Application of Cell Penetrating Peptides as a Promising Drug Carrier to Combat Viral Infections.

Novel effective drugs or therapeutic vaccines have been already developed to eradicate viral infections. Some non-viral carriers have been used for effective drug delivery to a target cell or tissue. Among them, cell penetrating peptides (CPPs) attracted a special interest to enhance drug delivery into the cells with low toxicity. They were also applied to transfer peptide/protein-based and nucleic acids-based therapeutic vaccines against viral infections. CPPs-conjugated drugs or vaccines were investigated in several viral infections including poliovirus, Ebola, coronavirus, herpes simplex virus, human immunodeficiency virus, hepatitis B virus, hepatitis C virus, Japanese encephalitis virus, and influenza A virus. Some studies showed that the uptake of CPPs or CPPs-conjugated drugs can be performed through both non-endocytic and endocytic pathways. Despite high potential of CPPs for cargo delivery, there are some serious drawbacks such as non-tissue-specificity, instability, and suboptimal pharmacokinetics features that limit their clinical applications. At present, some solutions are utilized to improve the CPPs properties such as conjugation of CPPs with targeting moieties, the use of fusogenic lipids, generation of the proton sponge effect, etc. Up to now, no CPP or composition containing CPPs has been approved by the Food and Drug Administration (FDA) due to the lack of sufficient in vivo studies on stability, immunological assays, toxicity, and endosomal escape of CPPs. In this review, we briefly describe the properties, uptake mechanisms, advantages and disadvantages, and improvement of intracellular delivery, and bioavailability of cell penetrating peptides. Moreover, we focus on their application as an effective drug carrier to combat viral infections.

Novel Lung Targeting Cell Penetrating Peptides as Vectors for Delivery of Therapeutics

Rationale: Cell-penetrating peptides are able to cross membranes and deliver cargoes in a functional form. Our prior work identified a 12-amino acid, cardiac targeting peptide (APWHLSSQYSRT). Studies into its mechanism of transduction led to the identification of two lung targeting peptides (LTPs), S7A and R11A. Here we report on a) the comparative lung uptake of S7A versus R11A, b) complete biodistribution of R11A, c) show that cyclic versions are -100-fold more efficient than linear counterparts, d) uptake is via a non-endocytic pathway, and e) cyclic R11A's (cR11A) ability to deliver siRNA targeting structural proteins of SARS-CoV-2 and act as an anti-viral. Methods: Linear LTPs were synthesized with N-terminal labeled with Cyanine 5.5 (Cy5.5). Cyclic versions were synthesized with lysine added to the N-terminus, cyclized through a peptide bond, with a side NH-group labeled with Cy5.5. cR11A was conjugated to siRNA duplexes via a DTME linker. Wild-type, CD1 mice, were injected with S7A or R11A at 10, 5, and 1mg/Kg, peptides allowed to circulate for 15mins, mice euthanized, lung along with multiple other organs dissected and imaged using In Vivo Imaging Systems (IVIS, Perkin-Elmer) followed by confocal microscopy. CD1 mice were injected with R11A, 5mg/Kg, and euthanized at different time intervals for biodistribution studies. Endocytosis studies were done using serum-starved human bronchial epithelial cells (HBEC) incubated with fluorescently labeled transferrin and LTP-S7A or LTP- R11A. Lastly, anti-viral activity was tested in HBECs pre-treated with cR11A-siRNA followed by viral infection. Results: Mice injected with LTP-S7A or LTP-R11A showed robust uptake of the peptides by lung tissue, with R11A showing an increasingly favorable lung:liver ratio with decreasing dose. Lung uptake of R11A peaked at 120mins with complete dissipation of fluorescence by 24 hours. In Vitro studies in HBECs showed no co-localization of transferrin with LTPs, ruling out endocytosis as a mechanism of uptake. Comparison of linear versus cyclic peptides using FACS showed cyclic peptides had -100-fold increased transduction efficiency over their linear counterparts. cR11A conjugated to ant-spike, and anti-envelop proteins showed an anti-viral effect with EC90 of 0.6uM and 1.0μM, respectively. Conclusions: We have identified two novel lung-targeting peptides capable of acting as delivery vectors. Peak uptake of R11A occurred at 120mins. Furthermore, this uptake was not via endocytosis, and cyclic versions were -100-fold more efficiently taken up. Lastly, as proof of concept, we show cR11A acts as a vector and delivers siRNA to HBECs in a functional form, and act as anti-virals.

