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Comparative Study
. 2020 Jun 2;25(11):2581.
doi: 10.3390/molecules25112581.

Comparative Molecular Transporter Properties of Cyclic Peptides Containing Tryptophan and Arginine Residues Formed through Disulfide Cyclization

Eman H M Mohammed  1   2 , Dindyal Mandal  1 , Saghar Mozaffari  1 , Magdy Abdel-Hamied Zahran  2 , Amany Mostafa Osman  2   3 , Rakesh Kumar Tiwari  1 , Keykavous Parang  1
Affiliations
Comparative Study

Comparative Molecular Transporter Properties of Cyclic Peptides Containing Tryptophan and Arginine Residues Formed through Disulfide Cyclization

Eman H M Mohammed et al. Molecules. .

Abstract

We have previously reported cyclic cell-penetrating peptides [WR]5 and [WR]4 as molecular transporters. To optimize further the utility of our developed peptides for targeted therapy in cancer cells using the redox condition, we designed a new generation of peptides and evaluated their cytotoxicity as well as uptake behavior against different cancer cell lines. Thus, cyclic [C(WR)xC] and linear counterparts (C(WR)xC), where x = 4-5, were synthesized using Fmoc/tBu solid-phase peptide synthesis, purified, and characterized. The compounds did not show any significant cytotoxicity (at 25 µM) against ovarian (SK-OV-3), leukemia (CCRF-CEM), gastric adenocarcinoma (CRL-1739), breast carcinoma (MDA-MB-231), and normal kidney (LLCPK) cells after 24 and 72 h incubation. Both cyclic [C(WR)5C] and linear (C(WR)5C) demonstrated comparable molecular transporter properties versus [WR]5 in the delivery of a phosphopeptide (F'-GpYEEI) in CCRF-CEM cells. The uptake of F'-GpYEEI in the presence of 1,4-dithiothreitol (DTT) as the reducing agent was significantly improved in case of l(C(WR)5C), while it was not changed by [C(WR)5C]. Fluorescence microscopy also demonstrated a significant uptake of F'-GpYEEI in the presence of l(C(WR)5C). Cyclic [C(WR)5C] improved the uptake of the fluorescent-labeled anti-HIV drugs F'-d4T, F'-3TC, and F'-FTC by 3.0-4.9-fold. These data indicate that both [C(WR)5C] and linear (C(WR)5C) peptides can act as molecular transporters.

Keywords: cancer; cell-penetrating peptide; cellular uptake; cytotoxicity; disulfide bridge; drug delivery; phosphopeptide.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Chemical structures of synthesized peptides.
Scheme 1
Scheme 1
Synthesis of l(C(WR)4C) (1) and c[C(WR)4C] (3) as representative examples.
Figure 2
Figure 2
MTS assay of peptides (5–50 µM) on LLC-PK1 cell lines. (A) 24 h incubation and (B) 72 h incubation (n = 3) (if p < 0.05 then *, if p < 0.01 then **, if p < 0.001 then ***, if p < 0.0001 then ****). Error bars are SD.
Figure 3
Figure 3
MTS assay of peptides (5–50 µM) on CCRF-CEM cell lines: (A) 24 h incubation and (B) 72 h incubation (n = 3) (if p < 0.05 then *, if p < 0.01 then **, if p < 0.001 then ***, if p < 0.0001 then ****). Error bars are SD.
Figure 4
Figure 4
MTS assay of peptides (5–50 µM) on CRL-1739 cell lines: (A) 24 h incubation and (B) 72 h incubation (n = 3) (if p < 0.05 then *, if p < 0.01 then **, if p < 0.001 then ***, if p < 0.0001 then ****). Error bars are SD.
Figure 5
Figure 5
MTS assay of peptides (5–50 µM) on SKOV-3 cell lines: (A) 24 h incubation and (B) 72 h incubation (n = 3). (Results are not statistically significant). Error bars are SD.
Figure 6
Figure 6
MTS assay of peptides (10–50 µM) on MDA-MB-231 cell lines’ incubation at 24 h and 72 h (n = 3) (results are not statistically significant). Error bars are SD.
Figure 7
Figure 7
Fluorescence-activated cell sorter (FACS) analysis of F′-GpYEEI (5 μM) using l(C(WR)5C) and c[C(WR)5C] at 25 µM in CCRF-CEM cells after 3 h incubation (n = 3) (if p < 0.05 then *, if p < 0.01 then **, if p < 0.001 then ***, if p < 0.0001 then ****). Error bars are SD.
Figure 8
Figure 8
FACS analysis of the uptake of F′-GpYEEI (5 μM) using synthesized peptides l(C(WR)5C) and c[C(WR)5C] at 25 µM in MDA-MB-231 cells after 3 h incubation in the presence and absence of DTT. NT stands for “No treatment”, n = 3, if p < 0.05 then *, if p < 0.01 then **, if p < 0.001 then ***, if p < 0.0001 then ****. Error bars are SD.
Figure 9
Figure 9
FACS analysis of the uptake of F′-d4T, F′-3TC, F′-FTC, and F’-d4T (5 μM) using synthesized peptides l(C(WR)5C) and c[C(WR)5C] at 25 μM in MDA-MB-231 cells after 3 h incubation (n = 3, if p < 0.05 then *, if p < 0.01 then **, if p < 0.001 then ***, if p < 0.0001 then ****). Error bars are SD.
Figure 10
Figure 10
FACS analysis of the uptake of F′-GpYEEI (5 μM) using synthesized peptide l(C(WR)5C) (25 µM) in the presence of different endocytic inhibitors in MDA-MB-231 cells after 3 h incubation (n = 3, if p < 0.05 then *, if p < 0.01 then **, if p < 0.001 then ***, if p < 0.0001 then ****). Error bars are SD.
Figure 11
Figure 11
Fluorescence microscope images of peptides l(C(WR)5C) and c[C(WR)5C]) after 3 h in the CCRF-CEM cells.
See this image and copyright information in PMC

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