Molecular Docking Studies of Natural Immunomodulators Indicate Their Interactions With CD40 and CSF1 of Microglia

Natural products have proved bene�cial in reducing neuroin�ammation in neurological diseases. Their impacts have also been associated with the activities of microglia, responsible for brain-specic immunity. Recent studies have shown involvement of the number of microglia-specic proteins in the regulation of brain-specic immunity. However, molecular targets of natural products and their mechanisms of interactions with microglia-specic proteins are elusive. Since the genetic signature of microglia offers many potential targets for drug discovery, molecular docking followed by Molecular Dynamics (MD) simulations of Cluster of Differentiation 40 (CD40) and Colony-stimulating factor 1 receptor (CSF1R) proteins with some known neuro-immunomodulators (Curcumin, Cannabidiol, Ginsenosides, Resveratrol, and Sulforaphane) has been evaluated. Curcumin and Cannabidiol were observed likely to modulate CD40L and expression of cytokines and entry of in�ammatory cells. Resveratrol and Cannabidiol may affect CSF1R and activation of microglia. Our �nding suggests that Curcumin, Cannabidiol, and Resveratrol may serve speci�c drug ligands in regulating microglia-mediated brain immunity.


Introduction
Natural products are de ned as small molecules produced by living organisms.The major advantages of using these natural products are their novel structure, multi-target molecular mechanism, potent e ciency, and safe pharmacodynamics.Recent researches have shown the use of natural products like Curcumin, Cannabidiol, Ginsenosides, Resveratrol, and Sulforaphane on brain immunity and neurodegenerative diseases [1][2][3][4].However, natural products mediated molecular targets of microglia are still elusive.
Microglia, resident immune cells of the immune-privileged brain, regulate brain homeostasis in response to pathogen and injury by accumulating at the site of in ammation or degeneration.They facilitate induction of a wide variety of cytokines, regulate neurogenesis, synaptic pruning, laminar structure of cortex, and neurite formation [5][6][7][8][9][10][11][12].The local microenvironment of neurons and astrocytes also affects phenotypes of microglia by interaction with both soluble and membrane-bound mediators [13].
Depending on the strength of injury, microglia may have neuroprotective or neuroin ammatory functions.
In case of short signal, microglia secretes growth factors to heal damaged neurons, cause phagocytosis of cell debris indicating neuroprotective phenotype whereas cause neuronal damage by secreting proin ammatory cytokines, reactive oxygen species, and nitric oxide in response to an acute and chronic signal which further recruits lymphocytes from blood to damage site [9].Upregulated expression of CD40 on the surface of activated microglia and its ligation with soluble ligands leads to increased secretion of numerous pro-in ammatory mediators by microglia [14].The CSF1R, a key signaling node, regulates the development, proliferation, migration, differentiation, and survival of microglia [15].Anti-in ammatory neuropeptide, pituitary adenylyl cyclase-activating polypeptide (PACAP), lovastatin, and glucocorticoids down-regulate the expression of CD40 in activated microglia [16][17][18].The CSF1R inhibitors (PLX3397 and PLX5622) cause depletion of microglia and prevent plaque formation in brain parenchyma in the 5xFAD mouse model of AD [19][20].Another CSF1R inhibitor JNJ-40346527 (JNJ-527) blocks the proliferation of microglial and attenuation of tau-induced neurodegeneration [21].
Therefore, it is aimed to identify the possible microglia-speci c molecular target of natural products (Curcumin, Cannabidiol, Ginsenosides, Resveratrol, and Sulforaphane) using in silico docking studies.

Drug likeness of the natural products
The drug likeliness properties of the ve natural products (Curcumin, Cannabidiol, Resveratrol, Ginsenosides Rg1, and Sulfopharane) were evaluated with the help of Lipinski drug lter rule of 5 (molecular mass, number of hydrogen bond donors, and acceptors, logP and molar refractivity) from supercomputing facility for bioinformatics and computational biology (http://www.scfbioiitd.res.in/software/drugdesign/lipinski.jsp)[23].Based on the Lipinski rule of ve which includes parameters such as (i) molecular mass should be less than 500 Dalton, (ii) hydrogen bond donor should be less than 5, (iii) hydrogen bond acceptor should be less than 10, (iv) molar refractivity must lie between 40 and 130 (v) high lipophilicity (expressed as LogP) should be less than 5, the drug-like or nondrug like properties of the molecule can be calculated.

Retrieval of the 3D structure of proteins and analysis for drug pocket predictions
The PDB le of 3D structure of CD40 (PDB ID: 1ALY) and CSF1R (PDB ID: 6T2W) was retrieved from Protein Data Bank (https://www.rcsb.org/).The DoGSiteScorer server was used to visualize potential drug binding pockets on protein structure where drug score determines the druggability of the pocket [25].

