20% mobile phase B. The solvent ratio was then returned to 85% phase B before re-equilibrating for 5 min. For MS, ionization was performed using ESI in positive mode with nebulizer pressure of 50 psig and a nitrogen drying gas flow rate of 5 l min−1 at 300 °C. The capillary, nozzle and fragmentor voltages were set to 1,500 V, 2,000 V and 100 V, respectively. Data were analysed as above with glutamine levels normalized to the sum of all the extracted metabolite intensities for each sample.
Generation of CRISPRi-mediated knockdown strains
DNA fragments encoding small guide RNAs (sgRNAs) that specifically target purF were cloned into the plRL2 plasmid as previously described4. In brief, complimentary single-stranded oligos, obtained from an online database of sgRNA sequences (pebble.rockefeller.edu), were annealed then cloned into plRL2 (Addgene) via BsmBI restriction sites using Golden Gate Assembly (Supplementary Table 1). Resulting colonies were Sanger sequenced to confirm the presence of the target sequence. Targeting and empty vector plRL2 plasmids (500 ng) were electroporated into WT M. tuberculosis H37Rv. Colonies were selected from 7H10 agar plates containing 50 µg ml−1 kanamycin. Inducible transcript knockdown using 100 ng ml−1 ATc was confirmed using quantitative real-time PCR (rt–qPCR). CFU counts were taken on days 7, 14 and 21 by plating serial dilutions onto 7H10 agar before incubation at 37 °C for 3 ± 1 weeks to measure the effect of transcript knockdown on growth over time.
rt–qPCR
CRISPRi-mediated knockdown strains with and without the presence of 100 ng ml−1 ATc or JNJ-6640-treated H37Rv were grown to mid-log phase and diluted to an OD600 of 0.05 in 7H9. The empty vector strain was grown in the presence of ATc. After 4 days, RNA was extracted using the RNeasy kit (Qiagen) and genomic DNA was depleted using Turbo DNAse (Invitrogen). cDNA was generated using Superscript III reverse transcriptase (Invitrogen). Relative transcript knockdown of each gene was determined using SYBR green and gene-specific primers (Supplementary Table 1). rt–qPCR was performed using the Applied Biosystems 7500 Fast Real-Time PCR System with the following conditions: 50 °C for 20 s, 95 °C for 10 min then 40 cycles of 95 °C for 10 s, 60 °C for 1 min followed by 95 °C for 15 s, 60 °C for 1 min, 95 °C for 30 s and 60 °C for 15 s. Transcripts of interest were normalized against sigA. Analysis was performed using the comparative Ct method44.
Determination of nucleobases levels in TB-infected and uninfected mouse lung tissue
Mice were infected intranasally with 200 CFU per mouse for 7 days before euthanization and lung collection. Lung tissue was snap frozen in liquid nitrogen and kept at −80 °C until further processing. Frozen lungs where homogenized in refrigerated GentleMACS M tubes containing 2.5 ml 70:30 methanol:water, using the GentleMAC Octa Dissociator on program ‘RNA_02_01’. Lung homogenate aliquots (50 µl) were transferred to screwcap Micronic tubes filled with 450 µl refrigerated acetonitrile, vortexed for 10 s, incubated on ice for 10 min and stored at −80 °C until bioanalysis.
