Development of\nTunable Mechanism-Based Carbasugar\nLigands that Stabilize Glycoside Hydrolases through the Formation\nof Transient Covalent Intermediates

Mutations in many members of the set of human lysosomal\nglycoside\nhydrolases cause a wide range of lysosomal storage diseases. As a\nresult, much effort has been directed toward identifying pharmacological\nchaperones of these lysosomal enzymes. The majority of the candidate\nchaperones are active site-directed competitive iminosugar inhibitors\nbut these have met with limited success. As a first step toward an\nalternative class of pharmacological chaperones we explored the potential\nof small molecule mechanism-based reversible covalent inhibitors to\nform transient enzyme–inhibitor adducts. By serial synthesis\nand kinetic analysis of candidate molecules, we show that rational\ntuning of the chemical reactivity of glucose-configured carbasugars\ndelivers cyclohexenyl-based allylic carbasugar that react with the\nlysosomal enzyme β-glucocerebrosidase (GCase) to form covalent\nenzyme-adducts with different half-lives. X-ray structural analysis\nof these compounds bound noncovalently to GCase, along with the structures\nof the covalent adducts of compounds that reacted with the catalytic\nnucleophile of GCase, reveal unexpected reactivities of these compounds.\nUsing differential scanning fluorimetry, we show that formation of\na transient covalent intermediate stabilizes the folded enzyme against\nthermal denaturation. In addition, these covalent adducts break down\nto liberate the active enzyme and a product that is no longer inhibitory.\nWe further show that the one compound, which reacts through an unprecedented\nS<sub>N</sub>1′-like mechanism, exhibits exceptional reactivity–illustrated\nby this compound also covalently labeling an α-glucosidase.\nWe anticipate that such carbasugar-based single turnover covalent\nligands may serve as pharmacological chaperones for lysosomal glycoside\nhydrolases and other disease-associated retaining glycosidases. The\nunusual reactivity of these molecules should also open the door to\ncreation of new chemical biology probes to explore the biology of\nthis important superfamily of glycoside hydrolases.