Discovery of novel pyrazolo[1,5-a]pyrimidine derivatives as potent reversal agents against ABCB1-mediated multidrug resistance.

The P-glycoprotein (ABCB1)-mediated multidrug resistance (MDR) has emerged as a significant impediment to the efficacy of cancer chemotherapy in clinical therapy, which could promote the development of effective agents for MDR reversal. In this work, we reported the exploration of novel pyrazolo [1,5-a]pyrimidine derivatives as potent reversal agents capable of enhancing the sensitivity of ABCB1-mediated MDR MCF-7/ADR cells to paclitaxel (PTX). Among them, compound 16q remarkably increased the sensitivity of MCF-7/ADR cells to PTX at 5 µM (IC50 = 27.00 nM, RF = 247.40) and 10 µM (IC50 = 10.07 nM, RF = 663.44). Compound 16q could effectively bind and stabilize ABCB1, and does not affect the expression and subcellular localization of ABCB1 in MCF-7/ADR cells. Compound 16q inhibited the function of ABCB1, thereby increasing PTX accumulation, and interrupting the accumulation and efflux of the ABCB1-mediated Rh123, thus resulting in exhibiting good reversal effects. In addition, due to the potent reversal effects of compound 16q, the abilities of PTX to inhibit tubulin depolymerization, and induce cell cycle arrest and apoptosis in MCF-7/ADR cells under low-dose conditions were restored. These results indicate that compound 16q might be a promising potent reversal agent capable of revising ABCB1-mediated MDR, and pyrazolo [1,5-a]pyrimidine might represent a novel scaffold for the discovery of new ABCB1-mediated MDR reversal agents.

Strategies that regulate Hippo signaling pathway for novel anticancer therapeutics.

As a highly conserved signaling network across different species, the Hippo pathway is involved in various biological processes. Dysregulation of the Hippo pathway could lead to a wide range of diseases, particularly cancers. Extensive researches have demonstrated the close association between dysregulated Hippo signaling and tumorigenesis as well as tumor progression. Consequently, targeting the Hippo pathway has emerged as a promising strategy for cancer treatment. In fact, there has been an increasing number of reports on small molecules that target the Hippo pathway, exhibiting therapeutic potential as anticancer agents. Importantly, some of Hippo signaling pathway inhibitors have been approved for the clinical trials. In this work, we try to provide an overview of the core components and signal transduction mechanisms of the Hippo signaling pathway. Furthermore, we also analyze the relationship between Hippo signaling pathway and cancers, as well as summarize the small molecules with proven anti-tumor effects in clinical trials or reported in literatures. Additionally, we discuss the anti-tumor potency and structure-activity relationship of the small molecule compounds, providing a valuable insight for further development of anticancer agents against this pathway.

Discovery of novel arylamide derivatives containing piperazine moiety as inhibitors of tubulin polymerisation with potent liver cancer inhibitory activity.

In this work, a series of novel arylamide derivatives containing piperazine moiety were designed and synthesised as tubulin polymerisation inhibitors. Among 25 target compounds, compound 16f (MY-1121) exhibited low nanomolar IC50 values ranging from 0.089 to 0.238 µM against nine human cancer cells. Its inhibitory effects on liver cancer cells were particularly evident with IC50 values of 89.42 and 91.62 nM for SMMC-7721 and HuH-7 cells, respectively. Further mechanism studies demonstrated that compound 16f (MY-1121) could bind to the colchicine binding site of ß-tubulin and directly act on ß-tubulin, thus inhibiting tubulin polymerisation. Additionally, compound 16f (MY-1121) could inhibit colony forming ability, cause morphological changes, block cell cycle arrest at the G2 phase, induce cell apoptosis, and regulate the expression of cell cycle and cell apoptosis related proteins in liver cancer cells. Overall, the promising bioactivities of compound 16f (MY-1121) make the novel arylamide derivatives have the value for further development as tubulin polymerisation inhibitors with potent anticancer activities.

Discovery of a novel Coumarin-Dihydroquinoxalone derivative MY-673 as a tubulin polymerization inhibitor capable of inhibiting the ERK pathway with potent anti-gastric cancer activities.

