Studies on Calcineurin and Calcium ATPase PMR-1 Roles in Azole Drug Susceptibility in Neurospora Crassa, and their Interactions with Azoles and Phytochemical Compounds

Abstract

This thesis describes the effect of azoles in Neurospora crassa, and the molecular basis of interactions of the calcineurin and PMR-1 proteins with the fluconazole, itraconazole, and ketoconazole drugs. Azole treatments reduced ergosterol levels and increased reactive oxygen species (ROS) in the N. crassa calcineurin and pmr-1 mutant strains. In addition, field emission scanning electron microscopy (FESEM) analysis revealed altered hyphal morphology in the calcineurin RIP and Δpmr-1 mutants treated with the azole drug fluconazole. Furthermore, expression of the pmr-1 was upregulated under itraconazole treatment. Molecular docking and molecular dynamics (MD) simulations revealed the stable interactions of the azole drugs within the active site of the protein calcineurin and PMR-1 with efficient binding energies. Preliminary computational insights also show the critical amino acid residues for drug-target interactions. In addition, this study also delves into phytochemical compounds as potential inhibitors against Ca2+ signaling proteins pivotal in fungal pathogenicity, using computer-aided drug design (CADD). A total of 11 phytochemicals were assessed for their inhibitory potential, adhering to Lipinski's rule of drug-likeness, and in silico studies identified isorhamnetin as a promising phytochemical inhibitor for PMR-1 and CNA-1 proteins and dillenetin as a potential inhibitor of CNB-1 protein with the formation of the most stable complex. In conclusion, this thesis enhances our understanding of the molecular basis of azole susceptibility in N. crassa. The study also provides a promising future for the development of target-specific new antifungals targeting calcineurin and PMR-1.