Inhibition of Paracoccidioides lutzii Pb01 isocitrate lyase by the natural compound argentilactone and its semi-synthetic derivatives

Abstract

The dimorphic fungus Paracoccidioides spp. is responsible for paracoccidioidomycosis, the most prevalent systemic mycosis in Latin America, causing serious public health problems. Adequate treatment of mycotic infections is difficult, since fungi are eukaryotic organisms with a structure and metabolism similar to those of eukaryotic hosts. In this way, specific fungus targets have become important to search of new antifungal compound. The role of the glyoxylate cycle and its enzymes in microbial virulence has been reported in many fungal pathogens, including Paracoccidioides spp. Here, we show the action of argentilactone and its semi-synthetic derivative reduced argentilactone on recombinant and native isocitrate lyase from Paracoccidioides lutzii Pb01 (PbICL) in the presence of different carbon sources, acetate and glucose. Additionally, argentilactone and its semi-synthetic derivative reduced argentilactone exhibited relevant inhibitory activity against P. lutzii Pb01 yeast cells and dose-dependently influenced the transition from the mycelium to yeast phase. The other oxygenated derivatives tested, epoxy argentilactone and diol argentilactone-, did not show inhibitory action on the fungus. The results were supported by in silico experiments.

Figure 1. Structures and synthesis of compounds reduced argentilactone (2), epoxy argentilactone (3 and 3a) and diol argentilactone (4) from natural compound argentilactone (1).

Figure 2. Effect of argentilactone and its derivatives reduced argentilactone, epoxy argentilactone, and diol argentilactone on P. lutzii Pb01 yeast cells growth.

Samples containing 10⁵, 10⁴, and 10³ Paracoccidioides Pb01 yeast cells were spotted onto MMcM agar medium containing glucose or acetate and supplemented with argentilactone, reduced argentilactone, epoxy argentilactone, and diol argentilactone at different concentrations for seven days. A) argentilactone; B) reduced argentilactone; C) epoxy argentilactone; D) diol argentilactone. The growth of 10⁵ P. lutzii Pb01 yeast cells was observed by spectrophotometer (520 nm) in medium containing glucose (E) or acetate (F). *p<0,05.

Figure 3. Effect of argentilactone on P. lutzii Pb01 yeast cells growth.

Yeast cells were cultured at 36°C in the presence of argentilactone for 6 hours. Aliquots were taken and the cells were counted in a Neubauer chamber.*p<0,05.

Figure 4. Effect of argentilactone, reduced argentilactone, epoxy argentilactone, and diol argentilactone on P. lutzii Pb01 differentiation from mycelium to yeast.

The mycelium was incubated for 10 days at 36°C on MMcM agar containing glucose or acetate and supplemented with different concentrations of argentilactone, reduced argentilactone, epoxy argentilactone, and diol argentilactone. Cell morphology was observed by optical microscopy in medium containing glucose (A) or acetate (B). The P. lutzii Pb01 yeast cells were counted using a Neubauer chamber in medium containing glucose (C) or acetate (D). *p<0,05.

Figure 5. Dynamic profile for RMSD obtained in molecular dynamic simulations of PbICL after 20 ns.

Superimposition of the homology model (red) and molecular dynamic structure (yellow) of PbICL is shown. The molecular dynamic-model structure was obtained using the program g_cluster, which determined the conformation that best represents the last 10 ns of the trajectory.

Figure 6. Molecular surface representation of PbICL and the three-dimensional structures of A) 1, B) 3, C) 4, and D) isocitrate (native ligand) in the PbICL binding pocket and key interactions of PbICL with the best conformation (mode 1) of each compound (orange).

Ligplots of the compounds in binding pocket of PbICL. Pink circles indicate residues involved in hydrogen-bond polar or charged interactions; green circles indicate residues involved in van der Waals interactions. Dashed lines represent stronger interactions. The PbICL residues that interact with the compounds are shown, and the dashed lines represent stronger interactions. Oxygen is colored red. The structures of the compounds shown refer to the lowest Fscore obtained in mode 1 from the docking simulations with AutoDock Vina. The grid was defined considering only the region of the PbICL site using sizex = sizey = sizez = 20 Å, centerx = 21.37 Å, centerx = 1.14 Å, and centerz = 10.83 Å.