Liquid chromatography was performed on an Acquity Premier system (Waters) and this was coupled to an Xevo TQ Absolute Triple Quadrupole Mass Spectrometer (Waters). For UHPLC analysis, a Cogent Diamond Hydride 4 u 2.1 × 150 mm column was used (MicroSolv Technology) at 30 °C. Solvent A consisted of 0.2% acetic acid in water and solvent B of 0.2% acetic acid in acetonitrile. Chromatographic separation of the nucleobases was obtained at a flow rate of 0.4 ml min−1 under the following conditions: gradient starting conditions were 95% solvent B with an isocratic hold for 2 min, then multiple linear gradient steps were applied each followed by an isocratic hold for 1 min, first to 80% solvent B in 3 min, followed by a linear gradient to 50% solvent B in 1 min and a final step to 20% solvent B in 0.5 min, afterwards the initial conditions were reached in 0.1 min. Total run time was 16 min to obtain optimal equilibration of the analytical column. The injection volume was 1 µl. Electrospray MS analysis was carried out on an Xevo TQ Absolute Triple Quadrupole Mass Spectrometer (Waters) operated in the positive ion mode. The instrument was optimized by flow injection for each nucleobase. The following internal standards were used: adenosine-13C10, 15N and xanthine-13C, 15N2. Selective reaction monitoring transitions were used as a quantifier: adenine (135.9 > 118.9), guanine (151.9 > 134.9), hypoxanthine (136.9 > 118.9), adenosine (268 > 118.9), guanosine (284.1 > 151.9), inosine (268.9 > 136.9) and xanthine (152.9 > 110); a second selective reaction monitoring transition was used as a qualifier adenine (135.9 > 92), guanine (151.9 > 110), hypoxanthine (136.9 > 109.9), adenosine (268 > 135.9), guanosine (284.1 > 134.9), inosine (268.9 > 110) and xanthine (152.9 > 135.9). For chromatographic evaluation, Waters TargetLynx software version 4.2 (Waters) was used. Calibration curves were plotted using appropriate log–log linear regression.
Human TB-infected and uninfected tissue homogenization and metabolite extraction
Wet mass of selected human lung specimens for homogenization ranged from 99 to 120 mg. Protocol for tissue homogenization was as follows: tissue sample was washed three times with 5 ml of refrigerated PBS (Gibco) and placed on adsorbent cloth to remove excess. Sample was then added to a refrigerated gentleMACs M tubes (Miltenyi Biotec) containing 1.3 ml of 70% methanol extraction solvent containing d4-alanine (Sigma) at a final concentration of 100 µg ml−1. Homogenization of tissue was performed using a gentleMACS Octo Tissue Dissociator (Miltenyi Biotec), using the ‘RNA_02_01’ protocol. Tubes were then centrifuged at 2,500g, 4 °C for 30 min and the supernatant transferred to a 70-µm nylon cell strainer (Falcon). The homogenate flow-through was then filter sterilized using 0.22-µm filter tubes (Costar Spin-X, Corning) at 13,000g at 4 °C for 10 min. Filtrates were then placed in a SpeedVac centrifugal concentrator (Thermo Scientific) and solvent removed overnight under vacuum. Dried residual solutes were reconstituted in high-purity water and placed in a Thermomixer (Eppendorf) at room temperature and shaken for 1 h (1,300 rpm). For LC–MS/MS analysis, samples were further diluted with acetonitrile (ACN) to a final solvent composition of 70% ACN.
LC–MS/MS analysis of human lung tissue
Metabolite extracts were analysed on a Thermo Q-Exactive mass spectrometer coupled to a Dionex Ultimate 3000 UPLC system. A Waters X-Bridge BEH Amide 2.1 × 100 mm HILIC column was used with mobile phase A (100% ACN) and mobile phase B (100% water), both containing 0.1% formic acid and run in gradient mode, at a flow rate of 200 μl min−1 and a temperature of 40 °C. The Q-Exactive parameters were as follows: ESI voltage of 3.5 kV was used, with a sheath gas flow rate of 45, aux gas of 10 and sweep gas of 2. Capillary temperature was kept at 250 °C. For high-resolution molecular ion scans, the range was from 50 to 750 m/z at a resolution of 70,000, in positive ion mode. For parallel reaction monitoring analysis, the basic conditions were the same, but collision energy values for each analyte were determined and were as follows: CE-40 for adenine, NCE-30 for, CE-38 for guanine, NCE-30 for guanosine, NCE-20 for inosine, CE-50 for hypoxanthine, NCE-35 for xanthine; for the internal control, d4-alanine, with a collision energy value of 10, was used, which was the minimum available and would suppress fragmentation. The gradient conditions for both methods were as follows: 0 min for 90–10% ACN to water then 15 min, 30–70% ACN to water, with a linear gradient and finally 15.1–26 min at 70% ACN. A solvent composition of 90% ACN to 10% water was used for column regeneration. Each sample was analysed twice: once using high-resolution molecular ion scan and a second time with the MS in parallel reaction monitoring mode where target molecular ions were fragmented and the spectra accumulated for further post-run analysis. Expected retention times on these methods were obtained from running high-purity, analytical standards (IROA Technologies, Thermo Scientific) for each analyte under the same conditions and used for molecule verification purposes. An external calibration series was prepared using the same high-purity, reference compounds for guanine, hypoxanthine, adenine, xanthine, guanosine, inosine and adenosine and used to calculate the concentration of analytes in the tissue samples. Peak area under curve values for analytes measured in samples were normalized to wet mass of the tissue. The RAW files obtained were processed using Skyline software (24.1.1.339) set up for this analysis and peak areas determined for both sample analytes and the external calibration standards. All solvents used for MS analysis were of highest purity.
