Novel Polymyxin-Inspired Peptidomimetics Targeting the SARS-CoV-2 Spike:hACE2 Interface.

Though the bulk of the COVID-19 pandemic is behind, the search for effective and safe anti-SARS-CoV-2 drugs continues to be relevant. A highly pursued approach for antiviral drug development involves targeting the viral spike (S) protein of SARS-CoV-2 to prevent its attachment to the cellular receptor ACE2. Here, we exploited the core structure of polymyxin B, a naturally occurring antibiotic, to design and synthesize unprecedented peptidomimetics (PMs), intended to target contemporarily two defined, non-overlapping regions of the S receptor-binding domain (RBD). Monomers 1, 2, and 8, and heterodimers 7 and 10 bound to the S-RBD with micromolar affinity in cell-free surface plasmon resonance assays (KD ranging from 2.31 µM to 2.78 µM for dimers and 8.56 µM to 10.12 µM for monomers). Although the PMs were not able to fully protect cell cultures from infection with authentic live SARS-CoV-2, dimer 10 exerted a minimal but detectable inhibition of SARS-CoV-2 entry in U87.ACE2+ and A549.ACE2.TMPRSS2+ cells. These results validated a previous modeling study and provided the first proof-of-feasibility of using medium-sized heterodimeric PMs for targeting the S-RBD. Thus, heterodimers 7 and 10 may serve as a lead for the development of optimized compounds, which are structurally related to polymyxin, with improved S-RBD affinity and anti-SARS-CoV-2 potential.

Total Chemical Synthesis of a SARS-CoV-2 Miniprotein Inhibitor LCB1.

Native chemical ligation is a widely used technique for peptide fragment condensation in aqueous solutions, which has broken through the length limitation of traditional solid-phase peptide synthesis. It can achieve high-efficient chemical synthesis of proteins containing more than 300 amino acid residues. Peptide hydrazide, as a valuable reagent equivalent to a thioester peptide, can be easily and efficiently prepared by the Fmoc-based SPPS method and has been widely used in native chemical ligation. Here we take the chemical synthesis of a SARS-CoV-2 miniprotein inhibitor LCB1 as an example to describe the detailed procedure of hydrazide-based native chemical ligation.

Synthesis, Physicochemical Characterization, In Vitro 2D/3D Human Cell Culture, and In Vitro Aerosol Dispersion Performance of Advanced Spray Dried and Co-Spray Dried Angiotensin (1-7) Peptide and PNA5 with Trehalose as Microparticles/Nanoparticles for Targeted Respiratory Delivery as Dry Powder Inhalers.

The peptide hormone Angiotensin (1-7), Ang (1-7) or (Asp-Arg-Val-Tyr-Ile-His-Pro), is an essential component of the renin-angiotensin system (RAS) peripherally and is an agonist of the Mas receptor centrally. Activation of this receptor in the CNS stimulates various biological activities that make the Ang (1-7)/MAS axis a novel therapeutic approach for the treatment of many diseases. The related O-linked glycopeptide, Asp-Arg-Val-Tyr-Ile-His-Ser-(O-ß-D-Glc)-amide (PNA5), is a biousian revision of the native peptide hormone Ang (1-7) and shows enhanced stability in vivo and greater levels of brain penetration. We have synthesized the native Ang (1-7) peptide and the glycopeptide, PNA5, and have formulated them for targeted respiratory delivery as inhalable dry powders. Solid phase peptide synthesis (SPPS) successfully produced Ang (1-7) and PNA5. Measurements of solubility and lipophilicity of raw Ang (1-7) and raw PNA5 using experimental and computational approaches confirmed that both the peptide and glycopeptide have high-water solubility and are amphipathic. Advanced organic solution spray drying was used to engineer the particles and produce spray-dried powders (SD) of both the peptide and the glycopeptide, as well as co-spray-dried powders (co-SD) with the non-reducing sugar and pharmaceutical excipient, trehalose. The native peptide, glycopeptide, SD, and co-SD powders were comprehensively characterized, and exhibited distinct glass transitions (Tg) consistent with the amorphous glassy state formation with Tgs that are compatible with use in vivo. The homogeneous particles displayed small sizes in the nanometer size range and low residual water content in the solid-state. Excellent aerosol dispersion performance with a human DPI device was demonstrated. In vitro human cell viability assays showed that Ang (1-7) and PNA5 are biocompatible and safe for different human respiratory and brain cells.

