Compound (SMILES): CC(C)(O)C#Cc1cccc(COc2cc(-c3cnc(C4CCN(C(=O)CCNC(=O)CCCCCCOc5cccc6c5C(=O)N(C5CCC(=O)NC5=O)C6=O)CC4)s3)cnc2N)c1
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): MAP4K1 (UniProt Q92918)
POI sequence: MDVVDPDIFNRDPRDHYDLLQRLGGGTYGEVFKARDKVSGDLVALKMVKMEPDDDVSTLQKEILILKTCRHANIVAYHGSYLWLQKLWICMEFCGAGSLQDIYQVTGSLSELQISYVCREVLQGLAYLHSQKKIHRDIKGANILINDAGEVRLADFGISAQIGATLARRLSFIGTPYWMAPEVAAVALKGGYNELCDIWSLGITAIELAELQPPLFDVHPLRVLFLMTKSGYQPPRLKEKGKWSAAFHNFIKVTLTKSPKKRPSATKMLSHQLVSQPGLNRGLILDLLDKLKNPGKGPSIGDIEDEEPELPPAIPRRIRSTHRSSSLGIPDADCCRRHMEFRKLRGMETRPPANTARLQPPRDLRSSSPRKQLSESSDDDYDDVDIPTPAEDTPPPLPPKPKFRSPSDEGPGSMGDDGQLSPGVLVRCASGPPPNSPRPGPPPSTSSPHLTAHSEPSLWNPPSRELDKPPLLPPKKEKMKRKGCALLVKLFNGCPLRIHSTAAWTHPSTKDQHLLLGAEEGIFILNRNDQEATLEMLFPSRTTWVYSINNVLMSLSGKTPHLYSHSILGLLERKETRAGNPIAHISPHRLLARKNMVSTKIQDTKGCRACCVAEGASSGGPFLCGALETSVVLLQWYQPMNKFLLVRQVLFPLPTPLSVFALLTGPGSELPAVCIGVSPGRPGKSVLFHTVRFGALSCWLGEMSTEHRGPVQVTQVEEDMVMVLMDGSVKLVTPEGSPVRGLRTPEIPMTEAVEAVAMVGGQLQAFWKHGVQVWALGSDQLLQELRDPTLTFRLLGSPRLECSGTISPHCNLLLPGSSNSPASASRVAGITGL
E3 ligase: CRBN (UniProt Q96SW2)
Ligase sequence: MAGEGDQQDAAHNMGNHLPLLPAESEEEDEMEVEDQDSKEAKKPNIINFDTSLPTSHTYLGADMEEFHGRTLHDDDSCQVIPVLPQVMMILIPGQTLPLQLFHPQEVSMVRNLIQKDRTFAVLAYSNVQEREAQFGTTAEIYAYREEQDFGIEIVKVKAIGRQRFKVLELRTQSDGIQQAKVQILPECVLPSTMSAVQLESLNKCQIFPSKPVSREDQCSYKWWQKYQKRKFHCANLTSWPRWLYSLYDAETLMDRIKKQLREWDENLKDDSLPSNPIDFSYRVAACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTSLCCKQCQETEITTKNEIFSLSLCGPMAAYVNPHGYVHETLTVYKACNLNLIGRPSTEHSWFPGYAWTVAQCKICASHIGWKFTATKKDMSPQKFWGLTRSALLPTIPDTEDEISPDKVILCL
Cell line: Jurkat (Homo sapiens)
Cell line description: African=0.4%; Native American=0%; East Asian, North=0.13%; East Asian, South=1.21%; South Asian=1.08%; European, North=70.73%; European, South=26.45%.
Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC
A*03
Instable (MSI-high).
In situ; Peripheral blood; 
Caucasian.
Space-flown cell line (cellonaut).
T-cell; CL=CL_0000084.
Cancer cell line
Assay: Western Blot
Dmax:  45 %
Assay description: S1: Jurkat cells were revived and cultured for 12 hours. Compounds were added at fixed concentrations (treatment times of 0, 3, 6, 12, 24, and 72 hours) or at fixed treatment times (compound concentrations of 0, 1, 10, 100, and 1000 nM).
S2: The culture medium was discarded, and the cells were washed with 3 ml of 4°C pre-chilled PBS. The wash solution was discarded. The appropriate amount of lysis buffer containing PMSF (100×) was added based on the cell size. The cells were mixed thoroughly and lysed on ice for 30 minutes. The cells were centrifuged at 12,000 rpm for 5 minutes at 4°C (pre-chilled). The supernatant was transferred to a fresh centrifuge tube for quantification.
S3: Pierce? BCA Protein Assay was used. The kit's BCA quantification reagent (200 μl/well) was then added with 20 μl of BSA of varying known concentrations to prepare a protein standard curve and the sample protein to be assayed. Avoid bubbles and incubate at 37°C in the dark for 30 minutes. Measure the OD at 562 nm. Add an appropriate amount of SDS-PAGE loading buffer to the protein sample and heat in a boiling water bath for 5 minutes to fully denature the protein. Store at -20°C until needed.

S4: Prepare an SDS-PAGE gel using a voltage of 90 V for the stacking gel and 120 V for the separating gel. Transfer the membrane at 100 V for 1 hour at 4°C. After transfer, immediately place the membrane in the pre-prepared 5% BSA solution, gently shake on a shaker, and block at room temperature for 1 hour.

S5: Reference HPK1 Antibody, GLK Rabbit mAb, SLP-76 Rabbit mAb, Phospho-SLP-76 XP Rabbit mAb, and β-actin Mouse Monoclonal Antibody. Follow the antibody instructions and dilute the primary antibody to the appropriate ratio. Incubate at 4°C with gentle shaking overnight, then wash three times with PBST (10 min each).

S6: Select either Goat Anti-Rabbit IgG (H&L)-HRP Conjugated or Goat Anti-Mouse IgG (H&L)-HRP Conjugated, depending on the primary antibody. Refer to the secondary antibody instructions and dilute the secondary antibody to the appropriate ratio. Incubate at room temperature with gentle shaking for 1 hour, then wash three times with PBST (10 min each).

S7: Add ECL luminescence solution and visualize the results using a developer. Quantify Western blotting results using Image J software. Calculate degradation rates (for HPK1 and GLK) or inhibition rates (for SLP76 Ser376 phosphorylation) by comparing with the control group without compound (degradation rate/inhibition rate = 1 - drug group/control group). The results are shown in the table below.
Reference: PROTAC SMALL MOLECULE COMPOUND AND APPLICATION THEREOF (patent)
Source database: TPDdb