Chagas disease is one of the most neglected tropical diseases in the world, affecting nearly 15 million people, primarily in Latin America. Only two drugs are used for the treatment of this disease, nifurtimox and benznidazole. These drugs have limited efficacy and frequently induce adverse effects, limiting their usefulness. Consequently, new drugs must be found. In this study, we demonstrated the in vitro trypanocidal effects of a series of four gallic acid derivatives characterized by a gallate group linked to a triphenylphosphonium (TPP⁺) moiety (a delocalized cation) via a hydrocarbon chain of 8, 10, 11, or 12 atoms (TPP⁺-C₈, TPP⁺-C₁₀, TPP⁺-C₁₁, and TPP⁺-C₁₂, respectively). We analyzed parasite viability in isolated parasites (by MTT reduction and flow cytometry) and infected mammalian cells using T. cruzi Y strain trypomastigotes. Among the four derivatives, TPP⁺-C₁₀ and TPP⁺-C₁₂ were the most potent in both models, with EC₅₀ values (in isolated parasites) of 1.0 ± 0.6 and 1.0 ± 0.7 μM, respectively, and were significantly more potent than nifurtimox (EC₅₀ = 4.1 ± 0.6 μM). At 1 μM, TPP⁺-C₁₀ and TPP⁺-C₁₂ induced markers of cell death, such as phosphatidylserine exposure and propidium iodide permeabilization. In addition, at 1 μM, TPP⁺-C₁₀ and TPP⁺-C₁₂ significantly decreased the number of intracellular amastigotes (TPP⁺-C₁₀: 24.3%, TPP⁺-C₁₂: 19.0% of control measurements, as measured by DAPI staining) and the parasite’s DNA load (C₁₀: 10%, C₁₂: 13% of control measurements, as measured by qPCR). Based on the previous mode of action described for these compounds in cancer cells, we explored their mitochondrial effects in isolated trypomastigotes. TPP⁺-C₁₀ and TPP⁺-C₁₂ were the most potent compounds, significantly altering mitochondrial membrane potential at 1 μM (measured by JC-1 fluorescence) and inducing mitochondrial transition pore opening at 5 μM. Taken together, these results indicate that the TPP⁺-C₁₀ and TPP⁺-C₁₂ derivatives of gallic acid are promising trypanocidal agents with mitochondrial activity.

Interest in tumor cell mitochondria as a pharmacological target has been rekindled in recent years. This attention is due in part to new publications documenting heterogenous characteristics of solid tumors, including anoxic and hypoxic zones that foster cellular populations with differentiating metabolic characteristics. These populations include tumor-initiating or cancer stem cells, which have a strong capacity to adapt to reduced oxygen availability, switching rapidly between glycolysis and oxidative phosphorylation as sources of energy and metabolites. Additionally, this cell subpopulation shows high chemo- and radioresistance and a high capacity for tumor repopulation. Interestingly, it has been shown that inhibiting mitochondrial function in tumor cells affects glycolysis pathways, cell bioenergy, and cell viability. Therefore, mitochondrial inhibition may be a viable strategy for eradicating cancer stem cells. In this context, medicinal chemistry research over the last decade has synthesized and characterized “vehicles” capable of transporting novel or existing pharmacophores to mitochondrial tumor cells, based on mechanisms that exploit the physicochemical properties of the vehicles and the inherent properties of the mitochondria. The pharmacophores, some of which have been isolated from plants and others, which were synthesized in the lab, are diverse in chemical nature. Some of these molecules are active, while others are prodrugs that have been evaluated alone or linked to mitochondria-targeted agents. Finally, researchers have recently described drugs with well-proven safety and efficacy that may exert a mitochondria-specific inhibitory effect in tumor cells through noncanonical mechanisms. The effectiveness of these molecules may be improved by linking them to mitochondrial carrier molecules. These promising pharmacological agents should be evaluated alone and in combination with classic chemotherapeutic drugs in clinical studies.

To evaluate the antifungal and antibiofilm activity of gallic acid derivatives TPP⁺-C₁₀ and TPP⁺-C₁₂ and their effects on mitochondrial function on two C. albicans reference strains (ATCC 90029 and ATCC 10231). First, we determined minimal inhibitory concentration using a microdilution assay. Both compounds exerted antifungal effects, and their minimal inhibitory concentrations (MICs) ranged from 3.9 to 13 μM, with no statistically significant differences between them (P > 0.05, t-test). These concentrations served as references for following assays. Subsequently, we measured oxygen consumption with a Clark electrode. Our observations revealed that both drugs inhibited oxygen consumption in both strains with TPP⁺-C₁₂ exerting a more pronounced inhibitory effect. We then employed flow cytometry with TMRE as a probe to assess mitochondrial membrane potential. For each strain assayed, the compounds induced a decay in transmembrane potential by 75% to 90% compared to the control condition (P < 0.05, ANOVA). Then, we measured ATP levels using a commercial kit. TPP⁺-C₁₂ showed a 50% decrease of ATP content (P < 0.05 ANOVA), while TPP⁺-C₁₀ exhibited a less pronounced effect. Finally, we assessed the antibiofilm effect using the MTT reduction assay. Both compounds were effective, but TPP⁺-C₁₂ displayed a greater potency, requiring a lower concentration to inhibit 50% of biofilms viability (P < 0.05, t-test). Derivatives of gallic acid linked to a TPP⁺ group exert antifungal and antibiofilm activity through impairment of mitochondrial function in C. albicans.

Indomethacin is a non-selective NSAID used against pain and inflammation. Although cyclooxygenase (COX) inhibition is considered indomethacin’s primary action mechanism, COX-independent ways are associated with beneficial effects in cancer. In colon cancer cells, the activation of the peroxisome proliferator-activated receptor-γ (PPAR-γ) is related to the increase in spermidine/spermine-N¹-acetyltransferase-1 (SSAT-1), a key enzyme for polyamine degradation, and related to cell cycle arrest. Indomethacin increases the SSAT-1 levels in lung cancer cells; however, the mechanism relying on the SSAT-1 increase is unclear. Thus, we asked for the influence of the PPAR-γ on the SSAT-1 expression in two lung cancer cell lines: H1299 and A549. We found that the inhibition of PPAR-γ with GW9662 did not revert the increase in SSAT-1 induced by indomethacin. Because the mRNA of SSAT-1 suffers a pre-translation retention step by nucleolin, a nucleolar protein, we explored the relationship between indomethacin and the upstream translation regulators of SSAT-1. We found that indomethacin decreases the nucleolin levels and the cyclin-dependent kinase 1 (CDK1) levels, which phosphorylates nucleolin in mitosis. Overexpression of nucleolin partially reverts the effect of indomethacin over cell viability and SSAT-1 levels. On the other hand, Casein Kinase, known for phosphorylating nucleolin during interphase, is not modified by indomethacin. SSAT-1 exerts its antiproliferative effect by acetylating polyamines, a process reverted by the polyamine oxidase (PAOX). Recently, methoctramine was described as the most specific inhibitor of PAOX. Thus, we asked if methoctramine could increase the effect of indomethacin. We found that, when combined, indomethacin and methoctramine have a synergistic effect against NSCLC cells in vitro. These results suggest that indomethacin increases the SSAT-1 levels by reducing the CDK1-nucleolin regulatory axis, and the PAOX inhibition with methoctramine could improve the antiproliferative effect of indomethacin.