2-((1-Phenyl-1H-1,2,3-triazol-4-yl)methyl)-2-azabicyclo[3.2.1]octan-3-one derivatives: Simplification and modification of aconitine scaffold for the discovery of novel anticancer agents.

The molecular chaperone heat shock protein 90 (Hsp90) is a promising target for cancer therapy. Natural product aconitine is a potential Hsp90 inhibitor reported in our previous work. In this study, we designed and synthesized a series of 2-((1-phenyl-1H-1,2,3-triazol-4-yl)methyl)-2-azabicyclo[3.2.1]octan-3-one derivatives as potent Hsp90 inhibitors by simplifying and modifying aconitine scaffold. Among these compounds, 14t exhibited an excellent antiproliferative activity against LoVo cells with an IC50 value of 0.02 µM and a significant Hsp90α inhibitory activity with an IC50 value of 0.71 nM. Molecular docking studies provided a rational binding model of 14t in complex with Hsp90α. The following cell cycle and apoptosis assays revealed that compound 14t could arrest cell cycle at G1/S phase and induce cell apoptosis via up-regulation of bax and cleaved-caspase 3 protein expressions while inhibiting the expressions of bcl-2. Moreover, 14t could inhibit cell migration in LoVo and SW620 cell lines. Consistent with in vitro results, 14t significantly repressed tumor growth in the SW620 xenograft mouse model.

An Integrated Network, RNA Sequencing, and Experiment Pharmacology Approach Reveals the Active Component, Potential Target, and Mechanism of Gelsemium elegans in the Treatment of Colorectal Cancer.

In this study, a combination of network pharmacology, bioinformatics analysis, molecular docking and transcriptomics was used to investigate the active ingredient and potential target of Gelsemium elegans in the treatment of colorectal cancer. Koumine was screened as the active component by targeting PDK1 through network pharmacology and reverse docking. RNA-Seq, enrichment analysis and validation experiment were then further employed to reveal koumine might function in inhibiting Akt/mTOR/HK2 pathway to regulate cell glycolysis and detachment of HK2 from mitochondria and VDAC-1 to activate cell apoptosis both in vitro and in vivo. In the present study, we provide a systematical approach for the identification of effective ingredient and potential target of herbal medicine. Our results have important implication for the intensive study of koumine as novel anticancer agents for colorectal cancer and could be supportive in its further structural modification.

Discovery of N-phenyl-(2,4-dihydroxypyrimidine-5-sulfonamido) phenylurea-based thymidylate synthase (TS) inhibitor as a novel multi-effects antitumor drugs with minimal toxicity.

Thymidylate synthase (TS) is a hot target for tumor chemotherapy, and its inhibitors are an essential direction for anti-tumor drug research. To our knowledge, currently, there are no reported thymidylate synthase inhibitors that could inhibit cancer cell migration. Therefore, for optimal therapeutic purposes, combines our previous reports and findings, we hope to obtain a multi-effects inhibitor. This study according to the principle of flattening we designed and synthesized 18 of N-phenyl-(2,4-dihydroxypyrimidine-5-sulfonamido)phenyl urea derivatives as multi-effects inhibitors. The biological evaluation results showed that target compounds could significantly inhibit the hTS enzyme, BRaf kinase and EGFR kinase activity in vitro, and most of the compounds had excellent anti-cell viability for six cancer cell lines. Notably, the candidate compound L14e (IC50 = 0.67 µM) had the superior anti-cell viability and safety to A549 and H460 cells compared with pemetrexed. Further studies had shown that L14e could cause G1/S phase arrest then induce intrinsic apoptosis. Transwell, western blot, and tube formation results proved that L14e could inhibit the activation of the EGFR signaling pathway, then ultimately achieve the purpose of inhibiting cancer cell migration and angiogenesis in cancer tissues. Furthermore, in vivo pharmacology evaluations of L14e showed significant antitumor activity in A549 cells xenografts with minimal toxicity. All of these results demonstrated that the L14e has the potential for drug discovery as a multi-effects inhibitor and provides a new reference for clinical treatment of non-small cell lung cancer.

The N-terminal zinc finger domain of Tgf2 transposase contributes to DNA binding and to transposition activity.

Active Hobo/Activator/Tam3 (hAT) transposable elements are rarely found in vertebrates. Previously, goldfish Tgf2 was found to be an autonomously active vertebrate transposon that is efficient at gene-transfer in teleost fish. However, little is known about Tgf2 functional domains required for transposition. To explore this, we first predicted in silico a zinc finger domain in the N-terminus of full length Tgf2 transposase (L-Tgf2TPase). Two truncated recombinant Tgf2 transposases with deletions in the N-terminal zinc finger domain, S1- and S2-Tgf2TPase, were expressed in bacteria from goldfish cDNAs. Both truncated Tgf2TPases lost their DNA-binding ability in vitro, specifically at the ends of Tgf2 transposon than native L-Tgf2TPase. Consequently, S1- and S2-Tgf2TPases mediated gene transfer in the zebrafish genome in vivo at a significantly (p < 0.01) lower efficiency (21%-25%), in comparison with L-Tgf2TPase (56% efficiency). Compared to L-Tgf2TPase, truncated Tgf2TPases catalyzed imprecise excisions with partial deletion of TE ends and/or plasmid backbone insertion/deletion. The gene integration into the zebrafish genome mediated by truncated Tgf2TPases was imperfect, creating incomplete 8-bp target site duplications at the insertion sites. These results indicate that the zinc finger domain in Tgf2 transposase is involved in binding to Tgf2 terminal sequences, and loss of those domains has effects on TE transposition.

Prokaryotic expression and purification of soluble goldfish Tgf2 transposase with transposition activity.

Goldfish Tgf2 transposon of Hobo/Activator/Tam3 (hAT) family can mediate gene insertion in a variety of aquacultural fish species by transposition; however, the protein structure of Tgf2 transposase (TPase) is still poorly understood. To express the goldfish Tgf2 TPase in Escherichia coli, the 2061-bp coding region was cloned into pET-28a(+) expression vector containing an N-terminal (His)6-tag. The pET-28a(+)-Tgf2 TPase expression cassette was transformed into Rosetta 1 (DE3) E. coli lines. A high yield of soluble proteins with molecular weight of ~80 kDa was obtained by optimized cultures including low-temperature (22 °C) incubation and early log phase (OD600 = 0.3-0.4) induction. Mass spectrometry analysis following trypsin digestion of the recombinant proteins confirmed a Tgf2 TPase component in the eluate of Ni(2+)-affinity chromatography. When co-injected into 1-2 cell embryos with a donor plasmid harboring a Tgf2 cis-element, the prokaryotic expressed Tgf2 TPase can mediate high rates (45 %) of transposition in blunt snout bream (Megalobrama amblycephala). Transposition was proved by the presence of 8-bp random direct repeats at the target sites, which is the signature of hAT family transposons. Production of the Tgf2 Tpase protein in a soluble and active form not only allows further investigation of its structure, but provides an alternative tool for fish transgenesis and insertional mutagenesis.