As a class of microtubule targeting agents, colchicine binding site inhibitors (CBSIs) are considered as promising drug candidates for cancer therapy. However, due to adverse reactions, there are currently no CBSIs approved by FDA for cancer treatment. Therefore, extensive efforts are still encouraged to find novel CBSIs with different chemical structures and better anticancer efficacies. In this work, we designed and synthesized a new coumarin-dihydroquinoxalone derivative, MY-673, and evaluated its anticancer potency in vitro and in vivo. We confirmed that MY-673 was a potent CBSI that it not only inhibited tubulin polymerization, but also exhibited significant inhibitory potency on the growth of 13 cancer cells with IC50 values from 11.7 nM to 395.9 nM. Based on the results of kinase panel screening, MY-673 could inhibit ERK (extracellular regulated protein kinases) pathways-related kinases. We further confirmed that MY-673 could inhibit ERK signaling pathway in MGC-803 and HGC-27 cells, and then affected the expression level of SMAD4 protein in TGF-ß (transforming growth factor ß) /SMAD (small mother against decapentaplegic) signaling pathway using the western blotting assay. In addition, compound MY-673 could effectively inhibit cell proliferation, migration and induce cell apoptosis. We also further confirmed the in vivo efficacy of MY-673 in inhibiting tumor growth using the MGC-803 xenograft tumor model. At 20 mg/kg, the TGI rate was 85.9%, and it did not cause obvious toxicity to the main organs of mice. Together, the results we report here indicated that MY-673 was a promising CBSI for cancer treatment, which was capable of inhibiting the ERK pathway with potent antiproliferative activities in vitro and in vivo.

Discovery of novel N-benzylarylamide-dithiocarbamate based derivatives as dual inhibitors of tubulin polymerization and LSD1 that inhibit gastric cancers.

In this work, N-benzylarylamide-dithiocarbamate based derivatives were designed, synthesized, and their biological activities as anticancer agents were explored. Some of the 33 target compounds displayed significant antiproliferative activities with IC50 values at the double-digit nanomolar level. The representative compound I-25 (also named MY-943) not only showed the most effective inhibitory effects on three selected cancer cells MGC-803 (IC50 = 0.017 µM), HCT-116 (IC50 = 0.044 µM) and KYSE450 (IC50 = 0.030 µM), but also exhibited low nanomolar IC50 values from 0.019 to 0.253 µM against the other 11 cancer cells. Compound I-25 (MY-943) effectively inhibited tubulin polymerization and suppressed LSD1 at the enzymatic levels. Compound I-25 (MY-943) could act on the colchicine binding site of ß-tubulin, thus disrupting the construction of cell microtubule network and affecting the mitosis. In addition, compound I-25 (MY-943) could dose-dependently induce the accumulation of H3K4me1/2 (MGC-803 and SGC-7091 cells) and H3K9me2 (SGC-7091 cells). Compound I-25 (MY-943) could induce G2/M phase arrest and cell apoptosis, and suppress migration in MGC-803 and SGC-7901 cells. In addition, compound I-25 (MY-943) significantly modulated the expression of apoptosis- and cycle-related proteins. Furthermore, the binding modes of compound I-25 (MY-943) with tubulin and LSD1 were explored by molecular docking. The results of in vivo anti-gastric cancer assays using in situ tumor models showed that compound I-25 (MY-943) effectively reduced the weight and volume of gastric cancer in vivo without obvious toxicity. All these findings suggested that the N-benzylarylamide-dithiocarbamate based derivative I-25 (MY-943) was an effective dual inhibitor of tubulin polymerization and LSD1 that inhibited gastric cancers.

Design, synthesis and biological evaluation of N-benzylaryl cinnamide derivatives as tubulin polymerization inhibitors capable of promoting YAP degradation with potent anti-gastric cancer activities.

