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| | Inhibition of Histone Demethylase Activity |
| Description: | Inhibition of LSD activity can be determined by a variety of both in vitro and in vivo methods known to one skilled in the art. For example, enzymatic activity can be determined in in vitro enzyme assay systems. In various embodiments, the enzymatic activity of LSD1 can be determined in a spectrophometric assay. Briefly, the assay is based on the multistep enzymatic reaction in which LSD1 first produces H2O2 during the demethylation of lysine 4 on a peptide corresponding to the first 21 amino acids of the N-terminal tail of histone H3. In the presence of horseradish peroxidase, the H2O2 produced reacts with ADHP to produce the highly fluorescent compound resorufin that can be analyzed with an excitation wavelength of 530-540 nm and an emission wavelength of 585-595 nm. The assay requires a source of LSD1 enzyme, either purified from natural sources (e.g. a tissue or cultured cells), isolated as a recombinantly expressed protein, or as a unpurified protein in whole cell extracts. In one embodiment, the disclosed compounds exhibit inhibition of LSD protein activity with an IC50 in an EMSA assay of less than about 300 mM, less than about 100 mM, less than about 50 mM, less than about 10 mM, less than about 1 mM, less than about 500 nM, or of less than about 100 nM. In another embodiment, the disclosed compounds exhibit inhibition of LSD1 protein activity with an IC50 in an EMSA assay of less than about 300 mM, less than about 100 mM, less than about 50 mM, less than about 10 mM, less than about 1 mM, less than about 500 nM, or of less than about 100 nM. In another embodiment, the disclosed compounds exhibit inhibition of LSD2 protein activity with an IC50 in an EMSA assay of less than about 300 mM, less than about 100 mM, less than about 50 mM, less than about 10 mM, less than about 1 mM, less than about 500 nM, or of less than about 100 nM. |
| Affinity data for this assay | |
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