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.

Natural product 1,2,3,4,6-penta-O-galloyl-ß-D-glucopyranose is a reversible inhibitor of glyceraldehyde 3-phosphate dehydrogenase.

Aerobic glycolysis, also known as the Warburg effect, is a hallmark of cancer cell glucose metabolism and plays a crucial role in the activation of various types of immune cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of D-glyceraldehyde 3-phosphate to D-glycerate 1,3-bisphosphate in the 6th critical step in glycolysis. GAPDH exerts metabolic flux control during aerobic glycolysis and therefore is an attractive therapeutic target for cancer and autoimmune diseases. Recently, GAPDH inhibitors were reported to function through common suicide inactivation by covalent binding to the cysteine catalytic residue of GAPDH. Herein, by developing a high-throughput enzymatic screening assay, we discovered that the natural product 1,2,3,4,6-penta-O-galloyl-ß-D-glucopyranose (PGG) is an inhibitor of GAPDH with Ki = 0.5 µM. PGG blocks GAPDH activity by a reversible and NAD+ and Pi competitive mechanism, suggesting that it represents a novel class of GAPDH inhibitors. In-depth hydrogen deuterium exchange mass spectrometry (HDX-MS) analysis revealed that PGG binds to a region that disrupts NAD+ and inorganic phosphate binding, resulting in a distal conformational change at the GAPDH tetramer interface. In addition, structural modeling analysis indicated that PGG probably reversibly binds to the center pocket of GAPDH. Moreover, PGG inhibits LPS-stimulated macrophage activation by specific downregulation of GAPDH-dependent glucose consumption and lactate production. In summary, PGG represents a novel class of GAPDH inhibitors that probably reversibly binds to the center pocket of GAPDH. Our study sheds new light on factors for designing a more potent and specific inhibitor of GAPDH for future therapeutic applications.

Design, synthesis, and biological evaluation of novel carbazole derivatives as potent DNMT1 inhibitors with reasonable PK properties.

The DNA methyltransferases (DNMTs) were found in mammals to maintain DNA methylation. Among them, DNMT1 was the first identified, and it is an attractive target for tumour chemotherapy. DC_05 and DC_517 have been reported in our previous work, which is non-nucleoside DNMT1 inhibitor with low micromolar IC50 values and significant selectivity towards other S-adenosyl-L-methionine (SAM)-dependent protein methyltransferases. In this study, through a process of similarity-based analog searching, a series of DNMT1 inhibitors were designed, synthesized, and evaluated as anticancer agents. SAR studies were conducted based on enzymatic assays. And most of the compounds showed strong inhibitory activity on human DNMT1, especially WK-23 displayed a good inhibitory effect on human DNMT1 with an IC50 value of 5.0 µM. Importantly, the pharmacokinetic (PK) profile of WK-23 was obtained with quite satisfying oral bioavailability and elimination half-life. Taken together, WK-23 is worth developing as DNMT1-selective therapy for the treatment of malignant tumour.

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.

Discovery of novel CBP bromodomain inhibitors through TR-FRET-based high-throughput screening.

The cAMP-responsive element binding protein (CREB) binding protein (CBP) and adenoviral E1A-binding protein (P300) are two closely related multifunctional transcriptional coactivators. Both proteins contain a bromodomain (BrD) adjacent to the histone acetyl transferase (HAT) catalytic domain, which serves as a promising drug target for cancers and immune system disorders. Several potent and selective small-molecule inhibitors targeting CBP BrD have been reported, but thus far small-molecule inhibitors targeting BrD outside of the BrD and extraterminal domain (BET) family are especially lacking. Here, we established and optimized a TR-FRET-based high-throughput screening platform for the CBP BrD and acetylated H4 peptide. Through an HTS assay against an in-house chemical library containing 20 000 compounds, compound DC_CP20 was discovered as a novel CBP BrD inhibitor with an IC50 value of 744.3 nM. This compound bound to CBP BrD with a KD value of 4.01 µM in the surface plasmon resonance assay. Molecular modeling revealed that DC_CP20 occupied the Kac-binding region firmly through hydrogen bonding with the conserved residue N1168. At the celluslar level, DC_CP20 dose-dependently inhibited the proliferation of human leukemia MV4-11 cells with an IC50 value of 19.2 µM and markedly downregulated the expression of the c-Myc in the cells. Taken together, the discovery of CBP BrD inhibitor DC_CP20 provides a novel chemical scaffold for further medicinal chemistry optimization and a potential chemical probe for CBP-related biological function research. In addition, this inhibitor may serve as a promising therapeutic strategy for MLL leukemia by targeting CBP BrD protein.

Discovery of alkoxy benzamide derivatives as novel BPTF bromodomain inhibitors via structure-based virtual screening.

