Design and synthesis of the first PARP-1 and proteasome dual inhibitors to treat breast cancer.

PARP-1 is a crucial factor in repairing DNA single strand damage and maintaining genomic stability. However, the use of PARP-1 inhibitors is limited to combination with chemotherapy or radiotherapy, or as a single agent for indications carrying HRR defects. The ubiquitin-proteasome system processes the majority of cellular proteins and is the principal manner by which cells regulate protein homeostasis. Proteasome inhibitors can cooperate with PARP-1 inhibitors to inhibit DNA homologous recombination repair function. In this study, we designed and synthesized the first dual PARP-1 and proteasome inhibitor based on Olaparib and Ixazomib. Both compounds 42d and 42i exhibited excellent proliferation inhibition and dual-target synergistic effects on cells that were insensitive to PARP-1 inhibitors. Further mechanistic evaluations revealed that 42d and 42i could inhibit homologous recombination repair function by down-regulating the expression of BRCA1 and RAD51. Additionally, 42i induced more significant apoptosis and showed better inhibitory effect on cell proliferation in clonal formation experiments in breast cancer cells than 42d. In summary, our study presented a new class of dual PARP-1/proteasome inhibitors with significant synergistic effects for the treatment of breast cancer.

Beyond Loading: Functions of Plant ARGONAUTE Proteins.

ARGONAUTE (AGO) proteins are key components of the RNA-induced silencing complex (RISC) that mediates gene silencing in eukaryotes. Small-RNA (sRNA) cargoes are selectively loaded into different members of the AGO protein family and then target complementary sequences to in-duce transcriptional repression, mRNA cleavage, or translation inhibition. Previous reviews have mainly focused on the traditional roles of AGOs in specific biological processes or on the molecular mechanisms of sRNA sorting. In this review, we summarize the biological significance of canonical sRNA loading, including the balance among distinct sRNA pathways, cross-regulation of different RISC activities during plant development and defense, and, especially, the emerging roles of AGOs in sRNA movement. We also discuss recent advances in novel non-canonical functions of plant AGOs. Perspectives for future functional studies of this evolutionarily conserved eukaryotic protein family will facilitate a more comprehensive understanding of the multi-faceted AGO proteins.

Discovery of novel reversible inhibitor of DprE1 based on benzomorpholine for the treatment of tuberculosis.

About a quarter of the world's population is infected with Mycobacterium tuberculosis, equivalent to about two billion people. With the emergence of multidrug-resistant tuberculosis, those existing anti-tuberculosis drugs no longer meet the demand for cure anymore; there is an urgent need for the development of new anti-tuberculosis drugs. Decaprenylphosphoryl-ß-D-ribose 2´-epimerase (DprE1) has been proven to be a potential antimycobacterial target, and several inhibitors have entered clinical trial. Herein, we designed and synthesized a series of compounds based on the indole and benzomorpholine by using the strategy of scaffold hopping. The preferred compound B18 showed strong antimycobacterial activity in H37Rv and drug-resistant clinical isolates. In addition, compound B18 did not exhibit antimycobacterial efficacy against other species of strains. Subsequently, the target of B18 was identified as DprE1 by analyzing spontaneous compound-resistant mutation data, and a docking study was performed to illustrate the binding mode between B18 and DprE1. In general, compound B18 is compatible to current DprE1 inhibitors, even higher phosphodiesterase 6C selectivity and plasma protein binding rate, which represent a new type of effective reversible DprE1 inhibitor. IMPORTANCE Drug therapy remains the cornerstone of tuberculosis (TB) treatment, yet first-line anti-tuberculosis drugs are associated with significant adverse effects that can compromise patient outcomes. Moreover, prolonged and widespread use has led to an alarming rise in drug-resistant strains of Mycobacterium tuberculosis, including multidrug-resistant [MDR-tuberculosis (TB)] and extensively drug-resistant (XDR-TB) forms. Urgent action is needed to develop novel anti-tuberculosis agents capable of overcoming these challenges. We report that compound B18, a decaprenylphosphoryl-ß-D-ribose 2´-epimerase inhibitor with a benzomorpholine backbone, exhibits potent activity against not only the non-pathogenic strain H37Ra, but also the pathogenic strain H37Rv and clinical MDR and XDR strains. Preliminary druggability studies indicate that B18 possesses high safety and acceptable pharmacokinetic properties, rendering it a promising candidate for further development as a novel anti-tuberculosis agent.

