An injectable hydrogel containing <i>N</i> -acetylglycine for the treatment of Gaucher disease

Gaucher disease (GD) is a rare inherited genetic disorder resulting from a recessive mutation in the GBA1 gene, which encodes the glucocerebrosidase (GCase) enzyme, resulting in the accumulation of glycolipids within the lysosomes of various body organs. Recently, small molecules that act as stabilizers for mutant proteins have become popular as a potential treatment for genetic disorders. Small-molecule stabilizers improve the stability of mutant proteins by facilitating proper folding, allowing them to regain their functional conformation. This prevents the proteins from entering the proteosomal degradation pathway, ultimately restoring their biological functions. Exploration of novel small-molecule stabilizers of GCase through rational process pipelines using <i>in vitro</i> and <i>in vivo</i> studies is quite expensive and time-consuming. A comprehensive process pipeline was developed for the pre-clinical identification of new GCase stabilizers using a stepwise funnel selection approach. Initially, small molecules were screened through a high-throughput molecular docking approach to repurpose these already existing molecules known for their protein stabilizing ability and evaluate their potential to stabilize mutant GCase, for the treatment of GD. Based on the intermolecular interactions and docking scores, the top two leads, <i>N</i>-acetylglycine (NAG) and deoxycholic acid (DCA), were further evaluated for their ability to stabilize the GCase enzyme through extensive molecular dynamics simulations and binding free energy analyses. Following detailed <i>in vitro</i> and <i>in vivo</i> toxicity studies, NAG was selected as the lead for further studies in <i>in vitro</i> and <i>in vivo</i> GD models. NAG increased the GCase activity in both chemically and siRNA-induced gene knockdown GD cell models, indicating NAG as a GCase stabilizer. The enhancement in GCase activity and improved motor behavior observed in the CBE-induced Gaucher mouse model following treatment with NAG confirmed its stabilizing efficacy. Finally, a single dose of NAG containing injectable hydrogel was developed and effectively used to treat GD and improve overall survivability.