Regulators of oxidative stress, such as Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), decrease with age and contribute to the development of neurodegenerative diseases. The interaction between Nrf2 and Kelch-like ECH-Associating protein 1 (Keap1), a sensor of oxidative stress, is central to cellular redox homeostasis; whereby inhibition of Keap1 leads to Nrf2 activation of the antioxidant responsive element (ARE) pathway. This protein-protein interaction (PPI) has been difficult to target with small molecules, and thus peptide therapeutics have been of interest. Keap1 inhibitory peptides have been identified, yet clinical translation is limited by poor pharmacokinetic properties and bioactivity. There is a need to develop a therapeutic modulator of the Keap1/Nrf2 interaction which could benefit age-related pathologies and serve a neuroprotective role. To address this, Keap1-inhibiting peptides were packaged as high-density brush polymers. These structures, termed protein-like polymers (PLPs), when compared to peptide drugs demonstrate resistance to proteolysis, improved pharmacokinetics and bioactivity. In a fluorescence polarization competitive inhibition assay, Keap1-inhibiting PLPs where shown to have 100 fold superior IC<sub>50</sub> values compared to free peptides while scrambled controls were unable to generate an inhibition curve. Cy5.5 labeled PLPs successfully showed cell uptake via confocal microscopy and flow cytometry in HepG2 cells. Keap1-inhibition was determined by assessing activation in an ARE-Luciferase Reporter HepG2 cell line. Only the Keap1-Inhibiting PLP showed dose dependent ARE activation while the free peptide and scramble control PLPs did not have significant activation. In summary, we successfully synthesized and characterized a novel biomaterial capable of overcoming the current challenges facing peptide therapeutics by achieving cellular uptake, enhanced binding affinity, and demonstrated bioactivity. This work is a step forward in the development of methods for direct inhibition of intracellular PPIs and has potential for clinical translation.

Development of protein-like polymer therapeutics for modulating the Nrf2/Keap1 protein-protein interaction

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ACS Fall 2022

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