Compound (SMILES): COc1cc(N2CCN(C(=O)CN3CCC(c4ccc5c(N6CCC(=O)NC6=O)nn(C)c5c4)C(F)(F)C3)CC2)c2cc1Nc1ncc(Br)c(n1)Nc1ccc(cc1N(C)S(C)(=O)=O)OCCCCC2
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): EGFR (UniProt Q01279)
POI sequence: MRPSGTAGAALLALLAALCPASRALEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRRRHIVRKRTLRRLLQERELVEPLTPSGEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLITQLMPFGCLLDYVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAARNVLVKTPQHVKITDFGLAKLLGAEEKEYHAEGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKPYDGIPASEISSILEKGERLPQPPICTIDVYMIMVKCWMIDADSRPKFRELIIEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYRALMDEEDMDDVVDADEYLIPQQGFFSSPSTSRTPLLSSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYSSDPTGALTEDSIDDTFLPVPEYINQSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCVNSTFDSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVAPQSSEFIGA
E3 ligase: CRBN (UniProt Q8C7D2)
Ligase sequence: MAGEGDQQDAAHNMGNHLPLLPAESEEEDEMEVEDQDSKEAKKPNIINFDTSLPTSHTYLGADMEEFHGRTLHDDDSCQVIPVLPQVMMILIPGQTLPLQLFHPQEVSMVRNLIQKDRTFAVLAYSNVQEREAQFGTTAEIYAYREEQDFGIEIVKVKAIGRQRFKVLELRTQSDGIQQAKVQILPECVLPSTMSAVQLESLNKCQIFPSKPVSREDQCSYKWWQKYQKRKFHCANLTSWPRWLYSLYDAETLMDRIKKQLREWDENLKDDSLPSNPIDFSYRVAACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTSLCCKQCQETEITTKNEIFSLSLCGPMAAYVNPHGYVHETLTVYKACNLNLIGRPSTEHSWFPGYAWTVAQCKICASHIGWKFTATKKDMSPQKFWGLTRSALLPTIPDTEDEISPDKVILCL
Cell line: Ba/F3 (Mus musculus)
Cell line description: Originally thought to originate from a BALB/c mouse but is in fact from a C3H mouse.
IL3 dependent.
C3H.
In situ; Bone marrow; 
Pro-B cell; CL=CL_0000826.
Factor-dependent cell line
Assay: HTRF
DC50:  64.3 nM
Assay description: Test Example 2: Determination of Total EGFR Protein Degradation in BaF3/EGFR L858R-T790M-C797S Cells by Compounds of the Present Disclosure
2. Experimental Methods
2.1. Cell Plating (Day 1)
a. Observe the cells under a microscope.
b. Collect the cell suspension into a 15 mL centrifuge tube and centrifuge at 300 g for 4 minutes. Resuspend the cells in an appropriate amount of fresh culture medium.
c. Take 20 μL of the resuspended cell suspension and count the cells to ensure cell viability is greater than 90%.
d. Based on the cell count results, dilute the cells to the appropriate plating density using 1640 medium supplemented with 10% FBS. Plate the cells in a 96-well culture plate at a density of 10,000-20,000 cells per well, using 25 μL per well.
2.2. Drug Addition (Day 1)
a. The initial compound concentration was 10 mM. Each compound was serially diluted 5-fold with DMSO to a total of 10 concentration points. For example, in a 96-well round-bottom plate, serially dilute 3 μL of compound into 12 μL of DMSO.
b. Dilute each compound concentration 167-fold into the corresponding volume of 1640 μL of cell culture medium.
c. Add 5 μL of the diluted compound solution to 25 μL/well of the cell suspension in each plate.
d. Incubate the plate with the drug in a 37°C, 5% CO2 incubator for 6 hours.
2.3. Sample Collection and Antibody Incubation (Day 1)
a. Remove the 96-well plate and add 10 μL of (4×)* supplemental lysis buffer (1 part blocking buffer + 24 parts 4× lysis buffer) to each well as directed in the Total EGFR Cell Kit instructions. Apply a transparent film, cover the plate, briefly shake the plate, and shake at room temperature for 60 minutes (800 rpm). b. Transfer 16 μL of the lysed solution to a 384-well plate and add 4 μL of premixed antibodies (d2 antibody:Eu antibody = 1:1 premix). Apply aluminum foil, cover the plate, shake at room temperature for 1 minute, briefly shake, and incubate at room temperature overnight.
2.4. HTRF Assay (Day 2)
a. Remove the 384-well plate from the incubator, shake at room temperature for 1 minute, and briefly shake.
b. Read the fluorescence emission signal at two different wavelengths, 665 nm and 620 nm, on an EnVision microscope.
2.5. Calculation of DC50 Values and Maximum Degradation Rates
a. Calculate the total EGFR degradation rate at each compound concentration using the following formula. Use XLfit to perform curve fitting based on the logarithmic concentration of the compound and the degradation rate, and calculate the DC50 value.
b. Calculate the ratio, R, of the acceptor and donor emission signals in each well. c. Degradation Rate (%) = 100 - 100 × (R Compound - R Blank Control) / (R Vehicle Control - R Blank Control)
The total EGFR degradation activity of the disclosed compounds was determined from the above analysis. The calculated DC50 values are shown in Table 3 below.
Reference: MACROCYCLE FOR INHIBITING AND INDUCING EGFR DEGRADATION AND PREPARATION METHOD FOR AND USE OF MACROCYCLE (patent)
Source database: TPDdb