Bromodomain PHD finger transcription factor (BPTF), a bromodomain-containing protein, plays a crucial role in the regulation of downstream gene expression through the specific recognition of lysine acetylation on bulk histones. The dysfunction of BPTF is closely involved with the development and progression of many human diseases, especially cancer. Therefore, BPTF bromodomain has become a promising drug target for epigenetic cancer therapy. However, unlike BET family inhibitors, few BPTF bromodomain inhibitors have been reported. In this study, by integrating docking-based virtual screening with biochemical analysis, we identified a novel selective BPTF bromodomain inhibitor DCB29 with the IC50 value of 13.2 ±â€¯1.6 µM by homogenous time-resolved fluorescence resonance energy transfer (HTRF) assays. The binding between DCB29 and BPTF was confirmed by NMR and SPR. Molecular docking disclosed that DCB29 occupied the pocket of acetylated H4 peptide substrate and provided detailed SAR explanations for its derivatives. Collectively, DCB29 presented great potential as a powerful tool for BPTF-related biological research and further medicinal chemistry optimization.

Discovery of potent DOT1L inhibitors by AlphaLISA based High Throughput Screening assay.

DOT1L (the disruptor of telomeric silencing 1-like), through its methyltransferase activity of H3K79, plays essential roles in transcriptional regulation, cell cycle regulation, and DNA damage response. In addition, DOT1L is believed to be involved in the development of MLL-rearranged leukemia driven by the MLL (mixed-lineage leukemia) fusion proteins, which thus to be a crucial target for leukemia therapy. Hence, discovering of novel DOT1L inhibitors has been in a great demand. In this study, we initiated the discovering process from setting up the AlphaLISA based High Throughput Screening (HTS) assay of DOT1L. Combining with radioactive inhibition assay and Surface Plasmon Resonance (SPR) binding assay, we identified compound 3 and its active analogues as novel DOT1L inhibitors with IC50 values range from 7 µM to 20 µM in vitro. Together with the analysis of structure activity relationships (SAR) and binding modes of these compounds, we provided clues to assist in the future development of more potent DOT1L inhibitors. Moreover, compounds 3 and 9 effectively inhibited the proliferation of MLL-rearranged leukemia cells MV4-11, which could induce cell cycle arrest and apoptosis. In conclusion, we developed a HTS platform based on AlphaLISA method for screening and discovery of DOT1L novel inhibitor, through which we discovered compound 3 and its analogues as potent DOT1L inhibitors with promising MLL-rearranged leukemia therapeutic application.

Discovery and biological evaluation of thiobarbituric derivatives as potent p300/CBP inhibitors.

Histone acetyltransferases (HATs) relieve transcriptional repression by preferentially acetylation of ε-amino group of lysine residues on histones. Dysregulation of HATs is strongly correlated with etiology of several diseases especially cancer, thus highlighting the utmost significance of the development of small molecule inhibitors against this potential therapeutic target. In the present study, through virtual screening and iterative optimization, we identified DCH36_06 as a bona fide, potent p300/CBP inhibitor. DCH36_06 mediated p300/CBP inhibition leading to hypoacetylation on H3K18 in leukemic cells. The suppression of p300/CBP activity retarded cell proliferation in several leukemic cell lines. In addition, DCH36_06 arrested cell cycle at G1 phase and induced apoptosis via activation of capase3, caspase9 and PARP that elucidated the molecular mechanism of its anti-proliferation activity. In transcriptome analysis, DCH36_06 altered downstream gene expression and apoptotic pathways-related genes verified by real-time PCR. Importantly, DCH36_06 blocked the leukemic xenograft growth in mice supporting its potential for in vivo use that underlies the therapeutic potential for p300/CBP inhibitors in clinical translation. Taken together, our findings suggest that DCH36_06 may serve as a qualified chemical tool to decode the acetylome code and open up new opportunities for clinical intervention.

Design, synthesis and anti leukemia cells proliferation activities of pyrimidylaminoquinoline derivatives as DOT1L inhibitors.

A series of novel pyrimidylaminoquinoline derivatives 8(a-i) and 9(a-i) containing amino side chain, and the bisaminoquinoline analogs 3(b-e) have been designed and synthesized by structural modifications on a lead DOT1L inhibitor, 3a. All the compounds have been evaluated for their DOT1L inhibitory activities. The results showed that most of the compounds have strong anti DOT1L activities. Compounds 3e, 8h and 9e are the most potential ones from each category with the IC50 values of 1.06 ±â€¯0.35 µM, 5.72 ±â€¯1.56 µM and 3.55 ±â€¯1.28 µM, respectively. Such inhibitors expressed significant binding interactions with DOT1L by surface plasmon resonance (SPR)-based binding assay. The results of molecular docking experiments suggested that they could occupy the SAM binding pocket of DOT1L. Compounds 8h and 9e exhibited better inhibitory activities but poor selectivities against the both MLL-rearranged MV4-11 cells and the non MLL-rearranged Kasumi-1 cells than those of 3a and 3e, which suggested that the introduction of the amino side chain would be beneficial for their anti leukemia cells proliferation activities, possibly due to the improvement of the fat solubility. Additionally, the direct cellular inhibition activities were found that compound 9e could effectively down-regulate both the level of H3k79 methylation and MLL-rearranged leukemia gene expression of Hoxa9 and Meis1 in MV4-11 in the qRT-PCR and western blot studies. These observations suggested DOT1L was one of the potential targets but perhaps not the most pivotal one for these compounds, which made their poor selectivities against leukemia cells proliferation.

