Investigation of Nonhydrolyzable ATP Analogues and Cofilin-Derived Peptides for Inhibition of Cofilin-Actin Rod Formation

Abstract

Actin-Cofilin rods are an important marker of neurodegenerative disease and are commonly observed in the neurons of patients with Alzheimer’s disease. The goal of this research is to investigate peptide and small-molecules for their potential to inhibit actin-cofilin rod formation in cells. These inhibitors could form the basis for new Alzheimer’s disease therapeutics. Using fluorescence microscopy and an optogenetic system known as CofActor in both the HeLa and HEK 293T cell lines, we assessed changes in actin/ cofilin dynamics under energetic stress conditions in the presence of small molecules and cofilin mimicking peptides. Our small molecule strategy uses nonhydrolyzable ATP analogues to block the formation of actin-cofilin rods in cells undergoing energetic stress. Our peptide-based strategy uses cofilin-1, a primary binding protein to F-actin in neurons, as a template for the creation of peptides that target known cofilin-binding sites on the surface of actin. In our small molecule inhibitor strategy, we observed that ATP α-Sulfur analogue was the most promising actin-cofilin rod inhibitor of the compounds tested. We hypothesize that this compound inhibits actin-cofilin rod formation by binding to P2X and P2Y cell surface receptors. In our peptide inhibition strategy, we discovered three peptides that inhibit actin-cofilin rod formation in cells undergoing energetic stress. These peptides were found to significantly decrease the length of native actin-cofilin rods in stressed cells. In some cases, we observed inhibition of CofActor-induced cofilin-actin clusters in cells under energetic stress. We hypothesize that these peptide-based inhibitors function by binding directly to actin and blocking actin/cofilin interactions. These studies lay the foundation for future work, including incorporating our peptides into optogenetic cassettes to enable light-activated recruitment of proteins to actin and for advanced pharmacological evaluation of ATP α-Sulfur analogues as therapeutics for neurodegenerative disease.