ABSTRACT
Successful and selective inhibition of the cytosolic protein-protein interaction (PPI) between nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associating protein 1 (Keap1) can enhance the antioxidant response, with the potential for a therapeutic effect in a range of settings including in neurodegenerative disease (ND). Small molecule inhibitors have been developed, yet many have off-target effects, or are otherwise limited by poor cellular permeability. Peptide-based strategies have also been attempted to enhance specificity, yet face challenges due to susceptibility to degradation and lack of cellular penetration. Herein, these barriers are overcome utilizing a polymer-based proteomimetics. The protein-like polymer (PLP) consists of a synthetic, lipophilic polymer backbone displaying water soluble Keap1-binding peptides on each monomer unit forming a brush polymer architecture. The PLPs are capable of engaging Keap1 and displacing the cellular protective transcription factor Nrf2, which then translocates to the nucleus, activating the antioxidant response element (ARE). PLPs exhibit increased Keap1 binding affinity by several orders of magnitude compared to free peptides, maintain serum stability, are cell-penetrant, and selectively activate the ARE pathway in cells, including in primary cortical neuronal cultures. Keap1/Nrf2-inhibitory PLPs have the potential to impact the treatment of disease states associated with dysregulation of oxidative stress, such as NDs.
Subject(s)
Kelch-Like ECH-Associated Protein 1 , NF-E2-Related Factor 2 , Polymers , Protein Binding , Kelch-Like ECH-Associated Protein 1/metabolism , Kelch-Like ECH-Associated Protein 1/chemistry , NF-E2-Related Factor 2/metabolism , Polymers/chemistry , Humans , Animals , Peptides/chemistry , Peptides/metabolism , Peptides/pharmacology , Antioxidant Response Elements , Neurons/metabolism , Neurons/drug effectsABSTRACT
The development of novel approaches to signal amplification in aqueous media could enable new diagnostic platforms for the detection of water-soluble analytes, including biomolecules. This paper describes a fluorogenic polymerization approach to amplify initiator signal by the detection of visible fluorescence upon polymerization in real-time. Fluorogenic monomers were synthesized and co-polymerized by atom transfer radical polymerization (ATRP) in water to reveal increasing polymer fluorescence as a function of both reaction time and initiator concentration. Optimization of the fluorogenic ATRP reaction conditions allowed for the quantitative detection of a small-molecule initiator as a model analyte over a broad linear concentration range (pM to mM). Raising the reaction temperature from 30 °C to 60 °C facilitated sensitive initiator detection at sub-picomolar concentrations in as little as 1 h of polymerization. This method was then applied to the detection of streptavidin as a model biological analyte by fluorogenic polymerization from a designed biotinylated ATRP initiator. Taken together, these studies represent the first example of a fluorogenic ATRP reaction and establish fluorogenic polymerization as a promising approach for the direct detection of aqueous analytes and biomolecular recognition events.