Design, synthesis of novel benzimidazole derivatives as ENL inhibitors suppressing leukemia cells viability via downregulating the expression of MYC.

Eleven-Nineteen-Leukemia Protein (ENL) containing YEATS domain, a potential drug target, has emerged as a reader of lysine acetylation. SGC-iMLLT bearing with benzimidazole scaffold was identified as an effective ENL inhibitor, but with weak activity against mixed-lineage leukemia (MLL)-rearranged cells proliferation. In this study, a series of compounds were designed and synthesized by structural optimization on SGC-iMLLT. All the compounds have been evaluated for their ENL inhibitory activities. The results showed that compounds 13, 23 and 28 are the most potential ones with the IC50 values of 14.5 ± 3.0 nM, 10.7 ± 5.3 nM, and 15.4 ± 2.2 nM, respectively, similar with that of SGC-iMLLT. They could interact with ENL protein and strengthen its thermal stability in vitro. Among them, compound 28 with methyl phenanthridinone moiety replacement of indazole in SGC-iMLLT, exhibited significantly inhibitory activities towards MV4-11 and MOLM-13 cell lines with IC50 values of 4.8 µM and 8.3 µM, respectively, exhibiting ∼7 folds and ∼9 folds more potent inhibition of cell growth than SGC-iMLLT. It could also increase the ENL thermal stability while SGC-iMLLT had no obvious effect on leukemia cells. Moreover, compound 28 could downregulate the expression of target gene MYC either alone or in combination with JQ-1 in cells, which was more effective than SGC-iMLLT. Besides, in vivo pharmacokinetic studies showed that the PK properties for compound 28 was much improved over that of SGC-iMLLT. These observations suggested compound 28 was a potential ligand for ENL-related MLL chemotherapy.

A potent PGK1 antagonist reveals PGK1 regulates the production of IL-1ß and IL-6.

Glycolytic metabolism enzymes have been implicated in the immunometabolism field through changes in metabolic status. PGK1 is a catalytic enzyme in the glycolytic pathway. Here, we set up a high-throughput screen platform to identify PGK1 inhibitors. DC-PGKI is an ATP-competitive inhibitor of PGK1 with an affinity of K d = 99.08 nmol/L. DC-PGKI stabilizes PGK1 in vitro and in vivo, and suppresses both glycolytic activity and the kinase function of PGK1. In addition, DC-PGKI unveils that PGK1 regulates production of IL-1ß and IL-6 in LPS-stimulated macrophages. Mechanistically, inhibition of PGK1 with DC-PGKI results in NRF2 (nuclear factor-erythroid factor 2-related factor 2, NFE2L2) accumulation, then NRF2 translocates to the nucleus and binds to the proximity region of Il-1ß and Il-6 genes, and inhibits LPS-induced expression of these genes. DC-PGKI ameliorates colitis in the dextran sulfate sodium (DSS)-induced colitis mouse model. These data support PGK1 as a regulator of macrophages and suggest potential utility of PGK1 inhibitors in the treatment of inflammatory bowel disease.

Discovery of High-Affinity Inhibitors of the BPTF Bromodomain.

The dysfunctional bromodomain PHD finger transcription factor (BPTF) exerts a pivotal influence in the occurrence and development of many human diseases, particularly cancers. Herein, through the structural decomposition of the reported BPTF inhibitor TP-238, the effective structural fragments were synthetically modified to obtain our lead compound DC-BPi-03. DC-BPi-03 was identified as a novel BPTF-BRD inhibitor with a moderate potency (IC50 = 698.3 ± 21.0 nM). A structure-guided structure-activity relationship exploration gave rise to two BPTF inhibitors with much higher affinities, DC-BPi-07 and DC-BPi-11. Notably, DC-BPi-07 and DC-BPi-11 show selectivities 100-fold higher than those of other BRD targets. The cocrystal structures of BPTF in complex with DC-BPi-07 and DC-BPi-11 demonstrate the rationale of chemical efforts from the atomic level. Further study showed that DC-BPi-11 significantly inhibited leukemia cell proliferation.

