Screening of FDA-approved library of molecules against Mtb mc2 6206 reveals growth-inhibitory effect of semapimod, an anti-inflammatory small molecule.

a, Status of inhibitors showing activity against Mtb mc2 6206. Bar-graph depicts Mtb mc2 6206 inhibitors acting on different host metabolic pathways. b, MIC90 status of Mtb inhibitors. Bar-graph shows inhibitors with different MIC90 against Mtb mc2 6206, in vitro. Molecules with <0.025µM MIC90 (encircled) are tabulated in the inset. c, Determination of minimum dose of semapimod for complete inhibition of Mtb mc2 6206 growth by visual inspection. Isoniazid (INH) was used as a control in the 96-well plate-based assay. Drug concentration beyond which no visual growth is observed, is written in bold. d, Effect of semapimod on the in vitro growth of Mtb mc2 6206. Shown is the CFU enumeration of drug-treated and untreated (control) bacteria at the indicated time points. e, Effect of semapimod on the intracellular proliferation of Mtb mc2 6206. Intracellular growth of Mtb mc2 6206 in the THP1-derived macrophages was examined after 6 days of infection in the absence (UT) or the presence of 0.10-1.0µM semapimod. Treatment with 2.5µM INH was used as control. A significant reduction in CFU counts of Mtb mc2 6206 is observed in the presence of semapimod under in vitro culture conditions (d) as well as during intracellular (p< 0.005) growth (e). Data represent mean±s.d of at least n=2 replicates in d and n=4 replicates in e. Data in c are representative of n=2 independent experiments. p values in e were obtained after comparison of CFUs between the UT and semapimod-treated samples, as described in Materials and Methods.

Effect of semapimod treatment on the expression profile of Mtb mc2 6206 transcripts.

a, Volcano plot of differentially expressed genes in semapimod-treated Mtb mc2 6206. The plot shows distribution of genes that are differentially expressed via log2 fold-change and the –log p values. Broken vertical lines represent the cutoff of ≥1.0 log2 fold-change, and horizontal line represents the cutoff of >2.583 –log p values. Genes below the - log p cutoff are represented by grey dots. Downregulated genes are represented by red dots, and those showing upregulation in response to semapimod treatment are shown with green dots. b, Status of differentially accumulated transcripts. Heatmap representation of transcripts showing accumulation (left) or suppression (right) upon exposure to semapimod across three biological replicates. c, Functional categorization of differentially regulated genes. The butterfly chart shows distribution pattern of differentially regulated genes according to their function, as classified in the Mycobrowser database (https://mycobrowser.epfl.ch/genes/). Mean fold-change values from n=3 biological replicates are shown in a.

Semapimod treatment perturbs L-leucine uptake in Mtb mc2 6206.

a, Effect of semapimod on the in vitro growth of different mycobacterial species. Growth inhibitory effect of semapimod was assessed against slow-growing Mtb H37Rv and M. bovis BCG, and fast-growing M. abscessus and M. smegmatis, respectively, by visual inspection using 96-well plate-based assay. Rifampicin (Rif) and INH were used as control drugs against Mtb H37Rv. b, Estimation of intracellular leucine in Mtb mc2 6206. Intracellular leucine was estimated in untreated (UT) and semapimod-treated (Sem) bacteria, after 24 hours of drug treatment, by mass spectrometry as described in Materials and Methods. c–e, Effect of leuC-leuD and panC-panD expression in Mtb mc2 6206 on bacterial killing by semapimod. Time-(c) and dose- (d–e) dependent growth kinetics reveals loss of bactericidal effect of semapimod against Mtb mc2 6206 by leuC-leuD, and not by panC-panD expression. Percent viability in e was calculated with respect to untreated (UT) cultures after 2 weeks of exposure to different drug concentrations, using 96-well plate-based method as described in Materials and Methods. Data in a and c–d are representative of n=2 independent experiments. Mean±s.d values from n=4 replicates are shown in b.

