June 15, 2014 (Vol. 34, No. 12)
Rebecca LaRose Global Product Manager Cisbio
Thomas Roux R&D Project Manager Cisbio
A Universal, Adaptable Assay for Substrates, Enzymes, Inhibitors, and SAM Concentrations
Epigenetics is one of the fastest growing fields in research because of its potential to identify therapeutic targets in oncology, immune and inflammatory diseases, cardiovascular disease, and personalized medicine. There are close to 500 epigenetic proteins that could be studied. These proteins are often referred to as writers, erasers, and readers.
The writers and erasers are the enzymes that add or remove the epigenetic modifications that lead to changes in gene expression. The writers are enzymes that methylate, acetylate, and phosphorylate substrates. The readers do as their name suggests: They read the modifications made by the writers. Examples of readers, also known as binding domains, are bromodomains, chromodomains, MBT domains, and tudor domains.
Until a few years ago, most epigenetic research was focused on histone deacetylases, more commonly known as HDACs. In the past five years, the focus has shifted quickly, and the evaluation of methyltransferase has became a high priority for scientists. These enzymes play a key role in modifying a large number of substrates such as histones and other proteins (e.g., p53), DNA, RNA, and small molecules making them key regulators of epigenetic states and, consequently, of gene expression.
The abundance of methyltransferases, their activity in effecting gene expression, and their potential in reversing modifications are key factors driving scientists to further understand their role and how they could be potential therapeutic targets.
The study of these important enzymes is proving to be challenging, however, because of their generally slow activity, environmental requirements such as buffers and pH balances, and variability in the substrates they modify as well as the number of modifications they make. All of these factors have made it difficult to develop universal assay approaches.
Methyltransferases are naturally slow acting enzymes and their reactions are known to take a significant period of time, making biologically relevant assays extremely difficult to develop. Several market surveys have reported that scientists are struggling to further their research and to understand these targets because manufacturers are not currently offering assays that meet their needs.
Until recently, scientists had been using a combination of various methods to study these enzymes such as ELISA, mass spec, Westerns, radioactivity, biomarker readouts, PCR, and several other technology platforms because there was not one platform besides radioactivity that scientists could use to screen all the methyltransferases.
The EPIgeneous Methyltransferase Assay from Cisbio was introduced to meet scientists’ needs and it is now being quickly adopted by scientists.
This mix-and-read assay uses HTRF technology to provide high sensitivity, flexibility, and is universal with regard to substrates, SAM concentrations, enzymatic buffers, and methyltransferases. This assay, which directly quantifies the methyltransferase reaction product S-adenosylhomocysteine (SAH), has been successfully validated on a variety of enzymes including G9a, SET8, NSD2, SET7/9, PRMT1, SETD2, DOT1L, and DNMT1. Its use has also been successfully demonstrated with all substrates (peptides, histones, nucleosomes, RNA, DNA, small molecules, and other proteins such as p53), making it a truly universal assay.
Figure 1 shows an example of the development and optimization of DOT1L assay with the EPIgeneous Methyltransferase Assay.
Detection of DOT1L specific activity and identification of optimal enzyme concentration was achieved with human recombinant DOT1L, serially diluted to the indicated concentrations and the assay carried out with 10 ng/μL (= 77 nM) oligonucleosome as substrate and 2 μM SAM for 2 h at 30°C. The negative controls (no SAM or no nucleosome) show the measurement of the enzymatic-specific activity. A DOT1L concentration of 4.5 nM (EC80) was selected for further experiments. This concentration leads to 10% conversion of SAM into SAH.
Determination of optimal substrate and SAM concentrations was performed using oligonucleosome, which was titrated with several concentrations of SAM. DOT1L is used at 4.5 nM and incubated with SAM and substrate 2 h at 30°C. 0.5 μM of SAM, a concentration below reported Km of 0.67 μM (1), is selected for subsequent experiments. For oligonucleosome, 77 nM (EC80) is selected for further tests.
The assay was validated by measuring the activity of the SGC0946 inhibitor (Figure 2). The validation was performed using 0.5 μM SAM (EC40), 77 nM oligonucleosome (EC80), and 4.5 nM DOT1L (EC80). The enzymatic reaction was stopped with the detection reagents after a 2 h incubation at 30°C. IC50 of SGC0946 is in good agreement with the literature (Kd = 0.06 nM in Yu et al. Nature Comm, 2012). As expected, BIX01294, which is a G9a selective inhibitor, does not inhibit DOT1L. Controls of inhibitors without the enzyme show that they do not affect the detection reagents.
The assay robustness is demonstrated through Z’ factor determination. This part of the assay was performed using 0.5 μM SAM (EC40), 77 nM oligonucleosome (EC80), and 4.5 nM DOT1L (EC80). The Z’ factor was obtained with biological balanced conditions and underlies the robustness of the assay and its suitability for HTS in biological relevant conditions.
In a recent study performed in collaboration with AFMB (UMR7257 CNRS–Aix-Marseille University), the EPIgeneous Methyltransferase assay was compared side-by-side with radioactivity. The reference inhibitor titrations were performed on human N7 & West Nile 2’O methyltransferases using 1 µM SAM/2 µM RNA. The HTRF assay was then read on the Pherastar Flashlamp (BMG Labtech) and the results are shown in Figure 3. The experiments indicate a good correlation in inhibitor potencies (IC50) between HTRF and radioactive reference assays.
These studies demonstrate that scientists now have a universal approach to studying these challenging epigenetic proteins without compromising sensitivity and also reducing the need for radioactivity. The assay is universal and adaptable to meet the specific needs required to study each methyltransferase. It works for all substrates regardless of the location and number of modifications without the need for site-specific antibodies.
The range of SAM the assay is compatible with is 0.4–200 µM (S/B > 2 with 10% turnover). The assay is very robust with a S/B range of 2– (turnover @ 5–30%) and has a Z’ range of 0.57–0.78 (EC80 enzyme, SAM & substrate). Another key feature of the methyltransferase assay is the enzymatic buffer flexibility (HEPES, Tris, PO4, MgCl2, NaCl, DTT, Tween, EDTA, DMSO, Glycerol) because each enzyme is tricky to work with and requires optimal conditions.