Contribution of Shape and Charge to the Inhibition of a Family GH99 endo-α-1,2-Mannanase

Abstract

Inhibitor design incorporating features of the reaction coordinate and transition-state structure has emerged as a powerful approach for the development of enzyme inhibitors. Such inhibitors find use as mechanistic probes, chemical biology tools, and therapeutics. Endo-α-1,2-mannosidases and endo-α-1,2-mannanases, members of glycoside hydrolase family 99 (GH99), are interesting targets for inhibitor development as they play key roles in N-glycan maturation and microbiotal yeast mannan degradation, respectively. These enzymes are proposed to act via a 1,2-anhydrosugar “epoxide” mechanism that proceeds through an unusual conformational itinerary. Here, we explore how shape and charge contribute to binding of diverse inhibitors of these enzymes. We report the synthesis of neutral dideoxy, glucal and cyclohexenyl disaccharide inhibitors, their binding to GH99 endo-α-1,2-mannanases, and their structural analysis by X-ray crystallography. Quantum mechanical calculations of the free energy landscapes reveal how the neutral inhibitors provide shape but not charge mimicry of the proposed intermediate and transition state structures. Building upon the knowledge of shape and charge contributions to inhibition of family GH99 enzymes, we design and synthesize α-Man-1,3-noeuromycin, which is revealed to be the most potent inhibitor (KD 13 nM for Bacteroides xylanisolvens GH99 enzyme) of these enzymes yet reported. This work reveals how shape and charge mimicry of transition state features can enable the rational design of potent inhibitors.

Exploration of Strategies for Mechanism‐Based Inhibitor Design for Family GH99 endo‐α‐1,2‐Mannanases

“…Structural analysis of the Bx GH99– 1 complex (Figure A) revealed the piperidine ring in a 4 C 1 conformation, which matches that seen for complexes of the wild‐type enzyme with GlcDMJ and isofagomine‐based inhibitors as well as that of a disabled mutant with substrate . The 2‐amino group is situated appropriately to interact with the E333 residue, that which is proposed to act as a general base/acid through deprotonation of the 2‐hydroxy group.…”

“…[8] Furthermore, reintroductiono ft he "missing" 2-OH of 1,3-isofagomine( IFG) into ManIFG gave a-mannosyl-1,3-noeuromycin( ManNOE), which was shown to be five-fold more potent towards the B. thetaiotaomicron GH99 enzyme (K D = 0.03 mm). [17] These compounds bind in ag round-state 4 C 1 conformation, as seen in complexes of inactive enzyme with substrate and thus proposed for the conformation of substrate within the Michaelis complex (Figure 1A), which suggests that potent inhibition of GH99e nzymes can be achieved simply by mimicry of the chargei nt he transition state. [17] Separately,S piro and co-workerss howedt hat the neutral compound GlcGlucal ( Figure 1C)w as am odesti nhibitor of mammalian GH99 (rat Golgi preparation, IC 50 = 2.3 mm;f or GlcDMJ IC 50 = 1.7 mm); [14,18] the equivalent molecule targeting bacterialG H99, ManGlucal, wasa lso al igand with mildly potent affinity (K D = 15 mm for BtGH99).…”

“…Separately, Spiro and co‐workers showed that the neutral compound GlcGlucal (Figure C) was a modest inhibitor of mammalian GH99 (rat Golgi preparation, IC 50 =2.3 μ m ; for GlcDMJ IC 50 =1.7 μ m ); the equivalent molecule targeting bacterial GH99, ManGlucal, was also a ligand with mildly potent affinity ( K D =15 μ m for Bt GH99) . Computational free‐energy landscape analysis of the preferred conformation of d ‐glucal suggested that the inhibition of the glucal‐based inhibitors arises from mimicry of the proposed 4 E conformation of the transition state or the proposed 4 H 5 conformation of the 1,2‐anhydro sugar intermediate, but with no contribution from charge mimicry owing to the neutral nature of this compound …”

Conformational Analysis of the Mannosidase Inhibitor Kifunensine: A Quantum Mechanical and Structural Approach

“…Conformational free‐energy landscapes (FELs) are quantitative maps of the energy of the full suite of conformations of a molecule, and provide insight into locally and globally stable conformations as well as the barriers that must be crossed in order to achieve them. In order to understand the conformational landscape of kifunensine, and how this contributes to its GH47 specific inhibition, its conformational FEL was calculated by ab initio metadynamics according to methods we have developed and applied to a range of glycosidase inhibitors . The kifunensine FEL (Figure A) exhibits a strong preference for the “southern hemisphere” 1 C 4 conformation; this is consistent with the conformation observed in a small‐molecule, single‐crystal X‐ray structure .…”

“…In order to understand the conformational landscape of kifunensine, and how this contributes to its GH47 specific inhibition, its conformational FEL was calculated by ab initio metadynamics [19] according to methods we have developed and applied to ar ange of glycosidase inhibitors. [18,20] The kifunensine FEL (Figure 2A)e xhibits as trong preference for the "southern hemisphere" 1 C 4 conformation; this is consistentw ith the conformation observed in as mallmolecule, single-crystal X-ray structure. [8,21] Al ow energy zone is also seen aroundt he 1 S 3 / 1,4 B/ 1 S 5 region of the FEL, which, at approximately 7-8 kcal mol À1 higheri ne nergy and with am inimal barrier, can be considered energetically accessible.…”