Structure-based discovery of a new series of nucleoside-derived ring-opening PRMT5 inhibitors.

The ubiquitous methyltransferases employing SAM as the methyl donor have emerged as potential targets in many disease treatments, especially in anticancer. Therefore, developing SAM-competitive inhibitors of methyltransferases is of great interest to the drug research. To explore this direction, herein, we rationally designed a series of nucleoside derivatives as potent PRMT5 inhibitors with novel scaffold. The representative compounds A2 and A8 exhibited highly potent PRMT5 inhibition activity as well as good selectivity over other PRMTs and PKMTs. Further cellular experiments revealed that compounds A2 and A8 potently reduced the level of sDMA and inhibited the proliferation of Z-138 and MOLM-13 cell lines by inducing apoptosis. Moreover, compounds A8 which had favorable pharmacokinetic properties exhibited potent antitumor efficacy without the loss of body weight in a subcutaneous MOLM-13 xenograft model. In summary, our efforts provided a series of novel nucleoside analogs as potent PRMT5 inhibitors and may also offer a new strategy to develop SAM analogs as other methyltransferases' inhibitors.

An optimized protocol for isolation of murine pancreatic single cells with high yield and purity.

Here, we present a protocol for rapidly isolating single cells from the mouse pancreas, minimizing damage caused by digestive enzymes in exocrine cells. We guide you through steps to optimize the dissection sequence, enzyme composition, and operational procedures, resulting in high yields of viable pancreatic single cells. This protocol can be applied across a wide range of research areas, including single-cell sequencing, gene expression profiling, primary cell culture, and even the development of spheroids or organoids. For complete details on the use and execution of this protocol, please refer to Jiang et al. (2023).1.

Discovery of a PROTAC degrader for METTL3-METTL14 complex.

N6-methyladenosine (m6A) methylation is the most abundant type of RNA modification that is mainly catalyzed by the METTL3-METTL14 methyltransferase complex. This complex has been linked to multiple cancers and is considered a promising therapeutic target for acute myeloid leukemia (AML). However, only a few METTL3 inhibitors targeting the catalytic activity were developed recently. Here, we present the discovery of WD6305 as the potent and selective proteolysis-targeting chimera (PROTAC) degrader of METTL3-METTL14 complex. WD6305 suppresses m6A modification and the proliferation of AML cells, and promotes apoptosis much more effectively than its parent inhibitor. WD6305 also affects a variety of signaling pathways related to the development and proliferation of AML. Collectively, our study reveals PROTAC degradation of METTL3-METTL14 complex as a potential anti-leukemic strategy and provides desirable chemical tool for further understanding METTL3-METTL14 protein functions.

Discovery of a New-Generation S-Adenosylmethionine-Noncompetitive Covalent Inhibitor Targeting the Lysine Methyltransferase Enhancer of Zeste Homologue 2.

The first-generation enhancer of zeste homologue 2 (EZH2) inhibitors suffer from several limitations, such as high dosage, cofactor S-adenosylmethionine (SAM) competition, and acquired drug resistance. Development of covalent EZH2 inhibitors that are noncompetitive with cofactor SAM offers an opportunity to overcome these disadvantages. The structure-based design of compound 16 (BBDDL2059) as a highly potent and selective covalent inhibitor of EZH2 is presented in this context. 16 inhibits EZH2 enzymatic activity at sub-nanomolar concentrations and achieves low nanomolar potencies in cell growth inhibition. The kinetic assay revealed that 16 is noncompetitive with the cofactor SAM, providing the basis for its superior activity over noncovalent and positive controls by reducing competition with cofactor SAM and offering a preliminary proof for its covalent inhibition nature. Mass spectrometric analysis and washout experiments firmly establish its covalent inhibition mechanism. This study demonstrates that covalent inhibition of EZH2 can offer a new opportunity for the development of promising new-generation drug candidates.

Structural Modification and Pharmacological Evaluation of Substituted Quinoline-5,8-diones as Potent NSD2 Inhibitors.

The histone lysine methyltransferase NSD2 is overexpressed, translocated, or mutated in multiple types of cancers and has emerged as an attractive therapeutic target. However, the development of small-molecule NSD2 inhibitors is still in its infancy, and selective and efficacious NSD2 inhibitors are highly desirable. Here, in view of the structural novelty of the reported NSD2 inhibitor DA3003-1, we conducted a comprehensive structural optimization based on the quinoline-5,8-dione scaffold. Compound 15a was identified possessing both high NSD2 inhibitory activity and potent anti-proliferative effects in the cell. Meanwhile, compound 15a has an excellent pharmacokinetic profile with high oral bioavailability. Further, this compound was found to display significant antitumor efficacy with desirable safety profile in the multiple myeloma xenograft mice models, thus warranting it as a promising candidate for further investigation.

