Compound (SMILES): COc1cc(N2CCN(C(=O)CN3CCC(c4ccc(NC5CCC(=O)NC5=O)cc4)CC3)CC2)ccc1Nc1ncc(Cl)c(Nc2ccccc2C(C)=O)n1
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): EGFR (UniProt P00533)
POI sequence: MRPSGTAGAALLALLAALCPASRALEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRRRHIVRKRTLRRLLQERELVEPLTPSGEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLITQLMPFGCLLDYVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAARNVLVKTPQHVKITDFGLAKLLGAEEKEYHAEGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKPYDGIPASEISSILEKGERLPQPPICTIDVYMIMVKCWMIDADSRPKFRELIIEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYRALMDEEDMDDVVDADEYLIPQQGFFSSPSTSRTPLLSSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYSSDPTGALTEDSIDDTFLPVPEYINQSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCVNSTFDSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVAPQSSEFIGA
E3 ligase: CRBN (UniProt Q96SW2)
Ligase sequence: MAGEGDQQDAAHNMGNHLPLLPAESEEEDEMEVEDQDSKEAKKPNIINFDTSLPTSHTYLGADMEEFHGRTLHDDDSCQVIPVLPQVMMILIPGQTLPLQLFHPQEVSMVRNLIQKDRTFAVLAYSNVQEREAQFGTTAEIYAYREEQDFGIEIVKVKAIGRQRFKVLELRTQSDGIQQAKVQILPECVLPSTMSAVQLESLNKCQIFPSKPVSREDQCSYKWWQKYQKRKFHCANLTSWPRWLYSLYDAETLMDRIKKQLREWDENLKDDSLPSNPIDFSYRVAACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTSLCCKQCQETEITTKNEIFSLSLCGPMAAYVNPHGYVHETLTVYKACNLNLIGRPSTEHSWFPGYAWTVAQCKICASHIGWKFTATKKDMSPQKFWGLTRSALLPTIPDTEDEISPDKVILCL
Cell line: HCC827 (Homo sapiens)
Cell line description: African=1.62%; Native American=0.39%; East Asian, North=3.47%; East Asian, South=0%; South Asian=7.09%; European, North=19.48%; European, South=67.96%.
Flat-moded hypotriploid karyotype with 6% polyploidy (DSMZ=ACC-566).
Mutation; HGNC; HGNC Mutation; HGNC; HGNC
A*11 A*11
Stable (MSS) (Sanger).
In situ; Lung; 
Caucasian.
Cancer cell line
Assay: In-Cell Western
DC50:  15.56 nM
Assay description: As shown in Table 4, for DC50, AA ≤ 10 nM; 10 nM < A ≤ 100 nM; 100 nM < B < 1000 nM; C ≥ 1000 nM; for Dmax, 70% ≤ A ≤ 100%; 50% ≤ B < 70%; C < 50%; NA indicates not tested.

EGFR PROTAC In-cell Western Blot Assay
1. Experimental Materials
HCC827 cell culture medium was purchased from Corning. The HCC827 cell line was purchased from Beina Biotechnology. EGF Receptor (D38B1) XP Rabbit mAb and β-Actin (8H10D10) Mouse mAb were from CST. IRDye 800CW, IRDye 680RD, and Intercept blocking buffer (PBS) were purchased from Licor. Triton-100 was purchased from Sigma, and 96-well black cell culture imaging microplates were purchased from Agilent. 2. Experimental Methods
1) Count HCC827 cells and seed each well of a 96-well black cell culture imaging microplate at a density of 40,000 cells per well, using 100 μl per well. Incubate in a CO2 incubator overnight.
2) Day 1: Add serial dilutions of the test compound (starting at 3 μM, 9 concentrations, 1:3 dilutions, duplicate wells) to the cells in the culture plate, with a final DMSO concentration of 0.5%. A blank control is DMSO without compound. Incubate the plate in a cell culture incubator for a final 16 hours.
3) Remove the culture medium from the 96-well plate using a pipette, add 200 μl of PBS to each well, and wash the remaining medium on a shaker for 5 minutes. After removing the PBS, quickly add 150 μl of fresh fixation buffer (3.7% paraformaldehyde) to fix the cells and incubate at room temperature for 20 minutes. 4) Remove the fixation buffer and add 200 μl of Triton elution buffer (0.3% Triton-100). Wash the plate four times for 5 minutes each on a shaker.
5) Add 150 μl of LI-COR Odyssey blocking buffer and incubate at room temperature for 1.5 hours with gentle shaking on a shaker.
6) While blocking the sample, prepare the primary antibodies against EGFR and Actin at a 1:300 ratio in antibody dilution buffer. Pour off the blocking buffer and add the diluted primary antibodies (total volume 50 μl/well). Cover the plate and incubate overnight at 4°C.
7) Wash the plate four times for 5 minutes each with PBS + Triton X-100. 8) Dilute the fluorescently labeled secondary antibodies (Figure PCTCN2022132632-appb-000194) and (Figure PCTCN2022132632-appb-000195) in antibody dilution buffer (1:600, total volume 50 μl/well) and incubate at room temperature in the dark for one hour.
9) Wash the cell plate four times with PBS + Triton X-100 for 5 minutes each.
3. Calculation
After thoroughly aspirating the PBS, invert the plate and image on a Bio-Rad imager. Select the multiple exposure program, IRDye680RD Blot for the first channel, and IRDye800CW Blot for the second channel. Open the image in Image Studio Lite Ver5.2 and analyze the signal value for each well. Circle each well to display the EGFR and Actin signal values. The blank group was the DMSO group, and the EGFR value of the corresponding sample well was calculated using EGFR/Actin. Degradation rate = 100-100*EGFR signal/DMSO signal. The DC 50 and D max of the degraded EGFR protein were calculated using Prism software and a three-parameter curve fitting method.
Reference: COMPOUND FOR DEGRADING EGFR PROTEIN AND USE THEREOF (patent)
Source database: TPDdb