Compound (SMILES): COc1ncccc1-c1cn(CCCN)c2ccc(CN3CCC(CN4CCN(c5ccc6c(c5)C(=O)N(C5CCC(=O)NC5=O)C6=O)CC4)CC3)cc12
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): KRAS (UniProt P01116)
POI sequence: MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEKTPGCVKIKKCIIM
E3 ligase: CRBN (UniProt Q96SW2)
Ligase sequence: MAGEGDQQDAAHNMGNHLPLLPAESEEEDEMEVEDQDSKEAKKPNIINFDTSLPTSHTYLGADMEEFHGRTLHDDDSCQVIPVLPQVMMILIPGQTLPLQLFHPQEVSMVRNLIQKDRTFAVLAYSNVQEREAQFGTTAEIYAYREEQDFGIEIVKVKAIGRQRFKVLELRTQSDGIQQAKVQILPECVLPSTMSAVQLESLNKCQIFPSKPVSREDQCSYKWWQKYQKRKFHCANLTSWPRWLYSLYDAETLMDRIKKQLREWDENLKDDSLPSNPIDFSYRVAACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTSLCCKQCQETEITTKNEIFSLSLCGPMAAYVNPHGYVHETLTVYKACNLNLIGRPSTEHSWFPGYAWTVAQCKICASHIGWKFTATKKDMSPQKFWGLTRSALLPTIPDTEDEISPDKVILCL
Cell line: MDA-MB-231 (Homo sapiens)
Cell line description: African=1.95%; Native American=0%; East Asian, North=2.44%; East Asian, South=0%; South Asian=7.42%; European, North=28.16%; European, South=60.03%.
Gene deletion; HGNC; HGNC Gene deletion; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC
A*02/28; B*07,08; C*w02,w06. A*02 A*02 A*02
Stable (MSS).
Metastatic; Pleural effusion; 
Caucasian.
Triple negative breast cancer (TNBC) cell line.
Cancer cell line
Assay: Western Blot
DC50: > 10000 nM
Assay description: Western blot to evaluate the cellular potencies of RAS-PROTACs in KRAS protein degradation. For western blot experiments, BT-474 (KRASWT) and MIA PaCa-2 (KRASG12C) cells were cultured in DMEM medium with 10% FBS. BxPC3 (KRASWT) and KLM-1 (KRASG12D) cells were cultured in DMEM medium with 10% FBS. MDA-MB-231 (KRASG13D) cells were cultured in L15 medium with 10% FBS. BT-474, MIA PaCa-2, BxPC3 and MDA-MB-231 were established from the American Type Culture Collection. KLM-1 were established from the ExPASy.

On the assay day, two hundred thousand cells were pretreated with each of the test compounds for 24 hours. After 24 hours, the whole cell lysate was harvested by adding 2x SDS Sample Buffer. Proteins were separated by SDS-PAGE electrophoresis and transferred to PVDF membrane. Protein expression was detected using immunoblot with various primary antibodies and secondary antibodies following standard protocols. Antibody against KRAS was purchased from Abcam (Cambridge, UK). Anti-rabbit IgG, HRP-linked secondary antibodies was purchased from Cell Signaling Technology (Danvers, MA). Antibody against actin was purchased from Millipore (Burlington, MA). Immunoblots were revealed by chemiluminescence (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Fisher, Waltham, MA) and detected by ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA). Band intensities of western blot were also quantified by ChemiDoc MP Imaging System. Relative intensities of bands corresponding to the drug treatment group were compared to those of the untreated group.

The primary screening of bifunctional compounds 1-21 degraded KRAS protein in Calu-1, H358, H441, KLM-1, MDA-MB-231 and HCT-116 cell line were as shown in Tables 2 which tested under the conditions described above.
Reference: COMPOUNDS FOR MUTANT KRAS PROTEIN DEGRADATION AND USES THEREOF (patent)
Source database: TPDdb