Computational analysis identifies quinone oxidoreductase as a mitochondria-associated redox protein with putative role in drug resistance in Leishmania donovani
Description
Quinone oxidoreductase (QOR) is an enzyme with a critical role in cellular redox homeostasis by protecting against oxidative stress. In the present study, we employed a computational approach to find out the structural features, evolutionary conservation, functional interaction network, ligand binding interaction of parasite QOR. The crystal structure of QOR was validated by confirming the stable architecture and favourable stereochemical conformations for subsequent computational analyses. Relative sequence alignment and phylogenetic analysis of QOR from fourteen Leishmania species demonstrated strong evolutionary conservation, indicating an essential functional role across the genus. Binding pocket prediction showed several surface-accessible cavities, with a dominant central pocket displaying structural features for ligand accommodation. Docking analysis with the clinically utilized antileishmanial drugs showed that amphotericin B, miltefosine, pentamidine, and paromomycin interacted with the residues Phe44, Tyr48, Cys127, Arg268, and Lys181. Pharmacokinetic analysis of drug-ligand interaction showed that miltefosine exhibited the most favorable ADMET profile, while other drugs displayed poor metabolic interactions. Protein–protein interaction network (PPI) analysis further placed QOR within a redox-linked metabolic module which is strongly associated with mitochondrial pathways. Transcriptomic analysis also revealed altered expression of QOR under drug-resistant conditions. Together, these findings highlight the importance of QOR in parasite redox metabolism and as a critical drug target for antileishmanial drug discovery.
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Steps to reproduce
1. Download quinone oxidoreductase crystal structure (PDB ID: 8J6E, 2.10 Å) from RCSB PDB and sequence (UniProt: A0A451EJQ8) from UniProt. Cross-check annotations in MMDB. Assess quality with SAVES v6.0 (ERRAT, Verify3D, PROCHECK Ramachandran plot) and physicochemical/secondary structure properties with ProtParam. (Outputs: Supplementary File 1) 2. Retrieve quinone oxidoreductase FASTA sequences from 14 Leishmania species (UniProt). Align with Clustal Omega, visualize conservation in ESPript 3.0. Identify domains via MMDB/InterProScan. Build phylogenetic tree using Phylogeny.fr One Click mode. (Outputs: Supplementary File 2) 3. Visualize 8J6E in PyMOL v2.5.5 or RasMol v2.7.5 (secondary structure, cofactor sites, geometry). Prepare by removing non-essential waters, adding hydrogens, protonating at pH 7.4 (OpenBabel v3.1.1, manual histidine check in PyMOL). Map B-factors in PyMOL to identify flexible regions. 4. Run fpocket web server on prepared 8J6E (default parameters). Rank pockets by score, volume, polarity. Parse PQR output with Python (NumPy/CSV) to compute pocket centroids; export as CSV. Visualize in PyMOL and select top 5 pockets based on size, accessibility, and location. (Supplementary File 3) 5. Submit the validated 8J6E structure to the iMODS server (Cα coarse-grained representation, edNMA force field, default parameters). Compute per-residue deformability and mobility across the first 20 non-trivial normal modes; derive NMA-predicted B-factors and compare with experimental crystallographic B-factors. Perform covariance analysis of Cα fluctuations to identify correlated/anticorrelated motions. (Supplementary File 4) 6. Dock amphotericin B, miltefosine, pentamidine, paromomycin, and antimonial derivative to 8J6E using CB-Dock2 (AutoDock Vina backend). Use automatic cavity detection. Select best pose in main conserved cavity. Analyze interactions (H-bonds, hydrophobic, salt bridges, π-stacking) with PyMOL and PLIP/LigPlot+. Cross-validate with fpocket pockets. (Outputs: Supplementary File 5) 7. Get SMILES from PubChem for the five drugs. Predict properties with SwissADME (lipophilicity, TPSA, GI absorption, BBB, drug-likeness rules; BOILED-Egg model) and pkCSM (ADMET/toxicity endpoints). (Supplementary File 6) 8. Construct PPI for quinone oxidoreductase in STRING v12.0 (L. donovani complex via L. infantum proxy; confidence ≥0.500, all channels). Export TSV. Analyze in Python 3.10 + NetworkX v3.1: build undirected graph, compute centrality (degree, betweenness, closeness, eigenvector), clustering coefficient, density, communities (greedy modularity). Visualize in NetworkX/Cytoscape. (Supplementary File 7) 9. Download GEO microarray datasets for QOR (LdBPK_030550.1) under drug-resistant conditions: antimony resistance (GSE144659) and miltefosine resistance (GSE45496, GPL16867 platform). Extract normalized probe values. Plot expression trends qualitatively (dot plots, means) without re-normalization. (Outputs: Supplementary File 8).
Institutions
- SRM University, Andhra PradeshAndhra Pradesh, Mangalagiri