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, was 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.

Figure 1. Overview of the SPA-based HTS approach

(A) The ³H-methyl residue of [³H-Me]-SAM is enzymatically transferred to biotinylated PMT substrates; (B) The methylated reaction can proceed through two pathways: inactivated, in which the compound inhibits the reaction or active, in which the compound leads to no apparent effect. The peptide is then immobilized onto streptavidin-conjugated SPA beads. The proximity between β-particles and bead-coated scintillation fluid generates strong scintillation signal, which is suppressed in the inactive pathway.

Figure 1. Overview of the SPA-based HTS approach

(A) The ³H-methyl residue of [³H-Me]-SAM is enzymatically transferred to biotinylated PMT substrates; (B) The methylated reaction can proceed through two pathways: inactivated, in which the compound inhibits the reaction or active, in which the compound leads to no apparent effect. The peptide is then immobilized onto streptavidin-conjugated SPA beads. The proximity between β-particles and bead-coated scintillation fluid generates strong scintillation signal, which is suppressed in the inactive pathway.

Figure 2. Optimization of assay parameters

(A) Enzyme concentration. The assays were carried out for 16 h with the varied concentrations of (▲) SET7/9, (●) SET8, (■) SETD2 and (◆) EuHTMase1.The final concentrations of 150 nM for SET7/9, 1.5 μM for SET8, 250 nM for SETD2, and 10 nM for EuHMTase1 were chosen for HTS; (B) Reaction time. Using these final concentrations, the maximal conversions were reached in 3 h for SET7/9 (▲), 8 h for SET8 (●), 4 h for SETD2 (■), and 1 h for 10 nM EuHMTase1 (◆); (C) DMSO tolerance. The reactions were carried out with 150 nM SET7/9 for 3 h, 1.5 μM SET8 for 8h, 250 nM SETD2 for 4h, and 10 nM EuHMTase1 for 1 h in the presence of a varied amount of DMSO: without (white), 1% (gray), 2% (horizontal lines), 3% (diagonal lines), 5% (black). The four PMTs were tolerant up to 5% DMSO; (D) Enzyme stability. Experiments were performed with 150 nM SET7/9, 1.5 μM SET8, 250 nM SETD2, and 10 nM EuHMTase1. All enzymes were pre-incubated at ambient temperature for 0 (black), 6 (gray), and 16 h (diagonal lines) prior to the assay start. The unchanged reactivity indicated that the four PMTs are stably functional under our assay conditions for at least 16 h; (E) SPA readout under high and low controls. The reactions were carried out under the optimized assay conditions with either active PMTs (black) or HClO4-treated PMTs (white). Upon mixing with 0.2 mg SPA beads, the scintillation signals were recorded with LEADseeker^™ Multimodality Imaging System. The ratios of the high-to-low controls of 8 for SET7/9, 3 for SET8, 6 for SETD2, and 30 for EuHTMase1, display an excellent signal-to-noise separation for the present assay format (10 repeats for each data set). Each assay point was performed in triplicate (n=3), and SDs were plotted unless otherwise mentioned.

Figure 3. Assay robustness for chemical screening

(A) Repeated (n=3) 384-well plate high- to-low controls were performed for SET7/9, SET8, SETD2, and EuHTMase1. For the high controls, the wells contained 2 μl of 9:1 H2O/DMSO prior to the addition of PMTs, substrate and cofactor. In comparison, the wells of the low controls contained 2 μl of 100 mM HClO4. Lowering pH inactivated PMTs. (B) Assay for the four enzymes. SET7/9, SET8, SETD2 and EuHMTase1 were evaluated on percent coefficient of variation (%CV) and Z′ factor.

Figure 4. Scatter plot analysis of the duplicate values of percentage inhibition for each compound in the pilot screening for SET7/9, SET8, SETD2, and EuHMTase1

(A) Each validation plate contained high and low controls (n=6). The high controls contained 1% DMSO (v/v) and low controls included 10 mM HClO4. (B) The assay for the four enzymes SET7/9, SET8, SETD2, and EuHMTase1 were evaluated by percent coefficient of variation (%CV) and Z′ factor. (C) Libraries of 6,912 compounds for both SET7/9 (upper left) and SETD2 (lower left), and 5,632 for SET8 (upper right), and EuHMTase1 (lower right) were examined (n=2). The scatter plot compares our two data sets in order to examine the correlation between percent inhibitions for each compound. The upper-right region represents potential inhibitors (red) and the compounds that cloud the central region (black) indicate that the majority of the examined compounds are inert against the tested PMTs.

Figure 4. Scatter plot analysis of the duplicate values of percentage inhibition for each compound in the pilot screening for SET7/9, SET8, SETD2, and EuHMTase1

(A) Each validation plate contained high and low controls (n=6). The high controls contained 1% DMSO (v/v) and low controls included 10 mM HClO4. (B) The assay for the four enzymes SET7/9, SET8, SETD2, and EuHMTase1 were evaluated by percent coefficient of variation (%CV) and Z′ factor. (C) Libraries of 6,912 compounds for both SET7/9 (upper left) and SETD2 (lower left), and 5,632 for SET8 (upper right), and EuHMTase1 (lower right) were examined (n=2). The scatter plot compares our two data sets in order to examine the correlation between percent inhibitions for each compound. The upper-right region represents potential inhibitors (red) and the compounds that cloud the central region (black) indicate that the majority of the examined compounds are inert against the tested PMTs.

Figure 5. Identification of inhibitors (A) Heat map representation of inhibition of our chemical library against SET7/9, SET8, SETD2, and EuHMTase1

The scale ranges from yellow (no inhibition) to red (100% inhibition). (B) Overlap analysis of SET7/9, SET8, SETD2, and EuHMTase1. With a threshold of 30% inhibition, we identified 80 positives between the four assays: 5 positives for SET7/9, 18 positives for SET8, 67 positives for SETD2, and 5 positives for EuHMTase1. 24 compounds hit across multiple enzymes and 5 of them were active across all four enzymes, including biotin (twice) and SPA-quenching dyes (Gentian violet, Evans blue, and methylene blue). We discerned potential specific inhibitors through pilot screening: 9 compounds for SET7/9, 4 for SET8 and 43 for SETD2.

Figure 6. Dose-response curves against SET7/9 (▲), SET8 (●), SETD2 (■), and EuHMTase1 (◆)

(A) The IC50 values for Biotin in the SPIA-bead assay were 1.30 ± 0.04 μM for SET7/9, 1.7 ± 0.1 μM for SET8, 1.3 ± 0.2 μM for SETD2, and 1.33 ± 0.03 μM for EuHMTase1 (B) Using the filter-paper assay to determine IC50 values for Biotin no effect observed, as expected; (C) Merbromin showed IC50 values of 28 ± 1 μM for SET7/9, 4.2 ± 0.4 μM for SET8, 2.4 ± 0.1 μM for SETD2, and 43 ± 15 μM for EuHMTase1 using the filter-paper assay; (D) NF-279, the specific inhibitor against SET7/9 and SETD2, was also tested using the filter-paper assay. It showed IC50 values of 1.9 ± 0.2 μM for SET7/9 and 1.1 ± 0.2 μM for SETD2. Each assay point was performed in triplicate (n=3), and SD is plotted.