Epigenetics
Figure 5: Assay technologies that have proven useful in epigenetic enzyme assays
Fluorimetric
(Fluorescent Intensity) TR-FRET (HTRF)
ELISA (eg with chemiluminescent readout)
FP (Fluoresence Polarisation) LabChip separation HCS Methods/Imaging Mass-Spec (Label Free) AlphaScreen/AlphaLISA Radiometric
Target Gene Readouts Colorimetric
0% © HTStec 2011
Epigenetic enzyme classes/proteins of greatest interest
Figure 6: Substrates most commonly used for epigenetic enzyme assays
Surrogate substrates Core Histone Peptides
Histone Octamers Substrate Independent Nucleosomes
Other © HTStec 2011 6%
0% 10% 20% 30% 40% 50% 60% % Responding
55% 34% 28% 26%
13% 13%
The epigenetic enzyme classes/proteins ranked of greatest interest to respondents were deacetylation – histone deacetylases (HDAC & Sirtuins). This was followed closely by histone methylation – his- tone methyltransferases (HKMT & HRMT), and then demethylation – histone demethylases (HDM). Ranked of least interest was dephospho- rylation – phosphatases in relation to histones (Figure 4).
Assay formats proven most useful for epigenetic screening
Figure 7: Most important challenges in epigenetic enzyme assay development for HTS
Specificity
Reproducibility/ Signal Window
Purity Activity
Sourcing raw materials 3.02
1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 RANKED ORDER of importance, where 1 = least or no important and 5 = greatest importance
© HTStec 2011
3.61 3.63
3.36 4.00
The assay formats/detection technologies that have proven most useful to date in epigenetic enzyme assays by respondents were fluorimetric (fluores- cent intensity), followed by TR-FRET (HTRF), ELISA and then radiometric. Least useful to date were target gene readouts and colorimetric assays (Figure 5).
Epigenetic enzymes assay substrates The identification and selection of appropriate sub- strates for epigenetic enzyme assays is subject to varying opinion and investigation. The survey showed respondents most commonly used peptides as substrates for their epigenetic enzyme assays (55% using). This was followed by core histone (34% using); surrogate substrates (28% using) and then nucleosomes (26% using) (Figure 6).
Main challenges to epigenetic enzyme assay development
A lot of effort is currently going into development of new epigenetic assays. Specificity was ranked as
42 Drug Discovery World Spring 2011
10% 10%
5% 10% 15% 20% 25% 30% 35% 40% % Responding
25%
20% 20%
17% 14% 12% 27% 32% 36%
will remain challenging until the biology and screening tools catch up with the hype’ with 41% responding. Only 8% of respondents had more negative perceptions about epigenetic screening (Figure 2).
Therapeutic areas targeting epigenetic enzyme assays
The majority (72%) of respondents were targeting, using or planning to use epigenetic assays within the oncology therapeutic area. Second greatest use/planned use of epigenetic assays was within the metabolic disease/diabetes therapeutic area (38% targeting). This was followed by CNS/neurodegen- eration/pain therapeutic area (30% targeting), inflammatory disease/autoimmune therapeutic area (29% targeting) and then cardiovascular ther- apeutic area (28% targeting) (Figure 3).
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