Assembly of constructs for enzymatic assays
Gene synthesis and cloning were performed at Epoch Life Sciences. DNA encoding MtPurF (UniProt P9WHQ7) residues 35–527 was optimized for E. coli expression, synthesized and inserted into a custom-engineered pET28a vector. DNA encoding Homo sapiens PPAT (UniProt Q06203) residues 1–517 was optimized for Sf9 expression, synthesized and inserted into the baculovirus transfer vector pVL1393. The open reading frame of codon-optimized DNA sequences are documented in Supplementary Fig. 4.
Expression of MtPurF
BL21(DE3) chemically competent cells (New England Biolabs) were transformed with a pET28a vector containing a MtPurF expression cassette with a C-terminal tag for affinity purification. After transformant selection on LB agar plates supplemented with kanamycin (50 µg ml−1), a 500-ml starter culture was inoculated with a single colony and incubated overnight with shaking at 37 °C. The following day, the starter culture was diluted (1:50) into fresh medium with 50 µg ml−1 kanamycin for expression. Cultures were grown with shaking to mid-log phase (OD600 ~ 1) and induced with 1 mM isopropylthio-β-galactoside. After induction, cultures were transferred to a 16 °C shaker and expression was allowed to proceed overnight. The next day, cells were harvested by centrifugation (5,000g for 15 min at 4 °C). The pellet was transferred to a sterile bag and frozen at −80 °C. The soluble expression yield, evaluated by western blot, was estimated to be more than 25 mg l−1.
Expression of H. sapiens PPAT
Human PPAT was expressed in insect Sf9 cells using a baculoviral expression system. In brief, recombinant baculovirus was generated using the BestBac 2.0 system (Expression Systems) following the manufacturer’s instructions. Linearized Δv-cath/chiA baculovirus DNA was co-transfected with a pVL1393 transport vector containing a human PPAT expression cassette with C-terminal sortase, FLAG and 8-His tags into Sf9 cells using Cell Fectin II Reagent (Thermo Fisher) in a plate-based format. P0 virus was harvested and P1 baculovirus-infected insect cells (BIICs) were generated following protocol based on a method previously described45. Before large-scale expression, an additional round of amplification was performed to generate P2 BIICs. BIICs were stored in liquid nitrogen. Expression of human PPAT was carried out in Sf9 cells at an effective multiplicity of infection (MOI) of 0.3. Cells were expanded to reach desired volume and split back to approximately 2 × 106 cells per millilitre on the day of infection. P2 BIICs were incubated at 27 °C until almost thawed then diluted slowly in a small volume of expression medium. The diluted P2 BIICs were then added directly to the culture, and expression was allowed to proceed for approximately 72 h. After expression, cells were collected by centrifugation (1,000g for 20 min at 4 °C). The pellet was transferred to a sterile bag and frozen at −80 °C. The soluble expression yield, evaluated by western blot, was estimated to be 1–5 mg l−1.