Molecular Docking and analysis of protein-ligand interaction
The molecular docking was performed by AutoDock 4.2 [26] using AutoDock Tools-1.5.6.Brie y, the PDB le of protein was opened in AutoDock Tools.The water molecules and any bound ligand were deleted followed by the addition of Kollman Charges on the protein molecule.The PDB le of natural products was converted to a PDBQT le using Open Babel software and then created PDBQT le was opened into the AutoDock Tools.The grid parameters were selected to cover the whole protein structure for the analysis.Then, docking was performed using genetic algorithm (GA) as a search parameter for 50 runs and a population size of 300.After docking, the docking complex was retrieved from AutoDock Tools in PDBQT le format which was further converted to PDB le format using Open Babel.Interaction between ligand and targets was calculated on the basis of binding energy (Kcal/mol), inhibition constant (µM), and contacting receptor residues.The protein-ligand interaction and analysis of hydrogen bond formation were analyzed by LigPlot2 [27] and Protein-Ligand Interface Pro ler (PLIP) server [28].The docking results were further validated by protein with ligand simulation for 100ns using GROMAC simulation package by online WEBGRO server [29] of Simlab (https://simlab.uams.edu/).The best binding pose of protein-ligand complex obtained through docking was taken as input for Molecular Dynamics (MD) simulations.System and structural stability were monitored by measuring the RMSD, RMSF, and Rg of the protein-ligand complex (Supplementary Figure 1-6).The stability of ligand inside the binding pocket of protein was assessed by measuring the RMSD of ligand.The effect of ligand on protein mobility or movement was measured through RMSF.Effect of ligand-protein compactness was measured through Rg with respect to time.

Results
Curcumin, Cannabidiol, and Resveratrol obeys Lipinski rule of 5 indicating their drug-like property Out of ve compounds, only Curcumin, Cannabidiol and Resveratrol obeyed the Lipinski rule of 5 (viz.molecular mass should be less than 500 Dalton, high lipophilicity expressed as LogP less than 5, hydrogen bond donor should be less than 5, less than 10 hydrogen bond acceptor, molar refractivity must lie between 40  ADMET studies of all the ve natural products were executed to screen the most e cacious pharmacologically active compound.Based on the ADME toxicity investigation result, it was found that all the ve natural products have the ability to absorb in the blood-brain barrier.Out of ve, Curcumin, Cannabidiol, Resveratrol, and Ginsenoside RG1 showed oral bioavailability, non-toxic and noncarcinogenic properties (Table 2).

Molecular docking showed potential interaction of Curcumin and Cannabidiol with CD40
The analysis of CD40 protein showed presence of 5 drug pockets (Table 3) which can be used for drug binding.The protein-ligand interaction using Curcumin and Cannabidiol as ligand showed its binding to drug pocket 0 of CD40 with a volume of 269.18 Å 3 and surface area of 471.12 Å 2 and drug score of 0.62 whereas Resveratrol does not show binding on predicted drug binding sites (Figure 2).The visualization of CD40 interaction with Curcumin showed 2 hydrogen bonds between Asp243 and Ser245 residue of CD40 with Curcumin has a distance of 2.85 and 2.87 Å, respectively and binding energy of -4.69 Kcal/Mol and inhibition constant of value 367.48 µM.Similarly with Cannabidiol, the binding energy obtained was -4.22 Kcal/Mol and an inhibition constant value of 805.16 µM.The Ser245 amino acids of CD40 form 2 hydrogen bond with Cannabidiol with distances between them are 3.08 and 3.10 Å, respectively.The resveratrol shows hydrogen binding with 4 amino acids (Val175, Ala173, Ile171, Gly227) of CD40 where Ile171 forms 2 hydrogen bonds of 3.24 and 2.46 Å whereas Val175, Ala173, and Gly227 form one hydrogen bond with resveratrol of 3.01, 2.74, and 2.66 Å each (Figure 3; Table 4).

Resveratrol and Cannabidiol seems promising for targeting CSF1R protein kinase domain
The analysis of CSF1R protein showed presence of 13 drug pockets that can be used for drug binding (Table 5).The protein-ligand interaction using Cannabidiol and Resveratrol as ligand showed its binding to drug pocket 0 of CSF1R with a volume of 1254.85Å 3 , surface area of 1374.39Å 2 , and drug score of 0.82.However, binding of Curcumin was observed away from the predicted drug pocket of CSF1R (Figure 4).Further, docking of CSF1R with Curcumin showed binding energy of -4.95 Kcal/Mol and an inhibition constant of value 236.48 µM.The 4 hydrogen bond between Arg579, Gly603, Gly605, and His655 residues of CSF1R with Curcumin has a distance of 3.07, 3.13, 2.56, and 2.96 Å, respectively.Similarly with Cannabidiol, the binding energy obtained is -4.75 Kcal/Mol, and the inhibition constant value of 328.22 µM.The Asp670 amino acid of CSF1R forms 2 hydrogen bonds with Cannabidiol with a distance between them are 3.23 and 2.69 Å, respectively and Leu588 amino acid forms a single hydrogen bond with a distance of 2.83 Å with Cannabidiol.The Resveratrol shows hydrogen binding with 4 amino acids (Lys616, Cys666, Gly669, and Phe797) of CSF1R with a distance of 3.18, 2.85, 3.53, 3.99 Å, respectively, binding energy obtained is -5.91 Kcal/Mol and inhibition constant value of 46.94 µM (Figure 5; Table 6).

