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0INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
SUITABILITY OF FTR2 (IRON PERMEASE) FOR SCREENING OF ANTIFUNGAL COMPOUNDS AGAINST CANDIDA
ALBICANS
1Sana Akhtar Usmani, 2Khushboo Arya, 3Shikha Chandra, 4Saumya Chaturvedi, 5Deeksha
Jattan, 6Nitin Bhardwaj, 7Ashutosh Singh
1Research Scholar-Department of Biochemistry, University of Lucknow, Lucknow, Uttar Pradesh, India 226024, 2Research Scholar-Department of Biochemistry, University of Lucknow,
Lucknow, Uttar Pradesh, India 226024, 3Research Scholar- Department of Biochemistry, University of Lucknow, Lucknow, Uttar Pradesh, India 226024, 4Research Scholar- Department of Biochemistry, University of Lucknow, Lucknow, Uttar Pradesh, India 226024, 5Research Scholar- Department of Biochemistry, University of Lucknow, Lucknow, Uttar Pradesh, India 226024, 6Department of Zoology and Environmental Science, Gurukula Kangri (Deemed University), Haridwar, 249404, Uttarakhand, India, 7Assistant Professor - Department of Biochemistry, University of Lucknow, Lucknow, Uttar Pradesh, India 226024. https://doi.org/10.5281/zenodo.13831907 Abstract: In recent years, there has been a concerning surge in fungal infections, primarily attributed to the limited efficacy of existing antifungal treatments and the emergence of multidrug-resistant strains. Consequently, an urgent and compelling demand existsfor innovative inhibitors to combat these infections, driving researchers to explore alternative therapeutic pathways for treating C. albicans. A study has focused on targeting iron pathways to discover a novel inhibitor within this context. This approach is rooted in previous research indicating that iron deprivation can substantially alter the pathogenic nature of the fungus. By specifically concentrating on iron acquisition protein Ftr2, this study aims to identify innovative inhibitors using in silico studies. Keywords: Candida, multidrug resistance, iron permeases, molecular docking ADMET, Lipinski Rule
Introduction
Iron is vital for the functioning of all living organisms as it is a cofactor for various enzymatic and biochemical reactions (3). Iron competition significantly affects survival and pathogenicity in the interaction between microbial organisms and their hosts. To combat this, hosts use a defence mechanism called nutritional immunity to limit iron availability to microbes. Candida albicans, a commensal organism, employs multiple strategies to obtain iron, including the reductive pathway, which involves the coordinated action of reductases, permeases, and multicopper oxidases (4). Ftr2, an iron permease of 382 amino acids, is involved in iron acquisition. Ftr2 is downregulated in response to iron limitation, suggesting that it may function in conditions with sufficient iron and might be involved in acquiring iron in specific niches under certain conditions. The expression of Ftr2 is tightly regulated by iron availability and potentially other environmental signals, reflecting the intricate iron homeostasis mechanisms in C. albicans. Recently, there has been a shift in focus
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" _25-26 SEPTEMBER, 2024_
towards natural compounds with various antimicrobial activity types. Research on medicinal plants has the potential to lead to the discovery of new therapeutic agents against diseases to which conventional medicines have become resistant. Natural products provide a safer and biodegradable source for new drug development. Medicinal plants also offer the potential for novel biologically active compounds. Numerous plant-derived compounds, such as essential oils, have been discovered to have antifungal properties (7). These natural products have multiple advantages over synthetic drugs, including fewer side effects and lower rates of drug resistance. We have used Molecular Docking techniques to target the Ftr2 protein using natural metabolites to identify a novel inhibitor (6). Methodology
Preparation of Protein structure
The Ftr2 crystal structure is unavailable on the Protein Data Bank (PDB) iron permease Ftr2 accession no sequence AOW27022 (NCBI Reference Sequence: XP_715020.1, strain C. albicans SC5314) was retrieved in FASTA format from the NCBI. We prepared the homology model using the SWISS-MODEL (https://swissmodel.expasy.org/). Further, the model was validated using UCLA-DOE LAB—SAVES v6.0 (https://saves.mbi.ucla.edu/). Homology Modelling of Ftr2
