Novel 4-Aryl-4H-chromene derivative displayed excellent in vivo anti-glioblastoma efficacy as the microtubule-targeting agent.

In this study, a series of novel 4-Aryl-4H-chromene derivatives (D1-D31) were designed and synthesized by integrating quinoline heterocycle to crolibulin template molecule based on the strategy of molecular hybridization. One of these compounds D19 displayed positive antiproliferative activity against U87 cancer cell line (IC50 = 0.90 ± 0.03 µM). Compound D19 was verified as the microtubule-targeting agent through downregulating tubulin related genes of U87 cells, destroying the cytoskeleton of tubulins and interacting with the colchicine-binding site to inhibit the polymerization of tubulins by transcriptome analysis, immune-fluorescence staining, microtubule dynamics and EBI competition assays as well as molecular docking simulations. Moreover, compound D19 induced G2/M phase arrest, resulted in cell apoptosis and inhibited the migration of U87 cells by flow cytometry analysis and wound healing assays. Significantly, compound D19 dose-dependently inhibited the tumor growth of orthotopic glioma xenografts model (GL261-Luc) and effectively prolonged the survival time of mice, which were extremely better than those of positive drug temozolomide (TMZ). Compound D19 exhibited potent in vivo antivascular activity as well as no observable toxicity. Furthermore, the results of in silico simulation studies and P-gp transwell assays verified the positive correlation between compound D19's Blood-Brain Barrier (BBB) permeability and its in vivo anti-GBM activity. Overall, compound D19 can be used as a promising anti-GBM lead compound for the treatment of glioblastoma.

Novel Chalcone-Phenazine Hybrids Induced Ferroptosis in U87-MG Cells through Activating Ferritinophagy.

Thirty-seven novel chalcone-phenazine hybrid molecules (C1∼C13 and F1∼F24) with 1,2,3-triazole or ethyl group as linkers were designed and synthesized in this study. Some compounds exhibited selective cytotoxicity against U87-MG cancer cell lines in vitro, in which compound C4 were found to have the best antiproliferative activity. SAR study indicated 1,2,3-triazole group may be crucial for enhancing compounds' cytotoxicity. C4 was verified to induce ferroptosis in U87-MG cells by transcription, lipid peroxidation, lipid ROS assays. Furthermore, C4 was up-regulated LC3-II, degradated FTH1, and then increasing iron resulted in the down-regulation of NCOA4. Together, all above evidences highlighted the potential of compound C4 that triggered ferroptosis by activating ferritinophagy against U87-MG cells.

Nanoaggregates of Disulfide-Decorated TrxR Inhibitor Promote Cellular Uptake, Selective Targeting, and Antitumor Efficacy.

Three self-assembled nanoaggregates (CPUL1-LA NAs, CPUL1-DA NAs, and CPUL1-AA NAs) were constructed through lipoic acid (LA), dithiodipropionic acid (DA), and adipic acid (AA) decorated TrxR inhibitor (CPUL1), respectively. Measurements of DLS, TEM, UV-vis, fluorescence, 1H NMR, ITC, and MTT assays verified disulfide-containing CPUL1-LA NAs and CPUL1-DA NAs spontaneously assembled carrier-free nanoparticles in aqueous solution, which possessed high drug contents, excellent stability, improved cytotoxicity against HUH7 hepatoma cells, and potential biosafety because of low cytotoxicity against L02 normal cells. In contrast, disulfide-free CPUL1-AA NAs happened to aggregate and precipitate after 48 h, which showed distinct instability in aqueous solution. Thus, disulfide units seemed to be crucial for constructing controllable and stable nanoaggregates. While measuring the reduction of nanoaggregates by TrxR/NADPH and GSH/GR/NADPH, cyclic disulfide of LA and linear disulfide of DA were verified to endow the nanoaggregates with targeting ability to respond specifically to TrxR over GSH. Furthermore, by tests of flow cytometry, fluorescence images, and CLSM, both CPUL1-LA NAs and CPUL1-DA NAs displayed a faster cellular uptake characteristic to be internalized by cancer cells and could generate more abundant ROS to induce cell apoptosis than that of free CPUL1, resulting in significantly improved antitumor efficacy against HUH7 cells in vitro.

Phenazine derivatives attenuate the stemness of breast cancer cells through triggering ferroptosis.

Breast cancer stem cells (BCSCs) are positively correlated with the metastasis, chemoresistance, and recurrence of breast cancer. However, there are still no drugs targeting BCSCs in clinical using for breast cancer treatment. Here, we tried to screen out small-molecule compounds targeting BCSCs from the phenazine library established by us before. We focused on the compounds without affecting cell viability and screened out three potential compounds (CPUL119, CPUL129, CPUL149) that can significantly attenuate the stemness of breast cancer cells, as evident by the decrease of stemness marker expression, CD44+/CD24- subpopulation, mammary spheroid-formation ability, and tumor-initiating capacity. Additionally, these compounds suppressed the metastatic ability of breast cancer cells in vitro and in vivo. Combined with the transcriptome sequencing analysis, ferroptosis was shown on the top of the most upregulated pathways by CPUL119, CPUL129, and CPUL149, respectively. Mechanistically, we found that these three compounds could trigger ferroptosis by accumulating and sequestering iron in lysosomes through interacting with iron, and by regulating the expression of proteins (IRP2, TfR1, ferritin) engaged in iron transport and storage. Furthermore, inhibition of ferroptosis rescued the suppression of these three compounds on breast cancer cell stemness. This study suggests that CPUL119, CPUL129, and CPUL149 can specifically inhibit the stemness of breast cancer cells through triggering ferroptosis and may be the potential compounds for breast cancer treatment.

Multifunctional Nanoaggregates Composed of Active CPUL1 and a Triphenylphosphine Derivative for Mitochondria-Targeted Drug Delivery and Cell Imaging.

We report that active substance (CPUL1) and triphenylphosphine (TPP) derivative could self-assemble into multifunctional nanoaggregates (CPUL1-TPP NAs) through electrostatic and π-π stacking interactions. CPUL1 was wrapped tightly inside the nanoparticles as well as CPUL1 and TPP derivative self-assembled into stable and compact nanoparticles in water. The positive surface charge of CPUL1-TPP NAs made them much easier to be endocytosed to enter cytoplasm, accumulate in the mitochondria and induce cell apoptosis based on their mitochondria targeting ability, fluorescence property and fast cell uptake characteristic, which showed better antitumor efficacy on HUH7 hepatoma cells in vitro than that of free CPUL1.

Design, synthesis and biological evaluations of diverse Michael acceptor-based phenazine hybrid molecules as TrxR1 inhibitors.

A series of novel phenazine derivatives (1~27) containing the Michael acceptor scaffolds were designed and synthesized in this study. Some compounds exhibited selective cytotoxicity against Bel-7402 cancer cell line in vitro, in which compound 26 were found to have the best antiproliferative activity. Meanwhile, compound 26 showed no obvious cell toxicity against human normal liver epithelial L02 cells, which means this compound possessed a better safety potential. In the following research, compound 26 was verified to inhibit TrxR1 enzyme activity, ultimately resulting in cellular molecular mechanism events of apoptosis including growth of intracellular ROS level, depletion of reduced Trx1, liberation of ASK1 and up-regulation of p38, respectively. Together, all these evidences implicated that compound 26 acted as the TrxR1 inhibitor against Bel-7402 cells, and could activate apoptosis through the ROS-Trx-ASK1-p38 pathway.