Histone Methyltransferase Assay Methods: What Works for HTS
Histone methyltransferases are proving much more difficult than kinases from an assay development standpoint. They are generally very slow enzymes, with turnovers in the range of less than 1 min-1 in many cases and they tend to have low Km values for SAM, many in the submicromolar range. These properties impose very high sensitivity requirements on assay methods, necessitating detection of low nanomolar levels of product under typical screening conditions (initial velocity conditions using Km concentrations of SAM.) Moreover, many HMTs function as complexes, with as many three or four proteins required for full activity, which results in high production costs – another driver for more sensitive detection methods.
All HMTs use S-adenosylmethionine (SAM or AdoMet), as a donor for methylation of lysine or arginine residues and generate S-adenosylhomocysteine (SAH) as a by-product. Detection of SAH is advantageous over detection of methylated products for a number of reasons, but not as sensitive with the current methods. Whereas kinases catalyze mono-phosphorylation, HMTs can add up to two methyl groups at arginines and up to three at lysines, resulting in a total of six possible methylation states. The products can be quantified non-selectively using radioassays (with 3H-SAM as donor) or separated and quantified using LC/MS. However neither of these approaches are accessible to many researchers, and immunodetection is more widely used for HTS. The diversity of methylated reaction products combined with variability in surrounding amino acids complicates development of immunochemical assay methods, as a single antibody generally does not recognize all of the products formed by a single HMT. Despite these drawbacks, immunoassays for methylated products have been formatted with a number of different HTS-friendly readouts, including ELISA, TR-FRET, and AlphaLISA (see table below.)
SAH detection avoids the complexities of detecting diverse methylation products and provides a universal assay method, which can reduce assay development costs by using a single set of assay reagents for all HMT targets. However, there are no commercially available reagents with sufficient selectivity for direct detection of SAH, which differs from SAM by just a single methyl group. Though BellBrook and others have developed antibodies capable of discriminating between SAH and SAM, technical and legal roadblocks have prevented their commercialization. The primary approach used in commercial assays is enzymatic conversion of SAH to a molecule that can be detected directly; i.e., enzyme-coupled assays. Though coupled enzyme assays are widely used for HTS because of their homogenous format, appropriate controls and/or counter screening are important to flag compounds that affect the coupling enzymes.
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There are several versions of the coupled enzyme assay (see table below). We developed our own version, in collaboration with Novartis, called the Transcreener EPIGEN Methyltransferase Assay that relies on detection of AMP using the Transcreener AMP Assay, and we have been collaborating with Reaction Biology to incorporate their purified HMT enzymes and substrates into turn-key methyltransferase assay kits. In another approach that has been commercialized, SAH is converted to homocysteine and adenosine using SAH hydrolase, and homocysteine is then detected using covalent reaction with a thiol-sensitive fluor. Alternatively, the adenine portion of SAH can be converted to urate by the sequential action of three coupling enzymes, with co-production of hydrogen peroxide in the final step. Hydrogen peroxide can be detected colorimetrically or fluorescently, via formation of resorufin. Recently, a luciferase-based coupled assay was developed that relies on the sequential conversion of SAH to adenine, AMP, and ATP, with the final step catalyzed by pyruvate phosphate dikinase.
The choice of assay method really depends on the specific goals and constraints of the screening program and the enzyme being used, and in many cases one approach may be used for primary screening and an orthogonal method used for hit confirmation and/or selectivity profiling. SAH detection is very simple to use and can be universally applied across all HMT targets, however sensitivity will become limiting in cases where very low SAM is used (less than 250nM) or with very slow enzymes. Detection of methylated products is generally more sensitive than the indirect SAH detection methods but requires the use of separate reagents for different HMTs, and suitable antibodies may not be available in all cases. Most important, the availability of an increasing number HMT assay methods suitable for HTS will facilitate epigenetics drug discovery programs.
| Representative HMT Assay Methods used for HTS | |||
| Product Detected | Assay method | Readout | Reference |
| SAH Detection (SAH) | Immunoassay – FPIA | FP | |
| SAH Detection (AMP) | 2 Coupling Enzymes, Immunoassay | FP | |
| SAH Detection (Homocysteine) | 1-2 Coupling Enzymes, Thiol-reactive fluor | FI | Anal Biochem 2005; 342: 86-92 |
| SAH Detection (H2O2) | 3 Coupling Enzymes, Resorufin | FI / Abs | |
| SAH Detection (ATP) | 3 Coupling Enzymes, Luciferase | Luminescent | |
| Methylated Products
| Flashplate, Filter-Based | Radioactive | |
| Methylated Product (Me-Arg) | Immunoassay – ELISA | Chemiluminescent | |
| Methylated Product (Me-Lys) | Immunoassay – Bead based | TR-FRET (LANCE), AlphaLISA | |
| Methylated Product (Me-Lys) | Immunoassay- Bead based | ALphaLISA | |
| Abbreviations used: FI – fluorescence intensity; FP – fluorescence polarization; FPIA – fluorescence polarization immunoassay; TR-FRET – time resolved Forster resonance energy transfer | |||
