Compound (SMILES): Cc1ncsc1-c1ccc(CNC(=O)[C@@H]2C[C@@H](O)CN2C(=O)[C@H](C)NC(=O)COCC(C)(COCCOCCOCCNC(=O)C[C@@H]2N=C(c3ccc(Cl)cc3)c3c(sc(C)c3C)-n3c(C)nnc32)COCCOCCOCCNC(=O)C[C@@H]2N=C(c3ccc(Cl)cc3)c3c(sc(C)c3C)-n3c(C)nnc32)cc1
Modality: PROteolysis-TArgeting Chimera (PROTAC)
Target protein (POI): BRD2 (UniProt P25440)
POI sequence: MLQNVTPHNKLPGEGNAGLLGLGPEAAAPGKRIRKPSLLYEGFESPTMASVPALQLTPANPPPPEVSNPKKPGRVTNQLQYLHKVVMKALWKHQFAWPFRQPVDAVKLGLPDYHKIIKQPMDMGTIKRRLENNYYWAASECMQDFNTMFTNCYIYNKPTDDIVLMAQTLEKIFLQKVASMPQEEQELVVTIPKNSHKKGAKLAALQGSVTSAHQVPAVSSVSHTALYTPPPEIPTTVLNIPHPSVISSPLLKSLHSAGPPLLAVTAAPPAQPLAKKKGVKRKADTTTPTPTAILAPGSPASPPGSLEPKAARLPPMRRESGRPIKPPRKDLPDSQQQHQSSKKGKLSEQLKHCNGILKELLSKKHAAYAWPFYKPVDASALGLHDYHDIIKHPMDLSTVKRKMENRDYRDAQEFAADVRLMFSNCYKYNPPDHDVVAMARKLQDVFEFRYAKMPDEPLEPGPLPVSTAMPPGLAKSSSESSSEESSSESSSEEEEEEDEEDEEEEESESSDSEEERAHRLAELQEQLRAVHEQLAALSQGPISKPKRKREKKEKKKKRKAEKHRGRAGADEDDKGPRAPRPPQPKKSKKASGSGGGSAALGPSGFGPSGGSGTKLPKKATKTAPPALPTGYDSEEEEESRPMSYDEKRQLSLDINKLPGEKLGRVVHIIQAREPSLRDSNPEEIEIDFETLKPSTLRELERYVLSCLRKKPRKPYTIKKPVGKTKEELALEKKRELEKRLQDVSGQLNSTKKPPKKANEKTESSSAQQVAVSRLSASSSSSDSSSSSSSSSSSDTSDSDSG
E3 ligase: VHL (UniProt P40337)
Ligase sequence: MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPRPVLRSVNSREPSQVIFCNRSPRVVLPVWLNFDGEPQPYPTLPPGTGRRIHSYRGHLWLFRDAGTHDGLLVNQTELFVPSLNVDGQPIFANITLPVYTLKERCLQVVRSLVKPENYRRLDIVRSLYEDLEDHPNVQKDLERLTQERIAHQRMGD
Cell line: RCC4/VHL (Homo sapiens)
Cell line description: Mutation; HGNC; HGNC
CVCL_0498 ! RCC4
In situ; Kidney; 
Caucasian.
Cancer cell line
Assay: Western Blot
DC50:  19 nM
Assay description: Figure 20 shows Table 3, which summarises the DCso values in nM of BRD2, BRD4_long and BRD4_short to quantify the potency of SIM1 and its analogue MN674 at degrading BRD2 and BRD4 isoforms. DC50 values are in nM and are obtained from the dose-dependent curves generated in Figure 22.
Compound MN674 was designed and synthesised as another analogue of SIM1. The structure of MN674 is the same as for SIM1, but contains one less PEG unit in the linkers to the JQ1 binders. one less PEG unit in the linkers to the JQ1 binders. MN674 was tested in the mammalian cell line RCC4- HA-VHL in a dose-response degradation assay. The assay involved immunoblots (see Figure 21 for raw western blot data and Figure 22 for dose-response curves) displaying 6 hour treatments of eight 5-fold serial dilutions from 500nM to 6.4pM of each compound. Quantification of protein levels of BRD2 and BRD4 were performed to measure DC50s values that are tabulated in Table 3 (Figure 20) Figure 21 shows the results of immunoblots demonstrating dose-dependent protein degradation of Brd2 and Brd4 using heterotrivalent PROTACs SIM1 and MN674 in RCC4-HA-VHL. RCC4-HA-VHL cells were exposed to eight 5-fold serial dilutions (from 500nM to 6.4pM) of heterotrivalent Bet-Bet-VHL PROTACs SIM1 and MN674 for 6 hour treatments, before lysates were harvested and analysed on immunoblots (Figure 21). A Bio-Rad MP imaging system was used for fluorescence detection of BRD2 (IRDye 800CW), BRD4_long (IRDye 800CW), BRD4_short (IRDye 800CW) and house-keeping gene Tubulin (Rhodamine). The intensities of the bands were determined using ImageLab software and all BET protein intensities normalised to Tubulin in order to convert them into a percentage band intensity rationalised to the 0.1% DMSO-only control. These intensities were then plotted in GraphPad Prism for Dose-Response [Inhibition] Variable slope (four parameter) analyses to generate the curves for BRD2 (Figure 22a) and BRD4 (Figure 22b), to determine the DC50 values presented in Table 3 (Figure 20). The experiment is representative of n=2 i.e. biological duplicate. Overall, MN674 was shown to be comparable in degradation potencies to SIM1, albeit with slightly differential outcome depending on the different cell lines.
Reference: Improved small molecules (patent)
Source database: TPDdb