Due to the lack of crystal structure data in the RCSB PDB database, a 3D model of Ftr2 was created using the Swiss model. The model was validated using various methods, including ERRAT, WHATCHECK, PROVE, VERIFY3D, and PROCHECK (5). It was also assessed using a Ramachandran plot to evaluate its stereochemical properties and stability. Preparation of Ligand
Based on literature and databases, anti-inflammatory and anti-cancer secondary metabolites were selected. The SDF files for the ligands were obtained from Zinc Databases (https://zinc.docking.org/) and prepared using Discovery Studio's 'Prepare ligand' version. The selected ligands were passed through ADMET analysis and Toxicity prediction (2). Further Drug likeness of the ADMET-screened secondary metabolites was done using DS by assessing four properties: H-Donor, H-Acceptor, molecular weight, and AlogP (partition coefficient). Protein-Protein interaction
Natural metabolites derived from various plant sources were carefully selected based on their chemical properties and structural characteristics. These metabolites were then subjected to molecular docking studies to understand their interactions with
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" 25-26 SEPTEMBER, 2024
Candida albicans' iron permease protein. The docking analyses were performed using the advanced 'Libdock' module of Discovery Studio, allowing for a comprehensive exploration of the binding affinities and potential modes of action of these natural compounds within the target protein's binding site (1). Result
Homology Modelling of Ftr2: Homology model of Ftr2 was generated as described in methods (Fig 1).
Fig 1. Structure of Ftr2
'hi \
Q ./
Allowed Region
86.2%
The
Residues in additional
loutâde
■ htardsphere
allowed region
Residues in the generously allowed region
Residues in disallowed regions
13.8%
0.0%
0.0%
Ftr2 protein illustrating allowed, disallowed, and additional disallowed regions.
Molecular docking of the selected natural metabolites with Ftr2
After preparing the model of the Ftr2 protein using the Swiss model, the 'Define and Edit Binding Site module' was used to anticipate the binding site of the Ftr2. Ftr2 did not interact with any of the tested natural metabolites (6), as no binding pocket was found in the Ftr2 model prepared.
generated model was validated by generating a Ramachandran plot to get an insight into the stereochemical properties of the protein model (Fig. 2). The number of
INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" 25-26 SEPTEMBER, 2024
residues present in allowed and this allowed region on the Ramachandran plot gives an idea about the stability of protein structure. A structure having more than 85% of residues in the allowed region is acceptable. Discussion
Candida albicans is the leading cause of mycoses globally, and is a severe and potentially life-threatening fungal infection that primarily affects individuals with compromised immune systems. With the increasing resistance to existing antifungal therapies, there is an urgent need to explore novel classes of antifungal drugs. Secondary metabolites, natural compounds produced by organisms, have shown promising antifungal properties. In this context, plants have emerged as a rich source of bioactive compounds with potential antifungal activity, making them a key focus in the search for new drug discoveries.
Our study focused on the iron pathway, a pivotal co-factor for many metabolic reactions in living organisms. We examined the behavior of Ftr2, a protein involved in iron transport, and its interaction with metabolites. Upon analysis, we made a significant observation-Ftr2 did not bind to any metabolite, indicating the absence of a binding pocket in the prepared Ftr2 model. Therefore, our next approach will be screen for other iron transport pathway proteins such as Ftr1, Ccc2 and Fet3 for their suitability as drug target. This discovery sheds light on the complexities of the iron pathway and its potential implications for drug development targeting fungal infections.
Acknowledgements: AS thanks support from ICMR (No.52/08/2019-BIO/BMS), DST-PURSE program (SR/PURSE Phase 2/29(C)), UP Higher Education (No. 10/2021/281/-4-Sattar-2021 -04(2)/2021 and No. 39/2024/242/Sattar-4-2024-001-4(33)/2023), DBT (BT/PR38505/MED/29/1513/2020) and DST (CRG/2022/001047) and the University of Lucknow.
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INTERNATIONAL SCIENTIFIC AND PRACTICAL CONFERENCE "STATUS AND DEVELOPMENT PROSPECTS OF FUNDAMENTAL AND APPLIED MICROBIOLOGY: THE VIEWPOINT OF YOUNG SCIENTISTS" 25-26 SEPTEMBER, 2024
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