Computational Approach to study the effect of point mutations in the development of antifungal resistance to Azoles and Flucytosine Drugs in Candida auris
Background: Post-discovery(2009), Candida auris is associated with invasive and severe candidemia, multi-drug resistance and high mortalities in over 30 countries across six continents. Misidentification and shortage of treatment services have made it difficult to control increasing threat. Azoles and Flucytosine are commonly used antifungal drugs due to their low toxicity and availability in both oral and intravenous formulations. Lanosterol alpha-demethylase (ERG11), Uracil phosphoribosyltransferase (FUR1) are two principal proteins involved in ergosterol biosynthesis and pyrimidine metabolism and act as drug targets for azole and flucytosine respectively. However, crystal structures of proteins from C. auris have not yet been established. In current study, we constructed structural model of ERG11 and FUR1 proteins for South-African Clade using homology modelling, molecular docking and molecular dynamics (MD) simulations. We applied same methods to ERG11 mutants (Y132F, K143R) and FUR1 mutants (F211I) to explain how point-mutations affects drug interaction. Method and Materials: Homology modelling was used to construct three-dimensional structure of proteins. Reliability of models was analysed by using validation tools. Molecular docking was used to study drug interaction in wild and mutant variants of proteins and binding free energy was calculated. Finally, we investigated structural significance of point-mutation in FUR1 through MD Simulation and how interactions differ in both variants of FUR1. Results: We established reliable structural model of proteins in complex with their drugs. Structural models variants of ERG11 and FUR1 were compared based on binding free energy and hydrogen bonding. Comparative study found that few azole compounds showed no effect of mutation on protein interaction. Further, on investigating structural significance of point-mutation in FUR1, it was found that binding affinity for 5-fluorocytosine decreases in the mutant variant. Moreover, MD Simulation of wild variant FUR1-5FC complex showed stabilisation till 7 ns while mutated complex was stable for 4.5 ns. Conclusion: Increased resistance of C. auris to conventional antifungal drugs poses significant danger to public health. Current study offers insight into how mutations influences drug-interaction. With better understanding of resistance mechanism, C.auris infection prevention can be strengthened and findings of this study could be employed in development of novel antifungal agents.
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1. Docking protocol of the protein-drug complexes: To investigate the protein-ligand interactions, ligand were docked into the specific site of protein using AutoDock Vina. Receptor Grid were centered based on the active residues mentioned by metaPocket metaserver on analysis the protein structure. Passive residues were automatically defined around active residues. Ligands were flexibly docked in the grid box and the positively docked molecules were ranked based on their docking score. The illustration and visualization of the final docked complex were completed with UCSF Chimera. While the interacting residues in the protein-ligand complex were analysed with LigPlot+. 2. Molecular Dynamics(MD) Simulation: Both wild and mutated varianted of docked FUR1 protein with 5-fluorocytosine(5FC) were subjected to MD Simulation with the CHARMM36 Force-field in GROMACS 5.1 Tool on UNIX System. The simulation was minimised using 5000 steps of Steepest Descent Minimisation Algorithm. The system was later equilibrated at a temperature of 300K and a pressure of 1 bar for 2 fs. Final production run in Protein-Ligand complex MD Simulation was run for 10 ns in both wild and mutated FUR1 protein complexed with 5FC to study the structural stability and difference in the interaction of both the complexes which were later analysed using RMSD, RMSF calculations for the protein-ligand complex.