In-silico comparison of cytochrome P450 inhibitory and dopaminergic activity of Piperine, Curcumin and Capsaicin

Abstract Psychiatric disorders are a heterogeneous group of mental disorders that manifest as abnormal mental or behavioral habits that cause the individual discomfort or disability. Dopamine imbalance plays a major role in many psychiatric disorders. Piperine, Curcumin and Capsaicin are CYP P450 3A4 & 2D6 inhibitors. The objective of this study is to determine the dopaminergic activity of Piperine, Curcumin and Capsaicin and also to compare cytochrome P450 3A4 and 2D6 inhibition activity by in-silico methods. In this in-silico study, we utilised compounds such as Piperine, Curcumin and Capsaicin were subjected to Lipinski’s rule of five, and ligands were also evaluated for toxicity profile and ADMET properties. Furthermore, the ligands were performed in docking studies. All three compounds were docked with three different targeted proteins (PDB IDs: 4D7D, 4WNW and 6LUQ). According to the docking result, Piperine has higher binding energy(-8.55 kcal/mol)(-8.1 kcal/mol)(-8.57 kcal/mol) when compared with Curcumin(-7.39 kcal/mol)(-5.61 kcal/mol)(-6.57 kcal/mol) and Capsaicin (-6.86 kcal/mol)(-6.57 kcal/mol)(-5.42 kcal/mol) and also with standard drug (-8.61 kcal/mol)(-7.65 kcal/mol)(-6.16 kcal/mol). The present study concluded that the bioactive compound Piperine has a better inhibitory activity of CYP 3A4, 2D6 enzymes and dopamine D2 receptor among the three compounds and also with the standard drug thioridazine. Graphical Abstract


Introduction
Dopamine, a neurotransmitter, is essential for brain activities ranging from pleasure and inspiration to motor control. Dopamine imbalances can result in various disorders, including ADHD, Schizophrenia, Parkinson's disease and addiction (Ju arez Olgu ın et al. 2016). In India, epidemiological studies show that the prevalence of psychiatric diseases ranges from 9.5 to 370/1000 population. These differences in mental disorder prevalence are not limited to Indian studies, but may also be found in global studies. The recent data from Indian studies suggest that one or more psychiatric diseases affect around 20% of the adult population in the community (Math and Srinivasaraju 2010). Herbal medicine is frequently utilised around the world to treat a variety of health problems (Ashour et al. 2017). Piperine, Curcumin, and Capsaicin are CYP 3A4 and 2D6 inhibitor. Inhibiting CYP450 enzymes has been found to reduce the metabolism of CYP450 substrates drugs and, as a result, change their pharmacokinetic profiles (Sabarathinam et al. 2021). The CYP450 enzyme system is widely known for its role in the metabolism of several drugs utilised in clinical practice (Rendic and Di Carlo 1997). CYP450 enzymes that are typically involved in drug metabolism include CYP1A2, 2C9, 2C19, 2D6, and 3A4. CYP3A4 is not only the most common CYP enzyme in the liver, but it is also used for metabolism and elimination by more than half of all medications on the market (Huang et al. 2007). The present study was intended to compare the CYP3A4 & 2D6 inhibition and dopaminergic activity of phytoconstituents such as Piperine, Curcumin, and Capsaicin by in-silico predictions like molecular modelling, ADMET studies, and toxicity prediction.

Results and discussion
In dopaminergic system, the ligands regulate the secretion of dopamine due to this mechanism it can be used for various psychiatric disorders with anti-psychiatric drugs. However, there is no in-silico evidence that directly compares the inhibition activity of Piperine, Curcumin and Capsaicin. Computational techniques are utilised to discover and optimise biologically effective compounds and optimising the molecular structure of lead compounds concerning the ADMET method has now become an essential aspect of the existing drug process. The free online computer programs such as molinspiration cheminformatics, OSIRIS property explorer and admetSAR were used in this study to evaluate the molecular properties, bioactivity score, toxicity and ADMET properties of Piperine, Curcumin and Capsaicin. Previously, Chembiodraw ultra was used to build the ligand structures of Piperine, Curcumin, and Capsaicin.

Drug-likeness prediction studies
The chemical components of all studied molecules were calculated with molinspiration cheminformatics, and the results are shown in Table S1. Lipinski's RO5 was utilised to assess the drug-likeness of all these compounds, which deals with four simple physiochemical parameters (Veber et al. 2002). The log p values of Piperine, Curcumin and Capsaicin in this in-silico study were found to be between 2.30 and 3.33 (within acceptable range 5). Piperine, Curcumin and Capsaicin have a molecular weight within the range 500. In this study, Piperine has a low molecular weight when compared to the other two compounds. In our study, it shows that Piperine (03) has a less rotatable bond than Curcumin (08) and Curcumin has a less rotatable bond than Capsaicin (09). None of the compounds present in this study violated Lipinski's criteria, making them potentially useful drug-like molecules.

Bioactivity score
The bioactivity score for selected compounds was calculated by molinspiration cheminformatics software and the results are shown in Table S2. The bioactivity score describes information about a binding cascade of compounds with various protein structures. It is commonly known that if the bioactivity score is greater than zero, the compounds exhibit greater biological activity (Shashank Shekhar et al. 2018). The bioactive data shows that the compounds in this study were active as enzyme inhibitors. Piperine and Capsaicin are active as G-Protein coupled receptor (GPCR) ligands, and Curcumin and Capsaicin are active as nuclear ligand receptor.

