Unveiling the Anti-HIV Potential of Cyclotides: A Computational Approach

Recent Trends in Science and Technology-2024 Bioinformatics www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788197073274, Page No.55 https://doi.org/10.5281/zenodo.17393857 Unveiling the Anti-HIV Potential of Cyclotides: A Computational Approach Saurav Kumar Mishra1, Elizabeth Varghese2,3, John J. Georrge1,2* 1 Department of Bioinformatics, University of North Bengal, District-Darjeeling, West Bengal-734013, India 2 Department of Bioinformatics, Christ College, Rajkot, Gujarat, India 3 Coventry University, Coventry, United Kingdom *Corresponding author: [email protected] Abstract: Cyclotides exhibit a range of therapeutic activities, including antiviral, anticancer, etc. Apart from their pharmacological activities, cyclotides have also been reported as having the potential to be used as biopesticides. In this study, the anti-HIV attributes of cyclotides toward their potential target were examined via an integrated in-silico and docking approach. The cyclotides sequence and their respective 3D structure were initially retrieved from the PDB database and used for similarity analysis via BLAST against different databases. These cyclotides were also used in protein-protein docking studies to interpret their structural affinity toward proven drug targets of HIV, and potential cyclotides were further examined. Based on the investigation, Cycloviolacin-y1 has shown the best docking score for inhibiting reverse transcriptase and integrase, while Circulin-F has shown the best score for inhibiting protease. This research aids in elucidating the molecular mechanism of cyclotides through different bioinformatics approaches, such as sequence analysis, structure analysis, and docking studies. Keywords: Cyclotides, Docking, Drug target, HIV, in-silico 1. Introduction Human immunodeficiency virus (HIV) is an ongoing global health concern; it affects approximately 39 million people worldwide as of 2023, according to the World Health Organization (WHO) (https://www.who.int/data/gho/data/themes/hiv-aids). Despite advances in antiretroviral therapy, the treatment of HIV is complicated by factors such as drug resistance, adverse effects, and the need for continuous medication protocols. The virus primarily attacks CD4+ T-cells, further debilitating the patient’s immune system, and the person remains prone to opportunistic infections and cancers. HIV replication includes a series of key phases that involve the binding of the virus to the CD4+ receptors, reverse transcription, incorporation into the host’s genetic material, replication, and the release of newly formed viral particles. All these phases could be exploited for therapeutic intervention during drug development (Bekker et al., 2023; Hokello et al., 2024). Furthermore, cyclotides represent a specific group of plant peptides that were identified. Due to their stability, they offer potential leads in drug research for addressing structural deformation and diverse biological activities. Such ribosomally synthesized peptides consist of a substantial number of cysteine residues. The characteristic feature is the cyclic cystine knot topology, which consists of multiple disulfide linkages arranged in a knotted configuration. Generally, Cyclotides have a core stabilized by disulfide bonds and typically include about 30 amino acids, which confer resistance to chemical, physical, and enzymatic degradation. Such structural stability places them into a promising class for therapeutic purposes, as these molecules retain activity under challenging biological conditions. In addition, the resistance of cyclotides to enzymatic degradation makes them highly amenable to oral delivery, a characteristic not found in most classical peptides, which usually have the drawbacks of low bioavailability and rapid degradation after oral administration (De Veer et al., 2019; Huang et al., 2021; Narayani et al., 2020). So far, Recent Trends in Science and Technology-2024 Bioinformatics www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788197073274, Page No.56 https://doi.org/10.5281/zenodo.17393857 cyclotides have been detected primarily in particular plant groups, specifically Violaceae and Rubiaceae. The Violaceae family is diverse, with about 22 genera and 900 species, and is wellrepresented in plants that produce cyclotides. These structural properties of cyclotides from these plants play a crucial role in providing stability for such a molecule. The cysteine residues involved in the knot formation (CysI-CysIV, CysII-CysV, and CysIII-CysVI) are essential for stability. This unique structural feature protects cyclotides from degradation and enhances their biological activity. Such properties make cyclotides valuable for various medical uses, ranging from protein design to drug design (De Veer et al., 2019; Narayani et al., 2020). Studies suggest that cyclotides have broad bioactivities, including uterotonic, hemolytic, anti-neurotensin, cytotoxic, antifouling, anticancer, and antiviral activities. A beautiful therapeutic potential of cyclotides is their ability to interfere with HIV infection. The interaction of cyclotides can occur at various stages in the lifecycle of HIV, such as preventing the entry and replication