Antimicrobial Effect of Cannabidiol on Intracellular Mycobacterium tuberculosis.

Introduction: Mycobacterium tuberculosis, the etiologic agent of tuberculosis (TB), has killed nearly one billion people during the last two centuries. Nowadays, TB remains a major global health problem ranked among the top 10 causes of death worldwide. One of the main challenges in developing new strategies to fight TB is focused on reducing the duration and complexity of drug regimens. Cannabidiol (CBD) is the main nonpsychoactive ingredient extracted from the Cannabis sativa L. plant, which has been shown to be biologically active against bacteria. The purpose of this work was to investigate the antimicrobial effect of CBD on M. tuberculosis intracellular infection. Materials and Methods: To assess the minimum inhibitory concentration (MIC) of CBD on mycobacterial strains, the MTT assay was performed on Mycobacterium smegmatis, and the Colony-Forming Unit (CFU) assay was conducted on MtbH37Rv. Additionally, the cytotoxic effect of CBD on THP-1 cells was assessed by MTT assay. Moreover, macrophages derived from the THP-1 cell were infected with MtbH37Rv (multiplicity of infection 1:10) to evaluate the intracellular activity of CBD by determining the CFU/mL. Results: Antimicrobial activity against M. smegmatis (MIC=100 µM) and MtbH37Rv (MIC=25 µM) cultures was exhibited by CBD. Furthermore, the effect of CBD was also evaluated on MtbH37Rv infected macrophage cells. Interestingly, a reduction in viable intracellular MtbH37Rv bacteria was observed after 24 h of treatment. Moreover, CBD exhibited a safe profile toward human THP-1 cells, since it showed no toxicity (CC50=1075 µM) at a concentration of antibacterial effect (selectivity index 43). Conclusion: These results extend the knowledge regarding the antimicrobial activity of CBD and demonstrate its ability to kill the human intracellular pathogen M. tuberculosis.

Cosolvent Sites-Based Discovery of Mycobacterium Tuberculosis Protein Kinase G Inhibitors.

Computer-aided drug discovery methods play a major role in the development of therapeutically important small molecules, but their performance needs to be improved. Molecular dynamics simulations in mixed solvents are useful in understanding protein-ligand recognition and improving molecular docking predictions. In this work, we used ethanol as a cosolvent to find relevant interactions for ligands toward protein kinase G, an essential protein of Mycobacterium tuberculosis (Mtb). We validated the hot spots by screening a database of fragment-like compounds and another one of known kinase inhibitors. Next, we performed a pharmacophore-guided docking simulation and found three low micromolar inhibitors, including one with a novel chemical scaffold that we expanded to four derivative compounds. Binding affinities were characterized by intrinsic fluorescence quenching assays, isothermal titration calorimetry, and the analysis of melting curves. The predicted binding mode was confirmed by X-ray crystallography. Finally, the compounds significantly inhibited the viability of Mtb in infected THP-1 macrophages.

Inhalable Mannosylated Rifampicin-Curcumin Co-Loaded Nanomicelles with Enhanced In Vitro Antimicrobial Efficacy for an Optimized Pulmonary Tuberculosis Therapy.

Among respiratory infections, tuberculosis was the second deadliest infectious disease in 2020 behind COVID-19. Inhalable nanocarriers offer the possibility of actively targeting anti-tuberculosis drugs to the lungs, especially to alveolar macrophages (cellular reservoirs of the Mycobacterium tuberculosis). Our strategy was based on the development of a mannose-decorated micellar nanoformulation based in Soluplus® to co-encapsulate rifampicin and curcumin. The former is one of the most effective anti-tuberculosis first-line drugs, while curcumin has demonstrated potential anti-mycobacterial properties. Mannose-coated rifampicin (10 mg/mL)-curcumin (5 mg/mL)-loaded polymeric micelles (10% w/v) demonstrated excellent colloidal properties with micellar size ~108 ± 1 nm after freeze-drying, and they remain stable under dilution in simulated interstitial lung fluid. Drug-loaded polymeric micelles were suitable for drug delivery to the deep lung with lung accumulation, according to the in vitro nebulization studies and the in vivo biodistribution assays of radiolabeled (99mTc) polymeric micelles, respectively. Hence, the nanoformulation did not exhibit hemolytic potential. Interestingly, the addition of mannose significantly improved (5.2-fold) the microbicidal efficacy against Mycobacterium tuberculosis H37Rv of the drug-co-loaded systems in comparison with their counterpart mannose-free polymeric micelles. Thus, this novel inhaled nanoformulation has demonstrated its potential for active drug delivery in pulmonary tuberculosis therapy.

Circulating Monocyte-Like Myeloid Derived Suppressor Cells and CD16 Positive Monocytes Correlate With Immunological Responsiveness of Tuberculosis Patients.

Alterations of myeloid cell populations have been reported in patients with tuberculosis (TB). In this work, we studied the relationship between myeloid-derived suppressor cells (MDSC) and monocytes subsets with the immunological responsiveness of TB patients. Individuals with active TB were classified as low responders (LR-TB) or high responders (HR-TB) according to their T cell responses against a cell lysate of Mycobacterium tuberculosis (Mtb-Ag). Thus, LR-TB, individuals with severe disease, display a weaker immune response to Mtb compare to HR-TB, subjects with strong immunity against the bacteria. We observed that LR-TB presented higher percentages of CD16 positive monocytes as compared to HR-TB and healthy donors. Moreover, monocyte-like (M-MDSC) and polymorphonuclear-like (PMN-MDSC) MDSC were increased in patients and the proportion of M-MDSC inversely correlated with IFN-γ levels released after Mtb-Ag stimulation in HR-TB. We also found that LR-TB displayed the highest percentages of circulating M-MDSC. These results demonstrate that CD16 positive monocytes and M-MDSC frequencies could be used as another immunological classification parameter. Interestingly, in LR-TB, frequencies of CD16 positive monocytes and M-MDSC were restored after only three weeks of anti-TB treatment. Together, our findings show a link between the immunological status of TB patients and the levels of different circulating myeloid cell populations.