Structure-activity relationship study of a series of nucleoside derivatives bearing sulfonamide scaffold as potent and selective PRMT5 inhibitors.

Protein arginine methyltransferase 5 (PRMT5) is a promising target for the treatment of malignant tumors. The discovery of nucleoside-derived inhibitors against PRMT5 with novel scaffold has been challenging. Herein, we report our effort on the design and synthesis of nucleoside derivatives bearing sulfonamide scaffold as potent PRMT5 inhibitors. The representative compound 23n was identified as a potent and selective PRMT5 inhibitor with an IC50 value of 8 nM. Molecular docking study demonstrated the binding mode of compound 23n and illustrated its inhibitory activity to PRMT5. The Trimethyl Lock prodrug strategy was used to afford prodrug 36 with lower polarity which could rapidly release the active compound 23n after entering the tumor cells. Cell-based assays revealed that the prodrug 36 restrained the proliferation of Z-138 and MOLM-13 cells and suppressed methylation of PRMT5 substrate more potently than 23n. Additionally, both compound 23n and 36 exerted antiproliferative effects against Z-138 cells mainly by inducing apoptosis effectively rather than arresting cell cycle. Thus, compounds 23n and 36 represent a series of potent PRMT5 inhibitor with novel scaffold.

Discovery of a potent and selective proteolysis targeting chimera (PROTAC) degrader of NSD3 histone methyltransferase.

Nuclear receptor binding SET domain protein 3 (NSD3) is an attractive potential target in the therapy for human cancers. Herein, we report the discovery of a series of small-molecule NSD3 degraders based on the proteolysis targeting chimera (PROTAC) strategy. The represented compound 8 induces NSD3 degradation with DC50 values of 1.43 and 0.94 µM in NCI-H1703 and A549 lung cancer cells, respectively, and shows selectivity over two other NSD proteins. 8 reduces histone H3 lysine 36 methylation and induces apoptosis and cell cycle arrest in lung cancer cells. Moreover, the RNA sequencing and immunohistochemistry assays showed that 8 downregulates NSD3-associated gene expression. Significantly, 8, but not 1 (a reported NSD3-PWWP antagonist) could inhibit the cell growth of NCI-H1703 and A549 cells. A single administration of 8 effectively decreases the NSD3 protein level in lung cancer xenograft models. Therefore, this study demonstrated that inducing NSD3 degradation is a more effective approach inhibiting the function of NSD3 than blocking the NSD3-PWWP domain, which may provide a potential therapeutic approach for lung cancer.

Discovery of novel benzimidazole derivatives as potent p300 bromodomain inhibitors with anti-proliferative activity in multiple cancer cells.

Adenovirus E1A-associated 300-kD protein (p300) bromodomain, which regulates gene expression by recognizing acetylated lysine (KAc) of histone, is a promising target for the treatment of cancer. Herein, a series of potent p300 bromodomain inhibitors with novel CBP30-based scaffolds was discovered through bioisosterism and conformational restriction strategies. The most promising compound 1u showed more potent inhibitory activity (IC50 = 49 nM) against p300 bromodomain and anti-proliferative activity in various cancer cell lines compared to CBP30. Moreover, 1u suppressed the expression of c-Myc and induced G1/G0 phase arrest and apoptosis in OPM-2 cells more potently than CBP30. This study provides new lead compounds for further research on the biological functions of p300.

Structure-Aided Design, Synthesis, and Biological Evaluation of Potent and Selective Non-Nucleoside Inhibitors Targeting Protein Arginine Methyltransferase 5.

PRMT5 is a major type II protein arginine methyltransferase and plays important roles in diverse cellular processes. Overexpression of PRMT5 is implicated in various types of cancer. Many efforts have been made to develop potent and selective PRMT5 inhibitors, the most potent of which is usually derived from nucleoside structures. Here, we designed a novel series of non-nucleoside PRMT5 inhibitors through the structure-aided drug design approach. SAR exploration and metabolic stability optimization led to the discovery of compound 41 as a potent PRMT5 inhibitor with good selectivity. Additionally, compound 41 exerted antiproliferative effects against A375 cells by inducing apoptosis and potently inhibited the methyltransferase activity of PRMT5 in cells. Moreover, it showed attractive pharmacokinetic properties and markedly suppressed the tumor growth in an A375 tumor xenograft model. These results clearly indicate that 41 is a highly potent and selective non-nucleoside PRMT5 inhibitor.

