Antifungal Effect and Inhibition of the Virulence Mechanism of D-Limonene against Candida parapsilosis.

Yeasts from the Candida parapsilosis complex are clinically relevant due to their high virulence and pathogenicity potential, such as adherence to epithelial cells and emission of filamentous structures, as well as their low susceptibility to antifungals. D-limonene, a natural compound, emerges as a promising alternative with previously described antibacterial, antiparasitic, and antifungal activity; however, its mechanisms of action and antivirulence activity against C. parapsilosis complex species have not been elucidated. Therefore, in the present study, we aimed to evaluate the antifungal and antivirulence action, as well as the mechanism of action of D-limonene against isolates from this complex. D-limonene exhibited relevant antifungal activity against C. parapsilosis complex yeasts, as well as excellent antivirulence activity by inhibiting yeast morphogenesis and adherence to the human epithelium. Furthermore, the apoptotic mechanism induced by this compound, which is not induced by oxidative stress, represents an important target for the development of new antifungal drugs.

Thiophene-thiosemicarbazone derivative (L10) exerts antifungal activity mediated by oxidative stress and apoptosis in C. albicans.

Reactive oxygen species (ROS) cause cell damage and death. To reverse these effects, cells produce substances such as reduced glutathione (GSH) that serve as substrates for antioxidant enzymes. One way to combat microbial resistance includes nullifying the effect of glutathione in microbial cells, causing them to die from oxidative stress. The compound 2-((5-nitrothiophen-2-yl)methylene)-N-(pyridin-3-yl) hydrazine carbothioamide (L10) is a new thiophene-thiosemicarbazone derivative with promising antifungal activity. The aim of this study was to evaluate its mechanism of action against Candida albicans using assays that evaluate its effects on redox balance. Treatment with L10 promoted significant changes in the minimum inhibitory concentration (MIC) values in ascorbic acid and GSH protection tests, the latter increasing up to 64-fold of the MIC. Using nuclear magnetic resonance, we demonstrated interaction of L10 and GSH. At concentrations of 4.0 and 8.0 µg/mL, significant changes were observed in ROS production and mitochondrial membrane potential. The cell death profile showed characteristics of initial apoptosis at inhibitory concentrations (4.0 µg/mL). Transmission electron microscopy data corroborated these results and indicated signs of apoptosis, damage to plasma and nuclear membranes, and to mitochondria. Taken together, these results suggest a possible mechanism of action for L10 antifungal activity, involving changes in cellular redox balance, ROS production, and apoptosis-compatible cellular changes.

5-Nitro-Thiophene-Thiosemicarbazone Derivatives Present Antitumor Activity Mediated by Apoptosis and DNA Intercalation.

BACKGROUND: Considering the need for the development of new antitumor drugs, associated with the great antitumor potential of thiophene and thiosemicarbazonic derivatives, in this work we promote molecular hybridization approach to synthesize new compounds with increased anticancer activity. OBJECTIVE: Investigate the antitumor activity and their likely mechanisms of action of a series of N-substituted 2-(5-nitro-thiophene)-thiosemicarbazone derivatives. METHODS: Methods were performed in vitro (cytotoxicity, cell cycle progression, morphological analysis, mitochondrial membrane potential evaluation and topoisomerase assay), spectroscopic (DNA interaction studies), and in silico studies (docking and molecular modelling). RESULTS: Most of the compounds presented significant inhibitory activity; the NCIH-292 cell line was the most resistant, and the HL-60 cell line was the most sensitive. The most promising compound was LNN-05 with IC50 values ranging from 0.5 to 1.9 µg.mL-1. The in vitro studies revealed that LNN-05 was able to depolarize (dose-dependently) the mitochondrial membrane, induceG1 phase cell cycle arrest noticeably, promote morphological cell changes associated with apoptosis in chronic human myelocytic leukaemia (K-562) cells, and presented no topoisomerase II inhibition. Spectroscopic UV-vis and molecular fluorescence studies showed that LNN compounds interact with ctDNA forming supramolecular complexes. Intercalation between nitrogenous bases was revealed through KI quenching and competitive ethidium bromide assays. Docking and Molecular Dynamics suggested that 5-nitro-thiophene-thiosemicarbazone compounds interact against the larger DNA groove, and corroborating the spectroscopic results, may assume an intercalating interaction mode. CONCLUSION: Our findings highlight 5-nitro-thiophene-thiosemicarbazone derivatives, especially LNN-05, as a promising new class of compounds for further studies to provide new anticancer therapies.

Synthesis, cytotoxicity and antifungal activity of 5-nitro-thiophene-thiosemicarbazones derivatives.

In the present work, twelve N-substituted 2-(5-nitro-thiophene)-thiosemicarbazones derivatives (L1-12) were synthesized, characterized and their in vitro cytotoxic and antifungal activities were evaluated against Candida sp. and Cryptococcus neoformans. The probable mechanisms of action have been investigated by sorbitol and ergosterol assays. Additionally, ultrastructural study by Scanning Electron Microscopy was performed with the L10 compound. All compounds were obtained in good yield and their chemical structures were characterized on basis of their physico-chemical and Nuclear Magnetic Resonance - NMR, Spectrophotometric Absorption in the Infrared - IR and High-resolution Mass Spectrometry - HRMS data. The results showed that all strains were more sensitive to the compound L10 except Candida tropicalis URM 6551. On the other hand, the cytotoxicity assay by incorporation of tritiated thymidine showed moderate cytotoxic activity on L8 of the 50 µg/mLat which had the best MIC-cytotoxicity relationship. Concerning the study of the possible mechanism of action, the compounds were not able to bind to ergosterol in the membrane, do not act by inhibiting the synthesis of fungal cell wall (sorbitol assay). However, the Scanning Electron Microscopy - SEM analysis shows significant morphological changes in shape, size, number of cells and hyphae, and cell wall indicating a possible mechanism of action by inhibition of enzymes related to the ergosterol biosynthesis pathway. Our results demonstrate that N-substituted 2-(5-nitro-thiophene)-thiosemicarbazones derivatives are potential antifungal agents with activity associated with inhibition of enzymes related to biosynthesis of ergosterol.