Acute viral illness known as dengue fever is brought on by the dengue virus (DENV), a member of the Flaviviridae family of viruses (Saur et al., 2021). The primary transmission vectors of this virus include Aedes mosquitoes. Dengue infection can appear clinically in numerous ways, ranging from an disease that is benign for dengue fever (DF) and also to the serious illness known as Dengue Shock Syndrome/Haemorrhagic Fever. The Aedes mosquito spreads the DENV mostly during the rainy season. The virus penetrates the skin of the host organism following an infection by an infected mosquito bite. The spread and course of DENV infection are influenced by a multitude of variables, including viral replication in macrophages, direct viral skin infection, and immunological and chemical-mediated pathways generated by host-viral interaction (Frischknecht, 2007). There are four different serotypes of the dengue virus which include, serotypes 1 (DENV-1), 2 (DENV-2), 3 (DENV-3), and 4 (DENV-4). Any serotype can cause dengue fever and can potentially progress to more serious instances like dengue hemorrhagic fever (Gubler, 1998) and dengue shock syndrome (Noisakran and Perng, 2008). The single-stranded RNA genome of DENV is positive-sense and approximately 11 kilobases long. The highly ordered 5′ and 3′ untranslated regions (UTRs) are arranged on either side of a single, long open reading frame (ORF).

An essential enzyme in the replication and pathogenesis of flaviviruses is the NS2B-NS3 protease. The cleavage of the viral polyprotein into distinct functional proteins required for viral replication is accomplished by this protease. The NS3 protein contains a domain of serine proteases that resembles trypsin, while the NS2B protein serves as a co-factor required for the protease action. NS2B and NS3, two viral proteins, combine to form the heterodimeric protease NS2B-NS3 of DENV, which cleaves the viral polyprotein during replication. Protease and helicase are the two primary enzymatic functions of NS3, a multifunctional protein. In order to synthesise unique functional proteins required for viral replication, the NS3 protease activity cleaves the viral polyprotein at specific places. The protease activity of NS3 is co-factored by NS2B. It is necessary for the NS3 protease domain to fold correctly and to activate. When NS2B and NS3 unite to form a stable complex, an active protease complex is generated that is crucial for the digestion of the viral polyprotein. In order to cleave the viral polyprotein into structural and non-structural proteins needed for viral maturation and assembly, NS2B and NS3 combine to form a functional protease complex. The reproduction cycle of the Dengue virus depends on this complex (Assenberg et al., 2009). However, there is still lack of FDA approved drugs against DENV (Kim et al., 2024, Lee et al., 2023) or any of its serotypes, making it to be a challenge and a forum to analyze the potential of different medicinal compounds against it to identify a potent inhibitor.

On the other hand, knot grass (Costea and Tardif, 2005), formally known as P. aviculare L., is a common weed that can be found in gardens, footpaths, and lawns. It is a social weed that thrives in areas that are constantly trampled and abused. Whereas, in context of regional habitats, this plant can be richly found in Middle Asia and Altai where it is used a source of traditional medicine (Luo et al., 2018, Salama and Marraiki, 2010). This was one of the strong foundations to select P. aviculare L. for this study to estimate its efficacy against DENV. Knot grass can be both annual and perennial, propagating through seeds typically between July and September (Hanna et al., 2015). A wide class of secondary plant compounds present in all plants are called flavonoids (Kumar and Pandey, 2013) which are also present in this plant. Numerous biological activities of flavonoids are known to exist, such as antioxidant (Pietta, 2000), anti-inflammatory (Maleki et al., 2019), antimicrobial (Cushnie and Lamb, 2005), anticancer (Kopustinskiene et al., 2020), cardioprotective (Testai, 2015), and neuroprotective effects (Hwang et al., 2012). Similarly, there are various studies which have reported therapeutic potential of P. aviculare L. in context of different illnesses which include hepatic, several chronic and anti-inflammatory infections (Leaf et al., 2024; The Investigating, 2024; Uçar, 2024; Jang et al., 2024). The potential health benefits and therapeutic qualities of these bioactive molecules have garnered great interest (Jucá et al., 2020). In plant biology, flavonoids are important because they affect many facets of growth and development. Furthermore, by functioning as detoxifying agents and scavenging dangerous concentrations of hydrogen peroxide (H2O2), reactive oxygen species (ROS), and toxic metals, flavonoids help shield plants from biotic and abiotic stresses. This helps to lessen oxidative stress brought on by external factors (Gill and Tuteja, 2010, Hossain et al., 2015). They also function as natural antibacterial agents and are produced by plants in response to microbial infections, known as phytoalexins (Hossain et al., 2015), which are essential to plant defence against pathogens. Though their precise function in protecting against viral infections particularly those resulting from plant viruses with single-stranded DNA (ssDNA) remains unclear, flavonoids may be able to counteract viral pathogens due to their broad-spectrum antiviral characteristics. Potential antiviral effects of several flavonoids, including (−)-epicatechin (Hossain et al., 2015), quercetin (Petrillo et al., 2022), kaempferol (Periferakis et al., 2023) and rutin (Luthar et al., 2020) have had their possible antiviral properties have been studied. As an illustration, it has been shown that quercetin inhibits HIV-1 entry and fusion with the cell, most likely via interacting with HIV-1 envelope glycoproteins (Hassan Khan and Ather, 2007).

Whereas, in this study multifaceted approaches are utilised to understand the interactions between NS3-NS2B protease and knot grass bioactive compounds. Protein Data Bank (PDB) was the source of the NS3-NS2B protease crystal structure, while Chemical Entities of Biological Interest (ChEBI) database was employed to retrieve the ligands. Structural quality validation via Ramachandran plot analysis ensured the model’s reliability in terms of no or lesser steric clashes. Molecular docking in the molecular operating environment (MOE) software predicted ligands-protein interactions, facilitated by ligand and protein preparation, ligand-binding active sites identification, and docking calculations. Analysis and visualisation of the docked complexes provided insights into binding modes and energetics, crucial for drug design and understanding therapeutic mechanisms. Afterwards, the flexibility and stability of the docked complex was performed which ensured the favourable results. Consequently, the in-silico drug testing was conducted in order to assess the drug-likeness characteristics of the screened-out compound i.e., (−)-epicatechin which also signified the favourable drug-likeness features.