NCTR25 fusion facilitates the formation of TRAIL polymers that selectively activate TRAIL receptors with higher potency and efficacy than TRAIL.

PURPOSE: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to death receptor (DR) 4 and DR5 and induces tumor-selective apoptosis. The fusion proteins NCTR25-TRAIL and NCTR25-TGF3L-TRAIL self-assembled into polymers and triggered super-active cancer cell killing. The role of TGF3L in self-assembly and super-activity was unclear. These multivalent TRAILs elicited apoptosis with great potency, but their specificity towards receptors and subsequent efficacy in signal activation were unclear. METHODS: NCTR25-TRAIL fusion was constructed and prokaryotically expressed. The size of fusion protein polymers was estimated. Their cytotoxicity was assessed in eight cancer cell lines and two noncancerous cell lines. Receptor binding and activation specificity were determined by antibody blockade. Apoptosis was evaluated, and the associated pathway was verified by quantifying caspase activity. The NF-κB signaling pathway was assessed by dual-luciferase assay. The in vivo antitumor activity was also evaluated in nude mice. RESULTS: NCTR25 fusion to TRAIL promoted its self-assembly into polymers and showed similar super-cytotoxicity to NCTR25-TGF3L-TRAIL in vitro. The multivalent TRAILs exclusively activated both DR4 and DR5 and showed a bias towards DR4 in mediating cytotoxicity in NCI-H460 cells. They activated caspase pathway and induced apoptosis with higher potency but in similar efficacy than TRAIL. A higher potency and a greater efficacy were observed in activating NF-κB pathway by NCTR25-TRAIL comparing to TRAIL. Both the polymers showed better in vivo antitumor activity than TRAIL. CONCLUSIONS: NCTR25 fusion alone facilitates the formation of TRAIL polymers. Multivalent TRAIL polymers bind and activate DR4 and DR5 specifically and exclusively, triggering the signaling pathways with higher potency, and greater efficacy than TRAIL.

Synthesis and biological evaluation of pyranocarbazole derivatives as Anti-tumor agents.

A series of pyrano[3,2-a]carbazole alkaloids were designed and synthesized as derivatives of Girinimbine. The anticancer activities of these derivatives (3, 4a-j, 5a, 5c, 5f, 5i, 6c, 7a, 7c, 7f, 7i) against 10 cancer cell lines were studied. Among them, compounds 3 and 7i with N-methyl piperazine showed significant anticancer activity against MCF-7 cell lines with the IC50 values of 1.77 and 4.32 µM, respectively. Furthermore, their effects on altering cell morphology, inducing cell cycle arrest and apoptosis in MCF-7 cells were studied in vitro. In addition, the molecular docking study was carried out by using Discovery Studio software to predict the interactions between these derivatives and tubulin. All in all, these consequences reveal that pyranocarbazole derivatives with N-methyl piperazine can be used as potential anticancer lead compounds and provide useful points for the further optimization of pyranocarbazole alkaloids.

Dimerization/oligomerization of the extracellular domain of the GLP-1 receptor and the negative cooperativity in its ligand binding revealed by the improved NanoBiT.

The glucagon-like peptide-1 receptor (GLP-1R), a family B G-protein coupled receptor (GPCR), regulates the insulin secretion following stimulation by ligands. The transmembrane domain (TM) mediates GLP-1R homodimerization, which modulates its ligand binding and signaling. We investigated the possible involvement of the N-terminal extracellular domain (NTD) in dimerization/oligomerization and dimer-associated ligand binding by NanoLuc Binary Technology (NanoBiT). With improved NanoBiT detection using a decreasing substrate concentration, the negative cooperativity of ligand binding to the NTD was confirmed by accelerated dissociation and Scatchard analysis. The dimerization/oligomerization of the isolated NTD was observed by NanoBiT and validated by analytical ultracentrifugation, deriving the comparable dimerization affinity (~105  M-1 ). The NTD was also involved in the dimerization/oligomerization of the full-length GLP-1R with mutated TM4 at the cellular level. In an analysis of the parameters of the NTD binding, the Kd for the probe GLP-1 (7-36, A8G) was similar (6-8 µM) in both the 1:1 binding model and the receptor dimerization model. Compared with GLP-1 and dulaglutide, exenatide showed two-site binding with Ki values of 1.4 pM and 8.7 nM. Our study indicates the involvement of NTD in the GLP-1R dimerization/oligomerization and suggests that further investigations on the role in other family B GPCRs are needed.

