Compound (SMILES): CON(C)C[C@@H](C)Oc1nc(N2C[C@@H]3C[C@H]2CN3)c2cc(C3CC3)c(-c3c(C)c(F)cc4[nH]ncc34)c(OCc3ccc(C4=CN([C@H](C(=O)N5C[C@H](O)CC5C(=O)N[C@@H](CO)c5ccc(-c6[se]cnc6C)cc5)C(C)C)NN4)cc3)c2n1
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): KRAS (UniProt P01116)
POI sequence: MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEKTPGCVKIKKCIIM
E3 ligase: VHL (UniProt P40337)
Ligase sequence: MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPRPVLRSVNSREPSQVIFCNRSPRVVLPVWLNFDGEPQPYPTLPPGTGRRIHSYRGHLWLFRDAGTHDGLLVNQTELFVPSLNVDGQPIFANITLPVYTLKERCLQVVRSLVKPENYRRLDIVRSLYEDLEDHPNVQKDLERLTQERIAHQRMGD
Cell line: AsPC-1 (Homo sapiens)
Cell line description: African=0.97%; Native American=0%; East Asian, North=3.23%; East Asian, South=0%; South Asian=0%; European, North=58.23%; European, South=37.57%.
TP53 mutation indicated incorrectly as being at c.818G>A in PubMed=1630814.
Gene deletion; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC
A*01 A*01
Stable (MSS) (Sanger).
Metastatic; Ascites; 
Caucasian.
Cancer cell line
DC50:  26.8 nM
Assay description: RPMI-1640 medium was purchased from GIbco, penicillin/streptomycin antibiotics were purchased from Vicente, and fetal bovine serum was purchased from Biosera. Protein primary and secondary antibodies were purchased from Cell Signaling Technology. AsPC-1/A375 and other cell lines were purchased from ATCC, and 4% paraformaldehyde and Triton-100 were purchased from Bio-Tech. Envision multi-label analyzer (PerkinElmer).
Experimental methods:
The cells were seeded in ultra-low attachment 96-well black-walled plates, with 80 μL of cell suspension per well, containing 100,000 cells. The cell plates were placed in a carbon dioxide incubator for overnight culture.
The compound to be tested was diluted 5-fold into 8 concentrations using a shotgun, i.e., from 2mM to 25.6nM, and a double-well experiment was set up. 78μL of culture medium was added to the middle plate, and then 2μL of the gradient diluted compound per well was transferred to the middle plate according to the corresponding position. After mixing, 20μL of each well was transferred to the cell plate. The concentration range of the compound transferred to the cell plate was 10μM to 0.128nM. After the cell plate was cultured in a carbon dioxide incubator for 24h, the cells were removed, the culture medium was discarded, and the cells were fixed with 4% PFA fixative for 30min, washed 3 times with PBS, permeabilized with 0.1% Triton for 20min, washed 3 times with PBS, blocked with 0.1% BSA solution at room temperature for 1h, incubated with protein primary antibody at 4°C overnight, washed 3 times with PBST, incubated with fluorescent secondary antibody at room temperature for 2h, washed 3 times with PBST,
Data Analysis:
Calculate the FITC/Cy5 in the sample well based on the measured fluorescence intensity
FITC/Cy5=(FITCsample-FITCblank)/(FITCsample-FITCblank)
The inhibition % under compound treatment was calculated as [1-(FITC/Cy5comp.)/(FITC/Cy5DMSO)]*100. The DC 50 value can be obtained by four-parameter curve fitting (obtained by "log(inhibitor)vs.response--Variable slope" mode in GraphPad Prism).
Reference: COMPOUND HAVING QUINAZOLINE STRUCTURE AND USE THEREOF (patent)
Source database: TPDdb