Compound (SMILES): CNc1cc(-n2ccc3cc(C#N)cnc32)ncc1-c1nnc(C2CC3(C2)CN(C(=O)CN2CCN4c5ccc(N6CCC(=O)NC6=O)cc5OC[C@H]4C2)C3)s1
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): IRAK4 (UniProt Q9NWZ3)
POI sequence: MNKPITPSTYVRCLNVGLIRKLSDFIDPQEGWKKLAVAIKKPSGDDRYNQFHIRRFEALLQTGKSPTSELLFDWGTTNCTVGDLVDLLIQNEFFAPASLLLPDAVPKTANTLPSKEAITVQQKQMPFCDKDRTLMTPVQNLEQSYMPPDSSSPENKSLEVSDTRFHSFSFYELKNVTNNFDERPISVGGNKMGEGGFGVVYKGYVNNTTVAVKKLAAMVDITTEELKQQFDQEIKVMAKCQHENLVELLGFSSDGDDLCLVYVYMPNGSLLDRLSCLDGTPPLSWHMRCKIAQGAANGINFLHENHHIHRDIKSANILLDEAFTAKISDFGLARASEKFAQTVMTSRIVGTTAYMAPEALRGEITPKSDIYSFGVVLLEIITGLPAVDEHREPQLLLDIKEEIEDEEKTIEDYIDKKMNDADSTSVEAMYSVASQCLHEKKNKRPDIKKVQQLLQEMTAS
E3 ligase: CRBN (UniProt Q96SW2)
Ligase sequence: MAGEGDQQDAAHNMGNHLPLLPAESEEEDEMEVEDQDSKEAKKPNIINFDTSLPTSHTYLGADMEEFHGRTLHDDDSCQVIPVLPQVMMILIPGQTLPLQLFHPQEVSMVRNLIQKDRTFAVLAYSNVQEREAQFGTTAEIYAYREEQDFGIEIVKVKAIGRQRFKVLELRTQSDGIQQAKVQILPECVLPSTMSAVQLESLNKCQIFPSKPVSREDQCSYKWWQKYQKRKFHCANLTSWPRWLYSLYDAETLMDRIKKQLREWDENLKDDSLPSNPIDFSYRVAACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTSLCCKQCQETEITTKNEIFSLSLCGPMAAYVNPHGYVHETLTVYKACNLNLIGRPSTEHSWFPGYAWTVAQCKICASHIGWKFTATKKDMSPQKFWGLTRSALLPTIPDTEDEISPDKVILCL
Cell line: OCI-Ly10 (Homo sapiens)
Cell line description: African=0.25%; Native American=0.93%; East Asian, North=0.48%; East Asian, South=0%; South Asian=0.05%; European, North=70.29%; European, South=28%.
Mutation; HGNC; HGNC Mutation; HGNC; HGNC
A*02
In situ; Lymph node; 
Caucasian.
Cancer cell line
Assay: HiBiT
Dmax: > 85 %
Assay description: Note: A represents DC50 ≤ 5 nM; B represents 5 nM < DC50 ≤ 50 nM.

In vitro IRAK4 degradation assay in OCI-LY10 cells based on the HiBiT assay.

Methods:
Cells were harvested from the logarithmic growth phase and resuspended in complete medium (IMDM + 20% FBS). Counted using a platelet counter, the cell concentration was adjusted to 1.5 x 106 cells/mL. The cells were seeded into a 96-well plate, and 50 μL of the cell suspension was added to each well. The test compound was diluted with DMSO to a 50 mM stock solution. Prior to the assay, the stock solution was diluted with DMSO to a 10 mM starting concentration. A 4-fold serial dilution was performed across eight concentrations. Starting from the starting concentration, 2 μL of the test compound was added to each concentration and diluted 100-fold in 198 μL of PBS, followed by mixing. Starting from the starting concentration, 5.5 μL of the diluted sample solution or culture medium was added to the cell suspension in a 96-well plate. Final compound concentrations per well were 10,000, 2,500, 625, 156.3, 39.1, 9.8, 2.4, 0.6, 0.15, and 0 nM, respectively. Two replicate wells were set for each sample concentration and incubated at 37°C, 5% CO2 for 4 or 24 hours. A mixture of Nano Glo HiBiT Lytic Buffer: Substate: Protein was added to each well at a ratio of 100:2:1, and the mixture was incubated at room temperature in the dark for 10 minutes. After gentle shaking, the cells were assayed on an Envision 2104 microplate reader and the protein degradation inhibition rate was calculated.
Data Analysis
Protein degradation inhibition rate (%) = (1-As/Ac) × 100.
As: OA of the sample (cells + test compound);
Ac: OA of the control cells (cells + DMSO). Graphpad Prism 6 software was used to fit the DC50 curve and calculate the DC50 value using the formula XY-analysis/Nonlinear regression (curve fit)/Dose response-Inhibition/log (inhibitor) vs. response-Variable slope (four parameters), where inhibitor is the protein degradation inhibition rate mentioned above.
Reference: BIFUNCTIONAL CHIMERIC HETEROCYCLIC COMPOUND OF INTERLEUKIN-1 RECEPTOR-ASSOCIATED KINASE 4, PREPARATION METHOD THEREFOR, PHARMACEUTICAL COMPOSITION THEREOF AND USE THEREOF (patent)
Source database: TPDdb