Discovery and Characterization of a Glycogen Synthase Kinase- 3 Small Molecule Activator

Abstract

Glycogen Synthase Kinase 3 (GSK-3) functions as a consistently active serine/threonine kinase that is positioned at the intersection of numerous signaling pathways, effectively regulating the activity of numerous substrates. Its activity is regulated by multiple upstream signals, leading to alterations in the substrates it regulates. Given its pivotal role in significant pathways such as cellular proliferation, cell cycle progression, metabolism, and adhesion, GSK-3 kinase activity is tightly regulated and modulated. Dysregulation in its signaling and, as such, its downstream targets can contribute to disease. In several cancers, including colorectal cancer (CRC), aberrant signaling due to mutations in upstream proteins regulating GSK-3 leads to pathological inhibition of its enzymatic activity. Loss of GSK-3 kinase activity can also increase resistance to specific chemotherapeutic and targeted therapies. Its central role in signaling pathways, downregulation in cancer, and its role (or lack thereof) in therapeutic resistance make GSK-3 an ideal therapeutic target. However, there are currently no known compounds that can directly activate GSK-3 kinase activity. Studies on GSK-3 suggest the possibility of allosterically activating its enzymatic activity. Thus, we hypothesize that a small molecule can enhance GSK-3 kinase activity, restoring its downstream function in conditions with pathologically low enzymatic activity. This study aims to identify a novel GSK-3 small molecule activator and explore the impact of restoring GSK-3 kinase activity in both normal and CRC conditions.In this study, we conducted a high-throughput screening of 50,000 small molecule compounds and established a systematic approach to screen and identify a compound capable of activating or enhancing GSK-3 enzymatic activity. Following multiple screens, we narrowed down our initial pool of compounds to the top 35 hits. We then utilized in-vitro kinase assays alongside cell-based assays measuring the expression of GSK-3 direct substrate and activation of Wnt signaling. This further refined our top hit to a single compound, GA33. Subsequent investigation into GA33 confirmed binding to GSK-3 in cells and its dose-dependent increase in GSK-3 activity. We found that GA33 enhanced GSK-3 mediate phosphorylation of multiple GSK-3 substrates, including β-catenin. GA33 exhibited a dose-dependent inhibition of β-catenin expression post-Wnt stimulation. In addition, GA33 treatment also inhibited Wnt signaling downstream of the GSK-3/β-catenin axis. GA33-mediated inhibition of β-catenin and Wnt signaling was also observed in CRC lines. While prolonged exposure to GA33 exhibited decreased cell viability at high doses in various CRC lines, a significant discovery was the enhanced sensitivity of CRC lines to an mTOR inhibitor, Rapamycin, upon GA33 treatment. Lastly, our study revealed that treating organoids with GA33 over an extended period influenced their growth and organization. In conclusion, the work presented in this dissertation unveiled a novel GSK-3 small molecule activator for the first time. This discovery holds importance not just for its potential therapeutic applications related to GSK-3 but also as a valuable tool for advancing our understanding of GSK-3’s structure and function.