APPLICATION NOTE
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of action for G9a could be an important step to treat cancer patients with such
phenotypes
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. To detect and then quantify the interactions between G9s and
the fragments, G9a was labeled with a red fluorescent probe and assays were
performed in 384-well microplates using the Dianthus NT.23PicoDuo system.
Results
For the single-dose assay, a library of 2,490 fragment containing molecules
ranging in size from ~40 Da to ~1 kDa, was screened. Each fragment (ligand)
was tested at a final concentration of 1 mM, while the target, G9a, was used at
a final concentration of 5 nM. Each ligand dilution was prepared and tested in
duplicate. S-adenosyl-L-methionine (SAM), a G9a natural ligand, was used as a
positive control at regular intervals during the screen at a final concentration of
100 µM. Data was analyzed with NanoTemper's DI.Screening Analysis so ware.
Figure 1 shows the signal amplitude obtained for each ligand and each
replicate of the positive control SAM (y-axis) plotted against the chronological
order in which they were measured (x-axis). The area of insignificant ΔFnorm
(pink shaded area) corresponds to three standard deviations from the signal
recorded from all DMSO references. Ligands with signals that fall within this
area are categorized as non-binders.
Screening a chemical library of such a broad variety of molecular masses,
reaching from ions to more complex organic molecules, requires a method
capable of detecting binding events independent of molecular mass and
binding site. Dianthus, in comparison to other biophysical technologies, does
this, making it an ideal tool for fragment based lead discovery.
