Bioinspired Bola-Type Peptide Dendrimers Inhibit Proliferation and Invasiveness of Glioblastoma Cells in a Manner Dependent on Their Structure and Amphipathic Properties

(1) Background: Natural peptides supporting the innate immune system studied at the functional and mechanistic level are a rich source of innovative compounds for application in human therapy. Increasing evidence indicates that apart from antimicrobial activity, some of them exhibit selective cytotoxicity towards tumor cells. Their cationic, amphipathic structure enables interactions with the negatively-charged membranes of microbial or malignant cells. It can be modeled in 3D by application of dendrimer chemistry. (2) Methods: Here we presented design principles, synthesis and bioactivity of branched peptides constructed from ornithine (Orn) assembled as proline (Pro)- or histidine (His)-rich dendrons and dendrimers of the bola structure. The impact of the structure and amphipathic properties of dendrons/dendrimers on two glioblastoma cell lines U87 and T98G was studied with the application of proliferation, apoptosis and cell migration assays. Cell morphology/cytoskeleton architecture was visualized by immunofluorescence microscopy. (3) Results: Dimerization of dendrons into bola dendrimers enhanced their bioactivity. Pro- and His-functionalized bola dendrimers displayed cytostatic activity, even though differences in the responsiveness of U87 and T98G cells to these compounds indicate that their bioactivity depends not only on multiple positive charge and amphipathic structure but also on cellular phenotype. (4) Conclusion: Ornithine dendrons/dendrimers represent a group of promising anti-tumor agents and the potential tools to study interrelations between drug bioactivity, its chemical properties and tumor cells’ phenotype.


Compound 13
Compound 14 Compound 15 Figure S2. Analytical HPLC profiles for dendrons 13-15. Figure S3. pro-apoptotic effects of ornithine dendrons/dendrimeres in T98G cell populations. Cells were seeded at the density of 2 × 10 5 /cm 2 . After 24 h of initial incubation, the tested agents were applied at the concentration of 100  M in the fresh portion of medium for the next 48 h. Subsequently, AnnexinV/Propidium iodide assay was performed (FITC AnnexinV Apoptosis Detection Kit, BD Pharminogen™) using ImageStreamX ® cytometer (Merck Millipore). Data representative for at least 3 independent experiments (n > 3) were analyzed with IDEAS ® 6.2 software (Merck Millipore). Figure S4. Exemplary plots showing the effect of ornithine dendrons/dendrimers on the motility of T98G cells. Cells were seeded at the density of 2 × 104/cm 2 . After 24 h of initial incubation, the tested agents were applied at the concentration of 10-100  M and cell movement was registered 48 h afterwards with time-lapse videomicroscopy. Cell trajectories are depicted in circular diagrams (axis scale in µm) drawn with the initial point of each trajectory placed at the origin of the plot (registered for 6 h; n > 50). Data representative for at least 3 independent experiments (n >3).
The Fluorenylmethyloxycarbonyl protecting group was removed by treating compounds with 20% piperidine/MeOH solution, followed by removing the solvent in vacuo. The products were washed with diethyl ether and the precipitate was dried in vacuo over P2O5.

