IntroductionTuberculosis (TB) is a severe and life-threatening infectious disease caused by a highly specialized human intracellular pathogen, Mycobacterium tuberculosis1. Standard chemotherapy and chemoprophylaxis treatment for drug-susceptible M. tuberculosis involves a six-month administration of a combination of first-line drugs, such as isoniazid (INH), ethambutol (ETH), rifampicin (RIF), and pyrazinamide (PZA), with a success rate of 85%2,3. Isoniazid is a prodrug activated by the enzyme catalase-peroxidase (KatG), forming adducts with nicotinamide adenine dinucleotide (NAD). These adducts inhibit the activity of enoyl-ACP reductase (InhA), leading to M. tuberculosis death4. However, the emergence of drug-resistant strains, such as those with genetic mutations in genes like KatG, thaws the efforts of the global campaign to end TB. Moreover, M. tuberculosis’s metabolic adaptation to the host lung microenvironment contributes to the development of drug-resistant strains5. This highlights the urgent requirement for alternative strategies to address the rapid increase in drug-resistant TB.The outcome of M. tuberculosis infection, whether the bacteria are drug-susceptible or drug-resistant, is determined by the ability of macrophages to modulate intracellular replication and the strategies employed by M. tuberculosis to avoid being killed by the macrophages6,7. Therefore, the progression of TB is influenced by the complex interaction between the macrophages and the Mycobacterium strain, both in terms of their phenotypic and genotypic features8,9. The production of cytokines and immune responses induced in both in-vitro and in-vivo models depends on the genotype and strain of M. tuberculosis. Therefore, it is essential to understand the gene expression of both the mycobacteria and host macrophages to comprehend the molecular mechanisms used by M. tuberculosis during an infection9,10. This knowledge is essential in developing strategies to combat TB disease. Repurposing apoptotic inducer drugs as potential M. tuberculosis agents may be an interesting approach to combat the pathogen and facilitate its clearance through apoptosis11,12,13. Previous research has shown that elesclomol, an apoptotic agent, exhibited a minimum inhibition concentration (MIC) of 4 mg/L against clinical isolates of extensively drug-resistant (XDR) M. tuberculosis14. Additionally, a combination of RIF and elesclomol effectively suppressed the intracellular growth of M. tuberculosis in bone marrow-derived macrophages. In another study, an anticancer drug with pro-apoptotic properties is also under investigation for its application as a host-directed therapeutic for treating TB15.In this study, 12 apoptotic inducer drugs from various classes were selected for repurposing as antitubercular agents. Minimum inhibition concentration (MIC) assays were conducted to determine the compounds whole-cell activity against Mycobacterium smegmatis mc2155 and M. tuberculosis H37Rv. This was followed by a host-directed evaluation of compounds to stimulate clearance of M. smegmatis mc2155 in infected THP-1 macrophage cells and the production of cytokines during treatment, which were measured using Luminex. The cell envelope of M. tuberculosis comprises mycolic acids and plays a critical role in the intricate immunomodulatory interaction between the bacterium and the host16,17,18. The biochemical pathways involved in the biosynthesis of mycolic acids comprise over 20 distinct multi-enzyme complexes that are well-characterized. One of the enzymes in the synthesis of mycolic acids is targeted by a prodrug called isoniazid. However, the increasing mutation rates of the katG, kasA, and inhA genes, which encode pro-drug activating enzymes, complicate TB treatment. Therefore, it is essential to develop new therapeutic agents that possess a specific mode of action and do not require prior activation19. Herein, the apoptotic inducer drugs were evaluated for potential strong binding dynamics to InhA using molecular docking, molecular dynamics simulations, and binding free energy calculations.Materials and methodsEvaluation of antimycobacterial activity against M. smegmatis mc2155 and M. tuberculosis H37Rv to determine MICsAntimycobacterial inhibitory assays were performed to evaluate the bioactivity of the selected 12 apoptotic inducer drugs in Fig. 1, Table S1, and Table S2 (Sigma-Aldrich, Missouri, United States) against M. smegmatis mc2155 and M. tuberculosis H37Rv.Fig. 1Compound structures of the 12 apoptotic inducer drugs investigated in this study.Full size imageThe drugs were initially dissolved in dimethyl sulfoxide (DMSO) to make a stock solution of 10 mg/mL and stored at -20 °C. Working stocks were prepared by diluting with Middlebrook 