Optimization of High-Throughput Methyltransferase Assays for the Discovery of Small Molecule Inhibitors

Abstract

Methyltransferases (MTases) play diverse roles in cellular processes. Aberrant methylation levels have been implicated in many diseases, indicating the need for the identification and development of small molecule inhibitors for each MTase. Specific inhibitors can serve as probes to investigate the function and validate therapeutic potential for the respective MTase. High-throughput screening (HTS) is a powerful method to identify initial hits for further optimization. Here, we report the development of a fluorescence-based MTase assay and compare this format with the recently developed MTase-Glo luminescence assay for application in HTS. Using protein N-terminal methyltransferase 1 (NTMT1) as a model system, we miniaturized to 1536-well quantitative HTS format. Through a pilot screen of 1428 pharmacologically active compounds and subsequent validation, we discovered that MTase-Glo produced lower false positive rates than the fluorescence-based MTase assay. Nevertheless, both assays displayed robust performance along with low reagent requirements and can potentially be employed as general HTS formats for the discovery of inhibitors for any MTase.

Development of a continuous fluorescence-based assay for N-terminal acetyltransferase D

“…To design suitable assay conditions for characterizing NatD inhibitors, we determined the steady-state kinetic parameters of both substrates for NatD using a continuous fluorescence assay. 18,19 Histone H4 peptide substrate was designed based on the first eight amino acids from N-terminus of H4 protein, which was synthesized using standard Fmoc chemistry. For the fluorescence assay, the concentration of CoA formed during the acetylation reaction was derived from a standard calibration curve generated with CoA and ThioGlo4 ( Figure 2D).…”

“…A fluorescence assay was adapted to study the kinetics of NatD, which monitors the formation of a ThioGlo4-thiol adduct that exhibits a strong fluorescence at 465 nm. 18,19 The optimal ThioGlo4, and 10 µM AcCoA. 18,19 The inhibitors in DMSO were 3-fold serially diluted from 300 µM to 0.03 µM.…”

“…18,19 The optimal ThioGlo4, and 10 µM AcCoA. 18,19 The inhibitors in DMSO were 3-fold serially diluted from 300 µM to 0.03 µM. After 10 min incubation at 37 °C, reactions were initiated by adding 10 µM H4-8.…”

“…CoA production by forming a fluorescent adduct with the thiol fluorescent probe IV (ThioGlo4). 18 Moreover, we optimized this fluorescence-based assay in a 384-well microplate with a Z'-factor of 0.77 and a coefficient of variation (CV) of 6%, demonstrating its applicability for HTS.…”

Development of A Continuous Fluorescence-Based Assay for N-Terminal Acetyltransferase D

“…Herein, we report the development of a continuous fluorescence-based assay to quantify CoA production by forming a fluorescent adduct with the thiol fluorescent probe IV (ThioGlo4) [ 18 ]. Moreover, we optimized this fluorescence-based assay in a 384-well microplate with a Z′-factor of 0.77 and a coefficient of variation (CV) of 6%, demonstrating its applicability for HTS.…”