Functional analysis of bipartite NRF2 activators that overcome feedback regulation for age-related chronic diseases.

Activating Nrf2 with small molecules is a promising strategy for countering aging, oxidative stress, inflammation, and various disorders, including neurodegeneration. The primary regulator of Nrf2 protein stability is Keap1, a redox sensor protein and an adapter in the Cullin III ubiquitin ligase complex, which labels Nrf2 for proteasomal degradation. The canonical Nrf2 activators either chemically modify sensor thiols in Keap1 or competitively displace Nrf2 from the ubiquitin ligase complex. The latter approach is considered the most suitable for continuous administration, as non-specific chemical modifiers of Keap1 thiols also modify active thiols on other cellular proteins, causing side effects. However, when transitioning from homogeneous cell-free to cell-based assays, genuine displacement activators show a significant loss in potency by several orders of magnitude. We demonstrate that this discrepancy arises due to higher micromolar concentrations of Keap1 in cell lines. The absolute amounts of Nrf2 and Keap1 determined in brain sub-regions show more than an order of magnitude excess of Keap1 over Nrf2. A potential solution could involve targeted delivery of an alkylating agent to Keap1 to achieve the desired specificity. Transcriptomic analysis of a cell-permeable Nrf2 peptide bearing an alkylating fumarate moiety indicates selective activation of the Nrf2 genetic program, confirming the high specificity of this approach. Activation of the Nrf2-genetic program has a built-in feedback regulatory mechanism through Bach1, an Nrf2 transcriptional repressor, whose levels are elevated in age-related neurodegeneration. Thus, a benign bipartite Nrf2 activator with Bach1 inhibition properties is needed for maximal benefits. The recently developed heterocyclic carboxamide, HPPE, exhibits overlap with the Nrf2 pathway activated by the fumarate-linked Nrf2 peptide, an Nrf2 activator, as well as with zinc and tin protoporphyrins, which are inhibitors of Bach1. Therefore, HPPE presents a promising and unique combination of the two desired activities that could be further optimized to treat age-related neurodegeneration.