Novel Assay Platform to Evaluate Intracellular Killing of Mycobacterium tuberculosis: In Vitro and In Vivo Validation

Abstract

One of the main hallmarks of tuberculosis (TB) is the ability of the causative agent to transform into a stage of dormancy and the capability of long persistence in the host phagocytes. It is believed that approximately one-third of the population of the world is latently infected with Mycobacterium tuberculosis (Mtb), and 5%–10% of these individuals can develop clinical manifestations of active TB even decades after the initial infection. In this latent, intracellular form, the bacillus is shielded by an extremely robust cell wall and becomes phenotypically resistant to most antituberculars. Therefore, there is a clear rationale to develop novel compounds or carrier-conjugated constructs of existing drugs that are effective against the intracellular form of the bacilli. In this paper, we describe an experimental road map to define optimal candidates against intracellular Mtb and potential compounds effective in the therapy of latent TB. To validate our approach, isoniazid, a first-line antitubercular drug was employed, which is active against extracellular Mtb in the submicromolar range, but ineffective against the intracellular form of the bacteria. Cationic peptide conjugates of isoniazid were synthesized and employed to study the host-directed drug delivery. To measure the intracellular killing activity of the compounds, Mtb-infected MonoMac-6 human monocytic cells were utilized. We have assessed the antitubercular activity, cytotoxicity, membrane interactions in combination with internalization efficacy, localization, and penetration ability on interface and tissue-mimicking 3D models. Based on these in vitro data, most active compounds were further evaluated in vivo in a murine model of TB. Intraperitoneal infectious route was employed to induce a course of slowly progressive and systemic disease. The well-being of the animals, monitored by the body weight, allows a prolonged experimental setup and provides a great opportunity to test the long-term activity of the drug candidates. Having shown the great potency of this simple and suitable experimental design for antimicrobial research, the proposed novel assay platform could be used in the future to develop further innovative and highly effective antituberculars.

New Salicylanilide Derivatives and Their Peptide Conjugates as Anticancer Compounds: Synthesis, Characterization, and In Vitro Effect on Glioblastoma

“…U87 cells were treated with Cf-peptides (25 μM) for 3 h. Before this analysis, a preliminary experiment was carried out to compare the intracellular localization on live and fixed cells (see Supporting Information, Figure S24 and description). Based on those and our previous results ,, for confocal microscopy, we have used fixed cells. The internalization pathway was studied by staining lysosomes with LysoTracker Deep Red.…”

Design and Characterization of a Multistage Peptide-Based Vaccine Platform to Target Mycobacterium tuberculosis Infection

“…More than 50% of the cells were 5(6)-FAM-positive after 45 min of incubation with as low as 5 μM Pal-CGHP concentration. To investigate how peptides interact with tissue barrier models, we developed a submerged, noncontact monolayer setup, representing different epithelial barriers. , …”

“…At the same time, 500 μL of the medium were added to the basolateral side. On day 4, the medium was changed, and epithelial cells were grown up to confluence, which was checked prior and after the experiments with CellTracker Green (CMFDA (5-chloromethylfluorescein diacetate), Invitrogen, C2925, data not shown, details see in refs , ). On day 4, detector cells (LCLC-103H) were seeded on a 24-well plate (1 × 10 5 cells in 1000 μL complete DMEM medium) and on day 5, the transwell inserts were placed above the detector cells (basolateral position).…”

“…To investigate how peptides interact with tissue barrier models, we developed a submerged, noncontact monolayer setup, representing different epithelial barriers. 52,53 Transwell inserts are permeable supports that fit well into common 24-well cell culture plates. They feature a porous membrane, and the inserts provide a simple support for culturing monolayers to mimic tissue barriers.…”

Identification of Chemical Scaffolds Targeting Drug-Resistant and Latent Mycobacterium tuberculosis through High-Throughput Whole-Cell Screening

“…Additionally, CFU assay was conducted to further understand the intracellular infection study. , Briefly, RAW 264.7 cells were initially seeded in six-well plates at a concentration of 4 × 10 5 cells/mL. After a 24 h incubation period, the cells were infected with Mtb -H37Rv at a multiplicity of infection (MOI) of 4.…”