7H9 broth (Becton, Dickinson and Company, New Jersey, United States), supplemented with 2% glycerol, 0.2% Tween-80 (v/v), and 10% Middlebrook oleic albumin dextrose catalase (OADC) (Becton, Dickinson and Company, New Jersey, United States).Cultures of M. smegmatis mc2155 and M. tuberculosis H37Rv were maintained as previously described Tapfuma et al.20. Inoculum for treatment was prepared using bacterial cultures at the exponential phase (OD600 nm = 0.4–0.6). The addition of drugs and serial micro-dilution on 96-well plates was then performed to obtain a range of concentrations. After adding the inoculum to 96-well plates, the final volume in each well was 200 µL with the concentration of bacteria at a calculated OD600 nm of 0.002–0.003. Treatments were incubated at 37 °C for 72 h for M. smegmatis mc2155 and 144 h for M. tuberculosis H37Rv. INH was used as the positive control. The MICs were determined by adding 0.2 mg/mL of resazurin dye as the colorimetric growth indicator. The experiments were conducted in three technical and biological replicates and the highest concentration tested for all the 12 drugs was 200 µg/mL.Thawing and culturing of THP-1 macrophage cellsThe THP-1 cells (TIB-202) were obtained from ATCC (https://www.atcc.org/). The THP-1 macrophage cells cryopreserved stock was thawed and washed to eliminate any dimethyl sulfoxide (DMSO) used for preservation. For the washing process, 1 mL of the stock was transferred to 5 mL pre-warmed RPMI 1640 with L-glutamine (Lonza, Sigma-Aldrich, Missouri, United States), supplemented with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Massachusetts, United States) (culture medium), followed by centrifugation at 180 × g for five minutes. The DMSO-free cells were then suspended in 5 mL of the culture medium and placed into a 25 cm2 tissue culture flask (Greiner, Lasec, South Africa). Incubation was conducted at 37 °C in a humidified incubator (ESCO Vivid Air, South Africa) with 5% CO2 for four days or until reaching 80% confluency. The culture was centrifuged at 180 × g and the pellet was resuspended and sub-cultured. An aliquot, 250 µL of the resuspended pellet of the THP-1 culture was transferred into 20 mL of fresh culture media in a 250 cm2 tissue culture flask (Greiner, Lasec, South Africa), followed by incubation at 37 °C until the culture reached 80% confluence.Differentiation of THP-1 macrophage cellsThe THP-1 cells from the confluent subculture were centrifuged and then suspended in fresh media. The cells were diluted to the desired density in culture media containing 50 µg/μL of phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, Missouri, United States) to differentiate the cells. Cells were seeded into either 96- or 24-well plates. The THP-1 macrophage cells were incubated for 72 h to facilitate their differentiation. After differentiation, the wells were washed twice with an equivalent volume of pre-warmed 1 × PBS, and the same volume of fresh media was added. Subsequently, the cells were incubated for 24 h to facilitate the recovery of THP-1 macrophages.Cytotoxicity analysis against THP-1 macrophage cellsFollowing the 24-h recovery phase, the cells were treated in triplicate with apoptotic inducer drugs at 25 µg/mL and then incubated for 24 h. A cytotoxicity assay was performed using 0.5 mg/mL of yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or MTT), which was incubated for 4 h. The media was then carefully replaced with DMSO. The intensity of the purple formazan crystals was subsequently measured using a spectrophotometer, and the data were analysed using GraphPad Prism (Version 8.0.1 for Windows, GraphPad Software, San Diego, California, United States).In-vitro infection of THP-1 macrophage cell with M. smegmatis mc2155THP-1 macrophage cells were grown and differentiated as follows: The THP-1 macrophage cells were diluted to 2 × 105 cells/mL and one mL was dispensed per well of a 24-well plate. The non-pathogenic M. smegmatis mc2155 was used as a surrogate for M. tuberculosis H37Rv. A cryopreserved M. smegmatis mc2155 stock was cultured in 10 mL of 7H9 OADC (Difco, Becton, Dickinson and Company, New Jersey, United States) until reaching an OD600 nm of 1.0. The bacterial culture was then washed twice through centrifugation at 4000 × g for 10 min in an equal volume of 1 × PBS. The pellets were resuspended in 5 mL of cell culture medium consisting of RPMI 1640 (Thermo Fisher Scientific, Massachusetts, United States) supplemented with 10% FBS. The resuspended cells were sonicated for 12 min at 37 °C to disrupt cell clumps. The bacterial cultures were filtered through a 40 µm filter, and the OD600 nm was measured. To attain a desired Multiplicity of Infection (MOI) of approximately 2:1, a conversion