LL37 antimicrobial peptide: Structure and function

The human cathelicidin LL-37 plays a major role in the innate immune system for protection against bacterial infections. LL-37 can interact with molecules of the cell wall, perforate cell membranes, and finally lead to bacterial cell death. Moreover, LL-37 participates in immune regulation, chemotaxis of immune cells, and tissue repair. This peptide is produced by white blood cells (mainly neutrophils) and different epithelial cells (in the testicles, epidermis, intestinal system, respiratory system), and may be detected all over the body. LL-37 peptide has dual effects: a) it can increase inflammation and immunological responses and possess anti-infective and anti-cancer properties;b) it can suppress inflammation and enhance carcinogenesis. LL-37 is related to the risk of autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis atherosclerosis, and psoriasis. This peptide binds to self-DNA, and acts as an autoantigen. On the other hand, targeting the antiviral and immunomodulatory activities of LL37 peptide can reduce virus transmission and pathogenicity such as human immunodeficiency virus (HIV-1) and the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). This peptide and its variants can directly attach to HIV-1 reverse transcriptase and inhibit its function in a dose-dependent approach. Moreover, in silico studies showed the potency of LL-37 as a therapeutic agent against SARS-CoV-2 infection. It is interesting that vitamin D, a candidate preventive molecule against coronavirus disease 2019 (COVID-19), can upregulate the expression of LL-37. The LL-37 cationic peptide can also penetrate the membrane of eukaryotic cells likely through lipid rafts. Indeed, it can transport the nucleic acid to endosomes, and facilitate oligonucleotide delivery to the cells. Regarding major functions of LL-37 peptide in body, this review will concentrate on its structure and functions as the only cathelicidin-derived defensive peptide identified in human.

Decoding the proteome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for cell-penetrating peptides involved in pathogenesis or applicable as drug delivery vectors.

Synthetic or natural derived cell-penetrating peptides (CPPs) are vastly investigated as tools for the intracellular delivery of membrane-impermeable molecules. As viruses are intracellular obligate parasites, viral originated CPPs have been considered as suitable intracellular shuttling vectors for cargo transportation. A total of 310 CPPs were identified in the proteome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Screening the proteome of the cause of COVID-19 reveals that SARS-CoV-2 CPPs (SCV2-CPPs) span the regions involved in replication, protein-nucleotide and protein-protein interaction, protein-metal ion interaction, and stabilization of homo/hetero-oligomers. However, to find the most appropriate peptides as drug delivery vectors, one might face several hurdles. Computational analyses showed that 94.3% of the identified SCV2-CPPs are non-toxins, and 38% are neither antigenic nor allergenic. Interestingly, 36.70% of SCV2-CPPs were resistant to all four groups of protease families. Nearly 1/3 of SCV2-CPPs had sufficient inherent or induced helix and sheet conformation leading to increased uptake efficiency. Heliquest lipid-binding discrimination factor revealed that 44.30% of the helical SCV2-CPPs are lipid-binding helices. Although Cys-rich derived CPPs of helicase (NSP13) can potentially fold into a cyclic conformation in endosomes with a higher rate of endosomal release, the most optimal SCV2-CPP candidates as vectors for drug delivery were SCV2-CPP118, SCV2-CPP119, SCV2-CPP122, and SCV2-CPP129 of NSP12 (RdRp). Ten experimentally validated viral-derived CPPs were also used as the positive control to check the scalability and reliability of our protocol in SCV2-CPP retrieval. Some peptides with a cell-penetration ability known as bioactive peptides are adopted as biotherapeutics themselves. Therefore, 59.60%, 29.63%, and 32.32% of SCV2-CPPs were identified as potential antibacterial, antiviral, and antifungals, respectively. While 63.64% of SCV2-CPPs had immuno-modulatory properties, 21.89% were recognized as anti-cancers. Conclusively, the workflow of this study provides a platform for profound screening of viral proteomes as a rich source of biotherapeutics or drug delivery carriers.