A New Benzothiazolthiazolidine Derivative, 11726172, Is Active In Vitro, In Vivo, and against Nonreplicating Cells of Mycobacterium tuberculosis

Abstract

Tuberculosis (TB) still poses a global menace as one of the deadliest infectious diseases. A quarter of the human population is indeed latently infected with Mycobacterium tuberculosis. People with latent infection have a 5 to 10% lifetime risk of becoming ill with TB, representing a reservoir for TB active infection. This is a worrisome problem to overcome in the case of relapse; unfortunately, few drugs are effective against nonreplicating M. tuberculosis cells. Novel strategies to combat TB, including its latent form, are urgently needed. In response to the lack of new effective drugs and after screening about 500 original chemical molecules, we selected a compound, 11726172, that is endowed with potent antitubercular activity against M. tuberculosis both in vitro and in vivo and importantly also against dormant nonculturable bacilli. We also investigated the mechanism of action of 11726172 by applying a multidisciplinary approach, including transcriptomic, labeled metabolomic, biochemical, and microbiological procedures. Our results represent an important step forward in the development of a new antitubercular compound with a novel mechanism of action active against latent bacilli. IMPORTANCE The discontinuation of TB services due to COVID-19 causes concern about a future resurgence of TB, also considering that latent infection affects a high number of people worldwide. To combat this situation, the identification of antitubercular compounds targeting Mycobacterium tuberculosis through novel mechanisms of action is necessary. These compounds should be active against not only replicating bacteria cells but also nonreplicating cells to limit the reservoir of latently infected people on which the bacterium can rely to spread after reactivation.

Keywords: Mycobacterium tuberculosis; antitubercular drug; copper; latency; nonreplicating cells.

FIG 1

Dose-dependent escalation of bactericidal activity of 11726172 for dormant nonreplicating M. tuberculosis H37Rv. The experiment was repeated three times with comparable results. The figure shows one representative experiment.

FIG 2

Metabolic labeling of M. tuberculosis H37Rv and M. tuberculosis H37Ra cells treated with compound 11726172. (A) Lipid analysis of M. tuberculosis H37Rv cells grown in the presence of 11726172. [¹⁴C]acetate (in a final concentration of 0.5 μCi/mL) was added to the culture in the presence of increasing concentrations of 11726172. The lipids were extracted and analyzed by TLC and detected by autoradiography. (B) Lipid analysis of M. tuberculosis H37Ra cells grown in the presence of 11726172. The cells were grown and metabolically labeled, and the lipids were analyzed as described above. The lipids were visualized using an Amersham Typhoon biomolecular imager. (C) Macromolecular synthesis assay with M. tuberculosis H37Ra treated with 11726172. [¹⁴C]acetate (blue), [¹⁴C]leucine (gray), [¹⁴C]uracil (orange), and [¹⁴C]methionine (green) were added as described above at a final concentration of 0.5 μCi/mL. After 24 h of incorporation, ¹⁴C was quantified by scintillation spectrometry. Data from two independent experiments are shown by two different color shades. TDM, trehalose dimycolates; TMM, trehalose monomycolates; PE, phosphatidylethanolamine; CL, cardiolipin; PIM, phosphatidylinositol mannosides; AcPIM, acylated forms of phosphatidylinositol mannosides; FAME, fatty acid methyl esters; MAME, mycolic acid methyl esters.

FIG 3

Transcriptome analysis of M. tuberculosis H37Rv cells exposed to 11726172. (A) Principal-component analysis (PCA) plot of M. tuberculosis H37Rv samples (control [not treated], 10× MIC, and 30× MIC). (B) Volcano plots of differentially expressed genes (10× MIC, 2.5 μg/mL). (C) Volcano plots of differentially expressed genes (30× MIC, 7.5 μg/mL). (D) Functional characterization of genes overexpressed in both treatment conditions. (E) Functional characterization of genes underexpressed in both treatment conditions.

FIG 4

Copper ions gradually increase the activity of 1726172 against M. tuberculosis.

FIG 5

Analysis of metal content in M. tuberculosis H37Rv cell lysates after treatment with 11726172. Determination of intracellular content of Cu²⁺, Ni²⁺, Co²⁺, Zn²⁺, and Fe²⁺ after incubation with a 50 μM concentration of the respective ion in the absence (−) or presence (+) of 7.5 μg/mL 11726172.

FIG 6

Identification of the potential 11726172 metabolites produced in M. bovis cells. (A) TLC analysis (hexane-ethyl acetate at a 6:4 ratio, visualized under UV light) of the compound (C) and of the metabolite mixture extracted in chloroform after incubation with mycobacterial culture (M). (B) TLC analysis of chromatographic fractions 1 to 6 from the cell culture extract. (C) Structure of potential 11726172 metabolites M 2a, M 2b, and M 4 identified by HESI-MS analysis (as shown in Fig. 7).

FIG 7

HESI-MS (positive-mode) direct infusion analysis of the silica gel chromatography fractions of 11726172-treated M. bovis culture extract. (A) Full mass of fraction 1; (B) fragmentation pattern of fraction 1; (C) full mass of fraction 2; (D) fragmentation pattern of M 2a; (E) full mass of fraction 3; (F) fragmentation pattern of M 3; (G) full mass of fraction 4; (H) fragmentation pattern of M 4.