Cytotoxic 9,19-cycloartane Triterpenoids from the Roots of Actaea dahurica.

Eight undescribed 9,19-cycloartane type triterpenoid glycosides (cimdalglnoside A-H) and ten known analogues were obtained from the phytochemical research on the roots of Actaea dahurica (syn. Cimicifuga dahurica). All compounds were characterised by spectroscopic experiments, and chemical method. All the compounds isolated were assayed for cytotoxicity to five human cancer cell lines. Cimdalglnoside G showed promising cytotoxicities against Hela, and MCF-7 cell lines with IC50 values at 7.7 and 12.2 µM.

Cytotoxic 9,19-cycloartane type triterpenoid glycosides from the roots of Actaea dahurica.

Ten undescribed 9,19-cycloartane type triterpenoid glycosides (cimdahxynoside A-J) and five known analogues were obtained from the phytochemical research on the roots of Actaea dahurica (syn. Cimicifuga dahurica). All compounds were characterised by spectroscopic experiments, chemical method and X-ray Single-crystal diffraction analysis. Cimdahxynoside A represented the first X-ray crystallography of 9,19-cycloartane type triterpenoid diglycoside. The cytotoxicity of all compounds were tested against five human cancer cell lines. Cimdahxynoside F showed significant cytotoxicity, with IC50 values between 6.6 and 9.9 µM.

TGF3L fusion enhances the antitumor activity of TRAIL by promoting assembly into polymers.

TRAIL, a promising antitumor immuno-agent, exerted limited efficacy in clinical trials. The third disulfide loop of TGF-α (TGF3L peptide) with a very low affinity for EGFR has been reported to enhance the activity of fused antigens or cytokines. We wondered whether fusion of this peptide could enhance TRAIL activity and what the underlying mechanism for this enhancement would be. The TGF3L-TRAIL showed greatly enhanced cytotoxicity in a variety of cancer cell lines while spared normal cells unharmed. Typical apoptosis and cellular caspase activation were potently induced by TGF3L-TRAIL at the concentration levels corresponding to its cytotoxicity. TGF3L-TRAIL was able to activate both DR4 and DR5 the same as TRAIL did. It induced complete cell death in Colo205 through only one receptor when the other one was blocked, different from TRAIL-induced cell death (through DR4 dominantly). TGF3L-TRAIL cytotoxicity was not reduced in some cell lines even if both receptors are blocked simultaneously. Surprisingly, TGF3L-TRAIL self-assembled into stable polymers, which was responsible for its enhanced cytotoxicity. In human tumor xenograft mouse models, TGF3L-TRAIL showed anti-tumor activity similar to or better than TRAIL in different cancer cell types, consistent with its differing enhancement of cytotoxicity in vitro. Taken together, TGF3L fusion of TRAIL obviously enhances the anticancer activity of TRAIL by promoting assembly into polymers, which presents a novel fusion strategy for improving TRAIL function.

Insulin chains as efficient fusion tags for prokaryotic expression of short peptides.

Insulin chains are usually expressed in Escherichia coli as fusion proteins with different tags, including various low molecular weight peptide tags. The objective of this study was to determine if insulin chains could facilitate the recombinant expression of other target proteins, with an emphasis on low molecular weight peptides. A series of short peptides were fused to mini-proinsulin, chain B or chain A, and induced for expression in Escherichia coli. All the tested peptides including glucagon-like peptide 1 (GLP-1), a C-terminal extended GLP-1, oxyntomodulin, enfuvirtide, linaclotide, and an unstructured artificial peptide were expressed with reasonable yields, identified by Tricine-SDS-PAGE and immunoblotting. All recombinant products were expressed in inclusion bodies. The effective accumulation of products was largely attributed to the insoluble expression induced by fusion with insulin chains, and was confirmed by the fusion expression of transthyretin. Insulin chains thus show promise as efficient fusion tags for mass production of heterologous peptides in prokaryotes.