Synthesis of meta-arylphenol derivatives via acid-promoted rearrangement of cyclohexadienones.

A highly effective strategy for the synthesis of meta-arylphenol derivatives through the selective rearrangement of 4-alkyl-4-aryl-2,5-cyclohexadienones under metal-free conditions was developed, in which acid-promoted [1,2]-migration of the aryl group at C-4 occurred exclusively when the alkyl group at C-4 was a methyl group. Treatment of 4-methyl-4-aryl-2,5-cyclohexadienones with 37% HCl in Ac2O at room temperature provided polysubstituted meta-arylphenyl acetates in 75-94% yields. The application of this protocol in the synthesis of polycyclic aromatic compounds was also described.

High-Throughput-Methyl-Reading (HTMR) assay: a solution based on nucleotide methyl-binding proteins enables large-scale screening for DNA/RNA methyltransferases and demethylases.

Epigenetic therapy has significant potential for cancer treatment. However, few small potent molecules have been identified against DNA or RNA modification regulatory proteins. Current approaches for activity detection of DNA/RNA methyltransferases and demethylases are time-consuming and labor-intensive, making it difficult to subject them to high-throughput screening. Here, we developed a fluorescence polarization-based 'High-Throughput Methyl Reading' (HTMR) assay to implement large-scale compound screening for DNA/RNA methyltransferases and demethylases-DNMTs, TETs, ALKBH5 and METTL3/METTL14. This assay is simple to perform in a mix-and-read manner by adding the methyl-binding proteins MBD1 or YTHDF1. The proteins can be used to distinguish FAM-labelled substrates or product oligonucleotides with different methylation statuses catalyzed by enzymes. Therefore, the extent of the enzymatic reactions can be coupled with the variation of FP binding signals. Furthermore, this assay can be effectively used to conduct a cofactor competition study. Based on the assay, we identified two natural products as candidate compounds for DNMT1 and ALKBH5. In summary, this study outlines a powerful homogeneous approach for high-throughput screening and evaluating enzymatic activity for DNA/RNA methyltransferases and demethylases that is cheap, easy, quick, and highly sensitive.

Discovery of a subtype-selective, covalent inhibitor against palmitoylation pocket of TEAD3.

The TEA domain (TEAD) family proteins (TEAD1‒4) are essential transcription factors that control cell differentiation and organ size in the Hippo pathway. Although the sequences and structures of TEAD family proteins are highly conserved, each TEAD isoform has unique physiological and pathological functions. Therefore, the development and discovery of subtype selective inhibitors for TEAD protein will provide important chemical probes for the TEAD-related function studies in development and diseases. Here, we identified a novel TEAD1/3 covalent inhibitor (DC-TEADin1072) with biochemical IC50 values of 0.61 ± 0.02 and 0.58 ± 0.12 µmol/L against TEAD1 and TEAD3, respectively. Further chemical optimization based on DC-TEAD in 1072 yielded a selective TEAD3 inhibitor DC-TEAD3in03 with the IC50 value of 0.16 ± 0.03 µmol/L, which shows 100-fold selectivity over other TEAD isoforms in activity-based protein profiling (ABPP) assays. In cells, DC-TEAD3in03 showed selective inhibitory effect on TEAD3 in GAL4-TEAD (1-4) reporter assays with the IC50 value of 1.15 µmol/L. When administered to zebrafish juveniles, experiments showed that DC-TEAD3in03 reduced the growth rate of zebrafish caudal fins, indicating the importance of TEAD3 activity in controlling proportional growth of vertebrate appendages.

Discovery of a novel 53BP1 inhibitor through AlphaScreen-based high-throughput screening.

Tumor suppressor p53-binding protein 1 (53BP1), a tantem tudor domain (TTD) protein, takes part in DNA Damage Repair (DDR) pathways through the specific recognition of lysine methylation on histones. The dysregulation of 53BP1 is closely related to the development of many diseases including cancer. Moreover, recent studies found that deficiency of 53BP1 could increase the efficiency of precise CRISPR/Cas9 genome editing. Thus, discovery of inhibitor is beneficial to the study of biological functions of 53BP1 and the application of CRISPR/Cas9 genome editing. UNC2170 and its derivatives have been reported as 53BP1 targeted small molecular inhibitors with modest activities. Hence, to discover better 53BP1 inhibitors, we conducted an AlphaScreen assay based high-throughput screening (HTS) and identified a novel and effective 53BP1-TTD inhibitor DP308 which disrupts the binding between 53BP1 and H4K20me2 peptide with an IC50 value of 1.69 ± 0.73 µM. Both Microscale Themophoresis (MST) and Surface Plasmon Resonance (SPR) assays confirmed the direct binding between DP308 and 53BP1-TTD protein with binding affinity (Kd) of about 2.7 µM. Molecular docking studies further suggested that DP308 possibly occupies the H4K20me2 binding pocket of the 53BP1-TTD aromatic cage. These results demonstrated that DP308 is a promising small molecule inhibitor for further optimization towards a more potent chemical probe of 53BP1. Additionally, it could be a potential valuable tool for applying to gene editing therapy by increasing the efficiency of CRISPR/Cas9 genome editing.