Compound (SMILES): C=C1CN2[C@H](COC(=O)NCC34CC(c5cn([C@H](C(=O)N6C[C@H](O)C[C@H]6C(=O)N[C@@H](C)c6ccc(-c7scnc7C)cc6)C(C)C)nn5)(C3)C4)CC[C@@]2(COc2nc(N3CCC[C@@](C)(O)C3)c3cnc(-c4cc(O)cc5ccc(F)c(CC)c45)cc3n2)C1
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:  23 nM
Assay description: 1) Observation under a microscope showed that the cells were in good condition, in the logarithmic growth phase, and the cell density was 80-90%.
2) Digest the cells with trypsin for 1-2 minutes, terminate the digestion with 5 mL of fresh culture medium, gently blow the cells to make a single cell suspension, and centrifuge at 1000 rpm for 3 min.
3) Add fresh culture medium to resuspend the cells, aspirate 10 μL of cell fluid, stain with trypan blue, and count the cell fluid density using a cell counter.
4) Prepare cell solution with fresh culture medium and adjust the cell density to 2.78×10? cells/mL (5×10? cells/180 μL). Add the cell solution to a 96-well black plate with a density of 180 μL/well and incubate overnight at 37°C in a 5% CO? incubator.
Preparation of drug-dosing compounds: Take 20 μL of 10 mM compound stock solution, add 30 μL DMSO, dilute to 4 mM, and use this as the highest concentration, dilute four times in a gradient to obtain 9 concentrations of compounds; then transfer 5 μL to 195 μL culture medium, mix well, and obtain the compound working solution; and transfer 20 μL of the working solution into the cells.
Experimental preparation: Place the frozen aliquots of 4% paraformaldehyde at room temperature to melt.
Cell fixation: 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.
During this period, PBS was used to prepare 0.5% Triton X-100 punching solution and 0.5% Tween 20 washing solution, the fixative was discarded, and PBS containing 0.5% Tween 20 was used to wash on a shaker for 5 minutes/4 times.
Punching: Fix with the above 0.5% Triton X-100 150 μL/well for 20 min.
Discard the punching solution and wash with PBS containing 0.5% Tween 20 on a shaker for 5 min/4 times.
Blocking: 150 μL/well blocking solution at room temperature for 1.5 h.
Primary antibody: 50 μL/well, incubate at 4°C overnight or at room temperature for 2 hr (blocking solution was added to the last column as background; KRAS G12D -1:200, GAPDH -1:400).
Discard the primary antibody and wash with PBS containing 0.5% Tween 20 on a shaker for 5 min/4 times.
Secondary antibody: 50 μL/well, incubate at room temperature for 1.5 h (anti-Rb 800:1/500; anti-Ms 680:1/500).
Discard the secondary antibody, wash with PBS containing 0.5% Tween 20 on a shaker for 5 min/4 times, invert and centrifuge at 1000 rpm for 1 min, and dry at room temperature for 3 hours before loading on the machine.
The measured Relative ICW800 signal values were imported into Prism software for fitting and calculation of DC50.
Reference: COMPOUND TARGETING PAN-KRAS PROTEIN DEGRADATION AGENT AND USE THEREOF (patent)
Source database: TPDdb