Structural investigations of the palmitoylated F13 envelope protein of Mpox virus

Dear Editor, In this Journal, Elnaz Khani et. al. has emphasized Mpox (monkeypox) as a global concern and listed various treatment approaches. The article also emphasized the importance of F13, a Mpox virus envelope protein, in membrane interaction, virion wrapping, and extracellular enveloped virus production. The F13 is a 372 amino acid protein that is a target for tecovirimat (CID: 16124688), a smallpox drug licensed by the Food and Drug Administration. The Mpox F13 lacks a transmembrane domain, however, cysteine residues 185 and 186 are palmitoylated. Li et al. emphasized that the lack of structural information hampers the proper understanding of F13‐drug interactions. While the F13's three‐dimensional (3D) structure has been modeled, palmitoylation details remain unknown. In this work, we modeled the palmitoylated structure of F13. The amino acid sequence of the envelope protein F13 of the Mpox virus was collected from the UniProt database (UniProt ID: Q5IXY0). The AlphaFold2 web service was used to model the initial 3D structure of the protein. The modeled structure was further evaluated with the Ramachandran Plot server (https://zlab. umassmed.edu/bu/rama/). The model showed 98.23% of the residues in the highly preferred region (Figure 1A). Next, The CHARM‐GUI server was used to generate the palmitoylated F13 protein and palmitoylated F13 protein‐membrane systems (Supporting Information: info 1). The cysteine residues 185 and 186 were palmitoylated as per the information from the literature. Both the systems along with a non‐palmitoylated form of F13 underwent 300‐ nanosecond production runs using molecular dynamics (MD) simulations. A stable root mean square deviation (RMSD) pattern was observed in the case of the palmitoylated F13 and membrane‐anchored palmitoylated F13 (Figure 1B). Using principal component analysis, the minimum energy conformations were extracted from the final 150 ns trajectory (Figure 1C,D). There is a small RMSD difference (1.26Å) between the membrane‐anchored and without membrane palmitoylated F13 conformations (Supporting Information: Figure S1). Tecovirimat, a previously approved smallpox drug, has been shown to be effective against the Mpox virus both in vitro and in vivo. Molecular docking was used to determine how tecovirimat interacts with palmitoylated F13. The simulated membrane‐ associated palmitoylated F13 model was selected as the starting model for docking. Tecovirimat interacted with palmitoylated F13 with a binding energy of −9.0 kcal/mol in the given grid box (Supporting Information: info 1). The residues, Thr 137 and Asn 312 formed hydrogen bonds (H‐bond). A total of 08 hydrophobic interactions were formed by the residues Phe 52, Ser 135, Ile 144, Leu 239, Thr 279, Lys 314, Ala 328, Asn 329 (Figure 1E). A recommended starting point for creating effective and safe antiviral drugs is screening natural compounds. In this work, 1048 filtered natural compounds obtained from the ZINC15 database were used for docking. The highest‐scoring compounds were selected using a binding energy cut‐off of −10 kcal/mol, higher than the tecovirimat binding energy (−9.0 kcal/mol). The top 05 compounds (Supporting Information: Figure S2) were selected based on their binding energy (Supporting Information: Table S1). Among the top 05 compounds, ZINC6028400 scored highest with a binding energy of −11.5 kcal/mol followed by ZINC3995616 (Ergoloid), ZINC4097766, ZINC42835355 (Antioquine), ZINC85493573 with binding energies of −11.5, −11.3, −11.3, −11.1 and −11.1 kcal/mol, respectively. ZINC6028400 formed a total of 03 H‐bonds and 14 hydrophobic interactions. Details of the interactions made by the top 05 compounds are given in Supporting Information: Table S1 and Supporting Information: Figure S3. The top five complexes were simulated for 100 ns each, including the tecovirimat complex. In comparison to tecovirimat, all five compounds displayed stable RMSD values in the MDS study (Figure 2A). According to post‐simulation binding free energy calculations, ZINC85493573 has a substantially stronger affinity for the F13 (–33.96 kcal/mol) than tecovirimat (–19.88 kcal/mol), followed by ZINC3995616 (–26.23 kcal/ mol) and ZINC6028400 (–20.48 kcal/mol). (Figure 2B and Supporting Information: Table S2). In summary, this work reported the first palmitoylated model of F13. It has been shown previously that palmitoylation is required for proper folding and stability of the transcription enhancer factor‐ associated domain. Similarly, the palmitoylated F13 showed greater stability than the non‐palmitoylated version (Figure 1B). Virtual screening was used to examine 1048 natural compounds, and the top three compounds with binding energy higher than the reference drug tecovirimat were reported. Although pre‐exposure immunization can prevent Mpox infection, therapeutic options post disease onset are limited due to a lack of approved medications. Due to their strong interaction with the F13 binding site and the stability the reported compounds are more likely to be efficient inhibitors. These results can help in further structural studies on the Mpox F13 protein as well as drug discovery endeavours.