Toxicity
The toxicity of all compounds was determined using the Osiris property explorer and is shown in Table S3. This software can also compute drug-likeness and drug scores. The compounds involved in this present study represent green which means do not have any toxicity like mutagenicity, tumorigenic, irritant, and reproductive effects which could help in developing non-toxic and effective compounds. The non-toxic compounds are then utilised in the molecular docking investigations.

ADMET properties
In-silico ADMET analysis is a rapid way to identify if a molecule has adequate pharmacokinetics and pharmacodynamics properties. In the present study, the bioavailability and toxicity risks for Piperine, Curcumin and Capsaicin were predicted. Further analysis exhibited CYP inhibitory promiscuity as Piperine (0.813), Curcumin (0.571) and Capsaicin (0.755) inhibited two cytochromes, including CYP450-3A4 and CYP450-2D6. The penetration through Blood Brain Barrier (BBB), compounds such as Piperine (0.996) and Capsaicin (0.621) cross the BBB, but Curcumin (0.616) does not cross the BBB. When it comes to P-glycoprotein inhibition, Piperine (0.821) and Capsaicin (0.785) are not inhibitors of p-glycoprotein except Curcumin (0.682). The compounds in this present study were noncarcinogenic and Piperine and Curcumin showed no AMES toxicity except Capsaicin. Piperine and Curcumin were found to be better ADMET properties when compared with Capsaicin (Table S4). In the biological system, poor pharmacokinetics and toxicity lead to drug development failure. Using an ADMET profile throughout drug discovery can exclude unsuitable molecules and also exhibits and plays a vital role in decreasing costs and effort. The activity of CYP450 enzymes is important since they are responsible for drug metabolism in biological systems and clearance from the body (Lagorce et al. 2017).

Docking
Molecular docking was performed on selected active constituents such as Piperine, Curcumin, and Capsaicin for cytochrome P450 3A4 activity on the binding site of (PDB: 4D7D) and cytochrome 2D6 activity on the binding site of (PDB: 4WNW) to find the inhibition activity of cytochrome 3A4 and 2D6. Then all three ligands were docked with the target protein (PDB: 6LUQ) to find the inhibition activity of dopamine D2 receptor. After the above optimisation, all three compounds were found to be nontoxic and they were docked with the target protein and rated based on the docking score. The AutodockV R tool version 1.5.6 was used to calculate the docking score. The results of docking calculation in terms of binding affinity (kcal/mol) of Piperine, Curcumin, and Capsaicin are displayed in Table S5. The Cytochrome P450 3A4 is bound to an inhibitor with the PDB ID: 4D7D. CYP3A4 plays a vital role in clinically associated xenobiotic metabolising enzymes in human beings. CYP3A4 has a huge and suitable active site that fixes with the wide variety of small molecules in which it will inhibit the metabolism. The binding energy for Piperine (-8.55 kcal/mol), Curcumin (-7.39 kcal/mol), and Capsaicin (-6.86 kcal/mol) [AQ]. The human cytochrome P450 2D6 thioridazine complex with the PDB ID:4WNW. The co-crystal structure was bounded by thioridazine complex, where the protonated piperidine nucleus of thioridazine forms stabilised hydrogen bond complexes at the active site. The docking score reveals Piperine(-8.1 kcal/mol), Curcumin(-5.61 kcal/mol), and Capsaicin(-6.57 kcal/mol) [AQ]. The ligands were docked with 6LUQ protein where the co-crystal ligand haloperidol bound D2 dopamine receptor inspired discovery of subtype-selective ligands. The dopamine receptors are most well-known therapeutic targets for neurological disorders. The D2 receptors activate Gai/o protein to inhibit cAMP levels which will modulate certain ion channels. DRD2 (dopamine receptor D2) is one of the well-established targets in psychiatry and neurology, where the selected inhibitors against D2 receptors showed the binding energy for Piperine (-8.57 kcal/mol), Curcumin (-6.57 kcal/mol) and Capsaicin (-5.42 kcal/mol). The interaction analysis of three docked compounds is examined in further detail. In the 4D7D protein, Piperine does not have hydrogenbond interaction, Curcumin is characterised by two hydrogen bonds with ARG105, PHE108, and Capsaicin with LYS208, TYR307. In 4WNW protein, Piperine with ARG101, LEU302, Curcumin with PHE427, GLN350, and Capsaicin with CYS443[AQ]. In 6LUQ protein with THR119, VAL115, Curcumin, and Capsaicin do not have hydrogen bond interaction ( Figures S5-S13). According to the above result, Piperine has better activity when compared with the other two ligands. Then Piperine was validated with the standard drug thioridazine in the targeted protein.
In 4D7D protein, it shows that binding energy for thioridazine (-8.61 kcal/mol). Then the standard drug was docked with 4WNW and 6LUQ protein, which shows that (-7.65 kcal/mol) and (-6.16 kcal/mol). From the above result, we concluded that Piperine has better activity.

Experimental
All experimental procedures are described in the supplementary material section.

Conclusion
The current study concludes that Piperine is a better inhibitor of all three targeted proteins when compared to Curcumin and Capsaicin. In ADMET properties, Capsaicin showed AMES toxicity. Piperine has shown better inhibitory activity on CYP3A4, CYP2D6 and dopamine D2 receptor when compared with Curcumin and Capsaicin. To support these encouraging results, we planned for an in-vitro study for the experimental validation of our findings.