An Approach to Evaluate the Effective Cytoplasmic Concentration of Bioactive Agents Interacting with a Selected Intracellular Target Protein

Self Cite

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.

Section: Discussionmentioning

confidence: 84%

Intracellular Degradation of SARS-CoV-2 N-Protein Caused by Modular Nanotransporters Containing Anti-N-Protein Monobody and a Sequence That Recruits the Keap1 E3 Ligase

Khramtsov,

Ulasov,

Lupanova

et al. 2023

Self Cite

“…As we showed earlier using the thermophoresis method in solution, the equilibrium dissociation constant of the MNT 1 complex with Keap1, K d , is equal to 7.9 ± 3.3 nM [ 38 ]. Thus, the preservation of the functional activity of the monobody module within the MNT 1 molecule has already been confirmed.…”

Section: Resultsmentioning

confidence: 95%

“…The interaction of MNT 1 with Keap1 in AML-12 cells was also studied using cellular thermal shift assays (CETSA) [ 34 , 35 , 36 , 37 , 38 ]. Using this method, a Keap1 melting curve in its natural protein environment was obtained in AML-12 cells ( Figure 4 , blue curve).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Modular Nanotransporters Delivering Biologically Active Molecules to the Surface of Mitochondria

Khramtsov,

Ulasov,

Slastnikova

et al. 2023

Self Cite

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 e6, demonstrated significantly higher photocytotoxicity than chlorin e6 alone. We assume that MNTs of this kind can improve efficiency of therapeutic photosensitizers and radionuclides emitting short-range particles.

“…These biomolecular condensates can be considered different phases in liquid–liquid separation [ 135 ]. In this regard, the possibility to assess the effective concentration of the delivered drugs acting on selected regulatory pathways can be very helpful for their further successful development [ 136 ].…”

Section: Intracellular Targets and Barriers On The Way To Themmentioning

confidence: 99%

Prospects of Using Protein Engineering for Selective Drug Delivery into a Specific Compartment of Target Cells

Rosenkranz

Slastnikova

2023

Self Cite

A large number of proteins are successfully used to treat various diseases. These include natural polypeptide hormones, their synthetic analogues, antibodies, antibody mimetics, enzymes, and other drugs based on them. Many of them are demanded in clinical settings and commercially successful, mainly for cancer treatment. The targets for most of the aforementioned drugs are located at the cell surface. Meanwhile, the vast majority of therapeutic targets, which are usually regulatory macromolecules, are located inside the cell. Traditional low molecular weight drugs freely penetrate all cells, causing side effects in non-target cells. In addition, it is often difficult to elaborate a small molecule that can specifically affect protein interactions. Modern technologies make it possible to obtain proteins capable of interacting with almost any target. However, proteins, like other macromolecules, cannot, as a rule, freely penetrate into the desired cellular compartment. Recent studies allow us to design multifunctional proteins that solve these problems. This review considers the scope of application of such artificial constructs for the targeted delivery of both protein-based and traditional low molecular weight drugs, the obstacles met on the way of their transport to the specified intracellular compartment of the target cells after their systemic bloodstream administration, and the means to overcome those difficulties.