In recent years, the world has witnessed the resurgence of several viral infections, including the Nipah virus, influenza virus (H1N1, H5N1), Ebola virus, Zika virus, and the recent coronavirus outbreaks (MERS-CoV and SARS-CoV-2; Mourya et al., 2019; Chadha et al., 2022c). The latest addition to this list is the monkeypox virus (MPXV), one of the four human pathogenic orthopoxvirus species, grouped along with variola, cowpox, and vaccinia virus (Lam et al., 2022). Monkeypox (MPX) is an emerging zoonotic disease caused by the MPXV, which was first discovered in 1958 following local outbreaks of a smallpox-like disease in monkey colonies maintained at research facilities in Africa and Denmark (Chadha et al., 2022b). However, the first clinical case of MPX (in humans) was reported in 1970 in the Equatorial province of the Democratic Republic of Congo (DRC; Marennikova et al., 1972). Since then, sporadic MPX outbreaks have been reported across several African countries, particularly the DRC, Republic of The Congo, Central African Republic, Nigeria, Liberia, Ghana, and Cameroon (Bunge et al., 2022). The MPXV has been endemic to sub-Saharan countries for decades, but in mid-2022 (amid the COVID-19 pandemic), an unexpected surge in MPX infections was recorded globally (WHO, 2022). Multi-country outbreak of MPX was reported across several non-endemic countries, raising concern among clinicians and epidemiologists (Chadha et al., 2022b). With the increasing global caseload, the World Health Organization also declared the ongoing multi-country outbreak of MPX as a Public Health Emergency of International Concern (PHEIC) on 23rd July 2022.

The MPXV is an enveloped virus with a dsDNA genome of nearly 190 kbp and harbors a dumbbell-shaped core with lateral bodies (Kugelman et al., 2014). The virus is known to infect various mammalian species, including primates, but its natural animal reservoir remains unknown. However, some investigations suggest that rodents are the primary reservoir of the MPXV (Reynolds et al., 2012). To date, two distinguishable clades of MPXV have been identified: West African and Central African (Congo Basin). The latter is highly transmissible among humans and exhibits potentially high virulence and case fatality rate (~ 11%) (Sklenovská and Van Ranst, 2018). MPX transmission has been strongly associated with animal bites, skin abrasions, prolonged or direct contact with bodily fluids of infected animals or humans, respiratory droplets, and contaminated inanimate objects (Ligon, 2004). However, the transmission of presently-circulating MPXV strains through sexual routes is still debated (Chadha et al., 2022b). The incubation period of MPXV ranges from 3 to 14 days, and the clinical manifestations are seldom mistaken for other viral infections like herpes simplex virus and chickenpox. Symptoms of MPX are similar to that of smallpox, including fever accompanied by blisters, muscle pain, rashes, chills, and respiratory illness (Titanji et al., 2022). The present-day treatment and clinical management of MPX involve the administration of repurposed antiviral drugs (cidofovir, brincidofovir, and tecovirimat) and preventive vaccines (ACAM2000 and JYNNEOS; Chadha et al., 2022b). Moreover, around 50 single-nucleotide polymorphisms have been identified in the genome of 2022 MPXV, which remained undetected in the previously circulating MPXV strains between 2018 and 2019 (Isidro et al., 2022). This increases the gravity of the ongoing multi-country outbreak, pointing toward the “accelerated evolution” of MPXV. Hence, there is a pressing need to develop novel therapeutics against the MPXV. In this direction, drug repurposing proves to be an effective alternative since it bypasses the need for drug development, thereby reducing the time frame for drug discovery (Gulati et al., 2021).

Due to their natural existence, easy availability, extensive antimicrobial, antiviral, antivirulence, and pharmacological properties, the application of plant-derived products and bioactive phytochemicals in modern medicine has gained momentum among researchers (Chadha et al., 2021, 2022a). Numerous in silico and pharmacoinformatics-based reports have predicted the effectiveness of phytochemicals and other drugs against SARS-CoV-2, Ebola, and Nipah virus (Kwofie et al., 2019; Pathania et al., 2019; Choudhury et al., 2021; Kumar A. et al., 2021). On similar lines, we employed a comprehensive in silico approach to identify phytochemicals that could potentially act as inhibitors against poxviruses for repurposing them against the MPXV. In a recent study, Lam et al. demonstrated a high sequence similarity among the structural proteins of viruses belonging to Poxviridae, especially between the MPXV and vaccinia virus (Lam et al., 2022). Contingent to this, we utilized the structural proteins of the vaccinia virus, which is a close relative of MPXV, as the drug target(s). The first step toward any successful viral infection is cellular entry, which is facilitated by the envelope proteins that are involved in the attachment of the virus to the host cell. Hence, drugs that target the envelope proteins could potentially inhibit viral infection at an early stage. Considering this fact, the phytochemicals included in this study were targeted against the three structural proteins (envelope) of poxviruses viz., D13, A26, and H3 (Figure 1) using molecular docking coupled with dynamic simulations and also evaluated for their pharmacokinetic properties and druglikeliness.