Design, synthesis and biological evaluation of a series of novel pyrrolo[2,3-d]pyrimidin/pyrazolo[3,4-d]pyrimidin-4-amine derivatives as FGFRs-dominant multi-target receptor tyrosine kinase inhibitors for the treatment of gastric cancer.

Gastric cancer is the second most lethal cancer across the world. With the progress in therapeutic approaches, the 5-year survival rate of early gastric cancer can reach > 95%. However, the prognosis and survival time of advanced gastric cancer is still somber. Therefore, more effective targeted therapies for gastric cancer treatment are urgently needed. FGFR, VEGFR and other receptor tyrosine kinases have recently been suggested as potential targets for gastric cancer treatment. We herein report the discovery of pyrrolo[2,3-d]pyrimidin/pyrazolo[3,4-d]pyrimidin-4-amine derivatives as a new class of FGFRs-dominant multi-target receptor tyrosine kinase inhibitors. SAR assessment identified the most active compounds 8f and 8k, which showed excellent inhibitory activity against a variety of receptor tyrosine kinases. Moreover, 8f and 8k displayed excellent potency in the SNU-16 gastric cancer cell line. Furthermore, 8f and 8k could inhibit FGFR1 phosphorylation and downstream signaling pathways as well as induce cell apoptosis. In vivo, 8f and 8k suppress tumor growth in the SNU-16 xenograft model without inducing obvious toxicity. These findings raise the possibility that compounds 8f and 8k might serve as potential agents for the treatment of gastric cancer.

A Facile Total Synthesis of Kilogram-Scale Production of SKLB1039: A Novel and Selective Hexahydroisoquinolin-Containing EZH2 Inhibitor.

BACKGROUND: SKLB1039 is a potent, highly selective, and orally bioavailable EZH2 inhibitor, which significantly inhibited breast tumor growth and metastasis in pre-clinical studies. In a previously reported synthesis of SKLB1039, the yields of several steps were low, which led to an overall yield of less than 10%. In addition, flash chromatography was required for the purification of several intermediates using this route. OBJECTIVE: To optimize the synthesis and establish an efficient commercial-scale method for the production of SKLB1039. METHODS: The reaction time, solvent, reactant ratio, temperature, and mode of addition of reactants in the reductive amination, hydrolysis, hexahydroisoquinoline formation, hydrogenolysis, condensation and Suzuki crosscoupling reactions were optimized. RESULTS: A chromatography-free seven-step process starting from a commercially available material was developed that afforded SKLB1039 in 36% overall yield with > 99% purity. CONCLUSION: A cost-effective, high-yielding, and convergent kilo-scale synthesis for the EZH2 inhibitor SKLB1039 was developed. The operation was simple, and the pure product was easily obtained without column chromatography. This method will be economical and convenient for the subsequent industrial scale-up production of SKLB1039, which will be conducive for this promising EZH2 inhibitor to enter clinical studies of its antitumor effects.

Design, synthesis and biological evaluation of 7-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)-2,3-dihydro-1H-inden-1-one derivatives as potent FAK inhibitors for the treatment of ovarian cancer.

Focal adhesion kinase (FAK) promotes tumor progression by intracellular signal transduction and regulation of gene expression and protein turnover, which is a compelling therapeutic target for various cancer types, including ovarian cancer. However, the clinical responses of FAK inhibitors remain unsatisfactory. Here, we describe the discovery of FAK inhibitors using a scaffold hopping strategy. Structure-activity relationship (SAR) exploration identified 36 as a potent FAK inhibitor, which exhibited inhibitory activities against FAK signaling in vitro. Treatment with 36 not only decreased migration and invasion of PA-1 cells, but also reduced expression of MMP-2 and MMP-9. Moreover, 36 inhibited tumor growth and metastasis, and no obvious adverse effects were observed during the in vivo study. These results revealed the potential of FAK inhibitor 36 for treatment of ovarian cancer.

