Compound (SMILES): COc1ccc(C(=O)N2CCC(N3CC(CN4CCN(c5c(C)cc(-c6cc7c(-c8cnn(C9CCN(C)CC9)c8)c[nH]c7nn6)cc5C)CC4)C3)CC2)cc1N1CCC(=O)NC1=O
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): MAP4K1 (UniProt Q92918)
POI sequence: MDVVDPDIFNRDPRDHYDLLQRLGGGTYGEVFKARDKVSGDLVALKMVKMEPDDDVSTLQKEILILKTCRHANIVAYHGSYLWLQKLWICMEFCGAGSLQDIYQVTGSLSELQISYVCREVLQGLAYLHSQKKIHRDIKGANILINDAGEVRLADFGISAQIGATLARRLSFIGTPYWMAPEVAAVALKGGYNELCDIWSLGITAIELAELQPPLFDVHPLRVLFLMTKSGYQPPRLKEKGKWSAAFHNFIKVTLTKSPKKRPSATKMLSHQLVSQPGLNRGLILDLLDKLKNPGKGPSIGDIEDEEPELPPAIPRRIRSTHRSSSLGIPDADCCRRHMEFRKLRGMETRPPANTARLQPPRDLRSSSPRKQLSESSDDDYDDVDIPTPAEDTPPPLPPKPKFRSPSDEGPGSMGDDGQLSPGVLVRCASGPPPNSPRPGPPPSTSSPHLTAHSEPSLWNPPSRELDKPPLLPPKKEKMKRKGCALLVKLFNGCPLRIHSTAAWTHPSTKDQHLLLGAEEGIFILNRNDQEATLEMLFPSRTTWVYSINNVLMSLSGKTPHLYSHSILGLLERKETRAGNPIAHISPHRLLARKNMVSTKIQDTKGCRACCVAEGASSGGPFLCGALETSVVLLQWYQPMNKFLLVRQVLFPLPTPLSVFALLTGPGSELPAVCIGVSPGRPGKSVLFHTVRFGALSCWLGEMSTEHRGPVQVTQVEEDMVMVLMDGSVKLVTPEGSPVRGLRTPEIPMTEAVEAVAMVGGQLQAFWKHGVQVWALGSDQLLQELRDPTLTFRLLGSPRLECSGTISPHCNLLLPGSSNSPASASRVAGITGL
E3 ligase: CRBN (UniProt Q96SW2)
Ligase sequence: MAGEGDQQDAAHNMGNHLPLLPAESEEEDEMEVEDQDSKEAKKPNIINFDTSLPTSHTYLGADMEEFHGRTLHDDDSCQVIPVLPQVMMILIPGQTLPLQLFHPQEVSMVRNLIQKDRTFAVLAYSNVQEREAQFGTTAEIYAYREEQDFGIEIVKVKAIGRQRFKVLELRTQSDGIQQAKVQILPECVLPSTMSAVQLESLNKCQIFPSKPVSREDQCSYKWWQKYQKRKFHCANLTSWPRWLYSLYDAETLMDRIKKQLREWDENLKDDSLPSNPIDFSYRVAACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTSLCCKQCQETEITTKNEIFSLSLCGPMAAYVNPHGYVHETLTVYKACNLNLIGRPSTEHSWFPGYAWTVAQCKICASHIGWKFTATKKDMSPQKFWGLTRSALLPTIPDTEDEISPDKVILCL
Cell line: Ramos (Homo sapiens)
Cell line description: EBV-negative.
Gene fusion; HGNC; HGNC Mutation; HGNC; HGNC
A*03 A*03 A*03 A*03
Stable (MSS).
In situ; Ascites; 
Caucasian.
B-cell; CL=CL_0000236.
Cancer cell line
Assay: Western Blot
DC50:  275 nM
Reported range: 50 to 500
Assay description: HPK1 protein degradation detection
WB method was used to test HPK1 protein degradation and DC50/Dmax to evaluate the in vitro degradation ability of the compound.
RAMOS cell line was selected as the degradation cell line, and the culture conditions were RMPI 1640 medium with 10% FBS, 37°C, 5% CO2.
Experimental steps:
(1) Cell plating: In a 6cm culture dish, 4ml culture medium was used, and the number of cells was 2×106/dish.
(2) Drug addition: The compound (stock solution concentration 10mM) was diluted with DMSO in a dilution plate, with the maximum starting concentration of the compound being 10uM, and 7 concentration gradient points (3-fold dilution). 4ul of each was added to the above culture dish and incubated for 16h.
(3) Cells were collected and lysed, centrifuged at 12000rpm/min for 20min, and the supernatant was collected. After quantification with the BCA quantitative kit, 5× loading buffer was added for denaturation for 10min. (4) Add 10 μg of denatured protein to the SDS-PAGE gel, run electrophoresis, transfer to the membrane, block with 5% BSA for 1 hour, incubate with primary antibody at 4°C overnight, wash the primary antibody, incubate with secondary antibody at room temperature for 1 hour, and wash the secondary antibody.
(5) Imaging: Scan the membrane using a dual-color fluorescence imaging system. Quantify the Western blot results using Image J software, and calculate DC50 and Dmax by comparing with the control group.
Reference: PROTAC CHIMERIC COMPOUND, PREPARATION METHOD THEREFOR AND USE THEREOF (patent)
Source database: TPDdb