Hydroxyl Groups on Annular Ring-B Dictate the Affinities of Flavonol–CCL2 Chemokine Binding Interactions

[Image: see text] Owing to the astounding biological properties, dietary plant flavonoids have received considerable attention toward developing unique supplementary food sources to prevent various ailments. Chemokines are chemotactic proteins involved in leukocyte trafficking through their interactions with G-protein-coupled receptors and cell surface glycosaminoglycans (GAGs). CCL2 chemokine, a foremost member of CC chemokines, is associated with the pathogenesis of various inflammatory infirmities, thus making the CCL2-Receptor (CCR2)/GAG axis a potential pharmacological target. The current study is designed to unravel the structural details of CCL2–flavonol interactions. Molecular interactions between flavonols (kaempferol, quercetin, and myricetin) with human/murine CCL2 orthologs and their monomeric/dimeric variants were systematically investigated using a combination of biophysical approaches. Fluorescence studies have unveiled that flavonols interact with CCL2 orthologs specifically but with differential affinities. The dissociation constants (K(d)) were in the range of 10(–5)–10(–7) μM. The NMR- and computational docking-based outcomes have strongly suggested that the flavonols interact with CCL2, comprising the N-terminal and β1- and β3-sheets. It has also been observed that the number of hydroxyl groups on the annular ring-B imposed a significant cumulative effect on the binding affinities of flavonols for CCL2 chemokine. Further, the binding surface of these flavonols to CCL2 orthologs was observed to be extensively overlapped with that of the receptor/GAG-binding surface, thus suggesting attenuation of CCL2-CCR2/GAG interactions in their presence. Considering the pivotal role of CCL2 during monocyte/macrophage trafficking and the immunomodulatory features of these flavonols, their direct interactions highlight the promising role of flavonols as nutraceuticals.