Lysine-targeting, covalent inhibitors of bromodomain BD1 of BET proteins in live cells and animals

Bromodomain (BRD) is an epigenetic reader of acetylated lysine. It has emerged as a therapeutic target for cancer and other diseases. Current nonselective BRD inhibitors (BRDis) face several adverse events (i.e. gastrointestinal toxicity and thrombocytopenia), making the development of target covalent inhibitor (TCI) for BD1/2 as a fresh avenue to overcome safety challenges. We report herein a set of activity-based probes (ABPs; P3-P7) based on various lysine-reactive covalent warheads capable of global profiling of ligandable lysine within BRDs in live cells and animals. Chemoproteomic experiments with P7 by utilizing 2-ethynylbenzaldehyde (EBA) identified 16 endogenous BRDs, thus giving a global landscape of ligandable lysines in BRDs. By further introducing EBA and salicylaldehyde into PLX51107 (a non-covalent BRDi), we generated novel irreversible (BDS1-4) and reversible (BDS5-6) lysine-reactive TCIs. Mass spectrometry and X-ray crystallography confirmed the successful covalent engagement between the covalent warhead and lysine near Kac binding site. BDS4 showed 104-fold selectivity for BD1 over BD2 with prolonged anti-cancer effect than non-covalent BRDi. Importantly, BDS4 retained robust activity against fibrosis in cells and animals in comparison to the marginal effect of BD2 inhibitor RVX-208. Our work serves as a useful tool to delineate the distinct function of BD1 and BD2. 3 × 105 LX-2 cells were evenly distributed into 6-well plates and incubated overnight at 37 °C in 5% CO2. Upon starvation for 24 h in DMEM containing 0.5% FBS, the cells were treated only with TGF-β (20 ng/mL, HZ-1011, Proteintech) or cotreated with TGF-β (20 ng/mL) and indicated compounds (5 μM) for 24 h. After incubation, the cells were collected for RNA sequencing carried out by Sinotech Genomics Co., Ltd. (Shanghai, China) at a cost, and the details are presented below. RNA of cells was isolated by using AG RNAex Pro Reagent (AG21102，Accurate Biology). RNA concentration and quality were determined with the Qubit®3.0 Fluorometer (Life Technologies, USA) and the Nanodrop One spectrophotometer (Thermo Fisher Scientific Inc., USA). Integrity of total RNA was assessed by using the Agilent 2100 Bioanalyzer (Agilent Technologies Inc., USA). One microgram of RNA was used as input material for the RNA sample preparations. Paired-end libraries were synthesized by using the Stranded mRNA-seq Lib Prep Kit for lllumina (RK20349, ABclonal) following Preparation Guide. Purified libraries were quantified by Qubit® 3.0 Fluorometer and validated by Agilent 2100 bioanalyzer to confirm the insert size and calculate the mole concentration. Cluster was generated by cBot with the library diluted to 10 pM and then were sequenced on the Illumina NovaSeq 6000 (Illumina, USA). FPKM values for known gene models were generated by Stringtie (version: 1.3.0).