A novel fixed-dose combination treatment for chronic hepatitis C, based on NS5A inhibitor fopitasvir and NS5B inhibitor sofosbuvir.

Drug resistance caused by the extreme genetic variability of zhe hepatitis C virus has rendered effective combinations of drugs indispensable in the treatment of chronic hepatitis C (CHC). Herein, we developed a fixed-dose combination (FDC) treatment containing the NS5B inhibitor sofosbuvir (SOF) and the NS5A inhibitor fopitasvir (FOP). Then the dissolution behavior of FOP in FOP/SOF FDC was improved by co-micronizing FOP with lactose. The enhanced dissolution rate of FOP in the FDC was in good agreement with the behavior of the FOP singledrug tablet. In addition, pharmacokinetic studies showed that both FOP and SOF in the FDC exhibited similar characteristics (area under the curve, Cmax, Tmax, and T1/2) as those of tablets containing FOP or SOF alone. These results revealed that the FOP/SOF FDC represents a potential therapeutic option for the treatment of CHC.

Design and Synthesis of EZH2-Based PROTACs to Degrade the PRC2 Complex for Targeting the Noncatalytic Activity of EZH2.

EZH2 mediates both PRC2-dependent gene silencing via catalyzing H3K27me3 and PRC2-independent transcriptional activation in various cancers. Given its oncogenic role in cancers, EZH2 has constituted a compelling target for anticancer therapy. However, current EZH2 inhibitors only target its methyltransferase activity to downregulate H3K27me3 levels and show limited efficacy because of inadequate suppression of the EZH2 oncogenic activity. Therefore, therapeutic strategies to completely block the oncogenic activity of EZH2 are urgently needed. Herein, we report a series of EZH2-targeted proteolysis targeting chimeras (PROTACs) that induce proteasomal degradation of PRC2 components, including EZH2, EED, SUZ12, and RbAp48. Preliminary assessment identified E7 as the most active PROTAC molecule, which decreased PRC2 subunits and H3K27me2/3 levels in various cancer cells. Furthermore, E7 strongly inhibited transcriptional silencing mediated by EZH2 dependent on PRC2 and transcriptional activation mediated by EZH2 independent of PRC2, showing significant antiproliferative activities against cancer cell lines dependent on the enzymatic and nonenzymatic activities of EZH2.

Design and synthesis of (E)-1,2-diphenylethene-based EZH2 inhibitors.

Enhancer of zeste homolog 2 (EZH2) serves as the catalytic subunit of the polycomb repression complex 2 (PRC2), which is implicated in cancer progression metastasis and poor prognosis. Based on our EZH2 inhibitor SKLB1049 with low nanomolar activity, we extended the "tail" region to get a series of (E)-1,2-diphenylethene derivatives as novel EZH2 inhibitors. SAR exploration and preliminary assessment led to the discovery of the potent novel EZH2 inhibitor 9b (EZH2WT IC50 = 22.0 nM). Compound 9b inhibited the proliferation of WSU-DLCL2 and SU-DHL-4 cell lines (IC50 = 1.61 µM and 2.34 µM, respectively). The biological evaluation showed that 9b was a potent inhibitor for wild-type EZH2 and greatly reduced the overall levels of H3K27me3 in a concentration-dependent manner. Further study indicated that 9b could significantly induce apoptosis of SU-DHL-4 cells. These findings indicated that 9b would be an attractive lead compound for further optimization and evaluation.

[A rapid method based on advanced polymer chromatography for analyzing the products of the acid-catalyzed depolymerization of lignosulfonate].

