Compound (SMILES): Nc1c(-c2nc3ccc(N4CCN(CC5CN(c6ccc(C7CCC(=O)NC7=O)cc6)C5)CC4)cc3[nH]2)c(=O)[nH]c2sccc12
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: Fully automated capillary immunoassay
DC50:  32 nM
Assay description: Detection of HPK1 protein expression in Jurkat cells. Jurkat cells obtained from ATCC were cultured in RPMI-1640 complete medium (supplemented with 10% FBS and 1% double-antibody) at 37°C and 5% CO2. Cells in the logarithmic growth phase were harvested, the cell density adjusted to 5 × 105 cells/well with culture medium, and plated at 1 mL/well into a 6-well cell culture plate. Test compounds were prepared to 2x the final concentration. 1 mL of each concentration was added to the treatment wells, while control wells were incubated with culture medium containing 0.2% DMSO. The cells were incubated at 37°C and 5% CO2 for 48 hours. Cells were harvested into a 1.5 mL centrifuge tube and 25 μL of RIPA lysis buffer (containing 1X protease inhibitor cocktail) was added. Lysis was performed on ice for 15 minutes, followed by centrifugation at 12,000 rpm at 4°C for 10 minutes. The supernatant was collected and protein content was determined using the BCA assay. The test protein sample was diluted to 0.2 mg/mL and HPK1 was detected using a fully automated protein expression quantitative analyzer (ProteinSimple). β-actin (antibodies were from CST) was used as the internal reference protein. Raw data were processed using the fully automated protein expression quantitative analyzer software (Compass for SW), and peak areas and HPK1 degradation rates relative to the control group were calculated. The HPK1 inhibition rate was calculated as A treatment well/A control well × 100%, where A treatment well represents the relative peak area of the treatment group and A control well represents the relative peak area of the vehicle control group. DC50 values were calculated using a four-parameter nonlinear fitting model in Graphpad 8.3.0 software.
Reference: COMPOUND FOR INHIBITING OR DEGRADING HPK1 KINASE AND MEDICAL USE THEREOF (patent)
Source database: TPDdb