Synthetic Procedure for Compound 4
The compound 1 (6.07 g, 12.3 mmol) was obtained by adding to Boc-Orn (Boc)-OH (4.5 g, 13.54 mmol) dissolved in 20 mL THF, HOSu (1.56 g, 13.54 mmol) and DCC (2.79 g, 13.54 mmol) dissolved in 10 mL THF and was stirred for 12 h at room temperature. So prepared active ester was added to phenylalanine methyl ester solution in 10 mL THF (Phe-OMe 2.92 g, 13.54 mmol) and TEA (8.22 g, 81 mmol) and stirred for 72 h at room temperature. The solvent was evaporated and the residue was dissolved in 100 mL of ethyl acetate and was washed 5 times respectively with 5% citric acid solution, saturated aqueous NaHCO3 and brine, dried over MgSO4, filtered and evaporated in vacuo. The compound was purified on Merck Kieselgel silica gel with the mobile phase consisted of 1-5% MeOH/DCM (yield: 90.8%). Subsequently, compound 1 was transformed into its octa-hydrochloride 2 by removing Boc groups with a saturated 1M HCl in AcOEt (yield: 98.2%).
Compound 3 (3.82 g, 4.1 mmol) was obtained by adding to Boc-Orn (Boc)-OH (2.92 g, 8.8 mmol) dissolved in 15 mL THF, HOSu (1 g, 8.8 mmol) and DCC (1.82 g, 8.8 mmol) dissolved in 10 mL THF and was stirred for 12 h at room temperature. So prepared active ester was added to a solution containing compound 2 (1.47 g, 4 mmol) in 10 mL THF and TEA (6.47 g, 64 mmol) and stirred for 72 h at room temperature. The solvent was evaporated and the residue was dissolved in 100 mL of ethyl acetate and was washed 5 times respectively with 5% citric acid solution, saturated aqueous NaHCO3 and brine, dried over MgSO4, filtered and evaporated in vacuo. The compound 3 was purified on Merck Kieselgel silica gel with the mobile phase consisted of 1-5% MeOH/DCM (yield: 94.1%). Subsequently, the phenylalanine methyl ester group was removed from compound 3 in the reaction with 1 M NaOH for 6 h at room temperature to give dendron4 (yield: 89.4%).
Synthesis of the core of the dendrimer (7)

Synthetic Procedure for Compound 7
The respective compound 4 (1.6g, 1.76mmol), HOSu (0.2g, 1.76mmol) and DCC (0.39g, 1.89 mmol) were dissolved in THF and stirred overnight. Then the active ester was added to a solution of 4,7,10-trioxa-1,13-tridecnediamine (5)(0.19g, 0.88 mmol) and TEA (0.356g, 3.52mmol) in THF and stirred for 96 h at room temperature. The mixture was evaporated and the residue was dissolved in ethyl acetate and washed with 5% citric acid solution, saturated aqueous NaHCO3 and brine, dried over MgSO4, filtered and evaporated in vacuo. The raw dendrimeric compound was purified by molecular filtration on the Sephadex LH-20 packing in MeOH and then by HPLC (yield: 66.9% for 7). Then the dendrimer was converted to its octahydrochloride by deprotection of Boc groups with HClsaturated AcOEt to give dendrimer 7a (yield: 98.6%).  The respective compound 4 (0 78 g, 0 86 mmol), DMAP (0.105g, 0.86 mmol) and DCC (0.185 g, 0.898 mmol) were dissolved in THF and a solution of tetraethylene glycol (6) (0.08 g, 0.43 mmol) in THF was added and stirred for 96 h at room temperature. The mixture was evaporated and the residue was dissolved in ethyl acetate and washed with 5% citric acid solution, saturated aqueous NaHCO3 and brine, dried over MgSO4, filtered and evaporated in vacuo. The raw dendrimeric compound was purified by molecular filtration on the Sephadex LH-20 packing in MeOH and then by HPLC (yield: 58.1% for 8) Then the dendrimer was converted to its octahydrochloride by deprotection of Boc groups with HCl-saturated AcOEt to give dendrimer 8a (yield: 96.2%).

Synthetic Procedure for Proline Decorated Bola-Dendrimer (9a)
Fmoc-Pro-OH (1.3g, 3.84 mmol) HOSu (0.442 g, 3.84 mmol) and DCC (0.824 g, 4 mmol) were dissolved in THF and stirred overnight. Then the active ester was added to a solution of 7a (0.32 g, 0.16 mmol) and TEA (0.518 g, 5.12 mmol) in THF and stirred for 126 h at room temperature. The mixture was evaporated and the residue was dissolved in ethyl acetate and washed with 5% citric acid solution, saturated aqueous NaHCO3 and brine, dried over MgSO4, filtered and evaporated in vacuo.

