The majority of lysosomal storage diseases (LSD) exist as late-onset variants with neurological involvement. In Tay-Sachs and Gaucher disease the affected enzymes, Hexosaminidase and Glucocerebrosidase, are unable to cleave the terminal monosaccharides from gangliosides leading to their accumulation in the lysosome. We and others have shown that the residual activity of the affected enzymes can be augmented by pharmacological chaperones; small molecule compounds that specifically target and stabilize the mutant. We have further validated this approach and shown that high throughput screening (NTS) of drug-like small molecule libraries can be used to identify novel compounds which inhibit the activity of purified hexosaminidase and that can enhance enzyme activity in cells. These and other pharmacological chaperones achieve specificity by targeting the active site of the enzyme. However, effective in the enhancing the activity of the enzyme, as active site inhibitors and depending on dosage and duration, they could in the log-term potentially inhibit the activity of the enzyme to be rescued. Our goal is to identify other specific """"""""chemical chaperones"""""""" that do not bind to functional sites on the mutant enzyme, i.e. are not inhibitory, but which also specifically bind to and stabilize the enzyme. Thus we aim to: 1) Develop cell based assays which continuously monitor the activity of lysosomal enzymes using 4-methylumbelliferone based substrates. 2) Use these cell based assays in a HTS for compounds which enhance the activity of the lysosomal enzyme in cells derived from patients with the lysosomal storage disease. 3) Select those compounds for further development, which enhance the activity of the mutant by specifically binding to the lysosomal enzyme, without compromising its activity. 4) Determine the area in the enzyme that binds each compound. In the future these results will be used to design more new effective compounds.
| Tropak, Michael B; Kornhaber, Gregory J; Rigat, Brigitte A et al. (2008) Identification of pharmacological chaperones for Gaucher disease and characterization of their effects on beta-glucocerebrosidase by hydrogen/deuterium exchange mass spectrometry. Chembiochem 9:2650-62 |
| Tropak, Michael B; Blanchard, Jan E; Withers, Stephen G et al. (2007) High-throughput screening for human lysosomal beta-N-Acetyl hexosaminidase inhibitors acting as pharmacological chaperones. Chem Biol 14:153-64 |
| Maegawa, Gustavo H B; Tropak, Michael; Buttner, Justin et al. (2007) Pyrimethamine as a potential pharmacological chaperone for late-onset forms of GM2 gangliosidosis. J Biol Chem 282:9150-61 |
