Compound (SMILES): O=C1CCC(c2coc3ccc4c(NC(=O)CN5CCN(C[C@H]6CC[C@H](n7cc(NC(=O)c8coc(-c9ccnc(NCC%10CC%10)c9)n8)c(C(F)F)n7)CC6)CC5)cccc4c23)C(=O)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: K-562 (Homo sapiens)
Cell line description: African=5.19%; Native American=0%; East Asian, North=8.4%; East Asian, South=0%; South Asian=0%; European, North=43.44%; European, South=42.97%.
Gene fusion; HGNC; HGNC Mutation; HGNC; HGNC
A*11 A*11; B*40,35/39; C*03,05. A*31
Stable (MSS).
In situ; Pleural effusion; 
Caucasian.
Cancer cell line
Assay: HiBiT
DC50:  9.6 nM
Assay description: Evaluation of Target Protein Degradation in K562 IRAK4-HiBiT Cells
Purpose: This experiment will examine the degradation of the target protein IRAK4 by the test compounds in K562 IRAK4-HiBiT cells.
Experimental plan: Day 1 1. Compound preparation (1). Dissolve the powder of the test compound in DMSO to 10 mM as the stock concentration, and manually pipette 9 μL of 10 mM test compound to columns 1 and 13 of the LDV plate; (2). Use a multidrop Combi to add 6 μL of DMSO to columns 2-12 and 14-24; (3). Use Bravo to dilute the test compound 3-fold (3 μL + 6 μL) to columns 1-11 and 13-23; (4). According to the plate layout, use Echo to transfer 25 nL of compound solution (columns 1-24 of the LDV plate) to the experimental plate; (5). Use Echo to transfer 25 nL of 1 mM positive control solution to the assay plate as a 100% degradation control (i.e., LC, HPE), and 25 nL of DMSO to the assay plate as a 0% control (i.e., HC, ZPE). 2. Cell plating
(1). Discard the cell culture medium, wash once with DPBS, trypsinize the cells, count the cells, and prepare a cell suspension of 2×10-5 cells/mL;
(2). Use a MultiDrop Combi at medium speed to add 25 μL/well of the cell suspension to the test plate containing the test compound;
(3). Return the test plate containing cells to the incubator at 37°C and 5% CO2 for 16-18 hours.
Day 2
(1). Use a MultiDrop Combi at high speed to add 25 μL/well of the detection reagent (NanoGlo lysis solution + substrate + LgBit protein) to the test plate and shake for 10 minutes;
(2). Centrifuge at 2000 rpm × 1 minute to remove bubbles;
(3). Read the plate using the Envision, US Luminescence detection method. 3. Data Analysis
The degradation rate (DR) of the test compound was calculated using the following formula: DR (%) = (RLU vehicle control - RLU compound) / (RLU vehicle control - RLU positive control) * 100%, where the vehicle control is the blank control. Degradation rates at different compound concentrations were calculated in Excel. XLFit software was then used to plot inhibition curves and calculate relevant parameters, including minimum degradation rate, maximum degradation rate, and DC50.
The test results are shown in Table 3.
Reference: 2,6-PIPERIDINEDIONE COMPOUND AND APPLICATION THEREOF (patent)
Source database: TPDdb