Difco LB broth (Becton, Dickinson and Company, Sparks, MD, USA) was autoclaved at 121 ^°C for 15 min and stored at room temperature. IPTG (Sigma-Aldrich, Bengaluru, India; now Merck KGaA, Darmstadt, Germany) was prepared as a 1 M stock in Milli-Q water and stored at -20 ^°C. Kanamycin (50 mg/mL) and ampicillin (100 mg/mL) stock solutions were prepared in Milli-Q water and stored at -20 ^°C. CAPS buffer was prepared as a 1 M stock in Milli-Q water, and pH was adjusted as required. NaCl (5 M) and glycerol (100% v/v) stock solutions were prepared in Milli-Q water and stored at room temperature. Resveratrol (Sigma-Aldrich) was prepared as stock solutions in an appropriate solvent (e.g., DMSO) and stored according to the manufacturer’s instructions.

The three-dimensional crystal structure of human MARK4 was retrieved from the RCSB Protein Data Bank (PDB ID: 5ES1) at a resolution of 2.8 Å. Prior to docking, the protein structure was prepared by removing co-crystallised ligands and water molecules, followed by the addition of hydrogen atoms and assignment of Kollman charges using MGL Tools to ensure proper geometry and charge distribution for molecular docking analysis (Mohammad et al., 2021). Molecular docking of MARK4 with resveratrol was carried out using InstaDock. Blind docking was carried out to explore the surface of the entire protein, applying exhaustiveness of 8. The resultant poses were further analysed on the basis of binding affinity and pattern of interaction. All the interactions between the protein and ligand, including hydrogen bonds and hydrophobic interactions, were studied with Discovery Studio Visualizer and PyMOL (BIOVIA, 2017; DeLano, 2002).

The protein MARK4 was expressed in M15 E.coli bacterial cells. The M15 strain contains the pREP4 plasmid, which has the lac repressor (lacI) gene, allowing regulation of protein expression under the T5 promoter in pQE vectors. The plasmid vector selected was pQE30 which is compatible with M15 and carried N-terminal polyHistidine tag, facilitating as a tag in purification and high yield. The recombinant plasmid with the protein of interest was transformed into E.coli-M15 cells, and protein purification was carried out with the previously established protocols (Atiya & others, 2023; Alrouji & others, 2023).

Enzyme here is a kinase, MARK4, which works by using ATP as a substrate for its activity, converting ATP to ADP, and the released phosphate is used by the kinase to phosphorylate its target. For the assay mentioned here, the principle is that the protein kinase is considered 100 per cent active by measuring the amount of phosphates that are released upon interaction with ATP. Keeping the concentration of protein constant, different concentrations of ligand are added to the protein and incubated, allowing the ligand to bind with the protein. Afterwards, ATP is added to the control, i.e. protein without ligand and protein with increasing concentration of ligand, and the released phosphate is estimated with a reagent known as BIOMOL green reagent. MARK4 and ATP stock solutions were prepared, and the reaction was set in a 96-well plate by mixing protein, ATP, buffer and ligands to obtain a final MARK4 concentration of 5 μ M and ATP of 200 μ M. The reaction mixtures were first set up without ATP and incubated with increasing concentrations of ligands for 30 minutes at 37^°C. Further, ATP was added and incubated at 37^°C for approximately 30 minutes. Following incubation, Biomol Green reagent was added according to the manufacturer’s instructions to terminate the reaction and enable detection of released inorganic phosphate. The mixtures were thoroughly mixed and incubated at room temperature in the dark to allow full color development. Absorbance was measured at 620 nm using a microplate reader.

The binding studies of RV with MARK4 were performed on the Jasco spectrofluorometer at 25^°C. The compound was prepared by dissolving it in DMSO, giving the desired 1 mM working concentration in the working buffer (pH 8.0). The binding studies were performed with a fixed concentration of MARK4 (4 μ M), and the ligand was added gradually in increasing concentration till saturation. The addition of ligand was mixed thoroughly and gently to ensure interaction between protein and ligand before measurements. The intrinsic fluorescence of protein, mainly contributed by the aromatic amino acids and predominantly Tryptophan, was measured by excitation at 280 nm, which generated an emission spectrum at 300–400 nm. The fluorescence quenching observed in the emission spectra was analysed using the modified Stern-Volmer equation (Dahiya & others, 2019).

In the given equation, F0 and F denote the fluorescence intensity of protein and protein ligand complex, respectively. K is the binding constant; n denotes number of binding sites and C for concentration of RV.

fracF0F = 1 + Ksv[C]

logleft(fracF0 - FFright) = log K + n log [C]

Cancer cells H1299 are a widely used human non-small cell lung carcinoma (NSCLC) cell line, specifically identified as a large cell carcinoma subtype. The cells were procured from NCCS, Pune, India. A culture medium was prepared for H1299 cells with antibiotics and heat-inactivated FBS. Using a 96-well culture plate, approximately 5 × 103 cells were plated per well following passages till passage number 20, with a confluency of 85%, and all the treatments were given. A compound stock solution was made with DMSO, with a maximum limit of 0.2% per treatment. The MTT stock was prepared as 5 mg/ml, and 10 μ L of the stock was incubated in all the treated wells at 37^°C with 5% CO2 for a few hours. The 96-well plate was read spectrophotometrically at 570 nm using a 96-well plate reader.

