Caulerpin, a bis-indole alkaloid isolated from Caulerpa racemosa, has several documented pharmacological activities, including antineoplastic and antiviral properties. This study aimed to evaluate the anti-inflammatory and anti-tubercular potentials of caulerpin and its analogues in RAW 264.7 macrophages infected with Mycobacterium spp. Additionally, we evaluated cytokine production and NLRP3 expression in this infection model. Toxicity tests were performed using Vero E6 and HepG2 cell lines and Artemia salina. Pre-incubation of RAW 264.7 cells with caulerpin and its analogues decreased internalized M. smegmatis and M. tuberculosis H37Ra. Furthermore, treatment of M. smegmatis-infected macrophages with caulerpin and its analogues reduced bacterial loads. Caulerpin reduced the CFU count of internalized bacilli in the M. tuberculosis H37Ra infection model. In addition, caulerpin and its diethyl derivative were notably found to modulate IL-1β and TNF-α production in the M. smegmatis infection model after quantifying pro-inflammatory cytokines and NLRP3. Caulerpin and its derivates did not affect the viability of Vero E6 and HepG2 cell lines or nauplii survival in toxicity studies. These findings demonstrate that caulerpin and its analogues exhibit anti-inflammatory activity against Mycobacterium spp. infection in RAW 264.7 macrophages and show promising potential for further efficacy and safety evaluation.

Cardiotonic steroids modulate various aspects of the inflammatory response. The synthetic cardiotonic steroid γ-benzylidene digoxin 15 (BD-15), a digoxin derivative, has emerged as a promising candidate with potential immunomodulatory effects. However, its biological activity remains largely unexplored. This study investigated the anti-mycobacterial and anti-inflammatory effects of BD-15 in an in vitro macrophage infection model with Mycobacterium spp. Unlike digoxin, which showed significant toxicity at higher concentrations, BD-15 exhibited no cytotoxicity in RAW 264.7 cells (a murine macrophage cell line). Both compounds were evaluated in Mycobacterium smegmatis-infected RAW 264.7 cells, reducing bacterial burden without direct bactericidal activity. Additionally, both modulated pro-inflammatory cytokine levels, notably by decreasing tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) levels. BD-15 specifically reduced NOD-, LRR-, and pyrin-domain-containing protein 3 (NLRP3) inflammasome expression and increased interleukin-10 (IL-10) production. Notably, BD-15 reduced colony-forming unit (CFU) counts in Mycobacterium tuberculosis-infected RAW 264.7 cells. Toxicity assays in HepG2 cells (a human liver cancer cell line) showed that BD-15 had minimal hepatotoxicity compared to digoxin, and both demonstrated negligible acute toxicity in an Artemia salina bioassay. These findings revealed the immunomodulatory effects of cardiotonic steroids in a bacterial infection model and highlighted BD-15 as a safer alternative to digoxin for therapeutic applications.

Selene-ethylenelacticamide derivatives have been suggested as promising scaffolds with leishmanicidal activity. In this work, we demonstrated, for the first time, the effectiveness of selene-ethylenelacticamide derivatives against mycobacteria. Firstly, selene-ethylenelacticamides inhibited the growth of laboratory strains of Mycobacterium tuberculosis with MIC values ranging from 10 to 20 µM. Importantly, three derivatives were active against two multi-drug-resistant clinical isolates of M. tuberculosis with MIC values similar to pan-sensitive strains. In addition, NC31 and NC34 displayed an improved activity compared to the group treated with isoniazid in the six-week nutrient-starved M. tuberculosis cultures. Moreover, in toxicity studies, NC34 did not significantly affect the viability of both Vero E6 and HepG2 cell lines. NC34 did not affect Artemia salina nauplii survival at concentrations lower than 100 µM. Importantly, NC34 displayed a synergistic effect when combined with rifampicin. Molecular docking simulations were used to evaluate Mycobacterium tuberculosis DprE1 and dihydrofolate reductase enzymes as putative targets of selene-ethylenelacticamides, mechanisms that could contribute to the antitubercular activity. Our findings reveal that NC34 may represent a hit for further drug optimization and for future preclinical development as a new anti-mycobacterial agent, especially in cases of resistant and/or dormant forms of tuberculosis.