Acylated flavonol monorhamnosides from Eriobotrya japonica as XIAP BIR3 inhibitors revealed by in silico and HPLC-SPE-NMR techniques

Abstract

Targeting the X-linked inhibitor of apoptosis proteins (XIAP), baculoviral IAP repeat (BIR) 3 groove represents an innovative strategy for increasing the sensitivity of resistant tumour cells to conventional chemotherapeutic drugs [1]. Using a previously generated pharmacophore model [2], 3D models of 122 reported constituents from the leaves of the medicinal plant Eriobotrya japonica Lindl. (Rosaceae) were virtually screened against this target. Based on the predicted hits, we focused on acylated flavonol monorhamnosides (AFMR) as promising phytochemical class. AFMRs identified in the crude methanol extract by LC-MS and enriched by chromatographic methods, showed chemosensitizing potential in combination with etoposide in XIAP-overexpressing Jurkat cells (Nicoletti assay [3]). Application of the HPLC-SPE-NMR hyphenated technique enabled the structure elucidation of two new AFMRs and three known ones. By means of preparative HPLC, one of the main constituents of the AFMR mixture, the virtual hit kaempferol-3-O-α-L-(2″,4″-di-E-p-coumaroyl)-rhamnoside (1), was isolated. Compound 1 sensitizes XIAP-overexpressing Jurkat cells towards the treatment with etoposide (Nicoletti assay), binding to the XIAP BIR3 groove with a dose-dependent affinity (IC50 7.69µM, fluorescence polarization based binding assay [4]). In accordance with the predicted structural requirements within the binding site, its substructures, kaempferol-3-O-α-L-rhamnoside and kaempferol, were inactive (IC50>100µM, resp.) revealing a major impact of the acid and sugar moieties of 1 on bioactivity. In conclusion, this study elucidates 1 as natural, small-molecular weight inhibitor of the XIAP BIR3 groove using a combination of in silico and HPLC-SPE-NMR techniques.