To understand the binding of RV with MARK4, molecular docking studies were carried out as shown in Figure 1. According to the docking research, RV interacts with Ile62 and Glu133 by typical hydrogen bonds, which are crucial for anchoring the ligand inside the binding cavity. The stability of ligand-protein complexes is greatly aided by hydrogen bonds, which are important factors in determining ligand specificity. RV also forms multiple hydrophobic and van der Waals interactions with several residues lining the MARK4 binding pocket, including Met132, Val116, Tyr134, Gly138, Glu139, Asp142, Ala135, and Leu185. Together, these interactions help keep the ligand inside the kinase's catalytic domain and stabilize the structure of the MARK4–RV complex. Moreover, π–alkyl interactions with Val70 and Ala83 were noted, suggesting stronger hydrophobic interactions between RV's aromatic ring and the nonpolar residues in the active site. Docking studies reveal RV occupies the catalytic pocket of MARK4 with multiple interactions (Fan et al., 2019; Shamsi & others, 2020; Anwar & others, 2022).

The aromatic amino acids in the protein structure are responsible for the intrinsic fluorescence of the protein. The major contributor amongst all aromatic amino acids is tryptophan (Trp), followed by tyrosine and phenylalanine. Trp has a high quantum yield, resulting in high protein fluorescence at 300–350 nm when excited at 280 nm. The intensity and spectral position of this emitted light provide information about the structural and environmental status of the protein (Valeur & Berberan-Santos, 2013; Eftink, 1997; Lakowicz & Weber, 1973). Trp fluorescence intensity and emission wavelength can be dramatically changed by changes in solvent polarity, conformational shifts, or ligand binding, which makes it an effective probe for researching protein structure, folding, and intermolecular interactions (Lakowicz, 2006; Vivian & Callis, 2001). As tryptophan fluorescence is highly sensitive to conformational rearrangements and binding-induced perturbations, it is widely used to investigate protein-ligand interactions, conformational changes, and to estimate binding constants (Taraska & Zagotta, 2010). In the current investigation, an increase in the ligand concentration resulted in decreased fluorescence intensity of the protein, as shown in Figure 2A. The quenching in the fluorescence of the protein can likely be due to the changes in the microenvironment near the tryptophan residues in the presence of RV. The binding constant of the interaction was estimated to be 7.0 × 104 M−1 using the modified Stern-Volmer equation (Figure 2B).

The kinase MARK4 is an important therapeutic target due to its association with various diseases, including cancer and neurodegeneration. MARK4 has a strong correlation with Alzheimer’s disease, as MARK4 is associated with pathological phosphorylation of tau protein, contributing to dementia and early onset of AD. MARK4 is upregulated in various cancers, including breast cancer, lung cancer and hepatocellular carcinoma (Mohammad et al., 2021; BIOVIA, 2017; DeLano, 2002). Therefore, MARK4 has emerged as an interesting therapeutic target in cancer, neurodegeneration and other diseases. RV is a naturally occurring compound with documented anti-inflammatory, anti-oxidant, anti-ageing, anti-cancer and neuroprotective activities. In order to study the influence of RV on kinase activity and to complement molecular docking and fluorescence-based binding studies, an enzyme inhibition assay was carried out.

A decrease in kinase activity of MARK4 was observed with increasing ligand concentration (Figure 4), indicating inhibition of the kinase activity of the protein, which can be due to the formation of protein-ligand association, as evident from docking studies. AAT Bioquest IC50 calculator was used to determine the half-maximal inhibitory concentration (IC50), which is the inhibitor concentration needed to decrease enzyme activity by 50% as shown in Figure 3. In line with earlier findings, the IC50 value for RV was found to be approx 7 μ M. (Dahiya & others, 2019; Fan et al., 2019). These findings validate RV's therapeutic potential and confirm its status as a strong MARK4 inhibitor. RV might be a promising addition to the expanding family of small-molecule kinase inhibitors that target MARK4, considering the pivotal role that kinases play in a variety of pathological situations, such as cancer and neurological diseases.

H1299 lung cancer cell lines were used to examine the cytotoxicity of RV against them. We performed a cell survival assay to verify the anti-cancerous activity of the compounds. The cell growth was observed for 48 hours following the treatment with increasing concentrations of RV. Compared to the DMSO vehicle control, RV showed significant cytotoxicity in the H1299 cell lines. The IC50 values of RV against H1299 were approx 114 μ M, as shown in Figure 4.