In this study, a fast and efficient method for the separation and analysis of the products in the acid-catalyzed depolymerization of commercially available sodium lignosulfonate has been developed. The depolymerized lignosulfonate products were well separated and characterized by advanced polymer chromatography (APC) employing four ACQUITY APC XT columns in series and a ultraviolet detector. The developed method enabled the detection of relative-low-molecular-mass lignin degradation products with peak molecular weights (Mp) of 720, 490, and 260 Da, and an extremely low polydispersity index (PDI) of 1, indicating almost complete conversion of lignosulfonate to smaller molecules. The effects of reaction temperature, time, and catalyst/lignin ratio on the reaction products were systematically investigated. High yields of depolymerization (>80%) could be obtained under the mild acid-catalyzed conditions at 130℃ for 60 min using a catalyst/lignin ratio of 2.334:1. Preliminary studies also indicated that the mild acid-catalytic mechanism is unaffected by the reaction time, temperature, or catalyst concentration, thus suggesting the specificity of the catalytic procedure employed.

Rapid determination of lignosulfonate depolymerization products by advanced polymer chromatography.

Depolymerized lignin products are very complex mixtures. Based on a depolymerization solution of commercially available sodium lignosulfonate under mild conditions, a fast and efficient method for the separation and direct characterization of the degree and efficiency of the acid-catalyzed depolymerization of lignin was developed in this study. Using an ultraviolet detector, the depolymerized lignosulfonate products were well separated and characterized according to the relative molar mass distribution on an advanced polymer chromatographic system with three ethylene-bridged hybrid columns having small pore sizes (45 Å) in series and tetrahydrofuran as the mobile phase. The developed advanced polymer chromatography method enabled the detection of low-molecular-weight lignin degradation products (Mn  = 260-1100 Da) with high peak resolutions in less than 7.2 min. Furthermore, preliminary advanced polymer chromatography studies to determine the influence of reaction temperature on the depolymerized products indicated that the depolymerized aromatics fell in several molecular weight ranges with an extremely low dispersity. This new approach can be used for the rapid analysis of lignin depolymerization products.

Synthesis and biological evaluation of benzomorpholine derivatives as novel EZH2 inhibitors for anti-non-small cell lung cancer activity.

The histone lysine methyltransferase EZH2 has been reported to play important roles in cancer aggressiveness, metastasis and poor prognosis. In this study, a series of benzomorpholine derivatives were synthesized and biologically evaluated as EZH2 inhibitors. The target compounds were obtained in good yields from 3-amino-5-bromo-2-hydroxybenzoic acid via cyclization, Suzuki coupling and amidation as the key steps. A preliminary optimization study led to the discovery of several potent novel EZH2 inhibitors (6b, 6c, 6x and 6y). Moreover, 6y inhibited the A549 and NCI-H1975 cell lines (IC50 = 1.1 µM and 1.1 µM, respectively). Further studies indicated that 6y can reduce EZH2 expression in intact cells and cause cell arrest in the G2/M phase.

Novel selective TOPK inhibitor SKLB-C05 inhibits colorectal carcinoma growth and metastasis.

The mitogen-activated protein kinase (MAPK) signaling pathway member T-LAK cell-originated protein kinase/PDZ-binding kinase (TOPK/PBK) is closely involved in tumorigenesis and progression. Its overexpression in colorectal carcinoma (CRC) exacerbates tumor malignancy, promotes metastasis and results in dismal prognosis. Therefore, targeting TOPK is a promising approach for CRC therapy. Here, we report the development of a TOPK selective inhibitor SKLB-C05, with subnanomolar inhibitory potency. In vitro, SKLB-C05 exhibited excellent cytotoxicity and anti-migration and invasion activity on TOPK high-expressing CRC cells and induced cell apoptosis. These activities could attribute to its inhibition of TOPK downstream signaling including extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and c-Jun N-terminal kinase 1, 2, and 3 (JNK1/2/3), as well as downregulation of FAK/Src- MMP signaling. Furthermore, SKLB-C05 disrupted cell mitosis and blocked CRC cell cycle. In vivo, oral administration of SKLB-C05 at concentrations of 20 and 10 mg kg-1·day-1 dramatically attenuated CRC tumor xenograft growth and completely suppressed hepatic metastasis of HCT116 cells, respectively. Thus, these findings suggest that SKLB-C05 is a specific TOPK inhibitor with potent anti-CRC oncogenic activity in vitro and in vivo.