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Background/Objectives: The study of oxidative stress in cells and ways to prevent it attract increasing attention. Antioxidant defense of cells can be activated by releasing the transcription factor Nrf2 from a complex with Keap1, its inhibitor protein. The aim of the work was to study the effect of the modular nanotransporter (MNT) carrying an R1 anti-Keap1 monobody (MNT R1 ) on cell homeostasis. Methods: The murine hepatocyte AML12 cells were used for the study. The interaction of fluorescently labeled MNT R1 with Keap1 fused to hrGFP was studied using the Fluorescence-Lifetime Imaging Microscopy-Förster Resonance Energy Transfer (FLIM-FRET) technique on living AML12 cells transfected with the Keap1-hrGFP gene. The release of Nrf2 from the complex with Keap1 and its levels in the cytoplasm and nuclei of the AML12 cells were examined using a cellular thermal shift assay (CETSA) and confocal laser scanning microscopy, respectively. The effect of MNT on the formation of reactive oxygen species was studied by flow cytometry using 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate. Results: MNT R1 is able to interact with Keap1 in the cytoplasm, leading to the release of Nrf2 from the complex with Keap1 and a rapid rise in Nrf2 levels both in the cytoplasm and nuclei, ultimately causing protection of cells from the action of hydrogen peroxide. The possibility of cleavage of the monobody in endosomes leads to an increase in the observed effects. Conclusions: These findings open up a new approach to specifically modulating the interaction of intracellular proteins, as demonstrated by the example of the Keap1-Nrf2 system.

Modular nanotransporters (MNTs) are drug delivery systems for targeted cancer treatment. As MNTs are composed of several modules, they offer the advantage of high specificity and biocompatibility in delivering drugs to the target compartment of cancer cells. The large carrier module brings together functioning MNT modules and serves as a platform for drug attachment. The development of smaller-sized MNTs via truncation of the carrier module appears advantageous in facilitating tissue penetration. In this study, two new MNTs with a truncated carrier module containing either an N-terminal (MNT N ) or a C-terminal (MNT C ) part were developed by genetic engineering. Both new MNTs demonstrated a high affinity for target receptors, as revealed by fluorescent-labeled ligand-competitive binding. The liposome leakage assay proved the endosomolytic activity of MNTs. Binding to the importin heterodimer of each truncated MNT was revealed by a thermophoresis assay, while only MNT N possessed binding to Keap1. Finally, the photodynamic efficacy of the photosensitizer attached to MNT N was significantly higher than when attached to either MNT C or the original MNTs. Thus, this work reveals that MNT's carrier module can be truncated without losing MNT functionality, favoring the N-terminal part of the carrier module due to its ability to bind Keap1.

The proper viral assembly relies on both nucleic acids and structural viral proteins. Thus a biologically active agent that provides the degradation of one of these key proteins and/or destroys the viral factory could suppress viral replication efficiently. The nucleocapsid protein (N-protein) is a key protein for the SARS-CoV-2 virus. As a bioactive agent, we offer a modular nanotransporter (MNT) developed by us, which, in addition to an antibody mimetic to the N-protein, contains an amino acid sequence for the attraction of the Keap1 E3 ubiquitin ligase. This should lead to the subsequent degradation of the N-protein. We have shown that the functional properties of modules within the MNT permit its internalization into target cells, endosome escape into the cytosol, and binding to the N-protein. Using flow cytometry and western blotting, we demonstrated significant degradation of N-protein when A549 and A431 cells transfected with a plasmid coding for N-protein were incubated with the developed MNTs. The proposed MNTs open up a new approach for the treatment of viral diseases.

Treatment of various diseases, in particular cancer, usually requires the targeting of biologically active molecules at a selected subcellular compartment. We modified our previously developed modular nanotransporters (MNTs) for targeting mitochondria. The new MNTs are capable of binding to the protein predominantly localized on the outer mitochondrial membrane, Keap1. These MNTs possessing antiKeap1 monobody co-localize with mitochondria upon addition to the cells. They efficiently interact with Keap1 both in solution and within living cells. A conjugate of the MNT with a photosensitizer, chlorin e 6 , demonstrated significantly higher photocytotoxicity than chlorin e 6 alone. We assume that MNTs of this kind can improve efficiency of therapeutic photosensitizers and radionuclides emitting short-range particles.

To compare the effectiveness of various bioactive agents reversibly acting within a cell on a target intracellular macromolecule, it is necessary to assess effective cytoplasmic concentrations of the delivered bioactive agents. In this work, based on a simple equilibrium model and the cellular thermal shift assay (CETSA), an approach is proposed to assess effective concentrations of both a delivered bioactive agent and a target protein. This approach was tested by evaluating the average concentrations of nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated-protein 1 (Keap1) proteins in the cytoplasm for five different cell lines (Hepa1, MEF, RAW264.7, 3LL, and AML12) and comparing the results with known literature data. The proposed approach makes it possible to analyze both binary interactions and ternary competition systems; thus, it can have a wide application for the analysis of protein-protein or molecule-protein interactions in the cell. The concentrations of Nrf2 and Keap1 in the cell can be useful not only in analyzing the conditions for the activation of the Nrf2 system, but also for comparing the effectiveness of various drug delivery systems, where the delivered molecule is able to interact with Keap1.

The development of epidermal growth factor receptor (EGFR)-targeting agents for the treatment of malignant melanoma requires cheap and easy animal tumor models for high-throughput in vivo screening. Thus, the aim of this study was to develop mouse syngeneic melanoma model that expresses human EGFR. Cloudman S91 clone M3 mouse melanoma cells were transduced with lentiviral particles carrying the human EGFR gene followed by a multistep selection process. The resulting M3-EGFR has been tested for EGFR expression and functionality in vitro and in vivo. Radioligand assay confirmed the presence of 13,900 ± 1500 EGF binding sites per cell at a dissociation constant of 5.3 ± 1.4 nM. M3-EGFR demonstrated the ability to bind and internalize specifically and provide the anticipated intracellular nuclear import of three different EGFR-targeted modular nanotransporters designed for specific anti-cancer drug delivery. Introduction of the human EGFR gene did not alter the tumorigenicity of the offspring M3-EGFR cells in host immunocompetent DBA/2J mice. Preservation of the expression of EGFR in vivo was confirmed by immunohistochemistry. To sum up, we successfully developed the first mouse syngeneic melanoma model with preserved in vivo expression of human EGFR.