Generation and characterization of semapimod-resistant strain of Mtb mc2 6206.

a, Propagation of putative semapimod-resistant (SemR) strain, but not the wild-type (WT) Mtb mc2 6206 in the presence of 50nM semapimod confirms drug-resistance in SemR. In contrast, both the strains exhibit substantial growth on 7H11-PLO agar plate in the absence of drug. Shown is the growth pattern of two different colonies– C1 and C2 of the respective strains. b, In vitro growth analysis of the WT and the SemR strains of Mtb mc2 6206. Comparative analysis of OD600 at different time points reveals substantially increased growth of SemR strain compared to WT under in vitro culture conditions. c, In vitro susceptibility of the WT and the SemR strains of Mtb mc2 6206 to vancomycin. Viability of WT and SemR Mtb strains was determined in the presence of vancomycin by 96-well plate-based assay, as described in Materials and Methods. Percent viability was calculated with respect to the untreated (UT) cultures after 2 weeks of exposure to different concentrations of antibiotic. Results show a substantial increase in susceptibility of SemR to vancomycin compared to WT. d, Whole genome map analysis of SemR. Shown is the genome map of SemR highlighting positions of genes undergoing substitutions, when compared with WT Mtb mc2 6206. Mutations leading to corresponding changes at the amino acid level in the respective protein are indicated alongside. Suspect genes presumably involved in providing semapimod resistance, are marked in red fonts. Data are representative of at least n=2 independent experiments in a–b. Mean±s.d values from n=3 biological replicates are shown in c.

Overexpression of ppsB alters susceptibility of SemR strain of Mtb mc2 6206 to semapimod and vancomycin.

a–b, In vitro susceptibility of different strains of Mtb mc2 6206 to vancomycin (a) and semapimod (b). Viability of WT and SemR Mtb strains in the presence of drugs was compared with those constitutively expressing ppsB by 96-well plate-based assay, as described in Materials and Methods. Percent viability was calculated with respect to UT cultures after 2 weeks of exposure to different concentrations of inhibitors. Remarkably, response of SemR to both semapimod and vancomycin is reversed upon overexpression of ppsB. Contrarily, ppsB expression in WT does not affect bacterial sensitivity to either of these drugs, thus indicating a specific effect of the PDIM biosynthesis gene in SemR. Mean values from n=3 biological replicates are shown in a–b.

Semapimod targets PDIM biosynthesis protein, PpsB.

a, Analysis of semapimod-PpsB interaction by BLI-Octet. Interaction of semapimod with Mtb PpsB was examined by optical interference–based biolayer interferometry from the Octet system (ForteBIO). Briefly, dialyzed 6xHis-PpsB was immobilized onto AR2G sensor up to a level of 2.1 nm. Binding was observed at the indicated concentrations of ligand (semapimod) to acquire differential graded response. Binding constant was calculated as per the standard steps, described in Materials and Methods. b, Analysis of cell-wall apolar lipids in the WT and SemR strains of Mtb mc2 6206. Cell-wall apolar lipids were extracted and analyzed by two-dimensional TLC as described in Materials and Methods, which reveals significant reduction in PDIMs of SemR when compared with the WT strain. Contrary to PDIMs, no change is observed in the triacyl glycerol (TAG) levels between the two strains. Position of PDIMs is marked by broken circle in both the images for clarity.

Effect of semapimod treatment on survival of Mtb H37Rv in the organelles of infected BALB/c mice.

a, Schematic of mouse infection. Infection was performed by aerosol route with the virulent Mtb H37Rv strain. After 21 days of infection, mice were divided into two groups: one receiving only 5% sucrose (sham) and others receiving 5mg/kg semapimod prepared in 5% sucrose. Intracellular bacterial load was determined by CFU plating of lung homogenates at days 1, 21 and 49, and in spleen homogenates prepared on day 49. b, Gross pathology of lungs. Images of lungs obtained from both the sham- and semapimod-treated groups of mice after 28 days of treatment (i.e., day 49 post-infection) are presented. Scale bar is shown for size reference. c–d, Effect of semapimod treatment on intracellular survival of Mtb H37Rv. Intracellular survival was determined by estimating the bacterial burden in lungs at the respective time points (c), and in spleen at day 49 post-infection (d), by CFU enumeration. Data represent mean ± s.d. values from n=3 animals in c–d. p values in c and d were obtained after comparison CFUs between the two groups, as described in Materials and Methods.

Proposed model describing the effect of semapimod on L-leucine uptake in Mtb mc2 6206.

a, Uptake of L-leucine is controlled by cell-wall PDIM. b, Semapimod targets PDIM biosynthesis protein (PpsB) causing altered PDIM profile which restricts L-leucine uptake in the auxotroph by an unknown mechanism (denoted by ‘?’), leading to death. c, L-leucine is freely accessible to SemR strain, owing to perturbation in the cell-wall PDIM. As a consequence, semapimod is ineffective against this strain. However, overexpression of wild-type ppsB in SemR reverts the bacterial susceptibility to semapimod. The illustration was created in BioRender. (Agarwal, N. (2025) https://BioRender.com/h04h035).