Compound (SMILES): O=C1CC[C@H](c2noc3ccc4c(NC(=O)CN5CCN(C[C@H]6CC[C@H](n7cc(NC(=O)c8coc(-c9ccnc(NCC%10CC%10)c9)n8)c(C(F)F)n7)CC6)CC5)cccc4c23)C(=O)N1
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): IKZF1 (UniProt Q13422)
POI sequence: MDADEGQDMSQVSGKESPPVSDTPDEGDEPMPIPEDLSTTSGGQQSSKSDRVVASNVKVETQSDEENGRACEMNGEECAEDLRMLDASGEKMNGSHRDQGSSALSGVGGIRLPNGKLKCDICGIICIGPNVLMVHKRSHTGERPFQCNQCGASFTQKGNLLRHIKLHSGEKPFKCHLCNYACRRRDALTGHLRTHSVGKPHKCGYCGRSYKQRSSLEEHKERCHNYLESMGLPGTLYPVIKEETNHSEMAEDLCKIGSERSLVLDRLASNVAKRKSSMPQKFLGDKGLSDTPYDSSASYEKENEMMKSHVMDQAINNAINYLGAESLRPLVQTPPGGSEVVPVISPMYQLHKPLAEGTPRSNHSAQDSAVENLLLLSKAKLVPSEREASPSNSCQDSTDTESNNEEQRSGLIYLTNHIAPHARNGLSLKEEHRAYDLLRAASENSQDALRVVSTSGEQMKVYKCEHCRVLFLDHVMYTIHMGCHGFRDPFECNMCGYHSQDRYEFSSHITRGEHRFHMS
E3 ligase: CRBN (UniProt Q96SW2)
Ligase sequence: MAGEGDQQDAAHNMGNHLPLLPAESEEEDEMEVEDQDSKEAKKPNIINFDTSLPTSHTYLGADMEEFHGRTLHDDDSCQVIPVLPQVMMILIPGQTLPLQLFHPQEVSMVRNLIQKDRTFAVLAYSNVQEREAQFGTTAEIYAYREEQDFGIEIVKVKAIGRQRFKVLELRTQSDGIQQAKVQILPECVLPSTMSAVQLESLNKCQIFPSKPVSREDQCSYKWWQKYQKRKFHCANLTSWPRWLYSLYDAETLMDRIKKQLREWDENLKDDSLPSNPIDFSYRVAACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTSLCCKQCQETEITTKNEIFSLSLCGPMAAYVNPHGYVHETLTVYKACNLNLIGRPSTEHSWFPGYAWTVAQCKICASHIGWKFTATKKDMSPQKFWGLTRSALLPTIPDTEDEISPDKVILCL
Cell line: MM1.S (Homo sapiens)
Cell line description: African=88.68%; Native American=0%; East Asian, North=4.01%; East Asian, South=0%; South Asian=0.43%; European, North=0%; European, South=6.87%.
Mutation; HGNC; HGNC Mutation; HGNC; HGNC
Produces IgA lambda (ATCC=CRL-2974). Sensitive to dexamethasone.
A*23,24; B*18,42; C*12,17. A*23
Instable (MSI-low) (Sanger).
CVCL_5801 ! MM.1
In situ; Peripheral blood; 
African American.
Plasma cell; CL=CL_0000786.
Cancer cell line
Assay: In-Cell Western
DC50:  27.38 nM
Assay description: In-cell Western analysis of IKZF1 and IKZF3 protein expression levels in MM.1S cells.
Purpose: This experiment examined the effects of test compounds on IKZF1 and IKZF3 protein expression levels in MM.1S cells to assess their ability to degrade IKZF1 and IKZF3 proteins.
Experimental Protocol:
1) Plate 1.2 × 10? cells per well in a 96-well plate in logarithmic growth phase and culture overnight.
2) The next day, add drug treatment starting at 300 nM, perform three-fold dilutions in triplicate, and incubate in a 10-well concentration gradient (containing DMSO). Incubate in a CO2 incubator for 24 hours.
3) Centrifuge, carefully remove the supernatant, and add 150 μL of 4% paraformaldehyde fixative along the sides of the wells, avoiding the cells at the bottom. Incubate at room temperature for 20 minutes.
4) Prepare permeabilization solution: Add 0.5 mL of 10% Triton X-100 to 49.5 mL of PBS and mix thoroughly.
5) Add 200 μL of permeabilization solution along the sides of the wells, avoiding the cells at the bottom. Incubate at room temperature on a shaker for 5 minutes.
6) Repeat the wash cycle four times.
7) Add 150 μL of Licor INERCEPT blocking solution along the sides of the wells. Buffer (500 μL), avoiding touching the cells at the bottom, and incubate on a shaker at room temperature for 1.5 hours.
8) Ikaros (D6N9Y) Rabbit mAb, Aiolos (D1C1E) Rabbit mAb, and GAPDH mouse mAb (Proteintech, 60004-1-Ig) were diluted 1:100 in antibody diluent.
9) 50 μL of the mixed antibody solution was added to each well in triplicate and incubated overnight at 4°C on a shaker.
10) PBST (PBS containing 0.1% Tween 20) was prepared.
11) Remove the primary antibody and add 200 μL of PBST along the sides of the wells, avoiding touching the cells at the bottom. Incubate on a shaker at room temperature for 5 minutes.
12) Repeat the wash cycle four times.
13) Prepare the secondary antibody diluent: Add 0.2% Tween 20 to Licor INTERCEPT blocking buffer. 20;
14) Dilute the fluorescent secondary antibody in the dark (1:800 dilution). Take 400 μL of the secondary antibody dilution solution and add 0.5 μL each of IRDye 800CW and IRDye 680CW (fluorescent secondary antibodies corresponding to the primary antibodies).
15) Add 50 μL of the diluted fluorescent secondary antibody to each well and incubate for 60 minutes at room temperature on a shaker in the dark.
16) Remove the secondary antibody and add 200 μL of PBST along the well walls, avoiding the cells at the bottom. Incubate for 5 minutes at room temperature on a shaker in the dark.
17) Repeat the wash cycle four times. Immediately after washing, analyze the cells using the Odyssey Gel Imaging System dual-color at 700 nm and 800 nm.
3. Data Analysis
Using GraphPad Prism 6 software, insert the inhibition data, fit the curve, and calculate the DC50 value. Protein inhibition rate = (1 - RLs/RLv) * 100%
RR (Raw Ratio) = 700nm/800nm
RLs = RR of sample-treated cells
RLv = RR of solvent-treated cells
Test results are shown in Table 6.
Reference: 2,6-PIPERIDINEDIONE COMPOUND AND APPLICATION THEREOF (patent)
Source database: TPDdb