Compound (SMILES): Cc1cccc(-c2cnc(Nc3cnn(C4CCN(CCNC(=O)COc5ccc6c(c5)C(=O)N(C5CCC(=O)NC5=O)C6=O)CC4)c3)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:  (Homo sapiens)
Assay: HTRF
DC50:  20 nM
Assay description: Cells (~50k) were plated in Cisbio 96-well low volume white plates. Compounds were dissolved in DMSO and a 3-fold serial dilution was performed using a TECAN D300E. Cells were incubated with the compound overnight. Total-IRAK3 HTRF kit from Cisbio was used for degradation analysis. Cryptate and D2 antibodies were diluted in Detection buffer as per manufacturers recommendation. Then 2 pL of each solution was added to 16 pL lysate. Buffer control (lysis buffer detection buffer), Cryptate control (lysis buffer + cryptate antibody + detection buffer), and Negative control (lysis buffer + cryptate antibody + D2 antibody) were made as per manufacturers recommendation. Post incubation with antibodies the HTRF signal was measured using a Perkin Elmer Envision reader and the HTRF signal was calculated using formula: (Emission at 665 nm/ Emission at 615 nm)* 10,000. All HTRF values were normalized to the average value of DMSO. The average value of the DMSO control wells were set to equal 100% of the relative IRAK3 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 normalized IRAK3 protein levels on the y-axis. The ECso value (the half-maximum effective concentration) of a compound for the degradation of the IRAK3 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 (EC50), D is the correlation coefficient, and A and B are the low and high limits of the fit, respectively). The Ymin was calculated by determining the lowest percentage of target protein remaining following compound treatment. Dmax was Calculated from Ymin (%Dmax = 100-Ymin).
Reference: SUBSTITUTED IMIDAZOPYRAZINE COMPOUNDS AS LIGAND DIRECTED DEGRADERS OF IRAK3 (patent)
Source database: TPDdb