Combinatorial approach toward rapid synthesis of iminosugar-based libraries: Development of new β-glucocerebrosidase inhibitors and pharmacological chaperones for Gaucher diseaseby Wei-Chieh Cheng

Molecular Genetics and Metabolism


Molecular Biology / Biochemistry / Endocrinology, Diabetes and Metabolism / Endocrinology / Genetics


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Combinatorial approach toward rapid synthesis of iminosugar-based libraries: Development of new β-glucocerebrosidase inhibitors and pharmacological chaperones for Gaucher disease

Wei-Chieh Cheng, Academia Sincia, Taipei, Taiwan

The rapid discovery of β-glucocerebrosidase (GC) inhibitors and pharmacological chaperones for Gaucher disease is described. The design and synthesis of new scaffolds and molecular libraries are often inspired by the skeletons of natural products or their mimics. Such a research approach referred to as “natural products-based combinatorial synthesis” has attracted a great deal of attention in modern drug discovery. Iminosugar-based alkaloids such as polyhydroxylated pyrrolidines, piperidines, and others have versatile biological or inhibitory activities against various sugar-processing enzymes, which are involved in diseases such as lysosomal disorders. In a rational design theory, the protonated nitrogen of the iminosugar may mimic the partial positive charge of the oxocarbenium transition-state analogs, to increase the interactions with enzyme residues. Through rapid diversification on the scaffold via an amide coupling with random carboxylic acids, structurally diverse bicyclic iminosugar-based libraries were prepared with substituent diversity, core diversity, and configuration diversity. Several members of this library were found to be nanomolar-range inhibitors of GC; the inhibition constant Ki of the most potent was found to be 71 nM. Although these new molecules showed reasonable chaperoning activity in the N370S fibroblast of

Gaucher patient-derived cell line, this was accompanied by a concomitant decrease in the cellular activity of lysosomal α-glucosidase, which might limit their further therapeutic potential. Next, lipophilic moieties were assembled with pyrrolidine-based scaffolds to generate new molecules for further evaluation. The new DMDP (2,5-dideoxy-2,5imino-d-mannitol)-based iminosugar was found to exhibit a satisfactory chaperoning activity to enhance GC activity by 2.2-fold in Gaucher

N370S cell line, without impairment of cellular α-glucosidase activity.

This discovery process allowed us to efficiently sieve out potent and specific glycosidase inhibitors and chemical chaperones.

References: (1) Cheng, T.-J. R.; Chan, T.-H.; Tsou, E.-L.; Chang S.-Y.;

Yun, W.-Y.; Yang, P.-J.; Wu, Y.-T.; Cheng, W.-C., From Natural ProductInspired Pyrrolidine Scaffolds to the Development of New Human

Golgi α-Mannosidase II Inhibitors. Chem. Asian J. 2013, 8, 2600-2604. (2) Cheng, W.-C.; Weng, C.-Y.; Yun, W.-Y.; Chang, S.-Y.; Lin, Y.-C.; Tsai,

F.-J.; Huang, F.-Y.; Chen, Y.-R., Rapid modifications of N-substitution in imino-sugars: Development of new β-glucocerebrosidase inhibitors and pharmacological chaperones for Gaucher disease. Bioorg. Med.

Chem. 2013, 21, 5021-5028.

Acknowledgements: We thank Academia Sinica, Taiwan and

Ministry of Science and Technology for financial support. doi:10.1016/j.ymgme.2014.12.051 50

Choroid plexus-directed viral gene therapy for α-mannosidosis, a prototypical lysosomal disease

Eun-Young Choia, Eva Bakerb, Stephen G. Kalera, aNICHD/NIH, Bethesda,

MD, USA, bNIH Clinical Center, Bethesda, MD, USA

The choroid plexuses are highly vascularized structures that project into the cerebrospinal fluid (CSF) of the four cerebral ventricles. The specialized polarized epithelia of choroid plexuses produce CSF by transporting water and ions into the ventricles, and turn over at an extremely slow rate. We hypothesized that remodeling these epithelia to secrete a missing lysosomal enzyme by one-time administration of a recombinant AAV (rAAV) gene therapy vector into the cerebrospinal fluid could be an attractive and efficacious approach for long-term treatment of lysosomal diseases (LSDs). Lysosomes function as the primary digestive units within cells and specific enzymes within lysosomes normally break down nutrients. Patients with LSD cannot metabolize certain nutrients, resulting in diminished lifespans and reduced quality of life. There are no ideal therapeutic options presently available for the neurological manifestations of LSD. Brain-directed recombinant enzyme replacement has shown promise for several LSD but requires repeated instillations due to short enzyme half-lives. In contrast, rAAV-mediated gene transfer to the choroid plexus would enable continuous synthesis and secretion of missing lysosomal enzymes into the CSF and steady penetration to the cerebral cortex and cerebellum. To evaluate this hypothesis, we obtained a mouse model of alpha-mannosidosis in which a targeted disruption of the lysosomal acid-mannosidase (LAMAN) gene is present. We cloned the human (hu) LAMAN cDNA into a rAAV shuttle plasmid and generated high titer rAAV5-huLAMAN expressing huLAMAN. We administered 1E+9 to 5E+10 viral particles to homozygous mutant mice (detected via a novel genotyping assay) by brain lateral ventricle injection on day 3 of life. We documented efficient and dose-dependent viral transduction in the brains of treated animals, and documented huLAMANmRNA expression in rAAV-treated mouse brains by RT-PCR using human

LAMAN specific primers. Brain biochemical analyses three weeks posttreatment illustrated increased LAMAN enzyme activity. If the choroid plexus viral gene therapy approach were successful, the largest current barriers to health for patients with many different LSD would be circumvented.We recently inaugurated a NIH clinical protocol (14-CH0106) to evaluate the natural history of alpha-mannosidosis and to identify unique clinical features and biochemical biomarkers in human subjectswith this illness, of potential value in a future clinical trial. Brain magnetic resonance spectroscopy in the initial subject, a 35 year old male, disclosed a diffuse abnormality in the white matter that was hyperintense on T2-weighted and FLAIR images, but not visible on T1weighted images. Incidental notewasmade of a large number of visible perivascular spaces. DTI demonstrated that diffusion was elevated in the areas of abnormal signal. Metal deposition in the usual locations (globus pallidus, substantia nigra, red nuclei, dentate nuclei) was much greater than typical for the patient's age. Therewas also excessivemetal deposition in the putamina and in the posterior portions of the thalami.