Introduction of C-alkyl branches to L-iminosugars changes their active site binding orientation.

L-ido-Deoxynojirimycin (L-ido-DNJ) itself showed no affinity for human lysosomal acid α-glucosidase (GAA), whereas 5-C-methyl-L-ido-DNJ showed a strong affinity for GAA, comparable to the glucose analog DNJ, with a Ki value of 0.060 µM. This excellent affinity for GAA and enzyme stabilization was observed only when methyl and ethyl groups were introduced. Docking simulation analysis revealed that the alkyl chains of 5-C-alkyl-L-ido-DNJs were stored in three different pockets, depending on their length, thereby the molecular orientation was changed. Comparison of the binding poses of DNJ and 5-C-methyl-L-ido-DNJ showed that they formed a common ionic interaction with Asp404, Asp518, and Asp616, but both the binding orientation and the distance between the ligand and each amino acid residue were different. 5-C-Methyl-L-ido-DNJ dose-dependently increased intracellular GAA activity in Pompe patient fibroblasts with the M519V mutation and also promoted enzyme transport to lysosomes. This study provides the first example of a strategy to design high-affinity ligands by introducing alkyl branches into rare sugars and L-sugar-type iminosugars to change the orientation of binding.

5-C-Branched Deoxynojirimycin: Strategy for Designing a 1-Deoxynojirimycin-Based Pharmacological Chaperone with a Nanomolar Affinity for Pompe Disease.

In recent years, the function of pharmacological chaperones as a "thermodynamic stabilizer" has been attracting attention in combination therapy. The coadministration of a pharmacological chaperone and recombinant human acid α-glucosidase (rhGAA) leads to improved stability and maturation by binding to the folded state of the rhGAA and thereby promotes enzyme delivery. This study provides the first example of a strategy to design a high-affinity ligand toward lysosomal acid α-glucosidase (GAA) focusing on alkyl branches on 1-deoxynojirimycin (DNJ); 5-C-heptyl-DNJ produced a nanomolar affinity for GAA with a Ki value of 0.0047 µM, which is 13-fold more potent than DNJ. The protein thermal shift assay revealed that 10 µM 5-C-heptyl-DNJ increased the midpoint of the protein denaturation temperature (Tm) to 73.6 °C from 58.6 °C in the absence of the ligand, significantly improving the thermal stability of rhGAA. Furthermore, 5-C-heptyl-DNJ dose dependency increased intracellular GAA activities in Pompe patient's fibroblasts with the M519V mutation. The introduction of C5 alkyl branches on DNJ provides a new molecular strategy for pharmacological chaperone therapy for Pompe disease, which may lead to the development of higher-affinity and practically useful chaperones.

Synthesis and glycosidase inhibition of 5-C-alkyl-DNJ and 5-C-alkyl-l-ido-DNJ derivatives.

5-C-Alkyl-DNJ and 5-C-alkyl-l-ido-DNJ derivatives have been designed and synthesized efficiently from an l-sorbose-derived cyclic nitrone. The DNJ and l-ido-DNJ derivatives with C-5 alkyl chains ranging from methyl to dodecyl were assayed against various glycosidases to study the effect of chain length on enzyme inhibition. Glycosidase inhibition study of DNJ derivatives showed potent and selective inhibitions of α-glucosidase; DNJ derivatives with methyl, pentyl to octyl, undecyl and dodecyl as C-5 branched chains showed significantly improved rat intestinal maltase inhibition. In contrast, most 5-C-alkyl-l-ido-DNJ derivatives were weak or moderate inhibitors of the enzymes tested, with only three compounds found to be potent α-glucosidase inhibitors. Docking studies showed different interaction modes of 5-C-ethyl-DNJ and 5-C-octyl-DNJ with ntMGAM and also different binding modes of 5-C-alkyl-DNJ and 5-C-alkyl-l-ido-DNJ derivatives; the importance of the degree of accommodation of the C-5 substituent in the hydrophobic groove and pocket may account for the variation of glycosidase inhibition in the two series of derivatives. The results reported herein are helpful in the design and development of α-glucosidase inhibitors; this may lead to novel agents for the treatment of viral infection and type II diabetes.