Compound (SMILES): C#Cc1c(F)ccc2cc(O)cc(-c3ncc4c(N5CC6CCC(C5)N6)nc(OCC5(CN6CCC(Cc7ccc(C(=O)N[C@H](C(=O)N8C[C@H](O)C[C@H]8C(=O)NCc8ccc(-c9scnc9C)cc8)C(C)(C)C)cc7)CC6)CC5)nc4c3F)c12
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): KRAS (UniProt P01116)
POI sequence: MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEKTPGCVKIKKCIIM
E3 ligase: VHL (UniProt P40337)
Ligase sequence: MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPRPVLRSVNSREPSQVIFCNRSPRVVLPVWLNFDGEPQPYPTLPPGTGRRIHSYRGHLWLFRDAGTHDGLLVNQTELFVPSLNVDGQPIFANITLPVYTLKERCLQVVRSLVKPENYRRLDIVRSLYEDLEDHPNVQKDLERLTQERIAHQRMGD
Cell line: AGS (Homo sapiens)
Cell line description: African=3.44%; Native American=32.25%; East Asian, North=5.38%; East Asian, South=0%; South Asian=0.23%; European, North=27.58%; European, South=31.12%.
Persistently infected with parainfluenza virus type 5 (PIV5) strain PIV5-AGS.
Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC
A*02 A*02
Stable (MSS) (Sanger).
In situ; Stomach; 
Caucasian.
Cancer cell line
Assay: Western Blot
DC50:  55 nM
Assay description: In this experiment, the ability of compounds to degrade KRAS G12D was detected by intracellular immunoblotting. The AGS cells in good growth condition and in the logarithmic growth phase were plated in a 96-well transparent bottom black plate, so that the number of cells in each well was 5×10?; cultured overnight at 37°C, 5% CO?.
On the second day, the compound was diluted to the corresponding concentration and administered so that the final concentration of the compound was 20000, 5000, 1250, 312.5, 78.1, 19.5, 4.9, 1.2, 0 nM (the final concentration of DMSO was 0.5%; the tenth column added DMSO of the corresponding concentration as the background well), and the plate after the addition of the drug was placed at 37°C, 5% CO? for 24 hours.
After the incubation time is over, discard the culture medium, gently add the prepared 4% paraformaldehyde into the plate along the well wall (150 μL/well), and fix at room temperature for 20 minutes; discard the fixation solution, wash with phosphate buffer solution containing 0.5% Tween 20 on a shaker for 5 minutes/4 times; then fix with 0.5% Triton X-100 150 μL/well for 20 minutes; Wash for 5 min/4 times; add 150 μL/well blocking solution to block at room temperature for 1.5 h; discard the blocking solution, and add 50 μL primary antibody diluent (KRASG12D-1:200, GAPDH-1:400, background well plus blocking solution), incubate at 4°C overnight or at room temperature for 2 hr.
After the primary antibody incubation, discard the primary antibody, wash with phosphate buffer containing 0.5% Tween 20 on a shaker for 5 min/4 times; add 50 μL of secondary antibody dilution (Rb secondary antibody 800:1/500; Ms secondary antibody 680:1/500), and incubate at room temperature for 1.5 hr; Centrifuge at 1000 rpm/1 min, and put it on the machine (if time permits, place the plate upside down in a dark place for a day, and the reading results will be more accurate the next day). Import the measured Relative ICW800 signal value into Prism software for fitting and calculation of DC50.
Reference: KRAS G12D DEGRADATION AGENT AND MEDICAL USE THEREOF (patent)
Source database: TPDdb