Doug Auld, Ph.D. Novartis Institutes for BioMedical Research

Researchers propose a SPA strategy that can be successfully implemented to identify novel and specific inhibitors of PMTs.

ASSAY & Drug Development Technologies offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a “Literature Search and Review” column that identifies published papers of note and discusses their importance. GEN presents one article that was analyzed in the “Literature Search and Review” column, a paper published in Combinatorial Chemistry & High Throughput Screening titled “A high throughput scintillation proximity imaging assay for protein methyltransferases.” Authors of the paper are Ibáñez G, Shum D, Blum G, Bhinder B, Radu C, Antczak C, Luo M, and Djaballah H.

Abstract from Combinatorial Chemistry & High Throughput Screening

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.


Scintillation proximity assays (SPAs) have been applied to adapt separation-based assays—for example, those in which substrate and products need to be separated—to assays that are suitable for high-throughput screening (HTS) using a simple “mix-and-read” format. In SPAs, a radiolabeled component is used that gives rise to a signal only when bound to beads that are impregnated with scintillate. SPAs have been widely applied to measure G-protein receptor–ligand binding, phosphorylation of peptide or protein substrates by kinases, as well as the quantification of a number of enzymatic products (for a review see Glickman et al., Assay Drug Dev Technol 2008;6:433–455.)

Non–separation-based heterogeneous assays that are not radioactive, such as AlphaScreen, in which donor and acceptor beads are brought into sufficient proximity to generate a chemiluminescent signal, have become increasingly popular. However, SPA remains an attractive assay format for HTS, particularly if the assay can be configured in a generic format in which a large target class can be addressed. This article describes a generic assay for protein methyl transferases (PMTs) using SPAs.

The PMT class of enzymes uses S-adenosyl-methionine (SAM) to transfer a methyl group to either lysine or arginine groups of proteins. The present article employs biotinylated peptides that are methylated with a [3H-Me]-SAM cofactor and streptavidin-coated SPA beads (see Figure). When histone H3 is used as a common substrate, the SPA format provides a generic read-out for PMTs. Assays for four PMTs (SET7/9, SET8, SETD2, and EuHMTase1) were developed using this approach.

The assay parameters were set using a three-step procedure. First, enzyme concentration, incubation time, dimethylsulfoxide tolerance, and stability were set in a standard filter-binding assay using a biotinylated-H3 substrate and [3H-Me]-SAM. Next, these conditions were applied to a heterogeneous assay format using streptavidin-coated sepharose beads from which the free label was removed by washing. Enzyme activity was then measured with standard scintillation counting. In the last step, the sepharose beads were replaced with SPA beads emitting a red chemiluminescence signal suitable for imaging on the LEADSeeker™ platform (imaging-based, SPIA).

The filter-based assay also served as a counter-screen for SPIA-related interference by compounds; for example, biotin mimetics that appear as inhibitors in SPIA due to competition with the streptavidin beads. Following optimization of the assay parameters, pilot library testing showed good performance in all four PMT assays. The use of a single detection technology allowed for rapid identification of inhibitors that are specific for a PMT. Although other assays have been described using specific antibodies to the methylated peptide product or detection of product S-adenosyl-L-homocysteine (SAH) by mass spectrometry, the SPA format described in this work provides a robust mix-and-read generic assay format for PMTs.

Figure. 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 ß-particles and bead-coated scintillation fluid generates strong scintillation signal, which is suppressed in the inactive pathway.

Doug Auld, Ph.D., is affiliated with the Novartis Institutes for BioMedical Research.

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