Purification of MtPurF
All buffers were extensively sparged with N2 to minimize oxidation of the 4Fe–4S cluster. All purification steps were performed at 4 °C unless noted. The cell pellet was resuspended in 5 ml g−1 of lysis buffer containing 25 mM HEPES pH 7.5, 500 mM NaCl, 10 mM MgCl2, 10% glycerol and 1 mM dithiothreitol. The suspension was lysed by sonication and clarified by centrifugation for 30 min at 35,000g. Clarified supernatant was incubated with 1 ml l−1 CaptureSelect C-tagXL Affinity Matrix (Thermo Fisher Scientific) for 1 h with gentle rolling. Slurry was filtered over a 2.5-cm Bio-Rad Econo-Column. Bound resin was washed with 60 column volume (CV) of lysis buffer. Lysis buffer was supplemented with 3 mM C-tag peptide (Biosynth International) to generate elution buffer. Resin was eluted with 5 × 1 CV of elution buffer; each elution was incubated with the resin for 5 min before collecting. Elution pool was concentrated to 0.5 ml using an Amicon Ultra 30 kDa MWCO centrifugal filter. Concentrated sample was resolved on a Superdex 200 10/300 size-exclusion chromatography column (Cytiva) in buffer containing 25 mM HEPES pH 7.5, 150 mM NaCl, 2 mM MgCl2 and 1 mM dithiothreitol. Size-exclusion fractions containing PurF were pooled, aliquoted and frozen at −80 °C for storage.
Purification of H. sapiens PPAT
All buffers were extensively sparged with N2 in attempt to minimize oxidation of the 4Fe–4S cluster. All purification steps were performed at 4 °C unless noted. The cell pellet was resuspended in 5 ml g−1 of lysis buffer containing 25 mM HEPES pH 7.5, 500 mM NaCl, 10 mM MgCl2, 2 mM AMP, 25 mM imidazole, 10% glycerol and 10 mM dithiothreitol. The suspension was lysed by sonication and clarified by centrifugation for 30 min at 35,000g. Supernatant was applied to a 5-ml HisTrapFF column (Cytiva) and washed with a stepwise gradient of lysis buffer supplemented with either 50 mM, 75 mM or 100 mM imidazole before eluting with buffer containing 25 mM HEPES pH 7.5, 500 mM NaCl, 10 mM MgCl2, 2 mM AMP, 500 mM imidazole, 10% glycerol and 10 mM dithiothreitol. Elution fractions containing PPAT had a light brown colour. The PPAT-containing fraction was pooled and subjected to ammonium sulfate precipitation to enhance purity. The sample was brought to an ammonium sulfate saturation of 40% and incubated for 30 min. The 40% ammonium sulfate precipitated sample was centrifuged for 5 min at 16,000g. After centrifugation, brown supernatant containing PPAT was removed from the white-precipitated pellet, calibrated to an ammonium sulfate saturation of 50% and incubated for 30 min. The 50% ammonium sulfate precipitated sample was then centrifuged for 5 min at 16,000g. Clear supernatant was removed, and the brown-precipitated pellet containing PPAT was resuspended in lysis buffer, aliquoted and frozen at −80 °C for storage. The supporting information for PPAT purification can be found in Supplementary Fig. 5.
Biochemical MtPurF inhibition
A schematic of PurF enzyme function is shown in Supplementary Fig. 6. PurF (20 nM) was incubated with varying concentrations of JNJ-6640 for 30 min in 25 mM Tris pH 8, 10 mM MgCl2, 0.5 mg ml−1 BSA and 0.001% Tween-20, before the addition of 70 mM glutamine and 2.5 mM phosphoribosyl pyrophosophate in a Revvity 384w ProxiPlate Plus for a total reaction volume of 4 µl. Reactions were carried out for 3 h at room temperature before quenching with 20 mM AMP. Glutamate oxidase (0.05 U ml−1) was added and allowed to incubate for 1 h at room temperature. An equal volume of HyPerBlu reagent was added and allowed to incubate for 30 min at room temperature before reading luminescence on a BMG Pherastar FSX.
Biochemical human PPAT inhibition
PPAT (5 nM) was incubated with varying concentrations of JNJ-6640 for 30 min in 25 mM Tris pH 8, 10 mM MgCl2, 0.5 mg ml−1 BSA and 0.001% Tween-20, before the addition of 200 µM glutamine and 30 µM phosphoribosyl pyrophosophate in a Revvity 384w ProxiPlate Plus for a total reaction volume of 4 µl. Reactions were carried out for 3 h at room temperature before quenching with 25 mM EDTA. Glutamate oxidase (0.05 U ml−1) was added and allowed to incubate for 1 h at room temperature. An equal volume of HyPerBlu reagent was added and allowed to incubate for 30 min at room temperature before reading luminescence on a BMG Pherastar FSX.
