Compound (SMILES): Cc1cccc(-c2cnc(Nc3ccc(N4CCN(C(=O)COc5ccc6c(c5)C(=O)N(C5CCC(=O)NC5=O)C6=O)CC4)cc3)c3nccn23)c1
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): IRAK3 (UniProt Q9Y616)
POI sequence: MAGNCGARGALSAHTLLFDLPPALLGELCAVLDSCDGALGWRGLAERLSSSWLDVRHIEKYVDQGKSGTRELLWSWAQKNKTIGDLLQVLQEMGHRRAIHLITNYGAVLSPSEKSYQEGGFPNILFKETANVTVDNVLIPEHNEKGILLKSSISFQNIIEGTRNFHKDFLIGEGEIFEVYRVEIQNLTYAVKLFKQEKKMQCKKHWKRFLSELEVLLLFHHPNILELAAYFTETEKFCLIYPYMRNGTLFDRLQCVGDTAPLPWHIRIGILIGISKAIHYLHNVQPCSVICGSISSANILLDDQFQPKLTDFAMAHFRSHLEHQSCTINMTSSSSKHLWYMPEEYIRQGKLSIKTDVYSFGIVIMEVLTGCRVVLDDPKHIQLRDLLRELMEKRGLDSCLSFLDKKVPPCPRNFSAKLFCLAGRCAATRAKLRPSMDEVLNTLESTQASLYFAEDPPTSLKSFRCPSPLFLENVPSIPVEDDESQNNNLLPSDEGLRIDRMTQKTPFECSQSEVMFLSLDKKPESKRNEEACNMPSSSCEESWFPKYIVPSQDLRPYKVNIDPSSEAPGHSCRSRPVESSCSSKFSWDEYEQYKKE
E3 ligase: CRBN (UniProt Q96SW2)
Ligase sequence: MAGEGDQQDAAHNMGNHLPLLPAESEEEDEMEVEDQDSKEAKKPNIINFDTSLPTSHTYLGADMEEFHGRTLHDDDSCQVIPVLPQVMMILIPGQTLPLQLFHPQEVSMVRNLIQKDRTFAVLAYSNVQEREAQFGTTAEIYAYREEQDFGIEIVKVKAIGRQRFKVLELRTQSDGIQQAKVQILPECVLPSTMSAVQLESLNKCQIFPSKPVSREDQCSYKWWQKYQKRKFHCANLTSWPRWLYSLYDAETLMDRIKKQLREWDENLKDDSLPSNPIDFSYRVAACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTSLCCKQCQETEITTKNEIFSLSLCGPMAAYVNPHGYVHETLTVYKACNLNLIGRPSTEHSWFPGYAWTVAQCKICASHIGWKFTATKKDMSPQKFWGLTRSALLPTIPDTEDEISPDKVILCL
Cell line: HEK293T (Homo sapiens)
Cell line description: Highly susceptible to infection by Zika virus (ZIKV).
CVCL_0045 ! HEK293
In situ; Fetal kidney; 
NCBI_TaxID; 28285; Human adenovirus C serotype 5. NCBI_TaxID; 1891767; Simian virus 40 (SV40) (Note=tsA).
Transformed cell line
Assay: InCELL Hunter Test
Dmax:  22 %
Assay description: IRAK3-ePL cellular dose response curve degradation assays were performed by the following protocol. Compounds to be tested were dispensed into a white 384-well tissue-culture treated plate using an acoustic liquid handler. Dilutions were prepared based on a 25 pL assay volume in duplicate 10 point 3 -fold serial dilutions starting with a 10 pM dose. Negative control wells were included, which only contain 0.2% DMSO to calculate 100% signal. Positive control wells containing 30 pM Ataluren (luciferase inhibitor) were included to calculate the background signal level. All wells were backfilled to a final DMSO concentration of 0.2% to ensure DMSO uniformity across wells. IRAK3-ePL expressing cells (IRAK3-ePL Lenti-X 293TCRBN/GSPT1 G575N) were washed, trypsinized, counted, and resuspended in fresh DMEM (Gibco) to give a cell concentration of 200,000 cells/mL. 25 pL of cells (5,000 cells/well) were dispensed into the wells of the 384-well plate prespotted with compounds in the previous step and incubated overnight at 37 °C/5% CO2. Following incubation, the 384-well plate was taken out of the incubator and left at room temperature for 30 min. InCELL hunter reagent was prepared according to manufacturer's instructions (EA reagent, lysis buffer, and substrate reagent in a 1 : 1 :4 ratio, Cat# 96-0002, DiscoverX), which was added to the 384-well plate 25 pL per well. Following the incubation of the plate for 1 h at room temperature, the luminescence signal was read using a ViewLux plate reader. Data was processed and analyzed in ActivityBase software. In short, the average luminescence values of the positive control wells were subtracted from the rest of the wells for background correction, and all luminescence values were normalized to the DMSO control wells. The average value of the DMSO control wells was set to equal 100% of the relative IRAK3-ePL protein levels. Normalized luminescence values were plotted on a graph as a function of compound concentration. Compound concentration was plotted on the x-axis and the corresponding relative IRAK3-ePL protein levels on the y-axis. The EC50 value (the half-maximum effective concentration) of a compound for the degradation of the IRAK3-ePL was calculated using a four-parameter logistic model (sigmoidal dose-response model) (FIT = (A + {(B - A)/l + [(C/x)D]})) where C is the inflection point (EC 50), D is the correlation coefficient, and A and B are the low and high limits of the fit, respectively).
The Dmax was calculated by determining the maximum percentage loss of target protein following compound treatment.
Reference: SUBSTITUTED IMIDAZOPYRAZINE COMPOUNDS AS LIGAND DIRECTED DEGRADERS OF IRAK3 (patent)
Source database: TPDdb