Compound (SMILES): COc1cc2nn(C3CCN(C4CCN(C(=O)c5ccc6c(c5)C(=O)N(C5CCC(=O)NC5=O)C6=O)CC4)CC3)cc2cc1NC(=O)c1cccc(C(F)(F)F)n1
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: > 1000 nM
Assay description: PROTAC-induced IRAK4 protein degradation in THP-1 cells

Based on a Western blot assay, THP-1 cell samples were stained with specific antibodies after gel electrophoresis. The IRAK4 protein degradation activity of the compounds in THP-1 cells was determined by analyzing the location and depth of staining. Compound concentrations included 0 μM, 0.3 μM, 1 μM, and 3 μM, and cells were treated for 8, 16, 24, and 48 hours. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control protein in this experiment.

THP-1 cells were plated in 6-well plates at a density of 1.5 x 10? cells per ml and incubated in a cell culture incubator set at 37°C and 5% CO? for 2 hours. Compounds were diluted in DMSO to 0.6 mM, 0.2 mM, and 0.06 mM, respectively. 10 μL of compound solution was added to the corresponding wells and incubated in the incubator for 16, 24, and 48 hours, respectively. Drug-treated THP-1 cells were harvested from the wells and lysed on wet ice for 30 minutes using 120 μL of RIPA lysis buffer containing protease inhibitors, phosphatase inhibitor solution II, and phosphatase inhibitor solution III. The cells were centrifuged at high speed for 5 minutes at low temperature, and the supernatant was collected. Protein concentrations of the cell samples were determined according to the Pierce? BCA Protein Assay Kit instructions.

Samples were adjusted to the same concentration using lysis buffer and LDS sample buffer containing 1 M DTT. Samples were heated at 95°C for 5 minutes and centrifuged at high speed at low temperature. 20 μL of the prepared protein sample and 4 μL of PageRuler Prestained Protein Ladder were added to the gel wells. The gel was run at 80 V for 0.5 hours, then the voltage was increased to 120 V and continued for 1.5 hours. Remove the gel and transfer the proteins to an IBlot? 2 transfer membrane stack at 20V. After successful transfer, cut out the bands between 65kDa and 40kDa, and between 40kDa and 30kDa, respectively. Block the membrane with 5% skim milk for 1 hour at room temperature. Wash the membrane three times with 1x TBST and incubate overnight at 4°C with IRAK4 antibody working solution. Discard the IRAK4 antibody working solution and wash the membrane three times with 1x TBST. Incubate the membrane with Goat Anti-Rabbit IgG H&L (HRP) working solution and Goat Anti-Mouse IgG H&L (HRP) working solution at room temperature for 1 hour. Discard the antibody working solution and wash the membrane three times with 1x TBST. Prepare a luminescent solution mixture by mixing equal volumes of the reagents in the SuperSignal? West Femto Maximum Sensitivity Substrate kit according to the kit instructions. Incubate the membrane for 1 minute before removing the membrane and exposing it to light. The concentration required for 50% degradation of IRAK4 by western blotting 24 hours after administration is shown in Table 4.
Reference: FIVE-MEMBERED RING-FUSED SIX-MEMBERED COMPOUND, PREPARATION METHOD THEREFOR, AND PHARMACEUTICAL COMPOSITION AND USE THEREOF (patent)
Source database: TPDdb