Synthesis and characterization of a SIRT6 open tubular column: Predicting deacetylation activity using frontal chromatography

Abstract

SIRT6 is a histone deacetylase that has been proposed as a potential therapeutic target for metabolic disorders and the prevention of age-associated diseases. Thus the identification of compounds that modulate SIRT6 activity could be of great therapeutic importance. We have previously reported on the identification of quercetin and vitexin as SIRT6 inhibitors, using SIRT6-coated magnetic beads. In this study, we have immobilized SIRT6 onto the surface of an open tubular capillary and characterized the quercetin binding site using frontal displacement chromatography. Structurally related flavonoids were tested for their activity on SIRT6, including apigenin, naringenin, luteolin and kaempferol. In addition to obtaining their binding activity using frontal affinity chromatographic techniques, we also ranked the compounds based on their ability to displace quercetin. The data suggest that a single displacement curve is representative of the enzymatic activity of the tested ligand. In addition, using the inhibition data obtained in this study, we developed a preliminary pharmacophore model that confirmed the experimental data.

N‐Acylethanolamines Bind to SIRT6

“…We have previously demonstrated that a series of flavanoids had significant inhibitory effects on the deacetylation activity of SIRT6 [13] , as a result, quercetin and luteolin were used as positive controls. Both quercetin and luteolin inhibited SIRT6 deacetylase activity in a concentration-dependent manner (Table 1), with an IC 50 value of 24 μM and 4.1 μM, respectively, consistent with previous reported values [13a, 17] . Further, they both inhibited SIRT6 deacetylation activity to a similar extent (38 and 30%, respectively).…”

“…Interestingly, while quercetin and luteolin both elicited a dose-dependent inhibition of SIRT6 deacetylation activity as previously reported [17] , they also displayed significant increase (6-10 fold) of SIRT6 deacetylation activity at higher doses. The identification of NAEs and the flavonoids as activators of SIRT6 expands the multiple roles of SIRT6.…”

“…The biological activity of endogenous fatty acid ethanolamides, N-acylethanolamines (NAEs), has recently been demonstrated to be mediated by PPAR-α [16-18a] and PPAR-γ [17] as well as through other targets. For example, anandamide bound to and induced PPAR-γ gene expression [14] , while oleoylethanolamide bound to and increased the transcriptional activity of PPAR-α [15] .…”

Development and characterization of the α3β4α5 nicotinic receptor cellular membrane affinity chromatography column and its application for on line screening of plant extracts

“…The extract was prepared by HerbPharm as previously described [12]. T. foenum-graecum seed was extracted by maceration for 3 weeks at 1:2.5 ratio at 72% ethanol, ratio expressed as mass raw plant material ( T. foenum-graecum seed) in weight (g) per volume (mL) of extraction solvent.…”

Cellular membrane affinity chromatography (CMAC) in drug discovery from complex natural matrices

“…Cytosolic proteins are usually covalently bonded using their N-or C-terminus groups forming the covalent bond with chemical moieties on the surface of the assay scaffold, e.g. the inside of the column or magnetic beads [9,10]. Many natural compounds target cell membrane proteins but the solutions developed for cytosolic proteins cannot be used to immobilize transmembrane proteins [7].…”