Compound (SMILES): N#Cc1ccc(CN2CCC(F)(CNC3CC3c3ccccc3)CC2)cc1
InChIKey: GPEDVCLQHOVQKX-UHFFFAOYSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: LSD1 In Vitro Activity Assay: Screening method: lysine-specific demethylase 1 (LSD1) activity screeningInstrument: microplate reader Envision (PerkinElmer, USA).MATERIALS: Human recombinant LSD1, the LSD1 protein fragment fused with GST (aa158-end) was expressed and purified by E. coli expression system by the laboratory in house.LSD1 Activity Detection Kit LANCE Ultra LSD1 Histone H3-Lysine 4 Demethylase Assay was purchased from Perkin Elmer;The H3 polypeptide substrate ARTK(me1)QTARKSTGGKAPRKQLA-GG-K(Biotin)-NH2 was synthesized by Jill Biochemical Company.Principle: LSD1 specifically removes the methylation modification at the K4 lysine on the H3 polypeptide substrate, making it a substrate without methylation modification. The method employs a histone H3 methylated polypeptide (1-24) as a substrate to introduce a biotin label in the C segment of the substrate. LSD1 initiates the reaction with the participation of FAD to remve the methylation modification on the substrate H3K4. The Eu-labeled H3K4 background antibody binds to the substrate by antigen-antibody reaction, while the streptavidin-labeled receptor is bounded by the specific interaction of streptavidin and biotin. This allows the Eu-labeled donor to interact with the streptavidin-labeled receptor. In fluorescence resonance energy transfer, when two fluorophores are brought close due to biomolecular interaction, part of the energy captured by the cryptate at the time of excitation will be released, the emission wavelength of which is 620 nm; the other part of the energy is transferred to the receptor (acceptor), the emission wavelength of which is 665 nm. The 665 nm emission is only produced by FRET caused by the donor. Therefore, when biomolecules interact, there are two excitation lights at 620 nm and 665 nm; when there is no interaction, there is only one excitation light at 620 nm. The LSD1 demethylation activity was reflected by detecting the ratio of the fluorescence signals at the two emission wavelengths of 665 nm and 620 nm. Meanwhile, a blank control was set to determine the strength of the enzyme activity. ORY-1001 and GSK-2879552 were employed as positive inhibitors in the experiment.Sample processing: Samples were dissolved in DMSO, stored at low temperature, and the concentration of DMSO in the final system was controlled within a range that won't affect the activity of the assay.The activity of the sample was tested by primary screening at a single concentration, for example 20 μM. For samples exhibiting activity under certain conditions, for example, the inhibition rate (% Inhibition) being greater than 50, the active dose-dependent relationship, i.e., the IC50 value, was obtained by nonlinearly fitting the sample activity vs sample concentration, the software used for the calculation was Graphpad Prism 5, the model used for fitting was sigmoidal dose-response (variable slope), and for most inhibitor screening models, the bottom and top of the fitted curve were set to 0 and 100.
Measurement: IC50 = 94 nM (pChEMBL 7.03000020980835)
Descriptors: MW 363.48, ALogP 4.01, HBA 3, HBD 1, PSA 39.06, QED 0.84