Elucidating the binding interaction of andrographolide with the plasma proteins: biophysical and computational approach

Abstract

The present study focuses on the interactions of andrographolide (ANDR) with plasma proteins, human serum albumin (HSA), and α-1-acid glycoprotein (AGP), and their biological importance. To understand the pharmacological role of ANDR, its anticancer activity was studied on a breast cancer cell line (MCF-7); it showed a dose-dependent inhibition of growth, and its IC₅₀ value was found to be 55 μM. Furthermore, to evaluate the binding mechanism of AGP and HSA with andrographolide, fluorescence emission quenching was observed as a static mechanism upon the binding of ANDR to plasma proteins. Additionally, active HSA sensor chip surfaces were prepared through an amine-coupling reaction protocol, and the equilibrium association constants for ANDR–HSA were then determined by surface plasmon resonance (SPR) analysis. The association constants of ANDR binding to HSA, obtained with fluorescence and SPR, were K_A(AGD) = 1.85 ± 0.02 × 10⁴ M⁻¹ and 3.1 ± 0.04 × 10³ M⁻¹, respectively. Similarly, the ANDR binding affinity with AGP was analyzed through fluorescence and SPR, and the calculated binding association values were 1.5 ± 0.01 × 10³ M⁻¹ and 1.3 ± 0.04 × 10³ M⁻¹, respectively. Molecular displacement and in silico docking shows that ANDR binds to subdomain IIB. Consequently, circular dichroism analysis showed that there is partial perturbation in the structure of HSA upon an increase in the concentration of ANDR. Moreover, molecular dynamics simulation revealed that the stability of the HSA–ANDR complexes reached an equilibration state at around 3000 ps, which clearly indicates the rigidity and stability of the HSA–ANDR complexes. Thus, our results provide evidence that both plasma proteins (HSA and AGP) can act as carrier proteins for ANDR.