of viruses that would otherwise decrease the general viral load. The peptides present a minimal level of toxicity; thus, they appear as potential agents for the development of new therapeutics for treating HIV. The great capacity of cyclotides to withstand passage through the gastrointestinal tract intact represents an outstanding ability. It confers significant advantages over most other therapeutic peptides, which are highly susceptible to digestion in the digestive tract. Additional supporting evidence for their therapeutic potential was derived from demonstrating the efficacy of engineered cyclic conotoxins, which had been established as orally active and effective in treating neuropathic pain (Colas et al., 2024; Ojeda et al., 2019; Sukmarini, 2022; Zhang et al., 2024). However, there is an urgent need for new and effective drugs because HIV is spreading worldwide. Natural products like cyclotides have a high potential to target the virus effectively with fewer side effects, making them an exciting alternative to traditional antiretroviral drugs. Currently, the computer-assisted approach and its associated application are the most suitable approach for therapeutics development (Balakrishnan et al., 2024; Georrge et al., 2025; Gurung et al., 2025; Mishra et al., 2024; Mishra et al., 2023; Mishra et al., 2025; Vaghasia et al., 2024; Vinjoda et al., 2024). With unique structural stability and bioactive properties, cyclotides are well-suited for developing new therapies to complement or replace existing treatments (Driche, 2024; Varghese et al., 2024). Cyclopeptides with anti-HIV properties are promising and attractive molecular templates for designing more potent analogues for medical applications. An in vitro XTT-based anti-HIV test was used to study the impact of cyclotides on virusinduced cell death in various HIV-infected cultures. Hence, the targets of cyclotides, which have anti-HIV properties, have not yet been discovered (Driche, 2024; Sukmarini, 2022; Zhang et al., 2024). Therefore, this study aims to explore the cyclotides’ activity toward HIV via a docking approach. 2. Materials and methods The research workflow comprises the steps mentioned below (Figure 1). Data acquisition The total protein sequences of cyclotides were collected via the CyBase database (https://www.cybase.org.au/) as a FASTA file (Wang et al., 2007). Hence, the biologically determined structures were retrieved from PDB (https://www.rcsb.org/) (Berman et al., 2007). The modelled structures were also retrieved and assembled from the CyBase database for structure analysis. The MODELLER tool (https://salilab.org/modeller/) has been employed to model cyclotides that were not experimentally confirmed. (Webb & Sali, 2016; Yu et al., 2024). Recent Trends in Science and Technology-2024 Bioinformatics www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788197073274, Page No.57 https://doi.org/10.5281/zenodo.17393857 Figure 1. The workflow of the computational analysis of anti-HIV cyclotides. Sequence analysis The sequences and structures of cyclotides with anti-HIV properties were manually screened from the sequence data. Further, these sequences were used for sequence analysis, which included sequence similarity studies, amino acid composition analysis, pattern construction, and pattern searching. The first and foremost step under sequence analysis was sequence similarity search using the BLAST tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul et al., 1990). The CAMP (Collection of Anti-Microbial Peptides) database has also been used for sequence similarity against anti-microbial peptides (Berman et al., 2007). BAGEL-2 (https://github.com/hart-lab/bagel) performs a similarity search against bacteriocin sequences, and AntiBP-2 (http://crdd.osdd.net/raghava/antibp2/ ) is used for an antibacterial peptide sequence similarity search (De Jong et al., 2010; Lata et al., 2010; Thomas et al., 2010). In addition, PRATT (https://web.expasy.org/pratt/) was used to create and search for patterns conserved in a collection of similar unaligned protein sequences (Jonassen, 1997). Further, the ScanProsite tool (https://prosite.expasy.org/scanprosite/) was used to detect PROSITE signature similarities in protein sequences (De Castro et al., 2006). However, the sequences to be scanned (cyclotide sequences) and the Motif to scan for (constructed pattern from ScanProsite) were also submitted in the ScanProsite tool. Patterns were also searched against anti-microbial peptide sequences to identify the presence and extent of similarity with constructed cyclotide patterns containing AMP sequences. Furthermore, protein signature studies have also been carried out by InterProscan, which scans the input sequences against the protein blocks of the InterPro database (Zdobnov & Apweiler, 2001). However, the ProtParam tool was used to calculate the physical and chemical properties of given proteins (Gasteiger, 2005). Lastly, studies on functional interactions among proteins in the cell, using the STRING Recent Trends in Science and Technology-2024 Bioinformatics www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788197073274, Page No.58 https://doi.org/10.5281/zenodo.17393857 database (https://string-db.org/), have also been implemented to analyze