Design, synthesis and biological evaluation of (R)-5-methylpyrrolidin-2-ones as p300 bromodomain inhibitors with Anti-Tumor activities in multiple tumor lines.

p300/CBP bromodomain plays an important role in transcriptional regulation, and its overexpression is closely related to various diseases such as cancers. Two inhibitors of this target are currently in clinical trials but only CCS1477 (A1) have been published with the chemical structure. Herein, we modified the structure of CCS1477 based on the principle of bioisosterism and reasonable scaffold hopping, and discovered a series of new p300 bromodomain inhibitors with improved potency. More tumor cell lines sensitive to p300/CBP bromodomain inhibition were also identified. Among our new inhibitors, (R)-5-methylpyrrolidin-2-one derivitive B4 was the most potent one which showed comparable inhibitory activity against p300 (IC50 = 0.060 µM) as lead A1 (IC50 = 0.064 µM) at molecular level, and performed more potent proliferation inhibitory activities on various tumor cells than A1. Further we found that compound B4 had the high cell permeability and overcame the defect of the high efflux rate of A1, which could also explain the possible reason why B4 showed more potent inhibitory activities on sensitive tumor cells than lead A1. Western blotting analysis proved the target effects that B4 could suppress the expression of c-Myc and reduce H3K27 acetylation significantly. Liver microsomal metabolic stability assay and hERG channel inhibition evaluation illustrate compound B4 is metabolic stabilizable in human liver microsomes and has no hERG risk, which further demonstrate the good drug-likeness of B4. Therefore, compound B4 is a promising compound for further optimization and development.

Structure-Based Discovery of Potent CARM1 Inhibitors for Solid Tumor and Cancer Immunology Therapy.

CARM1 is a protein arginine methyltransferase and acts as a transcriptional coactivator regulating multiple biological processes. Aberrant expression of CARM1 has been related to the progression of multiple types of cancers, and therefore CARM1 was considered as a promising drug target. In the present work, we report the structure-based discovery of a series of N1-(3-(pyrimidin-2-yl)benzyl)ethane-1,2-diamines as potent CARM1 inhibitors, in which compound 43 displays high potency and selectivity. With the advantage of excellent tissue distribution, compound 43 demonstrated good in vivo efficacy for solid tumors. Furthermore, from the detailed immuno-oncology study with MC38 C57BL/6J xenograft model, we confirmed that this chemical probe 43 has profound effects in tumor immunity, which paves the way for future studies on the modulation of arginine post-translational modification that could be utilized in solid tumor treatment and cancer immunotherapy.

5-Aminonaphthalene derivatives as selective nonnucleoside nuclear receptor binding SET domain-protein 2 (NSD2) inhibitors for the treatment of multiple myeloma.

Approximately 20% of multiple myeloma (MM) are caused by a chromosomal translocation t (4; 14) that leads to the overexpression of the nuclear receptor binding SET domain-protein 2 (NSD2) histone methyltransferase. NSD2 catalyzes the methylation of lysine 36 on histone H3 (H3K36me2) and is associated with transcriptionally active regions. Using high-throughput screening (HTS) with biological analyses, a series of 5-aminonaphthalene derivatives were designed and synthesized as novel NSD2 inhibitors. Among all the prepared compounds, 9c displayed a good NSD2 inhibitory activity (IC50 = 2.7 µM) and selectivity against both SET-domain-containing and non-SET-domain-containing methyltransferases. Preliminary research indicates the inhibition mechanism of compound 9c by significantly suppressed the methylation of H3K36me2. Compound 9c specifically inhibits the proliferation of the human B cell precursor leukemia cell line RS4:11 and the human myeloma cell line KMS11 by inducing cell cycle arrest and apoptosis with little cytotoxicity. It has been reported that the anti-cancer effect of compound 9c is partly achieved by completely suppressing the transcriptional activation of NSD2-targeted genes. When administered intraperitoneally at 25 mg/kg, compound 9c suppressed the tumor growth of RS4:11 xenografts in vivo and no body weight loss was detected in the tested SCID mice.