ИСТИНА

A promising strategy for the treatment of neurodegenerative diseases involves pharmaco-logically-induced activation of a coordinated antioxidant genetic program to shift the intracellular redox balance towards a homeostatic state by means of expression of detoxifying proteins and anti-oxidant enzymes. A key transcription factor orchestrating antioxidant defense is Nrf2 (nuclear fac-tor erythroid 2–related factor 2), a member of the cap’n’collar family of basic leucine zipper tran-scription factors. Nrf2 is constitutively produced in the cell. However, in the absence of environ-mental stress, Nrf2 is sequestered in the cytoplasm by binding to an inhibitory protein, Keap1 (Kelch-like ECH-associated protein-1), which promotes continuous ubiquitinylation. Keap1 serves as a bridge between Nrf2 and the Cul3-Rbx1 E3 ubiquitin ligase, leading to polyubiquitinylation of the lysines positioned within the central α-helix of the Neh2 domain of Nrf2 under homeostatic conditions. Nrf2 is composed of six Neh domains, with Neh2 being the putative negative regulatory domain that interacts with Keap1. Two motifs in the Neh2 domain, ETGE (high affinity) and DLG (low affinity), are recognized by the Keap1 homodimer in a hinge-latch mode. Electrophilic stress leads to modification of reactive cysteines within Keap1 that induces conformational changes re-sulting in detachment of the weak-binding DLG, resulting in Nrf2 stabilization. The Nrf2 protein is released from its complex with Keap1 and then translocates into the nucleus. There, it forms het-erodimers with other transcription regulators, such as small Maf proteins, and induces the expres-sion of antioxidant genes controlled by promoters with the antioxidant response element (ARE). We have developed a novel cell-based reporter system for identification of Nrf2 activators working via stabilization of Nrf2 protein. Our approach relies on constitutive expression of the minimum portion of the Nrf2 transcription factor, Neh-2 domain (sufficient for recognition by Keap1 and subsequent ubiquitinylation), fused to firefly luciferase. The minimum portion of Neh-2 domain sufficient for recognition by Keap1 has been identified by sequential cut-off of the Neh2-domain from N- and C-terminal sides: it has been found that 16-85 aa portion of Neh2 domain is the minimum necessary for the reporter to function properly. The Neh2-luc reporter system is at least 10-fold more efficient in the magnitude of reporter activation as compared to the commonly used ARE-luc, and more importantly, reporter activation can be monitored in real time just minutes after drug addition. The Neh2-luc reporter system, with appropriate controls, is a tool to directly monitor the effect of a particular compound on the first step controlling Nrf2 stability, i.e. Nrf2-Keap1 interaction. Nrf2 activation without covalent modification of Keap1 cysteines can be achieved via Nrf2 protein displacement from its complex with Keap1 either by a small molecule, or Nrf2 peptide. While peptides are not ideal drug candidates, they are very useful as chemical tools, particularly when conjugated to a cell-penetrating peptide sequence such as TAT-peptide derived from HIV. Our group examined a 16-mer version of ETGE peptide (YGRKKRRQRRRAQLQLDEETGE-FLPIQ) and found that the concentration of the wild-type ETGE-peptide necessary to trigger a sig-nificant response in 3 h has to be above 200 M, which is an order of magnitude higher than con-centrations of common Nrf2 activators of alkylating nature. Reporter activation peaks at 8-12 h, and then declines due to peptide degradation. To increase the peptide affinity for Kelch domain, we in-troduced a mutation Glu->Pro into ETGE motif, i.e. YGRKKRRQRRRAQLQLDPETGEFLPIQ, which was reported to increase the affinity of recombinant Keap1 for the mutant peptide by 50-fold. However, in the cell-based assay such mutation allowed us to improve EC50 by 3-4 fold and activa-tion magnitude by 2-3 fold. This result clearly points to the differences between in vitro affinity measurements with recombinant Keap1 and conditions of real competition with Nrf2 fusion protein (or endogenous Nrf2) in cell-based assays. The low potency of cell-permeable peptides in the cell-based reporter assay is not the result of its worsened characteristics in the in vitro assay: in frames of our ongoing collaboration with Dr.Y-H.Ahn (Wayne State Univ, Detroit, USA), the cell-permeable mutant Nrf2 peptide was tested in FP assay with recombinant Keap1, and its binding constant was estimated as ca. 50 nM (versus 84 nM for the wild-type Nrf2 peptide without TAT-sequence). Of note, the IC50 for the Neh2 domain of Nrf2, equals to 0.086 M in the in vitro assay. Thus, the presence of TAT-sequence did not compromise in vitro binding activity of the Nrf2 pep-tide, on the contrary, it made it better. Peptide sequence optimization for in vivo purposes requires further work on peptides testing in Neh2-luc cell-based assay, which eventually may generate a ver-sion competitive with the known alkylating activators of Nrf2. The work is supported by Russian Foundation for Basic Research (grant 17-04-01480а)