Compound (SMILES): Cc1cc(-c2cnc3[nH]cc(-c4ccc(C(=O)N(C)C[C@H](C)O)cc4)c3n2)cc2c1CN(CCC(=O)N1CCN(c3ccc(N4CCC(=O)NC4=O)cc3)CC1)CC2
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): MAP4K1 (UniProt Q92918)
POI sequence: MDVVDPDIFNRDPRDHYDLLQRLGGGTYGEVFKARDKVSGDLVALKMVKMEPDDDVSTLQKEILILKTCRHANIVAYHGSYLWLQKLWICMEFCGAGSLQDIYQVTGSLSELQISYVCREVLQGLAYLHSQKKIHRDIKGANILINDAGEVRLADFGISAQIGATLARRLSFIGTPYWMAPEVAAVALKGGYNELCDIWSLGITAIELAELQPPLFDVHPLRVLFLMTKSGYQPPRLKEKGKWSAAFHNFIKVTLTKSPKKRPSATKMLSHQLVSQPGLNRGLILDLLDKLKNPGKGPSIGDIEDEEPELPPAIPRRIRSTHRSSSLGIPDADCCRRHMEFRKLRGMETRPPANTARLQPPRDLRSSSPRKQLSESSDDDYDDVDIPTPAEDTPPPLPPKPKFRSPSDEGPGSMGDDGQLSPGVLVRCASGPPPNSPRPGPPPSTSSPHLTAHSEPSLWNPPSRELDKPPLLPPKKEKMKRKGCALLVKLFNGCPLRIHSTAAWTHPSTKDQHLLLGAEEGIFILNRNDQEATLEMLFPSRTTWVYSINNVLMSLSGKTPHLYSHSILGLLERKETRAGNPIAHISPHRLLARKNMVSTKIQDTKGCRACCVAEGASSGGPFLCGALETSVVLLQWYQPMNKFLLVRQVLFPLPTPLSVFALLTGPGSELPAVCIGVSPGRPGKSVLFHTVRFGALSCWLGEMSTEHRGPVQVTQVEEDMVMVLMDGSVKLVTPEGSPVRGLRTPEIPMTEAVEAVAMVGGQLQAFWKHGVQVWALGSDQLLQELRDPTLTFRLLGSPRLECSGTISPHCNLLLPGSSNSPASASRVAGITGL
E3 ligase: CRBN (UniProt Q96SW2)
Ligase sequence: MAGEGDQQDAAHNMGNHLPLLPAESEEEDEMEVEDQDSKEAKKPNIINFDTSLPTSHTYLGADMEEFHGRTLHDDDSCQVIPVLPQVMMILIPGQTLPLQLFHPQEVSMVRNLIQKDRTFAVLAYSNVQEREAQFGTTAEIYAYREEQDFGIEIVKVKAIGRQRFKVLELRTQSDGIQQAKVQILPECVLPSTMSAVQLESLNKCQIFPSKPVSREDQCSYKWWQKYQKRKFHCANLTSWPRWLYSLYDAETLMDRIKKQLREWDENLKDDSLPSNPIDFSYRVAACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTSLCCKQCQETEITTKNEIFSLSLCGPMAAYVNPHGYVHETLTVYKACNLNLIGRPSTEHSWFPGYAWTVAQCKICASHIGWKFTATKKDMSPQKFWGLTRSALLPTIPDTEDEISPDKVILCL
Cell line: Human PBMCs (Homo sapiens)
Assay: Flow Cytometry, FACS
DC50:  35 nM
Assay description: HPK1 Protein Degradation Assay in Human PBMC (FACS)
Human PBMCs (AllCells) was used in this study. Cells were thawed one day in advance and maintained in RPMI-1640 supplemented 10%fetal bovine serum, 50 units/mL penicillin and streptomycin and the mixture was kept at 37°C in a humidified atmosphere air containing 5%CO 2.3X10 6 PBMCs per well in 95 μL culture medium were seed in the 96-well plate, and the cells were treated with an appropriate dilution series of compounds. After 24 h compound treatment, 100 μL 1×Lyse/Fix buffer (BD, diluted with water, warm up in 37°C) was added to cells and the cells were incubated at 37 °C for 10 minutes. After fixed, the cells were transferred to V-bottom plates, and washed with FACS buffer (cold 2%FBS DPBS) . The cells were permeabilized by adding 100 μL pre-chilled BD Phosflow Perm Buffer III (BD) for 30 min on ice. After washed with FACS buffer twice, the cells were incubated with 50 μL antibody cocktail (HPK1 (CST) (1: 50) , anti-human CD4 BV421 (BD) (1: 25) , anti-human CD8 APC (BD) (1: 25) ) at room temperature for 1 h. After washed with FACS buffer twice, the cells were further incubated with 50 μL secondary antibody cocktail (Biolegend) (1: 50) at room temperature for 1 h. After washed with FACS buffer twice, the signals were detected by BD FACS Celesta. The data were analyzed by Excel and GraphPad Prism 8. The compounds disclosed herein showed the Dmax, DC50 and DC90 values as in Table 4.
Reference: PYRROLO [2, 3-b] PYRAZINE-BASED BIFUNCTIONAL COMPOUNDS AS HPK1 DEGRADERS AND THE USE THEREOF (patent)
Source database: TPDdb