Compound (SMILES): CCc1c(F)ccc2cc(O)cc(-c3ncc4c(N5CCOC[C@@](C)(O)C5)nc(OCC5(CN6CCC(CN7CCN(c8ccc9c(c8)CN([C@H]8CCC(=O)NC8=O)C9=O)CC7)CC6)CC5)nc4c3F)c12
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): KRAS (UniProt P01116)
POI sequence: MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEKTPGCVKIKKCIIM
E3 ligase: CRBN (UniProt Q96SW2)
Ligase sequence: MAGEGDQQDAAHNMGNHLPLLPAESEEEDEMEVEDQDSKEAKKPNIINFDTSLPTSHTYLGADMEEFHGRTLHDDDSCQVIPVLPQVMMILIPGQTLPLQLFHPQEVSMVRNLIQKDRTFAVLAYSNVQEREAQFGTTAEIYAYREEQDFGIEIVKVKAIGRQRFKVLELRTQSDGIQQAKVQILPECVLPSTMSAVQLESLNKCQIFPSKPVSREDQCSYKWWQKYQKRKFHCANLTSWPRWLYSLYDAETLMDRIKKQLREWDENLKDDSLPSNPIDFSYRVAACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTSLCCKQCQETEITTKNEIFSLSLCGPMAAYVNPHGYVHETLTVYKACNLNLIGRPSTEHSWFPGYAWTVAQCKICASHIGWKFTATKKDMSPQKFWGLTRSALLPTIPDTEDEISPDKVILCL
Cell line: SW620 (Homo sapiens)
Cell line description: African=2.02%; Native American=0%; East Asian, North=4.3%; East Asian, South=0%; South Asian=0%; European, North=50.01%; European, South=43.67%.
Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC
A*02 A*02,24; B*07,15; C*07 A*02
Stable (MSS).
Metastatic; Lymph node; 
Caucasian.
Cancer cell line
Assay: Western Blot
DC50:  2 nM
Assay description: Example 63: KRAS degradation in cells carrying endogenous KRAS mutants or wildtype (WT) KRAS and KRAS cells growth inhibition. SW620 (KRAS G12V homozygous), AsPC1 (KRAS G12D homozygous), MIA PaCa2 (KRAS G12C homozygous), A549 (KRAS G12S homozygous) and MCF7 (KRAS WT) cells were obtained from American Type Culture Collection (ATCC). Cells were plated in 24-well plates in the RPMI1640 growth medium containing 10% FBS and 1% Penicillin Streptomycin at the density of 1.5×105 (SW620, AsPC1, A549, MCF7) or 2x105 (MIA PACa2) cells/well, and then incubated at 37°C overnight. The following day, the test compound was administered to the cells by using 1000x compound stock solution prepared in DMSO at various concentrations. After administration of the compound, the cells were then incubated at 37°C for 24 hours. Upon completion, the cells were washed with PBS and protein was collected in Laemmli sample buffer (1x). Proteins in cell lysate were separated by SDS-PAGE and transferred to Odyssey nitrocellulose membranes with iblot dry blotting transfer system. Nonspecific binding was blocked by incubating membranes with Intercept Blocking Buffer for 1 hour at room temperature with gentle shaking. The membranes were then incubated overnight at 4 °C with Primary antibodies rabbit anti-KRAS (1:1,000) and mouse anti- GAPDH (1:5,000) diluted in Intercept Blocking Buffer containing 0.1% Tween 20. After washing 3 times with TBS-T, the membranes were incubated with IRDye 800CW goat anti-rabbit IgG (1:20,000) and IRDye 680RD goat anti-mouse IgG (1:20,000) for 1 hour. After TBS-T washes, membranes were rinsed in TBS and Western blot images were acquired by Odyssey CLx.
The results are shown in FIGs.1-5, which illustrate the pan-KRAS degradative activity of exemplary compound 1 of the present disclosure in cancer cell lines with various KRAS forms 24 hours after administration.
The signal intensity of target proteins was calculated using ImageStudio software. KRAS fluorescent intensity (FLU) from DMSO (vehicle) treatment was set as 100%. Relative KRAS expression level was determined by the following formula:
KRAS expression (%) = "FLUcompound" /"FLUDMSO" ×100 [00824] The percentage of KRAS expression was calculated using Microsoft Excel. The half- maximal degradation concentration (DC50) values were generated by GraphPad Prism (Version 9) using the dose-response equation of variable slope (four parameters).
The results in SW620 cells are shown in Table 3, which summarizes KRAS G12V degradation DC50 of exemplary compounds.
3D cell growth inhibition of SW620 cells carrying endogenous KRAS G12V mutation SW620 cells were obtained from American Type Culture Collection (ATCC) and maintained in the RPMI1640 growth medium containing 10% FBS and 1% Penicillin Streptomycin. For cell growth assay, SW620 cells were seeded in round bottom ultra-low attachment 96-well plates at 3000 cells/well in 90 μL of DMEM growth medium containing 10% FBS and 1% Penicillin Streptomycin, and then incubated at 37°C overnight. The following day, the test compound was administered to the cells by using 1000x compound stock solution prepared in DMSO at various concentrations.1000x compound stock solution was first diluted in culturing medium to 10x, then 10 μL compound medium was added to each well in the cell plates. After administration of the compound, the cells were then incubated at 37°C for 6 days. Upon completion, the plates were equilibrated at room temperature for approximately 10 minutes.100 μL of CellTiter-Glo 3D Reagent was added to each well. The plates were then incubated at room temperature for 30 minutes and luminescence was recorded by EnSpire plate reader. The results are shown in Table 3, which summarizes the growth inhibition (GI50) in SW620 cells with exemplary compounds of the present disclosure.
Reference: GLUTARIMIDE-CONTAINING PAN-KRAS-MUTANT DEGRADER COMPOUNDS AND USES THEREOF (patent)
Source database: TPDdb