Synthetic Procedure for Histidine Decorated Bola-Dendrimer (10a)
Fmoc-His(Boc)-OH (2.21 g, 3.84 mmol) HOSu (0.442 g, 3.84 mmol) and DCC (0.824 g, 4 mmol) were dissolved in THF and stirred overnight. Then the active ester was added to a solution of 7a (0.32 g, 0.16 mmol) and TEA (0.518 g, 5.12 mmol) in THF and stirred for 126 h at room temperature. The mixture was evaporated and the residue was dissolved in ethyl acetate and washed with 5% citric acid solution, saturated aqueous NaHCO3 and brine, dried over MgSO4, filtered and evaporated in vacuo.
Then the dendrimer was deprotected of Fmoc groups with 20% piperidine/MeOH solution The raw dendrimeric compound was purified by molecular filtration on the Sephadex LH-20 packing in MeOH and then by HPLC. Then the dendrimer was deprotected of Bocby treating with 1 M HCl in ethyl acetate (5 mL) for 6 h, followed by removing the solvent in vacuo. The product was washed with diethyl ether and the precipitate was dried in vacuo over P2O5 (yield: 41.9% for 10a).

Synthetic Procedure for Proline Decorated Bola-Dendrimer (11a)
Fmoc-Pro-OH (1.3g, 3.84 mmol) HOSu (0.442 g, 3.84 mmol) and DCC (0.824 g, 4 mmol) were dissolved in THF and stirred overnight. Then the active ester was added to a solution of 8a (0.3 g, 0.16 mmol) and TEA (0.518 g, 5.12 mmol) in THF and stirred for 126 h at room temperature. The mixture was evaporated and the residue was dissolved in ethyl acetate and washed with 5% citric acid solution, saturated aqueous NaHCO3 and brine, dried over MgSO4, filtered and evaporated in vacuo.
The raw dendrimeric compound was purified by molecular filtration on the Sephadex LH-20 packing in MeOH and then by HPLC. Then the dendrimer was deprotected of Fmoc groups with 20% piperidine/MeOH solution (yield: 26.11% for 11a).

Synthetic Procedure for Histidine Decorated Bola-Dendrimer (12a)
Fmoc-His(Boc)-OH (2.21 g, 3.84 mmol) HOSu (0.442 g, 3.84 mmol) and DCC (0.824 g, 4 mmol) were dissolved in THF and stirred overnight. Then the active ester was added to a solution of 8a (0.3 g, 0.16 mmol) and TEA (0.518 g, 5.12 mmol) in THF and stirred for 126 h at room temperature. The mixture was evaporated and the residue was dissolved in ethyl acetate and washed with 5% citric acid solution, saturated aqueous NaHCO3 and brine, dried over MgSO4, filtered and evaporated in vacuo.
Then the dendrimer was deprotected of Fmoc groups with 20% piperidine/MeOH solution The raw dendrimeric compound was purified by molecular filtration on the Sephadex LH-20 packing in MeOH and then by HPLC. Then the dendrimer was deprotected of Bocby treating with 1M HCl in ethyl acetate (5 mL) for 6 h, followed by removing the solvent in vacuo. The product was washed with diethyl ether and the precipitate was dried in vacuo over P2O5 (yield: 21.3% for 12a).

Synthetic Procedure for Proline Decorated Dendron (13)
Fmoc-Pro-OH (1.34 g, 3.96 mmol) HOSu (0.46 g, 3.96 mmol) and DCC (0.91 g, 4.4 mmol) were dissolved in THF and stirred overnight. Then the active ester was added to a solution of 3a (0.3 g, 0.33 mmol) and TEA (0.801 g, 7.92 mmol) in THF and stirred for 96 h at room temperature. The mixture was evaporated and the residue was dissolved in ethyl acetate and washed with 5% citric acid solution, saturated aqueous NaHCO3 and brine, dried over MgSO4, filtered and evaporated in vacuo.