Compound (SMILES): CC(OCC#Cc1cccc2c1n(C)c(=O)n2C1CCC(=O)NC1=O)C(=O)N1CCC2(CC1)CC(n1cc(NC(=O)c3cnn4ccc(N5C[C@H]6C[C@@H]5CO6)nc34)c(C(F)F)n1)C2
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): IRAK4 (UniProt Q9NWZ3)
POI sequence: MNKPITPSTYVRCLNVGLIRKLSDFIDPQEGWKKLAVAIKKPSGDDRYNQFHIRRFEALLQTGKSPTSELLFDWGTTNCTVGDLVDLLIQNEFFAPASLLLPDAVPKTANTLPSKEAITVQQKQMPFCDKDRTLMTPVQNLEQSYMPPDSSSPENKSLEVSDTRFHSFSFYELKNVTNNFDERPISVGGNKMGEGGFGVVYKGYVNNTTVAVKKLAAMVDITTEELKQQFDQEIKVMAKCQHENLVELLGFSSDGDDLCLVYVYMPNGSLLDRLSCLDGTPPLSWHMRCKIAQGAANGINFLHENHHIHRDIKSANILLDEAFTAKISDFGLARASEKFAQTVMTSRIVGTTAYMAPEALRGEITPKSDIYSFGVVLLEIITGLPAVDEHREPQLLLDIKEEIEDEEKTIEDYIDKKMNDADSTSVEAMYSVASQCLHEKKNKRPDIKKVQQLLQEMTAS
E3 ligase: CRBN (UniProt Q96SW2)
Ligase sequence: MAGEGDQQDAAHNMGNHLPLLPAESEEEDEMEVEDQDSKEAKKPNIINFDTSLPTSHTYLGADMEEFHGRTLHDDDSCQVIPVLPQVMMILIPGQTLPLQLFHPQEVSMVRNLIQKDRTFAVLAYSNVQEREAQFGTTAEIYAYREEQDFGIEIVKVKAIGRQRFKVLELRTQSDGIQQAKVQILPECVLPSTMSAVQLESLNKCQIFPSKPVSREDQCSYKWWQKYQKRKFHCANLTSWPRWLYSLYDAETLMDRIKKQLREWDENLKDDSLPSNPIDFSYRVAACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTSLCCKQCQETEITTKNEIFSLSLCGPMAAYVNPHGYVHETLTVYKACNLNLIGRPSTEHSWFPGYAWTVAQCKICASHIGWKFTATKKDMSPQKFWGLTRSALLPTIPDTEDEISPDKVILCL
Cell line: SU-DHL-2 (Homo sapiens)
Cell line description: Mutation; HGNC; HGNC
In situ; Pleural effusion; 
Caucasian.
Cancer cell line
Assay: Flow Cytometry
DC50:  3 nM
Assay description: Degradation of IRAK4 by Compounds in SU-DHL-2 and OCI-LY3 Cells
(1) Experimental Purpose
Flow cytometry was used to detect the degradation level of IRAK4 protein in SU-DHL-2 cell lines.
Experimental Methods
Step 1: SU-DHL-2 cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% double antibody.
Step 2: On day 1, cells were seeded in 96-well plates at a volume of 2*105 per well. Compounds were dissolved in DMSO to a final concentration of 10 mM, diluted in culture medium at a concentration gradient of 1:5 for 10 times, and added to cells to achieve a maximum final concentration of 2 μM. The wells containing 0.1% DMSO were used as positive controls. The cells were cultured in an incubator at 37°C with 5% CO2 for 24 hours.
Step 3: After centrifugation, the cells were washed with FACS buffer. Cells were stained for live/dead cell differentiation using the LIVE/DEAD? Fixable Blue Dead Cell Stain Kit. Monocytes were surface-stained with PE Mouse Anti-Human CD14 and incubated at 4°C for 30 minutes.
Step 4: After centrifugation, cells were washed with FACS buffer. An equal volume of Fix Buffer I was added to the cultured cells and the cells were fixed at 37°C for 10 minutes.
Step 5: After centrifugation, cells were washed with FACS buffer. 150 μL of Perm III reagent was added and the cells were incubated at 4°C for 30 minutes to permeabilize the cell membrane. After centrifugation, cells were washed with FACS buffer.
Step 6: Cells were stained with Alexa Fluor 647 Mouse Anti-Human IRAK4 Antibody and incubated at 4°C for 1 hour. Flow cytometric analysis was performed.
Data processing: Flow cytometric data were analyzed using FlowJo software. Data display and DC50 (half-maximal degradation concentration) calculation were performed using Prism 8.0.2 (GraphPad). Experimental Results
The results of the degradation activity of the compounds of the present disclosure against IRAK4 are shown in Table 1.
The Dmax values of the compounds of the present disclosure against SU-DHL-2 and OCI-LY3 cells were generally above 50%, with the highest reaching approximately 90%.
The results demonstrate that the compounds of the present disclosure have strong degradation activity against IRAK4 in both SU-DHL-2 and OCI-LY3 cells.
Reference: IRAK4 DEGRADER AND USE THEREOF (patent)
Source database: TPDdb