Epigenetics
Figure 19: Invitrogen’s BacMam-enabled cellular assay work flow. Cells are incubated with BacMam reagent encoding a GFP-fusion protein. 24 to 48 hours later, the cells are lysed in the presence of a terbium labelled anti-modification (such as methylation as shown) specific antibody. The level of modification on the GFP-fusion protein is determined via the LanthaScreen® TR-FRET readout
labelled Histone H3 modification specific antibod- ies. To identify local regions of the genome associ- ated with specific protein modifications, Invitrogen has developed the MAGnify™ ChIP System. Based upon magnetic beads, it provides a faster and more reliable method than traditional ChIP protocols and is highly amenable to analysis by Next Gen Sequencing (ChIP-Seq) (Figure 19).
Rapid and sensitive assays to probe the activity of enzymes involved in histone modification are essen- tial to perform high-throughput screenings aimed at finding hits that could provide a starting point to new drug discovery. PerkinElmer’s (
www.perkin
elmer.com) LANCE® Ultra and AlphaLISA® assay platforms are non-radioactive, homogeneous, anti-
Figure 20: Summary of available PerkinElmer AlphaLISA and LANCE Ultra reagents used to measure modifications on histone 3. All detection reagents have been well characterised for their specificity at detecting the modification of interest and validated using different enzyme classes depicted on the figure. More information on the specificity of the mark detected is available online and reagents for other histone modifications should be available shortly
52
body-based technologies commonly employed for such applications. Using these platforms, PerkinElmer recently launched a series of reagents to monitor epigenetic modifications elicited on four highly studied histone H3 marks, H3K4, H3K9, H3K27 and H3K36, by writer and erasers of the his- tone code. Technical notes are currently available that demonstrate the use of these reagents to measure the in vitro catalytic activities of histone acetyltrans- ferases and deacetylases, and also methyltransferases (HMT) and demethylases (Figure 1). The assays are performed in two simple steps: the enzymatic reac- tion, followed by product detection with either an anti-mark europium-labelled antibody (LANCE Ultra), or anti-mark AlphaLISA Acceptor Beads. Anti-mark antibodies were selected on the basis of their specificity against the modification of interest. Of note is that both AlphaLISA and LANCE Ultra assays require low peptide substrate and enzyme concentrations (in the nanomolar range), which is sufficient to generate robust and highly reproducible assay signals. This represents a significant advantage over alternative, less sensitive technologies that gen- erally require micromolar substrate and enzyme con- centrations, and greatly simplifies working near cofactor Km concentrations when applicable. PerkinElmer’s AlphaLISA reagents have also been shown to allow measurement of HMT activity using full length purified histone H3 protein, giving added flexibility to choose the most appropriate substrate. With the all-in-one-well format, absence of washes, and small number of assay steps, the LANCE Ultra and AlphaLISA platforms allow straightforward automation of epigenetic assays for screening (hit finding) and orthogonal testing (hit-to-lead) of his- tone modifying enzymes (Figure 20).
Drug Discovery World Spring 2011
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80