Covalent conjugation and non-covalent complexation strategies for intracellular delivery of proteins using cell-penetrating peptides.

Therapeutic proteins provided new opportunities for patients and high sales volumes. However, they are formulated for extracellular targets. The lipophilic barrier of the plasma membrane renders the vast array of intracellular targets out of reach. Peptide-based delivery systems, namely cell-penetrating peptides (CPPs), have few safety concerns, and low immunogenicity, with control over administered doses. This study investigates CPP-based protein delivery systems by classifying them into CPP-protein "covalent conjugation" and CPP: protein "non-covalent complexation" categories. Covalent conjugates ensure the proximity of the CPP to the cargo, which can improve cellular uptake and endosomal escape. We will discuss various aspects of covalent conjugates through non-cleavable (stable) or cleavable bonds. Non-cleavable CPP-protein conjugates are produced by recombinant DNA technology to express the complete fusion protein in a host cell or by chemical ligation of CPP and protein, which ensures stability during the delivery process. CPP-protein cleavable bonds are classified into pH-sensitive and redox-sensitive bonds, enzyme-cleavable bonds, and physical stimuli cleavable linkers (light radiation, ultrasonic waves, and thermo-responsive). We have highlighted the key characteristics of non-covalent complexes through electrostatic and hydrophobic interactions to preserve the conformational integrity of the CPP and cargo. CPP-mediated protein delivery by non-covalent complexation, such as zippers, CPP adaptor methods, and avidin-biotin technology, are featured. Conclusively, non-covalent complexation methods are appropriate when a high number of CPP or protein samples are to be screened. In contrast, when the high biological activity of the protein is critical in the intracellular compartment, conjugation protocols are preferred.

Viral Prefusion Targeting Using Entry Inhibitor Peptides: The Case of SARS-CoV-2 and Influenza A virus.

In this study, peptide entry inhibitors against the fusion processes of severe acute respiratory syndrome coronavirus-2 (SCV2) and influenza A virus (IAV) were designed and evaluated. Fusion inhibitor peptides targeting the conformational shift of the viral fusion protein were designed based on the relatively conserved sequence of HR2 from SCV2 spike protein and the conserved fusion peptide from hemagglutinin (HA) of IAV. Helical HR2 peptides bind more efficiently to HR1 trimer, while helical amphipathic anti-IAV peptides have higher cell penetration and endosomal uptake. The initial sequences were mutated by increasing the amphipathicity, using helix favoring residues, and residues likely to form salt- and disulfide-bridges. After docking against their targets, all anti-SCV2 designed peptides bonded with the HR1 3-helical bundle's hydrophobic crevice, while AntiSCV2P1, AntiSCV2P3, AntiSCV2P7, and AntiSCV2P8 expected to form coiled coils with at least one of the HR1 strands. Four of the designed anti-IAV peptides were cell-penetrating (AntiIAVP2, AntiIAVP3, AntiIAVP4, AntiIAVP7). All of them interacted with the fusion peptide of HA and some of the residues in the conserved hydrophobic pocket of HA2 in H1N1, H3N1, and H5N1 subtypes of IAV. AntiIAVP3 and AntiIAVP4 peptides had the best binding to HA2 conserved hydrophobic pocket, while, AntiIAVP2 and AntiIAVP6 showed the best binding to the fusion peptide region. According to analyses for in-vivo administration, AntiSCV2P1, AntiSCV2P7, AntiIAVP2, and AntiIAVP7 were the best candidates. AntiSCV2 and AntiIAV peptides were also conjugated using an in vivo cleavable linker sensitive to TMPRSS2 applicable as a single therapeutic in coinfections or uncertain diagnosis. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10989-021-10357-y.

Design, synthesis, and biological evaluation of symmetrical azine derivatives as novel tyrosinase inhibitors.

A series of symmetrical azine derivatives containing different substituted benzyl moieties were designed, synthesized, and evaluated for their inhibitory activity against tyrosinase. The results showed that compounds 3e, 3f, 3h, 3i, 3j, and 3k possess effective tyrosinase inhibition with IC50 values ranging from 7.30 µM to 62.60 µM. Particularly, compounds 3f displayed around three-fold improvement in the potency (IC50 = 7.30 ± 1.15 µM) compared to that of kojic acid (IC50 = 20.24 ± 2.28 µM) as the positive control. Kinetic study of compound 3f confirmed uncompetitive inhibitory activity towards tyrosinase indicating that it can bind to enzyme-substrate complex. Next, molecular docking analysis was performed to study the interactions and binding mode of the most potent compound 3f in the tyrosinase active site. Besides, the cytotoxicity of 3f, as well as its potency to reduce the melanin content were also measured on invasive melanoma B16F10 cell line. Also, 3f exhibited above 82% cell viability in the A375 cell line at 10 µM. Consequently, compounds 3f could be introduced as a potent tyrosinase inhibitor that might be a promising candidate in the cosmetics, medicine, and food industry.

Viral 3CLpro as a Target for Antiviral Intervention Using Milk-Derived Bioactive Peptides.

