Antimicrobial activity of IDD-B40 against drug-resistant Mycobacterium tuberculosis

Abstract

The emergence of multi-drug resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis creates the urgency for new anti-tuberculosis drugs to improve the efficiency of current tuberculosis treatment. In the search for a new potential tuberculosis drug, we synthesized an isoindole based chemical library and screened a potential candidate with significant anti-tuberculosis activity. The compound named 2-hydroxy-4-(4-nitro-1,3-dioxoisoindolin-2-yl) benzoic acid (IDD-B40) showed strong activity against all the tested drug-susceptible and drug-resistant strains of M. tuberculosis, with the 50% minimum inhibitory concentrations (MIC₅₀) of 0.39 μg/ml both in culture broth and inside Raw 264.7 cells. Also, IDD-B40, in combination with rifampicin, exhibited a direct synergistic effect against both XDR and H37Rv M. tuberculosis. Besides, IDD-B40 showed a better post-antibiotic effect (PAE) than did some first-line drugs and showed no significant cytotoxicity to any cell line tested, with a selectivity index of ≥ 128. Although IDD-B40 showed a result similar to isoniazid in the preliminary mycolic acid inhibition assay, it did not exhibit any effect against other mycolic acid-producing nontuberculous mycobacterial strains (NTM), and different non-mycobacterial pathogenic strains, so further studies are required to confirm the mode of action of IDD-B40. Considering its results against M. tuberculosis, IDD-B40 is a potential anti-tuberculosis drug candidate. However, further studies are required to evaluate its potential in vivo effect and therapeutic potential.

Figure 1

Chemical structures of 2-Hydroxy-4-(4-nitro-1,3-dioxoisoindolin-2-yl) benzoic acid (IDD-B40).

Figure 2

Antimicrobial activities of IDD-B40 and control drugs, including isoniazid (INH) and rifampicin (RIF), against M. tuberculosis H37Rv (A,C,E) and extensively drug-resistant (XDR)-M. tuberculosis (B,D,F). Mycobacterial susceptibility was assessed by resazurin assay (A,B) as RFU/ml (relative fluorescence unit per ml), by microbial cell viability assay (C,D) as RLU/ml (relative luminescence unit per ml), and by CFU enumeration assay (E,F) as CFU/ml. These experiments were carried out in triplicate. Data are given as mean values and standard deviations. *Statistical significance were determined in Graph Pad Prism 8.2.0 between drug treated versus drug-free control using an unpaired Student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 3

50% Minimum inhibitory concentration (MIC₅₀) of IDD-B40 and control drugs [isoniazid (INH), rifampicin (RIF), streptomycin (STR) and pyrazinamide (PZA)] against clinically isolated (A) multidrug-resistant (MDR) and (B) extensively drug-resistant (XDR) strains of M. tuberculosis in Korea. The figures are shown as a box plot, in which the bottom and top edges of the box are the 25th and 75th percentiles, and the center horizontal line is the 50th percentile (median). *Statistical significance were determined in Graph Pad Prism 8.2.0 between IDD-B40 versus control drugs using an unpaired Student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 4

Cytotoxicity of IDD-B40 was evaluated at a wide range of concentrations (100–0.02 μg/ml) against six different mammalian cell lines (Raw 264.7, L929, A549, HEPG2, Sh-SY5Y, and THP1) by MTT assay.

Figure 5

Intracellular killing effect of IDD-B40, isoniazid (INH) and rifampicin (RIF) in M. tuberculosis-infected macrophages (Raw 264.7). (A) Relative survival of H37Rv in H37Rv-infected macrophages after IDD-B40, INH, and RIF treatment. (B) Relative survival of XDR-TB in XDR-TB-infected cells after IDD-B40, INH, and RIF treatment. Data represent the mean ± SD of three independent experiments done in triplicate. ***p < 0.001, ****p < 0.0001 by Student’s t test.

Figure 6

Post-antibiotic effect (PAE) from the growth of M. tuberculosis after pulse dosing with IDD-B40, isoniazid (INH), rifampicin (RIF), streptomycin (STR), and ethambutol (EMB) at a concentration of 10 µg/ml. RIF shows the lowest PAE value of 144 h, followed by STR (40 h), IDD-B40 (29 h), EMB (28 h), and INH (20 h).

Figure 7

TLC Picture of mycolic acid extracted from H37Rv M. tuberculosis cells after the 7 days treatment of different concentration of IDD-B40 and INH.

Figure 8

Changes in mRNA levels of M. tuberculosis gene expression ratio involved in (A–E) mycolic acid synthesis and (F) heat-shock protein during exposure to 10 × IDD-B40 and INH.