Targeting p300/CBP Attenuates Hepatocellular Carcinoma Progression through Epigenetic Regulation of Metabolism.

Targeting epigenetics in cancer has emerged as a promising anticancer strategy. p300/CBP is a central regulator of epigenetics and plays an important role in hepatocellular carcinoma (HCC) progression. Tumor-associated metabolic alterations contribute to the establishment and maintenance of the tumorigenic state. In this study, we used a novel p300 inhibitor, B029-2, to investigate the effect of targeting p300/CBP in HCC and tumor metabolism. p300/CBP-mediated acetylation of H3K18 and H3K27 increased in HCC tissues compared with surrounding noncancerous tissues. Conversely, treatment with B029-2 specifically decreased H3K18Ac and H3K27Ac and displayed significant antitumor effects in HCC cells in vitro and in vivo. Importantly, ATAC-seq and RNA-seq integrated analysis revealed that B029-2 disturbed metabolic reprogramming in HCC cells. Moreover, B029-2 decreased glycolytic function and nucleotide synthesis in Huh7 cells by reducing H3K18Ac and H3K27Ac levels at the promoter regions of amino acid metabolism and nucleotide synthesis enzyme genes, including PSPH, PSAT1, ALDH18A1, TALDO1, ATIC, and DTYMK. Overexpression of PSPH and DTYMK partially reversed the inhibitory effect of B029-2 on HCC cells. These findings suggested that p300/CBP epigenetically regulates the expression of glycolysis-related metabolic enzymes through modulation of histone acetylation in HCC and highlights the value of targeting the histone acetyltransferase activity of p300/CBP for HCC therapy. SIGNIFICANCE: This study demonstrates p300/CBP as a critical epigenetic regulator of glycolysis-related metabolic enzymes in HCC and identifies the p300/CBP inhibitor B029-2 as a potential therapeutic strategy in this disease.

Discovery of 8-Methyl-pyrrolo[1,2-a]pyrazin-1(2H)-one Derivatives as Highly Potent and Selective Bromodomain and Extra-Terminal (BET) Bromodomain Inhibitors.

The bromodomain and extra-terminal (BET) family proteins have recently emerged as promising drug targets for cancer therapy. In this study, identification of an 8-methyl-pyrrolo[1,2-a]pyrazin-1(2H)-one fragment (47) as a new binder to the BET bromodomains and the subsequent incorporation of fragment 47 to the scaffold of ABBV-075, which recently entered Phase I clinical trials, enabled the generation of a series of highly potent BET bromodomain inhibitors. Further druggability optimization led to the discovery of compound 38 as a potential preclinical candidate. Significantly, compared with ABBV-075, which exhibits a 63-fold selectivity for BRD4(1) over EP300, compound 38 demonstrates an excellent selectivity for the BET bromodomain family over other bromodomains, with an ∼1500-fold selectivity for BRD4(1) over EP300. Orally administered 38 achieves a complete inhibition of tumor growth with a tumor growth inhibition (TGI) of 99.7% accompanied by good tolerability.

Design, synthesis and biological evaluation of benzo[cd]indol-2(1H)-ones derivatives as BRD4 inhibitors.

Compound 1 bearing with benzo [cd]indol-2(1H)-one scaffold was identified as an effective BRD4 inhibitor through the AlphaScreen-based high-throughput screening and its high-resolution crystal structure with BRD4_BD1 protein. A series of 48 compounds were designed and synthesized by structural optimization on compound 1. All the compounds have been evaluated for their BRD4 inhibitory activities. The results showed that compounds 23, 24, 28 and 44 are the most potential ones with the IC50 values of 1.02 µM, 1.43 µM, 1.55 µM and 3.02 µM, respectively. According to their co-crystal structures in complex with BRD4_BD1 and the protein thermal shift assays, the binding modes were revealed that the additional indirect hydrogen bonds and hydrophobic interactions make such four compounds more active than 1 against BRD4. Furthermore, compounds 1, 23 and 44 were chosen to evaluate for their antiproliferative activities on the MLL-AF4-expression acute leukemia cell line (MV4-11), other cancer cell lines (MDA-MB-231, A549, 22Rv1) and the non-cancer cell lines (HUV-EC-C, MRC5, RPTEC). The results showed that these compounds exhibited good and selective inhibitory activities against MV4-11 cells with the IC50 values of 11.67 µM, 5.55 µM, and 11.54 µM, respectively, and could act on the cell proliferation by blocking cell cycle at G1 phase. They could markedly down-regulate the expressions of the c-Myc, Bcl-2 and CDK6 oncogenes in MV4-11 in the qRT-PCR and western blot studies, which further demonstrated that compound 1 and its derivatives could serve as a promising therapeutic strategy for MLL leukemia by targeting BRD4_BD1 protein.