Compound (SMILES): CC1(C)Cc2cc3n(c2C1)CCN(c1cccc(-c2ncnc4[nH]c(-c5ccc(N6CCN(CC7CCN(c8ccc(N9CCC(=O)NC9=O)cc8)CC7)CC6)nc5)cc24)c1)C3=O
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): BTK (UniProt Q06187)
POI sequence: MAAVILESIFLKRSQQKKKTSPLNFKKRLFLLTVHKLSYYEYDFERGRRGSKKGSIDVEKITCVETVVPEKNPPPERQIPRRGEESSEMEQISIIERFPYPFQVVYDEGPLYVFSPTEELRKRWIHQLKNVIRYNSDLVQKYHPCFWIDGQYLCCSQTAKNAMGCQILENRNGSLKPGSSHRKTKKPLPPTPEEDQILKKPLPPEPAAAPVSTSELKKVVALYDYMPMNANDLQLRKGDEYFILEESNLPWWRARDKNGQEGYIPSNYVTEAEDSIEMYEWYSKHMTRSQAEQLLKQEGKEGGFIVRDSSKAGKYTVSVFAKSTGDPQGVIRHYVVCSTPQSQYYLAEKHLFSTIPELINYHQHNSAGLISRLKYPVSQQNKNAPSTAGLGYGSWEIDPKDLTFLKELGTGQFGVVKYGKWRGQYDVAIKMIKEGSMSEDEFIEEAKVMMNLSHEKLVQLYGVCTKQRPIFIITEYMANGCLLNYLREMRHRFQTQQLLEMCKDVCEAMEYLESKQFLHRDLAARNCLVNDQGVVKVSDFGLSRYVLDDEYTSSVGSKFPVRWSPPEVLMYSKFSSKSDIWAFGVLMWEIYSLGKMPYERFTNSETAEHIAQGLRLYRPHLASEKVYTIMYSCWHEKADERPTFKILLSNILDVMDEES
E3 ligase: CRBN (UniProt Q96SW2)
Ligase sequence: MAGEGDQQDAAHNMGNHLPLLPAESEEEDEMEVEDQDSKEAKKPNIINFDTSLPTSHTYLGADMEEFHGRTLHDDDSCQVIPVLPQVMMILIPGQTLPLQLFHPQEVSMVRNLIQKDRTFAVLAYSNVQEREAQFGTTAEIYAYREEQDFGIEIVKVKAIGRQRFKVLELRTQSDGIQQAKVQILPECVLPSTMSAVQLESLNKCQIFPSKPVSREDQCSYKWWQKYQKRKFHCANLTSWPRWLYSLYDAETLMDRIKKQLREWDENLKDDSLPSNPIDFSYRVAACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTSLCCKQCQETEITTKNEIFSLSLCGPMAAYVNPHGYVHETLTVYKACNLNLIGRPSTEHSWFPGYAWTVAQCKICASHIGWKFTATKKDMSPQKFWGLTRSALLPTIPDTEDEISPDKVILCL
Cell line:  (Homo sapiens)
Assay: HTRF
DC50:  7.2 nM
Assay description: After 6 hours treatment, add 10 microliters 4x lysis buffer to each well; seal the plate and incubate 30 minutes at room temperature on a plate shaker; once the cells are lysed, transfer 16 microliters of cell lysate to a PE 384-well HTRF detection plate; add 4 microliters of pre-mixed HTRF antibodies to each well; cover the plate with a plate sealer, spin at 1000 rpm for 1 minute, incubate overnight at room temperature; read on BMG PheraStar with HTRF protocol (337nm-665nm-620nm). The inhibition (degradation) percentage of the compound was calculated by the following equation: Inhibition percentage of Compound equals 100 minus 100 multiplied by (Signal minus low control) divided by (High control minus low control), wherein Signal equals each test compound group, Low control equals only lysis buffer without cells indicating that BTK is completely degraded, High control equals cell group with added DMSO and without compound indicating microplate readings without BTK degradation, Dmax is the maximum percentage of inhibition (degradation). The IC50 (DC50) value of a compound can be obtained by fitting the following equation: Y equals Bottom plus (TOP minus Bottom) divided by (1 plus ((IC50 divided by X) raised to hillslope)), wherein X and Y are known values, and IC50, Hillslope, Top and Bottom are the parameters obtained by fitting with software. Y is the inhibition percentage (calculated from the equation), X is the concentration of the compound; IC50 is the concentration of the compound when the 50% inhibition is reached. The smaller the IC50 value is, the stronger the inhibitory ability of the compound is. Vice versa, the higher the IC50 value is, the weaker the inhibitory ability of the compound is; Hillslope represents the slope of the fitted curve, generally around 1; Bottom represents the minimum value of the curve obtained by data fitting, which is generally 0% plus or minus 20%; Top represents the maximum value of the curve obtained by data fitting, which is generally 100% plus or minus 20%. The experimental data were fitted by calculating and analyzing with Dotmatics data analysis software.
Reference: PROCESS FOR PREPARING PROTAC BTK DEGRADERS (patent)
Source database: TPDdb