Human serum albumin-specific recognition of the natural herbal extract of Stryphnodendron polyphyllum through STD NMR, hyphenations and docking simulation studies

Abstract

Over the last two decades, new and more advanced strategies that help in the rapid screening and identification of new ligands for a specific macromolecule have become an important domain. From this viewpoint, the effectiveness of STD NMR, Tr-NOESY, and STD-TOCSY has been utilized to evaluate the binding potential of the natural extract of Stryphnodendron polyphyllum, used as a herbal medicine in Brazil, towards human serum albumin. Moreover, 1D-DOSY experiments have also been carried out for the discriminations of different molecular weight compounds present in this extract. Following the STD, Tr-NOESY, and TOCSY analysis, a hyphenated system comprising LC-SPE-NMR was utilized to see the complete structural assignments through 2D spectra. The combined results from NMR spectroscopy and separation methods provided myricetin-3-O-rhamnopyranoside (1), quercetin-3-O-glucopyranoside (2), quercetin-3-O-xylopyranoside (3), and quercetin-3-O-rhamnopyranoside (4) as the active site blockers. Moreover, epitope results and additional Tr-NOESY cross peaks suggested the presence of the flattened conformations of these ligands within the ligand-HSA complex through the edge protons. Similarly, STD competition studies with the ligand-HSA complex were demonstrated by varying the concentration of spy molecule that selectively binds with Sudlow's site II. Finally, docking simulations targeting bothSudlow sites (I and II) were performed, which interestingly mimic the STD competition results and showed that these compounds (1-4) are more prone towards binding site-1 inhibition. Therefore, we suggest that the sequence of techniques presented in this study can be considered as a simple and fast analytical tool for screening natural extracts to get better leads against any specific target.

Probing the interaction of a quercetin bioconjugate with Bcl‐2 in living human cancer cells with in‐cell NMR spectroscopy

“…Particularly noteworthy is the fact that new Tr‐NOE cross‐peaks were detected between protons H2′–H5′ and H2′–H8, due to protein binding, which were not present in the spectrum of the free 3′ quercetin‐alanine. This suggests that the flexibility of 3′ quercetin‐alanine was reduced, and the conformation was changed in the bound state . At this point it should be noted that several crystal structures of quercetin in complexation with protein receptors have shown that ring B may adopt such orientation that protons H–8 and H–2′ can be located adjacent due to the rotation of ring B around the C2–C1′ bond .…”

High Resolution NMR Spectroscopy as a Structural and Analytical Tool for Unsaturated Lipids in Solution

“…NMR studies have not been published using isotopic labeling of specific protein aminoacids with 13 C and 15 N to investigate the effect of pH on fatty acid binding. Furthermore, to the best of our knowledge, saturation transfer difference (STD) [ 237 , 238 , 239 ] and Tr-NOESY [ 240 , 241 ] experiments for unsaturated lipids have not been applied. These experiments would allow the rapid characterization of the binding epitope of the ligand closest to the receptor and the conformational changes upon ligand binding without the need of selectively enriched lipids.…”

Evaluation of binding competition and group epitopes of acetylcholinesterase inhibitors by STD NMR, Tr-NOESY, DOSY and molecular docking: an old approach but new findings

“…In addition, there is no restriction on the protein size. Based on the aforementioned facts, it is the most routinely used technique for ligand/inhibitor recognition processes; of the larger known and unknown molecular libraries, 31,32 for binding epitope mapping, 31,32 and for calculating dissociation constants 33 of weakly binding molecules where most of the other analytical methods are unable to provide satisfactory answers.…”