MD simulation of CD40 and CSF1R-ligand complexes
It was observed that RMSD of CD40 in CD40-Cannabidiol complex showed equilibrium with average RMSD value of around 0.25 nm (Supplementary Figure 1A).The major oscillations in RMSD of Cannabidiol in CD40-Cannabidiol complex were observed at 0-50 ns.After 50 ns, RMSD of ligand was stabilized with an average value of approx.1.75 nm (Figure 1B) though major oscillations recurred during 70-75 ns (Figure 1B).The CD40-Cannabidiol complex showed minor uctuation between 0.2nm to 0.45nm during the entire simulation period (Figure 1C).It was observed that Rg of CD40 in the CD40-Cannabidiol complex did not show major uctuation with an average value of 1.53 nm (Figure 1D).

Discussion
Among analyzed natural products, Curcumin, Cannabidiol, and Resveratrol ful lled all 5 criteria of the Lipinski rule [30] which may prove as potential drug-like compounds.Observations also indicate that natural products Curcumin, Cannabidiol, and Resveratrol can be used as a drug target for microgliamediated neuroin ammation.They also obey ADMET (Absorption, distribution, metabolism, excretion, and toxicity) properties useful for early preclinical drug screening and development [24].
It was observed that the lower the binding energy between protein-ligand interactions, higher the a nity of a ligand for the protein where the presence of hydrogen bond and other hydrophobic interactions helps in forming good binding a nity [31].Further, the presence of amino acid residues Gly, Ser, His, Trp, Tyr, Arg, and Asp were more abundant at the ligand-binding site of a protein and were found involved in the formation of hydrogen bond, hydrophobic bond, and salt bridge interaction between protein and ligand [32].According to the DoGSiteScorer server for identi cation of drug pockets on protein, pocket with drug score of near to 1 and volume more than 500 Å are considered more likely to be perfect druggable site [25,33].Results are in agreement with other reports on inhibition of the protein 34-35 .
Curcumin and Cannabidiol have similar binding energy but the inhibition constant is lowest for Curcumin with CD40.Resveratrol forms the maximum hydrogen bond with CD40 amino acids and showed a positive high value of binding energy which indicates unfavorable interaction among them.The CD40, a 45 to 50 kDa type I membrane glycoprotein, gets upregulated under pro-in ammatory and HIV-infected brain [36][37].The Protein-ligand interaction study indicates that Curcumin and Cannabidiol could possibly bind to a druggable pocket of CD40 and may interfere with CD40-CD40L interactions, accelerating the expression of chemokines and entry of in ammatory cells in the CNS.
The interaction of natural products with colony-stimulating factor 1 receptor (CSF1R) would be useful to modulate survival, maintenance, proliferation, and activation of microglia or activated microglia-mediated functions in normal and disease conditions [38][39][40][41].The evaluation of results indicates that Resveratrol and Cannabidiol could be e cient in inhibiting the CSF1R mediated signaling which can further prevent microglial activation and even microglia-mediated demyelination in neurological diseases.Resveratrol showed the lowest inhibition constant value among all.Curcumin and Resveratrol form the maximum hydrogen bond with CSF1R amino acids followed by Cannabidiol but Curcumin does not show binding at the predicted druggable site.Resveratrol and Cannabidiol bind to the most likely druggable pocket as compared to other natural products.
Overall, values of RMSD, RMSF, Rg of protein-ligand complexes observed in permissible limit.MD simulation of CD40 and CSF1R-ligand complexes suggested that while the complex showed stable and consistent behavior of RMSD and Rg, uctuation at a particular region of protein helped to acquire the stable conformation during ligand binding.Our study indicates the possible involvement of Curcumin, Cannabidiol, and Resveratrol towards functions of CD40 and CSR1F on microglia (Figure 5).

Conclusion
Our nding suggests potential interaction between natural products and microglia-speci c proteins.Druggable pockets, binding energy, inhibition constant, and formation of stable hydrogen bonds between natural products and microglia proteins suggest that Curcumin, Cannabidiol, and Resveratrol can be used for targeting proteins involved in microglia activation during neuroin ammation and neurodegeneration.

Declarations Statements and Declarations
Funding: The authors declare that no funds, grants, or other support were received during the preparation of this manuscript Con ict of interest/Competing interests: The authors have no relevant nancial or non-nancial interests to disclose.

Figures Figure 1
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Table 1 :
Drug likeliness using Lipinski rule of 5 for natural products

Table 3 :
Summary of binding site prediction granularity: Properties and druggability for CD40