Compound (SMILES): COc1cc2c(Oc3ccc(NC(=O)c4c(C)n(C)c5ccc(OC(F)(F)F)cc5c4=O)cc3F)ncnc2cc1OCC(=O)N1CCC(CC(=O)Nc2ccc3c(c2)CN(C2CCC(=O)NC2=O)C3=O)CC1
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): AXL (UniProt P30530)
POI sequence: MAWRCPRMGRVPLAWCLALCGWACMAPRGTQAEESPFVGNPGNITGARGLTGTLRCQLQVQGEPPEVHWLRDGQILELADSTQTQVPLGEDEQDDWIVVSQLRITSLQLSDTGQYQCLVFLGHQTFVSQPGYVGLEGLPYFLEEPEDRTVAANTPFNLSCQAQGPPEPVDLLWLQDAVPLATAPGHGPQRSLHVPGLNKTSSFSCEAHNAKGVTTSRTATITVLPQQPRNLHLVSRQPTELEVAWTPGLSGIYPLTHCTLQAVLSDDGMGIQAGEPDPPEEPLTSQASVPPHQLRLGSLHPHTPYHIRVACTSSQGPSSWTHWLPVETPEGVPLGPPENISATRNGSQAFVHWQEPRAPLQGTLLGYRLAYQGQDTPEVLMDIGLRQEVTLELQGDGSVSNLTVCVAAYTAAGDGPWSLPVPLEAWRPGQAQPVHQLVKEPSTPAFSWPWWYVLLGAVVAAACVLILALFLVHRRKKETRYGEVFEPTVERGELVVRYRVRKSYSRRTTEATLNSLGISEELKEKLRDVMVDRHKVALGKTLGEGEFGAVMEGQLNQDDSILKVAVKTMKIAICTRSELEDFLSEAVCMKEFDHPNVMRLIGVCFQGSERESFPAPVVILPFMKHGDLHSFLLYSRLGDQPVYLPTQMLVKFMADIASGMEYLSTKRFIHRDLAARNCMLNENMSVCVADFGLSKKIYNGDYYRQGRIAKMPVKWIAIESLADRVYTSKSDVWSFGVTMWEIATRGQTPYPGVENSEIYDYLRQGNRLKQPADCLDGLYALMSRCWELNPQDRPSFTELREDLENTLKALPPAQEPDEILYVNMDEGGGYPEPPGAAGGADPPTQPDPKDSCSCLTAAEVHPAGRYVLCPSTTPSPAQPADRGSPAAPGQEDGA
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: < 100 nM
Assay description: Effects of Compounds on AXL Expression in MDA-MB-231 Cells and FLT3 Expression in MV4-11 Cells

Methods: Cell lines: MDA-MB-231 and MV4-11 cell lines were purchased from the American Type Culture Collection (ATCC). Western blotting was performed as follows. MDA-MB-231 and MV4-11 cells were seeded in 12-well plates at defined concentrations and allowed to adhere overnight in an incubator. Compounds were then added at defined concentrations for 24 hours, and cells were lysed with lysis buffer and sampled. An appropriate amount of sample was then subjected to SDS-PAGE electrophoresis. Following electrophoresis, the proteins were transferred to a nitrocellulose membrane using a semi-dry electrotransfer system. The membrane was blocked in blocking buffer (5% skim milk powder diluted in TBS containing 0.1% Tween 20) for 2 hours at room temperature. The membrane was then incubated in primary antibody solution (1:1000 diluted in TBS containing 0.1% Tween 20) overnight at 4°C. The membrane was washed three times with TBS containing 0.1% Tween 20, each for 15 minutes. The membrane was then incubated in secondary antibody solution (horseradish peroxidase-conjugated goat anti-rabbit IgG, diluted 1:1000 in TBS containing 0.1% Tween 20) at room temperature for 1 hour. After washing the membrane three times as above, the membrane was developed with ECL plus reagent and photographed using an Amersham Imager 600 system. The results in Figure 1 demonstrate that the compounds selectively degrade AXL protein in MDA-MB-231 cells in a dose-dependent manner. HS-10, HS-28, SZQ-1, SZQ-3, SZQ-4, and SZQ-11 exhibit the highest activity, significantly downregulating AXL protein levels at 16 nM or 33 nM.
The results in Figure 2 demonstrate that the compounds selectively degrade FLT3 protein in MV4-11 cells in a dose-dependent manner. HS-10, HS-28, SZQ-1, SZQ-3, SZQ-4, and SZQ-11 exhibit the highest activity, significantly downregulating AXL protein levels at 16 nM or 33 nM. Table 4 summarizes the quantitative analysis results of Figures 1 and 2. Among the above compounds, Examples 10, 28, 33, 35, 36, 43, etc. can significantly degrade the AXL protein level in MDA-MB-231 cells and the FLT3 protein level in MV4-11 cells, among which Example 28 has the best AXL and FLT3 protein degradation activity.
Reference: PROTEIN KINASE DEGRADANT AND USE THEREOF (patent)
Source database: TPDdb