Cell proliferation assay
Measurement of JNJ-6640 activity against a range of 93 cell lines was performed by Oncolead (Germany). In brief, the cell lines (see source data for Fig. 2h) were treated with varying concentrations (0.25 nM to 25 µM) of JNJ-6640, azathioprine, MMPR and MPA in vitro for 72 h. Growth inhibition was measured using sulforhodamin B, a protein staining assay, to calculate the pIC50 of the compounds in each cell line.
Timelapse microscopy experiments
WT M. tuberculosis (Erdman strain) transformed with pND257 expressing tdTomato was grown and seeded for imaging into a microfluidic device as previously described12,46. Imaging of the bacteria was carried out on an inverted wide-field fluorescent microscope (Thunder Imaging System, Leica Microsystems), equipped with an environmental chamber maintained at 37 °C (Okolab). The bacteria were imaged using a ×100/1.32 NA oil immersion objective (Leica Microsystems) on the phase and red (635 nm excitation and 642 nm emission) channels and images captured using a scientific CMOS K8 camera (Leica Microsystems). Imaging was carried out at 60-min intervals over a period of 13–16 days. The full timelapse movie can be found in Supplementary Video 1. Medium was pumped through the device with a flow rate of 10 µl min−1 using a syringe pump. When necessary, the medium was supplemented with 0.6 µM JNJ-6640 and/or 1× physiologically relevant nucleobase mix (final concentrations: 518 nM guanine, 5.2 µM hypoxanthine, 4.3 µM adenine, 1.42 µM guanosine, 15 µM inosine and 23 µM adenosine, reconstituted in DMSO; Supplementary Table 2). Experiments were carried out at least two times for each condition and about 20–30 distinct xy positions were imaged in each experiment. Images were acquired and assembled using the LAS-X software (Leica Microsystems) and analysed using FIJI software47. For imaging of M. tuberculosis-infected macrophages, mouse bone marrow-derived macrophages were isolated and differentiated as described earlier12. For infection, M. tuberculosis bacteria transformed with pND257 (expressing tdTomato) was grown to OD600 of 0.4–0.8, washed and resuspended in DMEM medium. Bacteria were filtered through a 5-μm filter to get rid of bacterial clumps and the filtrate was used to infect the macrophages at an MOI of 1:1, over a period of 4 h. Imaging of the infected macrophages was carried out on an inverted wide-field fluorescent microscope (Thunder Imaging System, Leica Microsystems), in an environmental chamber maintained at 37 °C and 5% CO2 (Okolab), using a ×20/0.8 NA dry objective (Leica Microsystems) on the brightfield and fluorescence channel 555 nm excitation and 594 nm emission) channels. Images were captured every 2 h over 7–10 days using a scientific CMOS K8 camera (Leica Microsystems). Full timelapse movies can be found in Supplementary Videos 2 and 3. At least 20 independent xy positions (3 ×1 μm z-steps) were imaged for each condition: no treatment, treatment with 3 μM JNJ-6640, treatment with 3 μM JNJ-6640 plus supplementation with 100 ng ml−1 IFNγ, treatment with 3 μM JNJ-6640 plus supplementation with 100 ng ml−1 IFNγ and 1× physiologically relevant nucleobases mix (Supplementary Table 2). Images were acquired and assembled using the LAS-X software (Leica Microsystems) and analysed using FIJI software47.
Rabbit caseum MBC assay
The rabbit caseum MBC assay was performed to assess the activity of JNJ-6640 against non-replicating bacteria in ex vivo rabbit caseum homogenate, as previously described16,17,18. In brief, a 50 mM stock solution was serially diluted in DMSO to achieve the final concentration range of 0.125–128 µM. The assay was conducted in a 96-well plate format, with a 7-day incubation at 37 °C. After incubation, caseum homogenate was sampled from each well and plated on 7H11 agar. Colony enumeration was performed after 4 weeks.
