In Silico Analysis of Natural Plant-Derived Cyclotides with Antifungal Activity against Pathogenic Fungi

Background: Fungal infections in plants, animals, and humans are widespread across the world. Limited classes of antifungal drugs to treat fungal infections and loss of drug efficacy due to rapidly evolving fungal strains pose a challenge in the agriculture and health sectors. Hence, the search for a new class of antifungal agents is imperative. Cyclotides are cyclic plant peptides with multiple bioactivities, including antifungal activity. They have six conserved cysteine residues forming three disulfide linkages (C^I-C^IV, C^II-C^V, C^III-C^VI) that establish a Cyclic Cystine Knot (CCK) structure, making them extremely resistant to chemical, enzymatic, and thermal attacks. Aim: This in silico analysis of natural, plant-derived cyclotides aimed to assess the parameters that can assist and hasten the process of selecting the cyclotides with potent antifungal activity and prioritize them for in vivo/ in vitro experiments. Objective: The objective of this study was to conduct in silico studies to compare the physicochemical parameters, sequence diversity, surface structures, and membrane-cyclotide interactions of experimentally screened (from literature survey) potent (MIC ≤ 20 μM) and non-potent (MIC > 20 μM) cyclotides for antifungal activity. Methodology: Cyclotide sequences assessed for antifungal activity were retrieved from the database (Cybase). Various online and offline tools were used for sequence-based studies, such as physicochemical parameters, sequence diversity, and neighbor-joining trees. Structure-based studies involving surface structure analysis and membrane-cyclotide interaction were also carried out. All investigations were conducted in silico. Results: Physicochemical parameter values, viz. isoelectric point, net charge, and the number of basic amino acids, were significantly higher in potent cyclotides compared to non-potent cyclotides. The surface structure of potent cyclotides showed a larger hydrophobic patch with a higher number of hydrophobic amino acids. Furthermore, the membrane-cyclotide interaction studies of potent cyclotides revealed lower transfer free energy (ΔG transfer) and higher penetration depth into fungal membranes, indicating higher binding stability and membrane-disruption ability. Conclusion: These in silico studies can be applied for rapidly identifying putatively potent antifungal cyclotides for in vivo and in vitro experiments, which will ultimately be relevant in the agriculture and pharmaceutical sectors.