In this work, we utilized the N-benzylaryl derivative 9 as a lead compound and employed the molecular hybridization strategy by introducing cinnamoyl fragments to successfully design and synthesize 33 novel N-benzylaryl cinnamide derivatives 15a∼15 ag. The in vitro antiproliferative activities were explored, and the preliminary analysis and summary of their structure-activity relationship were conducted. The majority of the compounds demonstrated significant inhibitory potency on MGC-803, HCT-116 and KYSE450 cells with IC50 values below 0.5 µM. Among them, compound 15e (MY-1076) exhibited the most effective effect on the proliferative inhibition of MGC-803, SGC-7901, HCT-116 and KYSE450 cells with IC50 values of 0.019, 0.017, 0.020 and 0.044 µM, respectively, which is more potent than colchicine and the lead compound 9. Additionally, compound 15e (MY-1076) still exhibited significant inhibitory proliferation activity against 13 other types of tumor cells (IC50 values < 0.1 µM). Further studies revealed that compound 15e (MY-1076) could effectively inhibit tubulin polymerization by acting on the ß-tubulin colchicine binding site, thereby disrupting microtubule network assembly and mitotic progression. Additionally, compound 15e (MY-1076) also demonstrated a notable inhibitory effect on the oncogenic protein YAP by inducing its degradation. Compound 15e (MY-1076) could dose-dependently induce G2/M phase arrest and cell apoptosis, effectively inhibit the colony formatting ability and cause morphological changes in MGC-803 and SGC-7901 cells. Compound 15e (MY-1076) exhibited significant regulatory effects on the expression levels of cell cycle and apoptosis-related proteins. Taken together, we here reported a novel N-benzylaryl cinnamide derivative 15e (MY-1076) as a tubulin polymerization inhibitor capable of promoting degradation of YAP, which held great potential as an anti-gastric cancer agent.

Discovery of N-benzylarylamide derivatives as novel tubulin polymerization inhibitors capable of activating the Hippo pathway.

Novel N-benzylarylamide saderivatives were designed and synthesized, and their antiproliferative activities were explored. Some of 51 target compounds exhibited potent inhibitory activities against MGC-803, HCT-116 and KYSE450 cells with IC50 values in two-digit nanomolar. Compound I-33 (MY-875) displayed the most potent antiproliferative activities against MGC-803, HCT-116 and KYSE450 cells (IC50 = 0.027, 0.055 and 0.067 µM, respectively) and possessed IC50 values ranging from 0.025 to 0.094 µM against other 11 cancer cell lines. Further mechanism studies indicated that compound I-33 (MY-875) inhibited tubulin polymerization (IC50 = 0.92 µM) by targeting the colchicine bingding site of tubulin. Compound I-33 (MY-875) disrupted the construction of the microtubule networks and affected the mitosis in MGC-803 and SGC-7901 cells. In addition, although it acted as a colchicine binding site inhibitor, compound I-33 (MY-875) also activated the Hippo pathway to promote the phosphorylation status of MST and LATS, resulting in the YAP degradation in MGC-803 and SGC-7901 cells. Due to the degradation of YAP, the expression levels of TAZ and Axl decreased. Because of the dual actions on colchicine binding site and Hippo pathway, compound I-33 (MY-875) dose-dependently inhibited cell colony formatting ability, arrested cells at the G2/M phase and induced cells apoptosis in MGC-803 and SGC-7901 cells. Moreover, compound I-33 (MY-875) could regulate the levels of cell cycle and apoptosis regulatory proteins in MGC-803 and SGC-7901 cells. Furthermore, molecular docking analysis suggested that the hydrogen bond and hydrophobic interactions made compound I-33 (MY-875) well bind into the colchicine binding site of tubulin. Collectively, compound I-33 (MY-875) is a novel anti-gastric cancer agent and deserves to be further investigated for cancer therapy by targeting the colchicine binding site of tubulin and activating the Hippo pathway.

Discovery of novel coumarin-indole derivatives as tubulin polymerization inhibitors with potent anti-gastric cancer activities.

