Compound (SMILES): Cc1nn(C2CCC(=O)NC2=O)c2cccc(N3CCCN(C[C@H]4CC[C@H](n5cc6cc(NC(=O)c7cnn8cccnc78)c(N(C)C)cc6n5)CC4)CC3)c12
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): IRAK4 (UniProt Q9NWZ3)
POI sequence: MNKPITPSTYVRCLNVGLIRKLSDFIDPQEGWKKLAVAIKKPSGDDRYNQFHIRRFEALLQTGKSPTSELLFDWGTTNCTVGDLVDLLIQNEFFAPASLLLPDAVPKTANTLPSKEAITVQQKQMPFCDKDRTLMTPVQNLEQSYMPPDSSSPENKSLEVSDTRFHSFSFYELKNVTNNFDERPISVGGNKMGEGGFGVVYKGYVNNTTVAVKKLAAMVDITTEELKQQFDQEIKVMAKCQHENLVELLGFSSDGDDLCLVYVYMPNGSLLDRLSCLDGTPPLSWHMRCKIAQGAANGINFLHENHHIHRDIKSANILLDEAFTAKISDFGLARASEKFAQTVMTSRIVGTTAYMAPEALRGEITPKSDIYSFGVVLLEIITGLPAVDEHREPQLLLDIKEEIEDEEKTIEDYIDKKMNDADSTSVEAMYSVASQCLHEKKNKRPDIKKVQQLLQEMTAS
E3 ligase: CRBN (UniProt Q96SW2)
Ligase sequence: MAGEGDQQDAAHNMGNHLPLLPAESEEEDEMEVEDQDSKEAKKPNIINFDTSLPTSHTYLGADMEEFHGRTLHDDDSCQVIPVLPQVMMILIPGQTLPLQLFHPQEVSMVRNLIQKDRTFAVLAYSNVQEREAQFGTTAEIYAYREEQDFGIEIVKVKAIGRQRFKVLELRTQSDGIQQAKVQILPECVLPSTMSAVQLESLNKCQIFPSKPVSREDQCSYKWWQKYQKRKFHCANLTSWPRWLYSLYDAETLMDRIKKQLREWDENLKDDSLPSNPIDFSYRVAACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTSLCCKQCQETEITTKNEIFSLSLCGPMAAYVNPHGYVHETLTVYKACNLNLIGRPSTEHSWFPGYAWTVAQCKICASHIGWKFTATKKDMSPQKFWGLTRSALLPTIPDTEDEISPDKVILCL
Cell line: THP-1 (Homo sapiens)
Cell line description: African=0%; Native American=0.3%; East Asian, North=77.63%; East Asian, South=20.4%; South Asian=1.67%; European, North=0%; European, South=0%.
Used in the human Cell Line Activation Test (h-CLAT), an in vitro skin sensitization test.
Not susceptible to infection by SARS coronavirus 2 (SARS-CoV-2) (COVID-19).
NRAS mutation indicated incorrectly as being p.Gly12Ser in PubMed=9379676.
Gene fusion; HGNC; HGNC Gene fusion; HGNC; HGNC Mutation; HGNC; HGNC Mutation; HGNC; HGNC
Can differentiate from monocytes into macrophage-like cells upon stimulation with PMA.
A*02; B*15 A*02 A*02 A*02
Stable (MSS).
In situ; Peripheral blood; 
Japanese.
Space-flown cell line (cellonaut).
Cancer cell line
Assay: Western Blot
DC50:  0.58 nM
Assay description: Seed THP-1 cells in 96-well cell culture plates, 5×10 4 cells per well, 100 μL medium. Incubate the cell culture plates in a 37°C, 5% CO 2 incubator overnight. Add 100 nL of prepared compound stock solution and compound-free DMSO stock solution to each well of cells. Incubate the cell culture plates in a 37°C, 5% CO 2 incubator for 24 hours. Add one tablet of protease inhibitor and one tablet of phosphatase inhibitor to 20 mL of cell lysis buffer and mix gently until completely dissolved. Add 200 mM DTT to the 4x sample preparation buffer to make a 4x sample preparation working solution. Dilute 20x electrophoresis buffer to 1x with ultrapure water. Dilute 20x transfer buffer to 1x with ultrapure water and add 20% methanol. Dilute 10x electrophoresis buffer to 1x with ultrapure water. IRAK4 protein primary antibody working solution: add 20μl of IRAK4 antibody to 20mL of blocking solution; β-Actin primary antibody working solution: add 2μl of beta-actin (13E5) Rabbit mAb antibody to 20mL of blocking solution. IRAK4 protein secondary antibody working solution: Donkey Anti-Goat IgG H&L (HRP) is diluted 1/5000 in blocking solution; β-Actin secondary antibody working solution: Anti-rabbit IgG, HRP-linked antibody is diluted 1/10000 in blocking solution. Centrifuge the cell culture plate at 3000rpm for 3min, carefully remove most of the culture medium, invert the cell plate, centrifuge at 300rpm for 30s; add 45μL of lysis buffer to each well, shake at 300rpm for 30s, stand on ice for 30min, and pipette 30 times at 20min; add 6μL of 4x sample preparation working solution to 18μL of protein supernatant for sample preparation, and heat at 70°C for 10min. The remaining protein samples were stored at -80°C. In the precast gel, 8 μl was loaded into each well and electrophoresis was performed at a constant voltage of 120 V for about 60 minutes. The membrane was transferred using a PVDF membrane at a constant current of 300 mA for 1 hour. After transfer, the membrane was blocked with a blocking solution at room temperature for 1 hour. The primary antibody working solution was incubated overnight at 4°C. The membrane was washed with 1×TBST buffer for 3×10 minutes. The secondary antibody working solution was incubated at room temperature for 1 hour. The membrane was washed with 1×TBST buffer for 3×10 minutes and exposed and developed with a color developing solution. The grayscale value of each band was calculated using ImagJ software, and semi-quantitative analysis was performed and DC 50 was calculated using GraphPad Prism8.0. The results are shown in Table 21.
Reference: AROMATIC COMPOUND, PHARMACEUTICAL COMPOSITION CONTAINING SAME, AND USE THEREOF (patent)
Source database: TPDdb