factor of OD600 nm of 1.0 = 1 × 108 bacteria was employed to dilute the bacterial cultures to obtain a MOI of 2:1. THP-1 cells were removed from the incubator, the spent medium was replaced with 1 mL of the freshly prepared culture, and media was added to the uninfected wells. The plates were then incubated at 37 °C in a humidified incubator (ESCO Vivid Air, South Africa) with 5% CO2 for three hours to facilitate the infection of THP-1 macrophage cells. Post-infection, the bacterial culture was removed, and THP-1 macrophage cells were treated with 1 mL of 1:100 penicillin (10,000 UI/mL, Sigma-Aldrich, Missouri, United States) diluted in RPMI medium. Plates were incubated at 37 °C for 40 to 60 min to allow the antibiotic to eliminate any extracellular bacteria. The wells were washed twice with 1 × PBS and treated with apoptotic inducer drugs for various time points.Sampling was performed for each time point (0, 6, and 12). The supernatant was collected from representative wells for each treatment, then centrifuged and stored for cytokine analysis. The media was removed from all the wells, replaced with penicillin, and incubated for 40 to 60 min to eliminate any extracellular bacteria. The plates were again washed twice with 1 × PBS. Subsequently, 500 µL dH2O was added to the wells to lyse THP-1 macrophages. Each sample of lysed macrophage cultures (100 µL) was transferred to 900 µL of 1 × PBS containing 0.05% Tween-80, and serial dilutions were prepared up to 10–4. From these dilutions, 100 µL was plated in quarters on Middlebrook 7H10 agar and incubated at 37 °C to determine the CFU/mL. An uninfected control was plated, and no bacterial growth was expected on these plates. Colonies of mycobacteria were counted from two days onward for M. smegmatis mc2155. The number of colonies was recorded as colonies forming units (CFU) and represented the number of intracellular mycobacteria that survived the apoptotic drug treatment.Cytokine analysis by Luminex technologySamples intended for cytokine analysis were processed according to the manufacturer’s instructions. A centrifugation speed of 4000 × g was chosen to precipitate the bacteria and macrophage debris, ensuring the retention of our proteins of interest in the supernatant. Cytokine production was measured using Luminex PROCARTAPLEX 13 PLEX (Thermo Fisher Scientific, Massachusetts, United States). The supernatants of THP-1 macrophage culture were collected during THP-1 infections with M. smegmatis mc2155, as mentioned above. The samples collected at each point were M. smegmatis mc2155 centrifuged at 4000 × g at 4 °C for 10 min to remove all bacteria and macrophage debris. The top layer of the supernatant was transferred into labelled tubes and stored at -80 °C until the day of cytokine analysis. Cytokines were quantified following the protocol as indicated by the manufacturer’s instructions.Molecular dockingX-ray structure of InhA corresponding to PDB:6R9W with a resolution of 1.75 Å was retrieved from the protein data bank (https://www.rcsb.org/structure/). The protein preparation was performed as described by Nyambo et al.21 in Schrödinger Release 2022–4. Hydrogen atoms were added, hydrogen-bond assignments were optimized, the loop was refined, and the OPLS4 (Optimized Potentials for Liquid Simulations 4) force field was used for energy minimization. The binding site was generated from the coordinates of the co-crystallized ligand (2 ~ {S})-1-(benzimidazol-1-yl)-3-(2,3-dihydro-1 ~ {H}-inden-5-yloxy)propan-2-ol using the Receptor Grid Generating module (Schrödinger Release 2022–4). The co-crystallized ligand (2 ~ {S})-1-(benzimidazol-1-yl)-3-(2,3-dihydro-1 ~ {H}-inden-5-yloxy)propan-2-ol, was used as the control. The apoptotic compounds were acquired from PubChem. (https://pubchem.ncbi.nlm.nih.gov/). The compounds were prepared using the LigPrep module (Schrödinger Release 2022–4) as previously described22. Briefly, the LigPrep energy minimized the compounds using the OPLS4 force field, generated ionization states at pH 7.0 ± 2.0 and generated multiple conformers per ligand. The control ligand was subjected to extra-precision (XP) molecular docking calculations against the selected target proteins using the Glide module (Schrödinger Release 2022–4)23. The root mean square deviation of the docked control ligand and undocked control ligand was calculated to verify the docking protocol. The apoptotic compounds were subjected to the verified XP docking protocol in the Glide module (Schrödinger Release 2022–4).Molecular dynamics simulationsThe dynamic stability and molecular interactions of protein–ligand complexes generated from the XP docking were evaluated by performing 400 ns (ns) molecular dynamics (MD) simulations using Desmond (Schrödinger