Integrated multi-spectroscopic and molecular simulation approaches to decipher inhibition mechanism of late-stage cross-linking glycation by the privileged scaffold quercetin from Celosia argentea

Abstract

Deciphering protein cross-linking glycation networks is crucial for understanding the mechanism of diabetic complications at the molecular level. An array of multi-spectroscopic biophysical studies and computational tools revealed quercetin, which is a privileged flavonoid scaffold derived from Celosia argentea herb, for its cross-link antiglycation potential. Quercetin essentially impeded hyperglycemia-induced late glycation (IC₅₀ 55 µM), involving proteinase-resistant cross-linked aggregates in a dose- and time-dependent manner. The site probe displacement study provided insights into the binding of quercetin to serum protein at glycation site I of subdomain IIA that induced overall conformational alteration, as evident from the CD experiment. The micro-environment around the glycation site was assessed based on synchronous, 3D fluorescence, red edge excitation shift and TCSPC, which indicated an excited state interaction in a dynamically constrained micro-environment. The suppression of cross-linked aggregates by quercetin was evaluated using ANS, OPA, ThT, gel electrophoresis, and MALDI analyses. DLS studies revealed an alteration in the hydrodynamic size distribution and zeta potential from gBSA to gBSA–quercetin system. All the spectroscopic studies indicated an innate molecular mechanism of quercetin binding, primarily through hydrogen bonds. The multi-spectroscopic outcomes, including the glycation site selection, mode of micro-environment binding and excited state exploration, dipolar relaxation, misfolding mechanism, and quercetin atom-to-amino acid residue interactions, were comprehensively corroborated with molecular docking and dynamic simulation studies. This study deciphers the inhibition mechanism of cross-linking glycation by quercetin and paves the way for the development of flavonoid scaffold-based AGE therapeutics.