α-Mannosidases and α-mannanases have attracted attention for the insight they provide into nucleophilic substitution at the hindered anomeric center of α-mannosides, and the potential of mannosidase inhibitors as cellular probes and therapeutic agents. We report the conformational itinerary of the family GH76 α-mannanases studied through structural analysis of the Michaelis complex and synthesis and evaluation of novel aza/imino sugar inhibitors. A Michaelis complex in an (O) S2 conformation, coupled with distortion of an azasugar in an inhibitor complex to a high energy B2,5 conformation are rationalized through ab initio QM/MM metadynamics that show how the enzyme surface restricts the conformational landscape of the substrate, rendering the B2,5 conformation the most energetically stable on-enzyme. We conclude that GH76 enzymes perform catalysis using an itinerary that passes through (O) S2 and B2,5 (≠) conformations, information that should inspire the development of new antifungal agents.

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a-Mannosidases and a-mannanases have attracted attention for the insight they providei nto nucleophilic substitution at the hindered anomeric center of a-mannosides, and the potential of mannosidase inhibitors as cellular probes and therapeutic agents.W ereport the conformational itinerary of the family GH76 a-mannanases studied through structural analysis of the Michaelis complex and synthesis and evaluation of novel aza/imino sugar inhibitors.AMichaelis complex in an O S 2 conformation, coupled with distortion of an azasugar in an inhibitor complex to ah igh energy B 2,5 conformation are rationalized through ab initio QM/MM metadynamics that show how the enzyme surface restricts the conformational landscape of the substrate,rendering the B 2,5 conformation the most energetically stable on-enzyme.W ec onclude that GH76 enzymes perform catalysis using an itinerary that passes through O S 2 and B 2,5°c onformations,information that should inspire the development of new antifungal agents.

The United States is in the midst of an unprecedented epidemic of opioid substance use disorder, and while pharmacotherapies including opioid agonists and antagonists have shown success, they can be inadequate and frequently result in high recidivism. With these challenges facing opioid use disorder treatments immunopharmacotherapy is being explored as an alternative therapy option and is based upon antibody−opioid sequestering to block brain entry. Development of a heroin vaccine has become a major research focal point; however, producing an efficient vaccine against heroin has been particularly challenging because of the need to generate not only a potent immune response but one against heroin and its multiple psychoactive molecules. In this study, we explored the consequence of regioselective deuteration of a heroin hapten and its impact upon the immune response against heroin and its psychoactive metabolites. Deuterium (H dAc ) and cognate protium heroin (H Ac ) haptens were compared head to head in an inclusive vaccine study. Strikingly the H dAc vaccine granted greater efficacy in blunting heroin analgesia in murine behavioral models compared to the H Ac vaccine. Binding studies confirmed that the H dAc vaccine elicited both greater quantities and equivalent or higher affinity antibodies toward heroin and 6-AM. Blood−brain biodistribution experiments corroborated these affinity tests. These findings suggest that regioselective hapten deuteration could be useful for the resurrection of previous drug of abuse vaccines that have met limited success in the past.

The non-hydrolyzable S-linked azasugars, 1,6-α-mannosylthio- and 1,6-α-mannobiosylthioisofagomine, were synthesized and shown to bind with high affinity to a family 76 endo-1,6-α-mannanase from Bacillus circulans. X-ray crystallography showed an atypical interaction of the isofagomine nitrogen with the catalytic acid/base. Molecular dynamics simulations reveal that the atypical binding results from sulfur perturbing the most stable form away from the nucleophile interaction preferred for the O-linked congener.