Modular Nanotransporters Capable of Binding to SARS-CoV-2 Virus Nucleocapsid Protein in Target Cells.

On the basis of literature data, an antibody-like molecule, monobody, was selected that is capable of interacting with the nucleocapsid protein (N protein) of the SARS-CoV-2 virus with a high affinity (dissociation constant 6.7 nM). We have previously developed modular nanotransporters (MNTs) to deliver various molecules to a selected compartment of target cells. In this work, a monobody to the N protein of the SARS-CoV-2 virus was inserted in the MNT using genetic engineering methods. In this MNT, a site for the cleavage of the monobody from the MNT in endosomes was also inserted. It was shown by thermophoresis that the cleavage of this monobody from the MNT by the endosomal protease cathepsin B leads to a 12-fold increase in the affinity of the monobody for the N protein. Cellular thermal shift assay showed the ability of the obtained MNT to interact with the N protein in A431 cells transfected with the SARS-CoV-2 N protein fused to the mRuby3 fluorescent protein.

Intracellular Delivery of an Antibody-Like Molecule Capable of Inhibiting c-Myc.

A modular nanotransporter (MNT) carrying the sequence of an antibody-like molecule, anti-c-Myc nanobody, was synthesized and characterized. It was demonstrated that the created MNT is able to interact with the target protein, c-Myc oncogene, with a dissociation constant of 46 ± 14 nM, internalize into target cells, change Myc-dependent expression, and exert an antiproliferative effect.

Selection of an Amino Acid Site with One of the Fastest Cleavage Kinetics by the Endosomal Protease Cathepsin B for Potential Use in Drug Delivery Systems.

On the basis of known published data, six peptide sequences were selected that are potentially capable of being rapidly cleaved by the endosomal protease cathepsin B. For comparison, the cleavage of common linker sequences, polyglycine and polyglycine-serine, by cathepsin B was also studied. Different ends of these peptides were labeled with sulfoCyanine3 and sulfoCyanine5 fluorescent dyes, between which Förster resonant energy transfer (FRET) is possible. The kinetics of cleavage of peptides by cathepsin B was studied on a multimodal plate reader by FRET signal reduction. FKFL and FRRG cleavage sites have been shown to be the most suitable for potential use in various drug delivery systems. These sites are much more efficiently cleaved under slightly acidic conditions of endosomes than at neutral extracellular pH.

Quantitative Description of the N-Protein of the SARS-CoV-2 Virus Degradation in Cells Stably Expressing It under the Influence of New Modular Nanotransporters.

Two eukaryotic cell lines, A549 and A431, with stable expression of the nucleocapsid protein (N-protein) of the SARS-CoV-2 virus fused with the red fluorescent protein mRuby3 were obtained. Using microscopy, the volumes of the cytoplasm and nucleus were determined for these cells. Using quantitative immunoblotting techniques, the concentrations of the N-mRuby3 fusion protein in their cytoplasm were assessed. They were 19 and 9 µM for A549 and A431 cells, respectively. Using these concentrations, the initial rate of N-protein degradation in the studied cells was estimated from the decrease in cell fluorescence. In A549 and A431 cells, it was the same (84 nM per hour). The approach of quantitatively describing the degradation process can be applied to analyze the effectiveness of a wide class of antiviral drugs that cause degradation of viral proteins.

Modular Nanotransporters Capable of Causing Intracellular Degradation of the N-Protein of the SARS-CoV-2 Virus in A549 Cells with Temporary Expression of This Protein Fused with a Fluorescent Protein mRuby3.

Modular nanotransporters (MNTs) containing an antibody-like molecule, monobody, to the N­protein of the SARS-CoV-2 virus, as well as an amino acid sequence that recruits the Keap1 E3 ligase (E3BP) were created. This MNT also included a site for cleavage of the E3BP monobody from the MNT in acidic endocytic compartments. It was shown that this cleavage by the endosomal protease cathepsin B leads to a 2.7-fold increase in the affinity of the E3BP monobody for the N-protein. Using A549 cells with transient expression of the N-protein fused with the fluorescent protein mRuby3, it was shown that incubation with MNT leads to a significant decrease in mRuby3 fluorescence. It is assumed that the developed MNTs can serve as a basis for the creation of new antiviral drugs against the SARS-CoV-2 virus.

Delivery of Antibody-Like Molecules, Monobodies, Capable of Binding with SARS-CoV-2 Virus Nucleocapsid Protein, into Target Cells.