Confirming dormancy in foamy macrophage assay
The transcript levels of Tgs1 (Rv3130c), hspX (Rv2031c) and Rv3290c, known to be upregulated in stationary or low-oxygen conditions20,48, were used to confirm the induction of dormancy in our assay. Total RNA was extracted 4 days post-infection after hypoxia-infected or normoxia-infected THP-1 from at least three different batches. In brief, to protect RNA from degradation, macrophages were rinsed with PBS, then scraped with Maxwell RSC miRNA Tissue Kit homogenization solution/thioglycerol (50/1) (Promega). Followed by 10 min of incubation with Maxwell RSC miRNA Tissue Kit lysis buffer (Promega), cells and bacteria were lysed by bead beating into matrix B tubes containing silica beads (MP Biomedical) with the Super-Fast Prep-1 instrument (MP Biomedical). Finally, samples were processed into a Maxwell RSC instrument for RNA extraction. Reverse transcription was performed with 100 ng total RNA using SuperScript IV VILO Master (Applied Biosystems) or without reverse transcriptase (−RT). qPCR amplifications were run with a QuantStudio 12K Flex system (Applied Biosystems) using the oligos in Supplementary Table 1. The mRNA content was normalized to 16S expression, and relative expression was calculated following the ΔCt method (ΔCt = Ct(gene) − Ct(16S)) and expressed as 2−ΔCt. Also note that no change in CFU was observed between days 0 and 4, further confirming non-replication.
Determining bactericidality in foamy macrophages
Human THP-1 cells (American Type Culture Collection TIB-202) maintained in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 1 mM pyruvate and 2 mM l-glutamine. A luminescent M. tuberculosis H37Rv reporter strain (expressing LuxABCDE) was grown in Middlebrook 7H9 broth supplemented with 10% ADC, 0.4% glycerol and 0.05% Tween 80 until the mid-log phase. THP-1 cells were infected at a MOI of 0:4 in antibiotic-free RPMI 1640 medium containing 10% FBS, 1 mM pyruvate, 2 mM l-glutamine and 40 ng ml−1 phorbol 12-myristate 13-acetate for 4 h at 37 °C with 5% CO2. Following a 4-h incubation period, infected cells were harvested, washed and plated onto 96-well plates containing compounds (up to 30 μM). Infected cells were incubated under hypoxia (using anaeroPouch, Biomerieux). After 4 days of incubation under hypoxic condition, cell luminescence was measured using an Envision plate reader. The medium was then replaced, and cells were incubated under normoxia for 1 day. The luminescence was measured a second time using an Envision plate reader. Results were calculated in percent inhibition and analysed with Genedata software using the equation shown for ‘Intramacrophage MIC90 assay with M. tuberculosis’ shown in the Supplementary Methods.
Pharmacokinetics and tolerability in mouse
The pharmacokinetics of JNJ-6640 was investigated in female BALB/c mice dosed as solution at 1 mg kg−1 intravenously. JNJ-6640 was also dosed orally at 5 and 50 mg kg−1 as a solution. For the intravenous and oral arms, three animals were used. Animals had free access to food and water through each study. Blood samples were taken at multiple timepoints up to 24 h for intravenous dosing. The test compound was also dosed at 1,500 mg kg−1 subcutaneously as an LAI aqueous suspension. Three animals were used. Animals had free access to food and water through each study. Blood samples were taken at multiple timepoints up to 672 h after subcutaneous dosing. The animals were observed for any clinical signs of toxicity and effect on body weights during the 28-day period after subcutaneous administration of 1,500 mg kg−1. At the end of 672 h, the blood samples were also collected for the clinical chemistry evaluation. Plasma samples were prepared by protein precipitation with acetonitrile, and the supernatant was analysed for concentrations of compound using a qualified LC–MS/MS method.
Animal models
All experiments used 6–8-week-old female BALB/cBy mice. Regarding sample size, various scenarios were evaluated of 4–10 animals per group. From the power analysis, it was concluded that considering 6 animals per group provided more than 80% power to detect all significant effects of 1.5 CFU (log10), assuming s.d. = 0.5, with 20 or less groups (including the reference group). No blinding or randomization was performed.