interacting protein partners in the system (Lian et al., 2024; Szklarczyk et al., 2010). Structure analysis and molecular docking The retrieved structures of cyclotides were first used for a structure similarity search with the BLAST tool, using the PDB as the database. Later, the approved drugs that are available for HIV treatment were searched against DrugBank, a unique bioinformatics/cheminformatics resource that integrates detailed drug (chemical) data with complete drug target (protein) information (Wishart et al., 2006). The targets’ crystal structures were also retrieved separately, along with drug complexes from PDB, to explore the mechanism, binding site information, etc. Literature studies were also conducted to understand the anti-HIV drug mechanism. Binding pocket determination was performed using MVD (Molegro Virtual Docker) (http://molexus.io/molegro-virtual-docker/) to predict protein-ligand interactions. Furthermore, downloaded 3D structures (both modelled and biologically determined) of cyclotides were used for protein-protein docking studies to interpret the structural affinity of cyclotides towards the proven drug targets of HIV by using the ClusPro online docking tool (https://cluspro.org/help.php) (Kozakov et al., 2010; Lian et al., 2024). 3. Results and discussion Data acquisition and sequence analysis In silico, sequence-structure analysis and ligand (cyclotide)- based drug design have been proven powerful methods in drug discovery and molecular elucidation of proteins. Ligandbased drug design identifies molecules that bind to the biological target of interest. Cyclotides are plant peptide toxins, which were considered ligands for this study. Sequence analysis methods, including sequence similarity searches using NCBI BLAST, were employed to investigate anti-HIV cyclotides, revealing similarities with other cyclotides. However, searches against specialized databases like Bagel-2 and CAMP showed no significant matches, confirming the unique nature of cyclotides. Further analysis using BLASTALL, with antimicrobial peptide sequences as the database, identified significant similarities between cyclotides and anti-microbial peptides. Additionally, predictions made using the AntiBP-2 tool suggested that anti-HIV cyclotides exhibit antibacterial activity. These findings indicate that anti-HIV cyclotides have the potential to function as natural antibiotics, serving a dual role as both anti-HIV and antibacterial agents. A specific pattern for anti-HIV cyclotides, defined as “({C-[AG]-E-[ST]-C-x(4,5)-Cx(3)-[AGILV]-x(2)-[CG]} )” was identified. Pattern searches across 318 cyclotide sequences yielded 339 matches, suggesting that other cyclotides may also exhibit anti-HIV properties. To validate this, functional assays such as XTT-based assays can be employed to confirm the antiHIV activity of these cyclotides. However, protein signature analysis using InterProScan yielded no significant similarities with proteins from other organisms. This strongly supports the conclusion that cyclotides possess a unique sequence arrangement. The ProtParam tool analysis provided insights into anti-HIV cyclotides’ physical and chemical properties, including their amino acid composition, theoretical pI, and molecular weight. The results revealed a prominent presence of cysteine residues across all cyclotides, with glycine, serine, and proline also highly conserved within their sequences (Figure 2). Classification based on amino acid groups highlighted a significant prevalence of sulfurcontaining amino acids and a notable dominance of aliphatic and hydroxyl groups within the cyclotide structures (Figure 3). Recent Trends in Science and Technology-2024 Bioinformatics www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788197073274, Page No.59 https://doi.org/10.5281/zenodo.17393857 Figure 2. The physical and chemical properties of anti-HIV cyclotides. Figure 3. Classification based on amino acids within the cyclotides. Functional interaction analysis of cellular proteins using the STRING database yielded no significant results, suggesting a lack of established interactions involving cyclotides. This underscores the need for further investigation into the potential protein-protein interactions of cyclotides within cellular systems. Molecular docking analysis The selected drug candidates were evaluated for their binding affinities to identify a familiar candidate capable of inhibiting the enzymes reverse transcriptase, integrase, and protease. Among the analyzed cyclotides with anti-HIV properties, Cycloviolacin-Y1 emerged as the most promising candidate, demonstrating the highest docking scores against reverse Recent Trends in Science and Technology-2024 Bioinformatics www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788197073274, Page No.60 https://doi.org/10.5281/zenodo.17393857 transcriptase and integrase. Additionally, Circulin-F was identified as a potent inhibitor of protease. The detailed docking scores are presented in Table 1. The docking scores of cyclotides against the HIV enzymes reverse transcriptase, integrase, and protease were analyzed to identify potential inhibitors, where Cyclotide-Y1 emerged as the top candidate, exhibiting the lowest docking energy scores for reverse transcriptase (-1162.8) and