Elucidating the active interaction mechanism of phytochemicals withanolide and withanoside derivatives with human serum albumin

Abstract

Withania somnifera (Ashwagandha) is an efficient medicinal plant known in Ayurveda and Chinese medicine since ancient times, whose extracts are consumed orally as food supplement or as a health tonic owing to its several restorative properties for various CNS disorders, inflammation, tumour, stress, rheumatism etc. In this study, we have analyzed the binding interaction of four derivatives of Withania somnifera (Withanolide A, Withanolide B, Withanoside IV and Withanoside V) with HSA because of their important pharmacological properties. To unravel the binding between derivatives of Withania somnifera and HSA, fluorescence spectroscopy was used. Binding studies were further studied by molecular docking and dynamics and results confirmed greater stability upon binding of derivatives with HSA. Circular dichroism data illustrated change in the secondary structure of protein upon interaction with these derivatives, particularly the helical structure was increased and β-sheets and random coils were decreased. Furthermore, morphological and topological changes were observed using AFM and TEM upon binding of ligands with HSA indicating that HSA-withnoside/withanolide complexes were formed. All the results cumulatively demonstrate strong binding of withanosides and withanolides derivatives with serum albumin, which should further be explored to study the pharmacokinetics and pharmacodynamics of these derivatives.

Fig 1

The structure of withanolide A (C₂₈H₃₈O₆-470.6Da), withanolide B (C₂₈H₃₈O₅-454.6Da), withanoside IV (C₄₀H₆₂O₁₅-782.9Da) and withanoside V (C₄₀H₆₂O₁₄-766.9Da).

Fig 2. Fluorescence spectroscopic studies of HSA with withanolide and withanoside molecules, indicating the interaction of the drug with plasma protein.

The association constant (K_S) and free energy change along with stern-volmer plots showing fluorescence quenching constant (kq) and plot of Fo/F against [Q] at λex = 285 nm and λem = 360 nm for (A) withanolide A, (B) withanolide B, (C) withanoside IV and (D) withanoside V.

Fig 3. Site displacement studies using site-specific markers; Phenylbutazone (PHEB) was used as marker for HSA domain IIA (Sudlow site I).

Fluorescence spectroscopic studies performed using HSA and Phenylbutazone at equal concentrations (1μM) and drugs with increasing concentrations (1μM ~ 9μM) (A) HSA-PHEB-withanolide A (B) HSA-PHEB-withanolide B (C) HSA-PHEB-withanoside IV and (D) HSA-PHEB-withanoside V.

Fig 4. Circular Dichroism studies of the free HSA and HSA–drug complexes.

The free HSA and HSA–drug complexes in aqueous solution with a protein concentration fixed at 1μM and with increasing drug concentrations at 2,4, and 6 μM. (A) withanolide A (B) withanolide B (C) withanoside IV and (D) withanoside V.

Fig 5. Atomic Force Microscopic (AFM) studies to visualize alteration in HSA molecule topology in presence of withanolide and withanoside derivatives at 10 μM resolution.

(A) Only HSA (B) HSA+withanolide A (C) HSA+withanolide B (D) HSA+withanolide IV and (E) HSA+withanolide V.

Fig 6. Transmission Electron Microscopic (TEM) studies to visualize alteration in HSA molecule topology in presence of withanolide and withanoside derivatives at 200 nM resolution.

(A) Only HSA (B) HSA+withanolide A, (C) HSA+withanolide B, (D) HSA+withanolide IV and (E) HSA+withanolide V.

Fig 7

Molecular docking studies between HSA and withanolide A, withanolide B, withanoside IV and withanoside V showed that the minimum binding energy conformer is very close to the experimentally determined values. (A, D, G, J) Cartoon model of HSA showing withanolide derivatives A, B, IV and V docked in the binding pocket using Autodock 4.2. (B, E, H, K) Pymol generated images showing withanolide A, withanolide B, withanoside IV and withanoside V binding in their specific binding site of HSA. The cavity of hydrophobic and hydrophilic amino acid residues surrounding the probe. (C, F, I, L) Ligplot showing the hydrophobic interactions of HSA with withanolide derivatives.

Fig 8

(A-D)-Time evolution of the radius of gyration (Rg) during 10 ns of MD simulation of unliganded HSA and HSA–drug derivatives complexes. (E-H)-Plot of RMSD values for unliganded HSA and HSA–drug derivatives complexes. (I-L)- Comparison of the RMSF of Calcium atoms along the sequence derived from the 10 ns simulations.