With the COVID-19 pandemic slowing down, humankind faces another potential threat due to the sudden emergence of MPXV. The multi-country MPX outbreak may not match the scale of the receeding pandemic but demands immediate global attention and surveillance. With the discontinuation of smallpox vaccine (1980) that provided cross-protection against the MPXV, a large fraction of the world population remains susceptible to MPX (Chadha et al., 2022b). Moreover, the situation has been aggravated by several cluster outbreaks being reported across non-endemic countries and the identification of 50 genomic substitutions within the 2022 MPXV (Chadha et al., 2022b). In view of these events, there is an urgent need to develop therapeutics against the circulating MPXV. In this regard, repurposing bioactive phytochemicals and existing drugs proves to be a promising alternative to combat the wave of MPX infections. Recently, phytochemicals with profound antiviral activity have been successfully repurposed against SARS-CoV-2 (Balkrishna et al., 2021; Lingwan et al., 2021). Hence, the present study was undertaken to virtually screen bioactive phytochemicals targeting the envelope proteins of poxviruses through comprehensive in silico analysis. The envelope proteins of vaccinia virus were used as a template for this study since its structural proteins share high sequence similarity with the MPXV (Lam et al., 2022). Although poxviruses possess multiple drug targets, the envelope proteins were chosen specifically in an attempt to prevent virus attachment to the host cell, consequently preventing viral infection at the first instance.

The D13 protein is an essential component of the viral capsid that provides rigidity to the structure of poxviruses, thereby playing an essential role in viral morphogenesis. It is a scaffold protein that forms a honeycomb-like lattice on a viral membrane and aids in forming pleomorphic immature virions in conjunction with A14 and A17 proteins (Van Vliet et al., 2009). Interestingly, the downregulation of the D13 protein results in irregular single bilayer membranes instead of immature virions (Zhang and Moss, 1992). It has been experimentally demonstrated that the D13 protein associates with the viral membrane assembly proteins to form membrane precursors (Maruri-Avidal et al., 2013). Vaccinia virus adopts two modes of entry into the host cell, which include (i) direct fusion with the plasma membrane and (ii) endocytosis. The A26 protein is an envelope protein known to interact with the components of the extracellular matrix of the host cell, thereby promoting acid-dependent endocytosis in vaccinia virus (Bengali et al., 2009). Studies have demonstrated that the loss of the A26 protein blocks the endocytic pathway, thereby triggering the alternate plasma membrane fusion-based route for viral entry into the host cells (Chang et al., 2019). Also, experimental findings by Chang et al. revealed that the loss of the first 75 amino acids from the N-terminal region of the A26 protein in vaccinia virus significantly reduced infectivity as compared to the wild-type strain (Chang et al., 2019). Another important envelope protein of poxviruses is the H3 protein that binds to heparin sulfate residues on the host cell surface, directly facilitating virus attachment (Singh et al., 2016). The crystal structure of the H3 protein shows that it harbors a glycosyltransferase fold and thus binds to UDP glucose (Singh et al., 2016). It is highly conserved and is an immunodominant antigen that is a major target of neutralizing antibodies. Considering the pivotal role of these envelope proteins in the pathogenesis of poxviruses, we selected them as suitable drug targets for this study. Rifampicin was used as a control ligand since it is known to arrest viral morphogenesis before membrane assembly (Grimley et al., 1970). Additionally, rifampicin has been shown to occupy the phenylalanine-rich pocket of the D13 trimer, thereby inhibiting its binding to the A17 protein and affecting the formation of the immature virions (Garriga et al., 2018). However, this binding is reversible, and the assembly of membrane proteins resumes upon withdrawal of the drug. Rifampicin has widely been used as an antibacterial drug as it targets DNA-dependent RNA polymerases. However, its use as an antiviral drug is skeptical in clinical settings because of its low potency and the emergence of resistant viruses.

