Avaliação da atividade antifúngica de 2-Cloro-N-fenilacetamida (a1cl) contra espécies de candida de origem clínica

Abstract

Candida and mycoses are a growing global public health concern. Candida albicans, Candida tropicalis and Candida parapsilosis are on the list of priority fungal pathogens created by the World Health Organization. Thus, the development of research is planned to generate new drugs in order to combat and prevent the development of resistance. In this sense, prospective studies with reaction intermediates with amide groups and chlorine atoms are a promising strategy. This work aimed to evaluate the antifungal activity of the synthetic molecule 2-Chloro- N-phenylacetamides (A1Cl) against C. albicans, C. tropicalis and C. parapsilosis. The minimum inhibitory concentration (MIC) was determined by microdilution, so that values ranged from 16 to 256 μg/mL for A1Cl and 8 to 512 μg/mL for fluconazole (positive control). The minimum fungicidal concentration was similar to the MIC, for both evaluated compounds, confirming the fungicidal effect of A1Cl. In the model of morphological changes (microculture technique), A1Cl and fluconazole reduced the formation of virulence structures compared to their respective controls, showing a concentration-dependent effect. The presence of sorbitol and exogenous ergosterol did not alter the MIC values, confirming that A1Cl has no effect interfering with the functionality and/or integrity of the cell wall and membrane, respectively. The association study (checkerboard) of A1Cl with fluconazole resulted in an antifungal effect of indifference. In the in vitro antibiofilm effect assay, A1Cl was able to inhibit biofilm formation by more than 45% and 80% (***p<0.001), and was able to promote preformed biofilm rupture by more than 35% and 60 % (***p<0.001), in sub and supra inhibitory concentrations, respectively, when compared to the respective control groups. However, for fluconazole to achieve 50% (***p<0.001) or more inhibition effect, suprainhibitory concentrations were required. Similarly, A1Cl (CIM) was able to inhibit by more than 50% (***p<0.001) the formation of ex vivo biofilms (nail fragments) and for fluconazole to achieve the same antibiofilm effect by more than 50% 4xCIM was required. A1Cl showed binding energy values greater than or close to fluconazole in at least one scoring function, for Dihydrofolate reductase (DHFR), Geranylgeranyltransferase-I (GGTase-I) and Aspartic Protease-2 (SAP-2), so that these enzymes, may be targets susceptible to the antifungal action of A1Cl. The RMSD of the evaluated complexes revealed stability around 2-3ns and maintained its stability above 10 ns. For the RMSF of the complexes, we observed interactions similar to those investigated in docking after dynamic simulations at times of 200 and 600ps and that A1Cl remained in the active site under the influence of solvents and structural flexibility. Given these results, it is possible to infer that A1Cl has an antifungal effect and that this activity is related to antibiofilm mechanisms and binding to DHFR and SAP-2.