integrase (-906). Circulin-F demonstrated the strongest binding affinity towards protease, with a docking score of -813. These results suggest that Cycloviolacin-Y1 and Circulin-F hold significant potential as enzyme-specific inhibitors. Cyclotides derived from the Viola genus, including Cycloviolacin-Y1, are known for their diverse bioactivities, such as nematocidal, molluscicidal, anticancer, and anti-HIV properties. Structural analysis using the Anolea protein structure and model assessment tool further characterized the interactions of Cycloviolacin-Y1 with the active sites of reverse transcriptase, integrase, and protease, alongside comparisons with existing HIV drugs (Figure 4). The analysis confirmed the strong affinity of Cycloviolacin-Y1 for reverse transcriptase and integrase, highlighting its multi-target potential. Circulin-F, which showed a high inhibitory effect on protease (Figure 5), also demonstrated moderate binding affinities to reverse transcriptase (-1064.6) and integrase (-773.4), underscoring its broad-spectrum potential. Other cyclotides, such as Circulin-D, Circulin-E, and Cycloviolin-D, exhibited relatively strong binding affinities but were less effective than Cycloviolacin-Y1 and Circulin-F. Overall, the docking study emphasizes the efficacy of Cycloviolacin-Y1 and Circulin-F as promising candidates for HIV therapy, with distinct enzyme-specific inhibition profiles warranting further experimental validation. Table 1. Docking scores of cyclotides obtained from molecular docking studies. Docking Score of Cyclotides Sl. No. Name Reverse Transcriptase Integrase Protease 1. Cycloviolacin-Y1 -1162.8 -906 -783.6 2. Circulin-F -1064.6 -773.4 -813 3. Circulin-D -1047.5 -775.4 -608.3 4. Circulin-E -1039.9 -797.4 -774.1 5. Cycloviolin-D -1039.6 -805.2 -763.6 6. Cycloviolacin-Y4 -977.5 -684.7 -764.2 7. Kalata-B8 -957.5 -726.7 -731 8. Kalata-S -953.4 -649.7 -666.9 9. Kalata-B17 -919.1 -669.1 -643.6 10. Circulin-A -910.5 -706.2 -760 11. ([Lys(Ac)]2[Arg(Chd) -891.2 -669.1 -643.6 12. Palicourin -870.2 -630.6 -598.5 13. Cycloviolacin-Y5 -865.2 -641.2 -600.5 14. Cycloviolin-B -861.3 -671.3 -698.4 15. Cycloviolacin-O12 -853.8 -715.1 -646.7 16. Circulin-B -847.4 -599.3 -698.7 17. Circulin-C -843.6 -581.4 -608.3 18. Cycloviolin-C -839.9 -711.3 -711.4 19. Cycloviolin-A -830.2 -645.1 -619.9 20. 2kcg -781 -613.3 -621.5 Recent Trends in Science and Technology-2024 Bioinformatics www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788197073274, Page No.61 https://doi.org/10.5281/zenodo.17393857 Figure 4. Binding interaction of cycloviolacin-y1 with HIV enzyme active sites. Figure 5. Multi-target inhibition potential of Cycloviolacin-Y1 and Circulin-F. Recent Trends in Science and Technology-2024 Bioinformatics www.christcollegerajkot.edu.in, © Christ College, Rajkot, India ISBN: 9788197073274, Page No.62 https://doi.org/10.5281/zenodo.17393857 After the structure assessment, the results were analyzed, proving that the active sites of approved drugs and cyclotides vary. Further, the docked complexes were analyzed using the structure assessment tool ProCheck. The Ramachandran plot of all the anti-HIV cyclotides with a score (Core + allowed region) ranges from 95%-97%. Hence, it is proven that the docked models have a high degree of HIV-inhibiting properties. The obtained results are shown below (Table 1). However, the HIV life cycle highlights reverse transcriptase, integrase, and protease as critical enzymatic targets for therapeutic intervention. Cyclotides, particularly Cycloviolacin-Y1 and Circulin-F, demonstrated strong binding affinities to these enzymes, as evidenced by their docking scores. 4. Conclusion Cyclotides offer immense potential in drug development with their unique cystine knot motif and remarkable stability against various degradative conditions. Their multifunctional biological activities, including anti-HIV and cell-penetrating properties, make them particularly promising for therapeutic applications. Utilizing in silico approaches, Cycloviolacin-Y1 and Circulin-F have been identified as potential inhibitors of key HIV enzymes such as reverse transcriptase, integrase, and protease. Furthermore, pattern analysis has revealed additional cyclotides with anti-HIV activity, suggesting an expansive repertoire of therapeutic candidates within this peptide family. However, future efforts should prioritize experimental validation of these computational findings through wet lab studies to confirm their anti-HIV efficacy. Exploring the functional mechanisms and therapeutic potential of cyclotides with predicted anti-HIV properties will be critical to advancing their application as novel and effective treatments for HIV. Funding None Data Availability Statement Not applicable. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethical approval Not applicable. References Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of molecular biology, 215(3), 403-410. Balakrishnan, A., Mishra, S. K., Sharma, K., Gaglani, C., & Georrge, J. J. (2024). Intersecting Peptidomics and Bioactive Peptides in Drug Therapeutics. Bekker, L.-G., Beyrer, C., Mgodi, N., Lewin, S. R., Delany-Moretlwe, S., Taiwo, B., Masters, M. C., & Lazarus, J. V. (2023). HIV infection. 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