In the short acute model, experiments were performed as previously detailed49,50. Mice were infected intranasally with 200 CFU M. tuberculosis H37Rv per mouse. To verify the infection level, a subgroup of six mice were euthanized 1 day after infection. After 1 week of infection, treatment was started. Either the LAI was administered (subcutaneous) on day 1 and day 7 or daily PO administration for 12 consecutive days. Mice were then euthanized 3 days after the last dose to prevent carry over effect. To monitor the evolution of the infection, a group of mice were euthanized 7 days post-infection, when treatment started, and 21 days post-infection, when treatment had ended. Negative control mice remained untreated.
In the chronic model, mice were infected intranasally with 200 CFU M. tuberculosis H37Rv per mouse. To verify the infection level, a subgroup of six mice were euthanized 1 day after the infection. Mice were infected 1 month before treatment start and treatment lasted for 8 weeks. Six mice were euthanized at the start of treatment as a pretreatment control. Negative control mice remained untreated.
In the high acute model, mice were infected intranasally with 10,000 CFU M. tuberculosis H37Rv per mouse51. To verify the infection level, a subgroup of six mice were euthanized 1 day after the infection. Mice were infected for 10 days before treatment start and treatment lasted for 2 weeks. Six mice were euthanized at the start of treatment as a pretreatment control. As the high infection levels would lead to severe clinical signs, no ‘vehicle control’ mice were used in this study. Mice were dosed by oral gavage (100 µl, drencher with rounded end straight, 0.9 mm × 25 mm, Socorex Swiss) except for the LAI formulation that was injected subcutaneously in the upper back (100 µl) using a needle (26 G × 13 mm, BD Microlance). At euthanization, whole lungs were aseptically collected in GentleMACS tubes (M tubes with strainer, Miltenyi Biotec) containing 2.5 ml of PBS and homogenized using the RNA_01_01 settings of GentleMACS Octo Dissociator (Miltenyi Biotec). Lung homogenate was diluted in PBS and plated in 7H10 charcoal agar plates containing antibiotics (100 µg ml−1 amphotericin, 25 µg ml−1 polymyxin B, 50 µg ml−1 carbenecillin and 20 µg ml−1 trimethoprim). Plates were incubated at 37 °C during 3–5 weeks. After that, CFU counts were recorded and data were expressed in the mean log CFU per lung for each group. Statistical analysis was done by one-way analysis of variance (ANOVA) with Sidak’s test for multiple comparisons or an unpaired t-test (GraphPad Prism). An outline of the three mouse models can be found in Supplementary Fig. 7.
Measurement of pairwise drug interactions
Pairwise drug interactions with JNJ-6640 were measured using a modified checkerboard assay with DiaMOND, with interactions quantified using the fraction inhibitory concentrations at the 50% growth inhibitory levels and calculated based on Loewe additivity as the null model. Culturing conditions, experimental design and analysis for drug interaction measurements with JNJ-6640 were as previously described52 so that drug interaction profiles with JNJ-6640 (measured in this study) could be directly compared with drug interaction profiles with moxifloxacin (measured previously52). In brief, M. tuberculosis were adapted to four different in vitro conditions (with butyrate or 0.2 mM cholesterol as carbon sources, a simple dormancy model and standard growth conditions) before drug treatment for the maximum length used in these assays (10, 24 and 5 days of treatment for butyrate, cholesterol and standard, respectively, and 7 days of treatment followed by 6 days of recovery for the dormancy model). Single and combination dose–response curves to calculate fractional inhibitory concentration at 50% inhibition (FIC50) values via DiaMOND were made in these growth conditions to model aspects of the microenvironments where M. tuberculosis are resident during infection52,53. Bedaquiline and pyrazinamide were sourced from Sigma, and pretomanid was sourced from APExBIO. Single-use aliquots of stock antibiotic solutions were prepared in DMSO and stored at −30 °C.
Material availability
All unique materials used in this article are readily available from the authors.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
