The long-term goals of this application are to develop selective inhibitors for an ER glycoprotein processing enzyme that plays a key role in quality control in the endoplasmic reticulum (ER) as broad-based therapeutics for glycoprotein misfolding diseases. This enzyme, ERalpha-mannosidase I (ERManl), acts as a key timer for ER residence for newly synthesized glycoproteins by initiating a rate-limiting step leading to a cascade of interactions that ultimately leads to the targeting of terminally misfolded glycoproteins for retrotranslocation to the cytoplasm and proteasomal disposal in a process known as """"""""ER-associated degradation"""""""" (ERAD). Many loss-of-function human genetic diseases result from mutations that cause delayed protein folding kinetics rather than generating terminally misfolded polypeptides. Recognition of the incompletely folded intermediates by the ERAD targeting machinery can lead to premature disposal of potentially functional glycoproteins and subsequently leads to pathology. Inhibition of the rate-determining steps in ERAD could provide a broad-based therapeutic approach for treatment of glycprotein misfolding diseases by delaying ERAD and providing sufficient time to complete the protein folding process. All of the known inhibitors of early mannose trimming steps, however, also have unacceptable serious side effects. They also inhibit glycan processing alpha-mannosidases in the Golgi complex and block maturation to complex type glycan structures on cell surface and secreted glycoproteins. Thus, the goals of this application are to identify selective ERManl inhibitors that can act to delay ERAD, rescue ER protein folding defects in human disease, and retain normal glycan maturation in the Golgi complex. The unique interdisciplinary team that we have assembled takes advantage of ongoing synergistic collaborative interactions between investigators at the University of Georgia and Baylor College of Medicine with expertise in the synthesis of selective glycosidase inhibitors (Boons), the biochemistry and structural biology of the ER and Golgi mannosidases (Moremen), and cell-based assays for a human glycoprotein misfolding disorder, alpha1-antitrypsin deficiency (Sifers). Promising leads will also be evaluated in established lysosomal storage disease models by collaborators (Amicus).
Three specific aims are proposed including 1) the directed rational and combinatorial synthesis of analogs of alpha-mannosidase inhibitors with selectivity toward ERManl, 2) high-throughput screens combined with detailed biochemical and structural analysis to assess selectivity and effectiveness of the inhibitor compounds in blocking ERManl but not Golgi glycan maturation, and 3) cell-based assays to assess chemical chaperone effects of mannosidase inhibitors in the rescue of mutant alpha1-antitrypsin secretion and lysosomal enzyme targeting and without blockage of N-glycan maturation. ? ? ?
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