A Fluorescence‐Based Supramolecular Tandem Assay for Monitoring Lysine Methyltransferase Activity in Homogeneous Solution

Abstract

The demand for practical and convenient enzyme assays for histone lysine methyltransferases (HKMTs) emerges along with the rapid development of this young class of enzymes. A supramolecular reporter pair composed of p-sulfonatocalix[4]arene (CX4) and the fluorescent dye lucigenin (LCG) has been used to monitor enzymatic trimethylation of lysine residues in peptide substrates. The assay affords a switch-ON fluorescence response and operates in a continuous, real-time, and label-free fashion. The underlying working principle relies on the higher affinity of the macrocycle towards the trimethylated product of the enzymatic reaction as compared to the substrate, which allows the assay to be carried out in the product-selective mode. The final product incorporates a trimethylammonium moiety, a known high-affinity binding motif for CX4. Two substrates corresponding to the H3 N-terminal tail, namely, S2 (RTKQTARKSTGGKAP) and S6 (QTARKSTGGS), were selected as model compounds for methylation with the Neurospora crassa Dim-5 enzyme and investigated by the newly developed supramolecular tandem HKMTs assay. Only the longer substrate S2 underwent methylation in solution. The potential of the assay for inhibitor screening was demonstrated by means of inhibition studies with 1,10-phenanthroline to afford an inhibition constant of (70±20) μM.

Inhibition of histone binding by supramolecular hosts

“…This assay was adapted from an indicator displacement assay [22,23]. LCG (lucigenin) dye was purchased from Invitrogen.…”

Site-Selective Sensing of Histone Methylation Enzyme Activity via an Arrayed Supramolecular Tandem Assay

“…A number of enzymatic targets have been monitored using small molecule FDAs, 9 such as cholinesterases, 9a decarboxylases, 9b–d protease, 9e,f histone deacetylase, 9f,g and lysine methyltransferase. 10 …”

“…13 These techniques have not yet been coupled to an enzyme assay, though. While monitoring KMT activity is possible with a CB-based tandem assay, 10 site-selective activity monitoring was not shown. Effective in vitro assays of methylation enzyme activity require both site-selective molecular recognition and an optical readout of the KMT or KDM activity.…”

Fluorogenic sensor platform for the histone code using receptors from dynamic combinatorial libraries

“…Current research has shown the application of single sensor IDAs to the study of enzymatic reactions, such as diamine oxidase 38 and lysine methyltransferase enzymes. 39 However, these assays typically rely on the design and optimization of a single receptor with sufficient discrimination between substrate and product to be effective. 38 A multiple sensor array, could be ‘trained’ to sense the product of the reaction and be rapidly applied to enzymatic assays, even with complex substrates bearing multiple modifications, as we would expect in studying the histone code.…”