Novel coumarin-indole derivatives were designed, synthesized and evaluated as tubulin polymerization inhibitors targeting the colchicine binding site. Among these compounds, compound MY-413 displayed the most potent inhibitory activities against gastric cancer cell line MGC-803 with an IC50 value of 0.011 µM. Furthermore, the IC50 values of compound MY-413 was less than 0.1 µM for other 17 cancer cell lines and less than 0.05 µM for other 8 cancer cell lines. Compound MY-413 effectively inhibited the tubulin polymerization (IC50 = 2.46 µM) by binding to the colchicine site. Screening for the inhibitory effects of compound MY-413 on 61 kinases, it was found that compound MY-413 could inhibit MAPK pathways-related kinases. Because of the inhibitory effects of compound MY-413 on tubulin polymerization and MAPK signaling pathway, compound MY-413 induced cell apoptosis, arrested the cell cycle in the G2/M phase, induced the inhibition of cell proliferation and migration in gastric cancer cells MGC-803 and HGC-27. In addition, compound MY-413 could significantly inhibit tumor growth in MGC-803 xenograft tumor models with tumor growth inhibition (TGI) rates of 70% (15 mg/kg) and 80% (30 mg/kg) without obvious toxicity. Consistent with the in vitro results, compound MY-413 also inhibited MAPK signaling pathway, and induced apoptosis and proliferation inhibition in vivo. In conclusion, this work indicated that compound MY-413 was a promising lead compound for the further investigation as a potential anti-gastric cancer agent.

A novel aromatic amide derivative SY-65 co-targeted tubulin and histone deacetylase 1 with potent anticancer activity in vitro and in vivo.

Given the essential role of Epigenetic regulation in many biological processes, targeted epigenetic drugs have been gradually applied to the treatment of tumors. Histone deacetylases (HDACs) are a class of epigenetic enzymes, which play key roles in chromosome structural modification and gene expression regulation. Targeted microtubules drugs have achieved great success in clinical application for decades. Development of novel agents with multitargeting capabilities specially dual-target has become a popular research field for the treatment of human cancers, which may provide synergistic anticancer effects. Here, we reported a novel aromatic amide derivative SY-65 co-targeted tubulin and histone deacetylase 1 with potent anticancer activity in vitro and in vivo. Compound SY-65 was identified as a dual inhibitor of tubulin/HDAC1 (IC50 = 3.64 and 0.529 µM, respectively) with excellent antiproliferative activity against MGC-803, HCT-116, KYSE-450, HGC-27, SGC-7901 and MKN-45 cells. Especially, compound SY-65 exhibited potent antiproliferative activity against MGC-803, HGC-27 and SGC-7901 cells with IC50 values <55 nM, which was better than that of Colchicine, MS-275 and SAHA. Compound SY-65 effectively inhibited tubulin polymerization and bound to the colchicine binding site of tubulin, as well as inhibited HDAC1 activity both intra/extracellularly. Molecular docking results suggested there were the well-defined binding modes of compound SY-65 in HDAC1 and tubulin. In addition, compound SY-65 inhibited colony formation, interfered with the cell cycle distribution, induced cell cycle arrest at the G2/M phase and apoptosis in MGC-803 and HGC-27 cells. Compound SY-65 also exhibited a good tumor inhibitory effect in vivo without obvious toxicity. Therefore, compound SY-65 could be developed as a novel tubulin/HDAC1 candidate inhibitor for future cancer therapeutics.

Discovery of 1,2,4-triazine dithiocarbamate derivatives as NEDDylation agonists to inhibit gastric cancers.

NEDDylation process regulates multiple physiological functions and signaling pathways, which are still in an equilibrium that favors the survival and proliferation of tumor cells. Unlike inhibitors, NEDDylation agonists are rarely studied. In this work, novel 1,2,4-triazine-dithiocarbamate derivatives were synthesized and evaluated for antiproliferative activity against MGC-803, PC-3 and EC-109 cells. Among them, compound K3 displayed the most potent activity MGC-803, PC-3 and EC-109 cells with IC50 values of 2.35, 5.71 and 10.1 µM, respectively, which were more potent than 5-FU. Further cellular mechanisms suggested that compound K3 inhibited the cell viability, induced proliferation inhibition, arrested cell cycle at G2/M phase and induced cell apoptosis in MGC-803 and HGC-27 cells. Importantly, compound K3 could interact with NAE1 to promote the NEDDylation of MGC-803 and HGC-27 cells. The promotion of NEDDylation resulted in the degradation of c-IAP and YAP/TAZ, which leads to the induction of cell apoptosis and inhibition of proliferation in MGC-803 and HGC-27 cells. Therefore, as a NEDDylation agonist, compound K3 could effectively inhibit gastric cancer cells. Here, we reported NEDDylation promotion induced by compound K3, which could inhibit the cancer cell lines MGC-803 and HGC-27 and induce the cancer cell apoptosis via prompting the degradation of c-IAP and YAP/TAZ.