Release 2022–4). A total of seven MD systems were prepared in Maestro (Schrödinger Release 2022–4) as previously described20. Briefly, the TIP3P hydration model explicitly solvated the protein–ligand complex in an orthorhombic box with a buffer boundary dimension of (10 × 10 × 10 Å). The system was neutralized by adding counter ions (Na+ and Cl-) and sufficient NaCl was added to produce a 0.15 M buffer solution. For long electrostatic forces, periodic grid conditions were automatically generated for Particle-mesh Ewald Fast Fourier Transform (FFT). The entire system was energy-minimized and equilibrated at constant pressure (1.01325 bar) and temperature (303.15 K). The MD simulations were performed with the NPT ensemble. The Nose–Hoover thermostat was used with a 1.0 ps interval, and Martyna-Tobias-Klein was used as the default barostat with a 2.0 ps interval by applying an isotropic coupling style. The systems were subjected to MD simulations for 400 ns, and the internal energy was stored for every 1000 ps of the actual frame. The structural and dynamic behavior of the protein–ligand complexes were calculated by the Simulation Interaction Diagram module in Maestro (Schrödinger Release 2022–4) and represented as the root mean fluctuation (RMSF) and the root means square deviation (RMSD). The post-molecular dynamic simulation analysis was conducted by calculating the protein–ligand binding free energies based on MD simulation trajectories. The molecular mechanics generalised Born surface area (MM-GBSA) (ΔGbind) (kcal/mol) binding free energies were computed based on (Molecular Mechanics + Implicit Solvent Energy Function)24.Statistical analysisStatistical analyses were conducted using GraphPad Prism v.8.0.1. For infection data, multiple t-tests followed by the Holm-Sidak correction were performed to determine statistical significance with an α level of 0.05. Each dataset was analyzed individually without assuming a consistent standard deviation, involving three separate t-tests. To address significant gaps between CFU values from technical and biological replicates, treatment efficacy was considered valid only if the ratio of post-treatment CFU to control CFU was 80% or higher, indicating 20% inhibition or less. Cytokine data were analyzed using ordinary two-way ANOVA in the same software.ResultsIn-vitro antimicrobial activity against M. smegmatis mc2155 and M. tuberculosis H37RvThe antimycobacterial activity assays performed using the 12 repurposed apoptotic inducer drugs revealed that four, namely, CEP (1.5 µg/mL), DIH (3.1 µg/mL), NUT (12.5 µg/mL), and MAR (25 µg/mL) showed the highest antimycobacterial activity against M. tuberculosis H37Rv (Table 1). The rest of the drugs recorded MICs > 200 µg/mL against M. smegmatis mc2155 and M. tuberculosis H37Rv.Table 1 Antimycobacterial activity of apoptotic inducer drugs against M. smegmatis mc2155 and M. tuberculosis H37Rv.Full size tableCytotoxicity of the apoptotic inducer drugs against differentiated THP-1 macrophage cellsIn the study, the cytotoxicity of apoptotic inducer drugs was evaluated against differentiated THP-1 macrophage cells, setting 80% cell viability as the minimum tolerable treatment concentration (Fig. 2). There was no significant difference between treated and untreated samples except for BV02, 17-AAG, MAR, and DIH treatments at 25 μg/mL.Fig. 2Cytotoxicity profile of apoptotic inducer drugs against differentiated THP-1 macrophage cells. The healthy THP-1 macrophages were treated with various concentrations of the drugs corresponding to previously recorded MIC values against M. smegmatis mc2155 and M. tuberculosis H37Rv. Data represents three technical and biological replicates where treated macrophages were analyzed against untreated controls. Data was interpreted as viable percentages and analyzed through ANOVA where p ≤ 0,05*.Full size imageGrowth inhibitory activity of selected apoptotic inducer drugs against M. smegmatis mc2155 infected THP-1 cellsIn this study, the efficacy of apoptotic inducer drugs against intracellular M. smegmatis mc2155 was evaluated using the untreated THP-1 macrophage cells as the control sample. The drug MAR was excluded from subsequent assays due to limited resources and reports that nucleophiles can readily displace chlorides on pyrrole rings and also react with serum25. The results demonstrated that the investigated drugs, DIH, CEP, CAR, and NUT relatively inhibited the growth of intracellular M. smegmatis mc2155 (Fig. 3). Particularly, CEP showed the maximum growth inhibition at 100% after 12 h of treatment at a concentration of 3.15 µg/mL (Fig. 3). A significant reduction in M. smegmatis mc2155 relative survival was observed in DIH (6.25 μg/mL, p = 0.0002) and CEP (3.15 μg/mL, p