Compound (SMILES): Cn1c(=O)n(C2CCC(=O)NC2=O)c2cccc(C#CCOC34CCC(CN5CCC6(CC5)CC(N5C=C(NC(=O)c7cnn8ccc(N9C[C@H]%10C[C@@H]9CO%10)nc78)C(C(F)F)N5)C6)(CC3)CC4)c21
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): IRAK4 (UniProt Q9NWZ3)
POI sequence: MNKPITPSTYVRCLNVGLIRKLSDFIDPQEGWKKLAVAIKKPSGDDRYNQFHIRRFEALLQTGKSPTSELLFDWGTTNCTVGDLVDLLIQNEFFAPASLLLPDAVPKTANTLPSKEAITVQQKQMPFCDKDRTLMTPVQNLEQSYMPPDSSSPENKSLEVSDTRFHSFSFYELKNVTNNFDERPISVGGNKMGEGGFGVVYKGYVNNTTVAVKKLAAMVDITTEELKQQFDQEIKVMAKCQHENLVELLGFSSDGDDLCLVYVYMPNGSLLDRLSCLDGTPPLSWHMRCKIAQGAANGINFLHENHHIHRDIKSANILLDEAFTAKISDFGLARASEKFAQTVMTSRIVGTTAYMAPEALRGEITPKSDIYSFGVVLLEIITGLPAVDEHREPQLLLDIKEEIEDEEKTIEDYIDKKMNDADSTSVEAMYSVASQCLHEKKNKRPDIKKVQQLLQEMTAS
E3 ligase: CRBN (UniProt Q96SW2)
Ligase sequence: MAGEGDQQDAAHNMGNHLPLLPAESEEEDEMEVEDQDSKEAKKPNIINFDTSLPTSHTYLGADMEEFHGRTLHDDDSCQVIPVLPQVMMILIPGQTLPLQLFHPQEVSMVRNLIQKDRTFAVLAYSNVQEREAQFGTTAEIYAYREEQDFGIEIVKVKAIGRQRFKVLELRTQSDGIQQAKVQILPECVLPSTMSAVQLESLNKCQIFPSKPVSREDQCSYKWWQKYQKRKFHCANLTSWPRWLYSLYDAETLMDRIKKQLREWDENLKDDSLPSNPIDFSYRVAACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTSLCCKQCQETEITTKNEIFSLSLCGPMAAYVNPHGYVHETLTVYKACNLNLIGRPSTEHSWFPGYAWTVAQCKICASHIGWKFTATKKDMSPQKFWGLTRSALLPTIPDTEDEISPDKVILCL
Cell line: Human PBMCs (Homo sapiens)
Assay: Flow Cytometry
DC50:  0.65 nM
Assay description: Compounds Degrade IRAK4 in Human PBMCs
Objective
Flow cytometry was used to assess the degradation of monocyte IRAK4 by compounds in human peripheral blood mononuclear cells (hPBMCs).
Methods
Frozen hPBMCs were thawed and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% Pen+Strep, then rehydrated at 37°C for 1 hour.
On day 1, 100 μL of cells were seeded per well of a 96-well plate at a density of 2*10^5.
Compounds were dissolved in DMSO to a final concentration of 10 mM, diluted 10 times in culture medium at a 1:5 ratio, and added to the cells, resulting in a maximum final concentration of 2 μM. A well containing cells supplemented with 0.1% DMSO served as a positive control.
The cells were cultured at 37°C in a 5% CO2 incubator for 24 hours.
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.
After centrifugation, the 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.
After centrifugation, the cells were washed with FACS buffer.
The cell membrane was permeabilized by adding 150 μl of Perm III reagent and incubated at 4°C for 30 minutes.
After centrifugation, the cells were washed with FACS buffer.
The 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 using a flow cytometer.
Data processing: Flow cytometric data were analyzed using FlowJo software. Data display and DC50 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 2.
The results demonstrate that the compounds of the present disclosure have significant degradation activity against the IRAK4 target in human PBMC cells.
Reference: IRAK4 DEGRADER AND USE THEREOF (patent)
Source database: TPDdb