Molecular tumor targeting of gelonin by fusion with F3 peptide

Abstract

Therapeutically potent macromolecular drugs have shown great promise for overcoming the limitations of small-molecule anti-cancer drugs. But tumor cell-selective intracellular delivery of the macromolecules remains a major hurdle for their successful clinical application. To overcome this challenge, we engineered a novel genetic fusion protein (F3-Gel) that composed of F3 peptide, a tumorhoming peptide, and gelonin, a plant-derived ribosome-inactivating protein (RIP), and then evaluated its anti-cancer activity in vitro and in vivo. The F3-Gel-encoding gene was synthesized by genetic recombination, and F3-Gel was successfully expressed in E coli. The anti-cancer activity of the produced F3-Gel was evaluated by various in vitro assays, which revealed that F3-Gel maintained equipotent protein synthesis inhibition activity (IC 50 =11 pmol/L) as unmodified gelonin (IC 50 =10 pmol/L). Furthermore, F3-Gel displayed enhanced cellular uptake into cancer cells (U87 MG, HeLa, LnCaP and 9L) than noncancerous cells (293 HEK and SVGp12). Compared with gelonin, F3-Gel exerted significantly higher cytotoxicity against these cancer cells. F3-Gel displayed significantly greater inhibition of protein translation in U87 MG cells: F3-Gel (0.5 μmol/L) was able to reduce the protein level to less than 50%, while gelonin (1 μmol/L) did not affect the intracellular protein level. In a U87 MG xenograft tumor-bearing mouse model, F3-Gel was accumulated in the tumor site at much higher levels and maintained for a prolonged time compared with gelonin. Administration of F3-Gel (0.5, 0.75 mol/kg, iv) caused 36% and 66%, respectively, inhibition of tumor growth in U87 MG xenograft mice, suggesting that it is a promising candidate drug for cancer treatment. Furthermore, this study demonstrates that fusion of F3 peptide to a potent macromolecule could provides an effective method for targeting tumors and eventually could improve their druggability.

Production of Recombinant Gelonin Using an Automated Liquid Chromatography System

“…The data points from four (rGel ref ) and six (rGel) separate experiments, respectively, were plotted in the same graph page and subjected to sigmoidal regression ( Figure 5 c). The associated IC 50 values of 104.1 pM (rGel ref ) and 65.4 pM (rGel) appears consistent with previous reports on unmodified recombinant gelonin documenting IC 50 values from 7.25 to 185 pM [ 6 , 9 , 11 , 26 , 36 , 37 ] and reveals that our rGel maintains equipotent protein synthesis inhibition activity.…”

Dendrimer-like supramolecular assembly of proteins with a tunable size and valency through stepwise iterative growth

“…Gelonin is a plant-derived N-glycosidase, which inactivates the 60S ribosomal subunit and is known to be unable to cross the cell membrane alone [34,39] . For this, eGFP was replaced with gelonin in the previous construct, yielding Gmono (indicating a monomeric cargo with gelonin) (Figure 4a).…”

Protein-Based Therapeutic Killing for Cancer Therapies

“…In TRX-F3-Gel, the active, N-terminal segment of the plant toxin gelonin is targeted by F3, a ligand of nucleolin overexpressed in several tumor cell lineages. The thioredoxin (TRX)-H6 segment, used to enhance solubility and for purification upon recombinant production, is removed in vitro by the Tev protease [84]. In T22-BAK-H6, the human pro-apoptotic BAK is targeted by T22, a ligand of the cell-surface tumor marker CXCR4.…”