Peptide-Based Antiviral Drugs.

Three types of chemical entities, namely, small organic molecules (organics), peptides, and biologics, are mainly used as drug candidates for the treatment of various diseases. Even though the peptide drugs are known since 1920 in association with the clinical use of insulin, only a limited number of peptides are currently used for therapeutics due to various disadvantages associated with them such as limited serum and blood stability, oral bioavailability, and permeability. Since, through chemical modifications and structure tuning, many of these limitations can be overcome, peptide-based drugs are gaining attention in pharmaceutical research. As of today, there are more than 60 peptide-based drugs approved by FDA, and over 150 peptides are in the advanced clinical studies. In this book chapter, the peptide-based lead compounds and drugs available for treating various viral diseases and their advantages and disadvantages when compared to small molecules drugs are discussed.

A nanotechnological approach in the current therapy of COVID-19: model drug oseltamivir-phosphate loaded PLGA nanoparticles targeted with spike protein binder peptide of SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) is today's most serious epidemic disease threatening the human race. The initial therapeutic approach of SARS-CoV-2 disease is based upon the binding the receptor-binding site of the spike protein to the host cell's ACE-2 receptor on the plasma membrane. In this study, it is aimed to develop a biocompatible and biodegradable polymeric drug delivery system that is targeted to the relevant receptor binding site and provides controlled drug release. Oseltamivir phosphate (OP) is an orally administered antiviral prodrug for primary therapy of the disease in biochemically activated carboxylate form (oseltamivir carboxylate OC). In the presented study, model drug OP loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) targeted with spike-binding peptide 1 (SBP1) of SARS-CoV-2 were designed to be used as an efficient and prolonged released antiviral drug delivery system. RY, EE, and DL values of the OP-loaded NPs produced by the solvent evaporation method were calculated to be 59.3%, 61.4%, and 26.9%, respectively. The particle size of OP-loaded NPs and OP-loaded NPs targeted with SBP1 peptide were 162.0 ± 11.0 and 226.9 ± 21.4 nm, respectively. While the zeta potential of the produced OP-loaded NPs was achieved negatively -23.9 ± 1.21 mV), the result of the modification with SBP1 peptide this value approached zero as -4.59 ± 0.728 mV. Morphological features of the OP-loaded NPs were evaluated using FEG-SEM. The further characterization and surface modification of the NPs were analyzed by FT-IR.In-vitrorelease studies of NPs showed that sustained release of OP occurred for two months that fitting the Higuchi kinetic model. By evaluating these outputs, it was reported that surface modification of OP-loaded NPs was significantly effective on characteristics such as size, zeta potential values, surface functionality, and release behavior. The therapeutic model drug-loaded polymeric formulation targeted with a specific peptide may serve as an alternative to more effective and controlled release pharmaceuticals in the treatment of COVID-19 upon an extensive investigation.

Downstream Processing of Therapeutic Peptides by Means of Preparative Liquid Chromatography.

The market of biomolecules with therapeutic scopes, including peptides, is continuously expanding. The interest towards this class of pharmaceuticals is stimulated by the broad range of bioactivities that peptides can trigger in the human body. The main production methods to obtain peptides are enzymatic hydrolysis, microbial fermentation, recombinant approach and, especially, chemical synthesis. None of these methods, however, produce exclusively the target product. Other species represent impurities that, for safety and pharmaceutical quality reasons, must be removed. The remarkable production volumes of peptide mixtures have generated a strong interest towards the purification procedures, particularly due to their relevant impact on the manufacturing costs. The purification method of choice is mainly preparative liquid chromatography, because of its flexibility, which allows one to choose case-by-case the experimental conditions that most suitably fit that particular purification problem. Different modes of chromatography that can cover almost every separation case are reviewed in this article. Additionally, an outlook to a very recent continuous chromatographic process (namely Multicolumn Countercurrent Solvent Gradient Purification, MCSGP) and future perspectives regarding purification strategies will be considered at the end of this review.