In the present study, phytochemicals (ligands) were reported to bind effectively to the envelope proteins of MPXV with significantly lower binding energies. The phytochemicals could easily occupy and fit into the active site (pocket) of vaccinia virus A26, D13, and H3 envelope proteins, thus acting as a roadblock for the virus attachment to the host cell. Additionally, the test phytochemicals and control (rifampicin) shared similar molecular interactions with the amino acid residues on the viral proteins. Moreover, with respect to a single protein, certain amino acid residues displayed common interactions with few phytochemicals. Among the 12 compounds screened, 11 displayed binding energies lower than-6.0 kcal/mol against all the three target proteins, except rosmarinic acid, which showed binding energy of −6.0 kcal/mol against the H3 protein. Among the lead phytochemicals, oleanolic acid and ursolic acid belong to the category of pentacyclic triterpenoids. Numerous studies have demonstrated the antiviral, antitumor, and antimicrobial prospects of triterpenoids (Yu et al., 2014; Xiao et al., 2018). Oleanolic acid, an essential component of olive oil, exhibits immunomodulatory effects on the human immune system and has been extensively used in cancer prevention by abrogating various signaling pathways (Ríos, 2010). Numerous studies have also illustrated the application of oleanolic acid and ursolic acid as antiviral agents against different viruses (Khwaza et al., 2018; Shan et al., 2021). Moreover, silibinin is a flavonolignan known to exhibit anti-neoplastic and antioxidative properties. It also inhibits prostate cancer by targeting multiple signaling pathways, inducing apoptosis, and inhibiting metastasis and angiogenesis (Deep and Agarwal, 2010). The existing literature and upcoming investigations collectively increase the foothold for repurposing phytochemicals as alternate therapy against emerging pathogens in clinical settings.

In a recently published study, FDA-approved drugs have also been evaluated for repurposing them against the viral capsid and replication proteins of the vaccinia virus through a comprehensive in silico approach (Lam et al., 2022). The role of hydrophobic interactions in stabilizing the protein-ligand complex has been well documented across various investigations (Patil et al., 2010; Ferreira De Freitas and Schapira, 2017). Moreover, hydrogen bondings at the drug-ligand interface are known to play a vital role in stabilizing intermolecular interactions within the complex, thereby promoting complex stiffness (Patil et al., 2010). In this study, a relatively greater number of hydrogen and hydrophobic bonds were formed by the test phytochemicals as compared to the rifampicin. Multiple hydrogen bonds between the phytochemicals and target protein(s) can possibly enhance the stability of the interacting complex. The findings from the molecular docking and counter verification by MD simulation and NMA studies studies collectively provided evidence that bioactive phytochemicals can effectively associate with viral envelope proteins and stimulate conformational changes that may interfere with binding to their cognate ligands on the host cell surface.

Altogether, our data sheds light on the possible molecular interaction(s) between bioactive phytochemicals and the envelope proteins of MPXV, thereby interfering with the functional mechanisms of viral entry into the host cell. The investigation generates preliminary findings that indicate the ability of oleanolic acid, ursolic acid, and silibinin to effectively target the D13, A26, and H3 envelope proteins of MPXV. Nevertheless, these in silico findings warrant further experimental validation for their application as alternative medicine against MPX in laboratory settings.

In summary, of all the phytochemicals tested, oleanolic acid, ursolic acid, and silibinin can be envisioned as the lead compounds for repurposing phytochemicals against poxviruses, including the MPXV. Apart from displaying efficacious binding through a plethora of hydrogen and hydrophobic bonds, these 3 phytochemicals were well-fitted in the pockets of all the three envelope proteins taken into consideration. Thus, these compounds dwell into the criteria of a multi-targeted drug delivery approach which utilizes distinct drug candidates that act synergistically against a particular target through different molecular mechanisms. Owing to their natural existence, these bioactive phytochemicals can be used as alternatives to existing chemical drugs that inflict adverse drug reactions and off-target effects. Hence, this study points toward the possible use of phytochemicals to combat the ongoing multi-country outbreak of MPX. However, this demands further validation through in vitro and in vivo testing.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

PG: idea conceptualization, perform molecular docking and MD simulation experiments, data analysis and curation, and manuscript writing. JC: idea conceptualization, experimental design, data curation, manuscript writing and editing, and critical analysis. KH: continuous motivation and formal supervision and analysis. SS: continuous motivation, supervision of the study, and manuscript editing. All authors read and approved the final version of this manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.