A High Throughput Scintillation Proximity Imaging Assay for Protein Methyltransferases

Abstract

Protein methyltransferases (PMTs) orchestrate epigenetic modifications through post-translational methylation of various protein substrates including histones. Since dysregulation of this process is widely implicated in many cancers, it is of pertinent interest to screen inhibitors of PMTs, as they offer novel target-based opportunities to discover small molecules with potential chemotherapeutic use. We have thus developed an enzymatic screening strategy, which can be adapted to scintillation proximity imaging assay (SPIA) format, to identify these inhibitors. We took advantage of S-adenosyl-L-[3H-methyl]-methionine availability and monitored the enzymatically catalyzed [3H]-methyl addition on lysine residues of biotinylated peptide substrates. The radiolabeled peptides were subsequently captured by streptavidin coated SPA imaging PS beads. We applied this strategy to four PMTs: SET7/9, SET8, SETD2, and EuHMTase1, and optimized assay conditions to achieve Z′ values ranging from 0.48 to 0.91. The robust performance of this SPIA for the four PMTs was validated in a pilot screen of approximately 7,000 compounds. We identified 80 cumulative hits across the four targets. NF279, a suramin analogue found to specifically inhibit SET7/9 and SETD2 with IC50 values of 1.9 and 1.1 μM, respectively. Another identified compound, Merbromin, a topical antiseptic, was classified as a pan-active inhibitor of the four PMTs. These findings demonstrate that our proposed SPIA strategy is generic for multiple PMTs and can be successfully implemented to identify novel and specific inhibitors of PMTs. The specific PMT inhibitors may constitute a new class of anti-proliferative agents for potential therapeutic use.

Kinetic isotope effects reveal early transition state of protein lysine methyltransferase SET8

“…The quality of the peptide was confirmed by MALDI-MS. The two H4K20 peptides were shown previously to be active substrates of SET8 with the biotinylated peptide used in the present work unless specified (63). SeAM and isotopically labeled SAM/SeAM were prepared according to previous methods (SI Materials and Methods).…”

Rapid and Sensitive Method To Identify Mycobacterium avium subsp. paratuberculosis in Cow's Milk by DNA Methylase Genotyping

“…The biotinylated PCR products were bound to streptavidin-coated Flash plates (New England Nuclear, Boston, MA) and 3 Hmethyl groups were detected on a scintillation counter (Packard TopCount microplate scintillation and luminescence counter model B9904: Packard Instrument Company, Meriden, CT) at 2 min/sample. These plates use scintillation proximity assay (SPA) technology (47), which allows detection of only ␤-rays emitted close to the plastic.…”

A High-Content Biosensor-Based Screen Identifies Cell-Permeable Activators and Inhibitors of EGFR Function: Implications in Drug Discovery

“…9 A549 cells were purchased from ATCC (catalogue #CCL-185). H2030, H3255 and HCC4011 Non Small Cell Lung Cancer Cells (NSCLC) were obtained from Dr. Romel Somwar (The Varmus Lab, MSKCC).…”

“…The “killer mix” used as a low control in viability assays consists of a proprietary mixture of cytotoxic compounds as previously described. 9-11 For the luminescence ADP production kinase assay, HEPES, ß-glycerol phosphate, MgCl 2 , TCEP, Sodium orthovanadate, Poly(Glu,Tyr) peptide and Tween 80 were purchased from Sigma-Aldrich (Saint Louis, MO). ADP-Glo Kinase Assay Kit and ATP were purchased from Promega (Madison, WI).…”