Click-Free Synthesis of a Multivalent Tricyclic Peptide as a Molecular Transporter

Abstract

The cellular delivery of cell-impermeable and water-insoluble molecules remains an ongoing challenge to overcome. Previously, we reported amphipathic cyclic peptides c[WR]4 and c[WR]5 consisting of alternate arginine and tryptophan residues as nuclear-targeting molecular transporters. These peptides contain an optimal balance of positive charge and hydrophobicity, which is required for interactions with the phospholipid bilayer to facilitate their application as a drug delivery system. To further optimize them, we synthesized and evaluated a multivalent tricyclic peptide as an efficient molecular transporter. The monomeric cyclic peptide building blocks were synthesized using Fmoc/tBu solid-phase chemistry and cyclization in the solution and conjugated with each other through an amide bond to afford the tricyclic peptide, which demonstrated modest antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli (E. coli) with a minimum inhibitory concentration (MIC) of 64–128 µg/mL. The tricyclic peptide was found to be nontoxic up to 30 µM in the breast cancer cell lines (MDA-MB-231). The presence of tricyclic peptide enhanced cellular uptakes of fluorescently-labeled phosphopeptide (F’-GpYEEI, 18-fold), anti-HIV drugs (lamivudine (F’-3TC), emtricitabine (F’-FTC), and stavudine (F’-d4T), 1.7–12-fold), and siRNA (3.3-fold) in the MDA-MB-231 cell lines.

Recent Advances of Cell-Penetrating Peptides and Their Application as Vectors for Delivery of Peptide and Protein-Based Cargo Molecules

“…Moreover, these bicyclics also selectively inhibited proliferation of cisplatinresistant ovarian cancer cells, as evidenced by their low μM IC50 values. [62]. This tricyclic enhanced cellular uptake of fluorescently labeled phosphopeptide (F'-GpYEEI), anti-HIV drugs (e.g., lamivudine, emtricitabine, stavudine), and siRNA by cells of the breast cancer cell line MDA-MB-231.…”

“…Reprinted with permission from[60]. Copyright 2018 American Chemical Society.Recently, research groups have synthesized tricyclic peptides, including a trimer of monocyclic peptide [WR] 4 named [WR] 4 -[WR] 4 -[WR]4[62]. This tricyclic enhanced cellular uptake of fluorescently labeled phosphopeptide (F'-GpYEEI), anti-HIV drugs (e.g., lamivudine, emtricitabine, stavudine), and siRNA by cells of the breast cancer cell line MDA-MB-231.…”

A Global Review on Short Peptides: Frontiers and Perspectives

“…Two bicyclic peptides containing tryptophan and arginine residues, namely [W The confocal microscopy exhibited that the peptides were localized in the nucleus and cytosol [219]. Recently, we designed a tricyclic peptide [WR] 4 -[WR] 4 -WR] 4 containing alternate tryptophan and arginine in each ring that improved the cellular uptake of F'-GpYEEI and fluorescently labeled anti-HIV drugs, lamivudine (3TC), emtricitabine (FTC), and siRNA in the breast cancer cell line MDA-MB-231 [221]. A combination of tryptophan and arginine residues in the design provided a diverse class of cyclic CPPs with differential cellular delivery properties.…”

Putative Repurposing of Lamivudine, a Nucleoside/Nucleotide Analogue and Antiretroviral to Improve the Outcome of Cancer and COVID-19 Patients

“…A recent study showed enhanced inhibition of proliferation of cancer cell lines by nanoparticle-encapsulated Lamivudine compared with the free Lamivudine ( 48 ). Other works had assayed some cysteine and arginine rich peptides ( 49 ) and multivalent tricyclic peptides ( 50 ) as vehicles to deliver Lamivudine into the target cells. In the first case, a number of cysteine and arginine containing linear and cyclic peptides were challenged to see whether these peptides could improve the intracellular transportation of two cargoes, a fluorescently-labeled and cell-impermeable phosphopeptide (F´-GpYEEI) and fluorescently-labeled Lamivudine (F-3TC).…”

“…In the second case, a trycyclic peptide was constructed with an optimal balance of positive charge and hydrophobicity to improve its interactions with the cell membranes and promote transport of the cargo; in this case fluorescently labeled anti-HIV drugs such as F´-Lamivudine (F´-3TC), F´-Emtricitabine (F´-TC), and F´-Stavudine (F´-d4T). In all cases, the trycyclic peptide used as molecular transporter enhanced the cellular uptakes of these fluorescently labeled pro-drugs ( 50 ). In summary, new delivery strategies are being optimized to improve the entrance of Lamivudine and other ANNAs into target cells ( Figure 1B ), which could support the repurposing of Lamivudine and other ANNAs to work as direct anti-cancer therapies in vitro and in vivo .…”