Viruses of the picornavirus-like supercluster mainly achieve cleavage of polyproteins into mature proteins through viral 3-chymotrypsin proteases (3Cpro) or 3-chymotrypsin-like proteases (3CLpro). Due to the essential role in processing viral polyproteins, 3Cpro/3CLpro is a drug target for treating viral infections. The 3CLpro is considered the main protease (Mpro) of coronaviruses. In the current study, the SARS-CoV-2 Mpro inhibitory activity of di- and tri-peptides (DTPs) resulted from the proteolysis of bovine milk proteins was evaluated. A set of 326 DTPs were obtained from virtual digestion of bovine milk major proteins. The resulted DTPs were screened using molecular docking. Twenty peptides (P1-P20) showed the best binding energies (ΔG b < - 7.0 kcal/mol). Among these 20 peptides, the top five ligands, namely P1 (RVY), P3 (QSW), P17 (DAY), P18 (QSA), and P20 (RNA), based on the highest binding affinity and the highest number of interactions with residues in the active site of Mpro were selected for further characterization by ADME/Tox analyses. For further validation of our results, molecular dynamics simulation was carried out for P3 as one of the most favorable candidates for up to 100 ns. In comparison to N3, a peptidomimetic control inhibitor, high stability was observed as supported by the calculated binding energy of the Mpro-P3 complex (- 59.48 ± 4.87 kcal/mol). Strong interactions between P3 and the Mpro active site, including four major hydrogen bonds to HIS41, ASN142, GLU166, GLN189 residues, and many hydrophobic interactions from which the interaction with CYS145 as a catalytic residue is worth mentioning. Conclusively, milk-derived bioactive peptides, especially the top five selected peptides P1, P3, P17, P18, and P20, show promise as an antiviral lead compound. Supplementary Information: The online version contains supplementary material available at 10.1007/s10989-021-10284-y.

Introducing a delivery system for melanogenesis inhibition in melanoma B16F10 cells mediated by the conjugation of tyrosine ammonia-lyase and a TAT-penetrating peptide.

Hyperpigmentation disorders negatively influence an individual's quality of life and may cause emotional distress. Over the years, various melanogenesis inhibitors (mainly tyrosinase inhibitors) have been developed, most of which with low efficacy or high toxicity. Although metabolic engineering by deviation in the flux of substrate is of considerable interest, trials to develop a melanogenesis inhibitor based on L-tyrosine (L-Tyr) restriction are missing. We propose a novel proteinaceous melanogenesis inhibitor called tyrosine ammonia-lyase (TAL), an enzyme that catalyzes the conversion of L-Tyr to p-coumaric acid and ammonia. Since the cell membrane can act as a barrier for intracellular protein delivery, we have covalently conjugated a recombinant TAL enzyme from Rhodobacter sphaeroides (RsTAL) to a trans-activator of transcription (TAT) cell-penetrating peptide (CPP) to afford the intracellular delivery. The heterologously expressed TAT-RsTAL fusion protein was delivered successfully into B16F10 melanocytes as confirmed by the direct fluorescence microscopy with increased intensity from 30 to 180 min. TAT-RsTAL showed sufficient intracellular activity of about 0.83 ± 0.04 and 0.34 ± 0.03 nmol•mg-1 •s-1 for the native and inclusion body-extracted conjugates, respectively. The conjugate inhibited melanin biosynthesis in B16F10 cells in a time-dependent manner. Melanin accumulation was inhibited by 12.7 ± 6.2%, 28.2 ± 5.7%, and 33.9 ± 2.9% compared to the nontreated control groups after 24, 48, and 72 hr of incubation, respectively. L-Tyr restriction had no significant effect on the cell viability up to a concentration of 100 µgml-1 even after 72 hr. According to the observed hypopigmentary effect of the conjugate in this study, TAT-RsTAL can be suggested as a melanogenesis inhibitor for further investigations.

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.

Considerations on the Rational Design of Covalently Conjugated Cell-Penetrating Peptides (CPPs) for Intracellular Delivery of Proteins: A Guide to CPP Selection Using Glucarpidase as the Model Cargo Molecule.

Access of proteins to their intracellular targets is limited by a hydrophobic barrier called the cellular membrane. Conjugation with cell-penetrating peptides (CPPs) has been shown to improve protein transduction into the cells. This conjugation can be either covalent or non-covalent, each with its unique pros and cons. The CPP-protein covalent conjugation may result in undesirable structural and functional alterations in the target protein. Therefore, we propose a systematic approach to evaluate different CPPs for covalent conjugations. This guide is presented using the carboxypeptidase G2 (CPG2) enzyme as the target protein. Seventy CPPs -out of 1155- with the highest probability of uptake efficiency were selected. These peptides were then conjugated to the N- or C-terminus of CPG2. Translational efficacy of the conjugates, robustness and thermodynamic properties of the chimera, aggregation possibility, folding rate, backbone flexibility, and aspects of in vivo administration such as protease susceptibility were predicted. The effect of the position of conjugation was evaluated using unpaired t-test (p < 0.05). It was concluded that N-terminal conjugation resulted in higher quality constructs. Seventeen CPP-CPG2/CPG2-CPP constructs were identified as the most promising. Based on this study, the bioinformatics workflow that is presented may be universally applied to any CPP-protein conjugate design.