Based on previous studies, two antibody-like molecules, monobodies, capable of high-affinity interaction with the SARS-CoV-2 nucleocapsid protein (dissociation constant of tens of nM) were selected. For delivery to target cells, genetically engineered constructs containing monobody and TAT peptide, placed either at the N- or C-terminus of the resulting polypeptide, were produced and expressed in E. coli. The construct with the highest affinity to the SARS-CoV-2 nucleocapsid protein was revealed with the use of thermophoresis technique. Cellular thermal shift assay demonstrated the ability of this construct to interact with the nucleocapsid protein within HEK293T cells transfected with the SARS-CoV-2 nucleocapsid protein fused to the mRuby3 fluorescent protein. Replacement of TAT peptide to S10 shuttle peptide, containing endosomolytic peptide, significantly improved the penetration of the construct into the target cells.

Among Antibody-Like Molecules, Monobodies, Able to Interact with Nucleocapsid Protein of SARS-CoV Virus, There Are Monobodies with High Affinity to Nucleocapsid Protein of SARS-CoV-2 Virus.

Seven amino acid sequences of antibody mimetics molecules, monobodies, capable of interacting with the nucleocapsid protein of the SARS-CoV virus, were taken from the literature. Nucleotide sequences of monobody genes were obtained by gene synthesis, which were expressed in E. coli and isolated using Ni-NTA chromatography. It was shown by thermophoresis that three of the seven selected antibody-like molecules can interact with high affinity (dissociation constant of tens of nM) with the nucleocapsid protein of the SARS-CoV-2 virus. For the remaining four monobodies, only low affinity binding with a dissociation constant of several µM was found.

New Recombinant Carriers Binding Specifically to the Epidermal Growth Factor Receptor.

New recombinant carriers-modular nanotransporters (MNTs)-with N-terminal ligand module to the epidermal growth factor receptor (EGFR) were developed and characterized. Human epidermal growth factor (hEGF) and antibody-like protein Z1907 were used as a ligand module. We demonstrated that MNTs are able to internalize in a receptor-specific manner into the target cancer cells and to accumulate in the target cell nuclei. Conjugation of MNTs with the Auger electron emitter 111In significantly enhanced the cytotoxic effect of 111In on the target cells. It was found that the transfer of EGF from the C-terminus to the N-terminus of the MNT enhanced the proliferation of target cells, whereas the use of Z1907 did not have a similar effect.

MNT Optimization for Intracellular Delivery of Antibody Fragments.

We studied the possibility of optimizing modular nanotransporters (MNTs) for the intracellular delivery of antibody fragments into the nuclei of cells of a specified type. Basic MNT with a reduced size retaining the desired functions was obtained, and the principal possibility of obtaining an MNT carrying an antibody fragment by microbiological synthesis was shown.

Modular Nanotransporter with P21 Fragment Inhibits DNA Repair after Bleomycin Treatment.

Modular nanotransporter (MNT) with C-terminal fragment of the p21 protein was synthesized and characterized, and its effect on DNA lesions was studied. This p21 fragment in MNT can significantly inhibit DNA repair in A431 human carcinoma cells after bleomycin treatment.

Study of Biodistribution of the Modular Nanotransporters after Systemic Administration in Murine Cloudman S91 Melanoma Model.

The distribution of modular nanotransporters (MNTs) that are used to deliver drugs into melanoma cell nuclei after their intravenous administration into mice with Cloudman S91 melanoma was studied. The modification of MNTs with polyethylene glycol (PEG) of different length and their administration during the treatment with docetaxel, nitroglycerin, and excess of nonspecific MNTs leads to an improved accumulation of MNTs in the tummor. Among the variants studied, the MNT with attached PEG with Mr 40 kDa exhibited the best properties.

Use of intracellular transport processes for targeted drug delivery into a specified cellular compartment.

Targeted drug delivery into the cell compartment that is the most vulnerable to effects of the corresponding drug is a challenging problem, and its successful solution can significantly increase the efficiency and reduce side effects of the delivered therapeutic agents. To accomplish this one can utilize natural mechanisms of cellular specific uptake of macromolecules by receptor-mediated endocytosis and intracellular transport between cellular compartments. A transporting construction combining the components responsible for different steps of intracellular transport is promising for creating multifunctional modular constructions capable of delivering the necessary therapeutic agent into a given compartment of type-specified cells. This review focuses on intracellular transport peculiarities along with approaches for designing such transporting constructions for new, more effective, and safer strategies for treatment of various diseases.