Compound (SMILES): Cc1cc(-c2c[nH]c3ncc(-c4cc(C)c5c(c4)CCN(CCNC(=O)C4CCN(c6ccc7c(c6)C(=O)N(C6CCC(=O)NC6=O)C7=O)CC4)C5)nc23)ccc1C(=O)N(C)C
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: Western Blot
DC50:  183 nM
Assay description: HPK1 Protein Degradation Assay in Human PBMC (Western Blot)
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 1 mL culture medium were seeded in the 12-well plate, and the cells were treated with an appropriate dilution series of compounds with a final concentration up to 10 mM. After 24 h of compound incubation, the medium was aspirated, the cells were washed with PBS, and then 30 μL of 1X protein lysis buffer containing protease inhibitors and phosphatase inhibitors was added. The cells were lysed. After centrifugation, the supernatants were quantified by BCA protein assay kit . 4X loading Buffer was added to equal amounts of total protein from each sample and heated at 95 °C for 5 minutes. 30-50 μg of cell lysate was loaded onto a 4%-12%NuPAGE Bis-Tris Gel and electro-transferred to NC membranes . The membranes were blocked for least 1 hour with blocking reagent, and then incubated overnight with anti-HPK1 antibodies and as loading control anti-β-actin or anti-GAPDH at 4°C with gentle shaking. The membranes were washed three times with TBST, and incubated at least 1 hour at room temperature with anti-mouse or anti-rabbit secondary fluorescent antibody. Then the membranes were washed three times in TBST, and one time in water. Immunoreactive bands were visualized by Odyssey CLx and analyzed by GraphPad Prism 8. The compounds disclosed herein showed the Dmax, DC50 and DC90 values as in Table 3.
Reference: PYRROLO [2, 3-b] PYRAZINE-BASED BIFUNCTIONAL COMPOUNDS AS HPK1 DEGRADERS AND THE USE THEREOF (patent)
Source database: TPDdb