Design and synthesis of biologically active carbaglycosylamines: From glycosidase inhibitors to pharmacological chaperones.

For over 50 years, our group has been involved in synthetic studies on biologically active cyclitols including carbasugars. Among a variety of compounds synthesized, this review focuses on carbaglycosylamine glycosidase inhibitors, highlighting the following: (1) the naturally occurring N-linked carbaoligosaccharide α-amylase inhibitor acarbose and related compounds; (2) the novel synthetic ß-glycosidase inhibitors, 1'-epi-acarviosin and its 6-hydroxy analogue as well as ß-valienaminylceramide and its 4'-epimer; (3) the discovery of the ß-glycosidase inhibitors with chaperone activity, N-octyl-ß-valienamine (NOV) and its 4-epimer (NOEV); and (4) the recent development of the potential pharmacological chaperone N-alkyl-conduramine F-4 derivatives.

Concise syntheses of potent chaperone drug candidates, N-octyl-4-epi-ß-valinenamine (NOEV) and its 6-deoxy derivative, from (+)-proto-quercitol.

Described are the efficient syntheses of ß-galactose-type unsaturated carbasugar amine, N-octyl-4-epi-ß-valienamine (1a, NOEV) and 6-deoxy NOEV (12), starting from (+)-proto-quercitol (2), which is readily provided by the bioconversion of myo-inositol. NOEV is a potent chemical chaperone drug candidate for G(M1)-gangliosidosis. An intermediate alkadiene benzoate was prepared from 2 in five steps, with the key step being a Wittig reaction with an enol ester. The 6-deoxy derivative 12 was conveniently synthesized from the versatile intermediate dibromo compound 6, which was also an intermediate in the synthesis of NOEV. Enzyme inhibition assays demonstrated that 12 possessed stronger inhibitory activity than the parent 1a, suggesting that the C-6 position of the 4-epi-ß-valienamine-type inhibitor could have hydrophobic interactions at the ß-galactosidase active site residues.

Therapeutic chaperone effect of N-octyl 4-epi-ß-valienamine on murine G(M1)-gangliosidosis.

Therapeutic chaperone effect of a valienamine derivative N-octyl 4-epi-ß-valienamine (NOEV) was studied in G(M1)-gangliosidosis model mice. Phamacokinetic analysis revealed rapid intestinal absorption and renal excretion after oral administration. Intracellular accumulation was not observed after continuous treatment. NOEV was delivered to the central nervous system through the blood-brain barrier to induce high expression of the apparently deficient ß-galactosidase activity. NOEV treatment starting at the early stage of disease resulted in remarkable arrest of neurological progression within a few months. Survival time was significantly prolonged. This result suggests that NOEV chaperone therapy will be clinically effective for prevention of neuronal damage if started early in life hopefully also in human patients with G(M1)-gangliosidosis.

Transformation of quercitols into 4-methylenecyclohex-5-ene-1,2,3-triol derivatives, precursors for the chemical chaperones N-octyl-4-epi-ß-valienamine (NOEV) and N-octyl-ß-valienamine (NOV).

(+)-proto-Quercitol (1) and (-)-vibo-quercitol (2), both of which could be readily prepared by the bioconversion of myo-inositol, were successfully converted into the corresponding 4-methylenecyclohex-5-ene-1,2,3-triol derivatives. These compounds were demonstrated to be suitable precursors, preserving their configurations, for bioactive carba-aminosugars such as the potent chemical chaperone drug candidates, N-octyl-4-epi-ß-valienamine (NOEV, 3) and N-octyl-ß-valienamine (NOV, 4).

Chemical chaperone therapy: chaperone effect on mutant enzyme and cellular pathophysiology in ß-galactosidase deficiency.

ß-Galactosidase deficiency is a group of lysosomal lipid storage disorders with an autosomal recessive trait. It causes two clinically different diseases, G(M1) -gangliosidosis and Morquio B disease. It is caused by heterogeneous mutations in the GLB1 gene coding for the lysosomal acid ß-galactosidase. We have previously reported the chaperone effect of N-octyl-4-epi-ß-valienamine (NOEV) on mutant ß-galactosidase proteins. In this study, we performed genotype analyses of patients with ß-galactosidase deficiency and identified 46 mutation alleles including 9 novel mutations. We then examined the NOEV effect on mutant ß-galactosidase proteins by using six strains of patient-derived skin fibroblast. We also performed mutagenesis to identify ß-galactosidase mutants that were responsive to NOEV and found that 22 out of 94 mutants were responsive. Computational structural analysis revealed the mode of interaction between human ß-galactosidase and NOEV. Moreover, we confirmed that NOEV reduced G(M1) accumulation and ameliorated the impairments of lipid trafficking and protein degradation in ß-galactosidase deficient cells. These results provided further evidence to NOEV as a promising chaperone compound for ß-galactosidase deficiency.

A new, convenient cell-based screening method for small-molecule glycolytic inhibitors.

To counteract active glycolysis in tumors, we developed a new, convenient cell-based screening system to identify an inhibitor of glycolysis. Using this system, we searched for an inhibitor in the synthetic Carbasugar library and found two candidates. It was found that both inhibited glycolysis by suppressing the glucose uptake step in tumor cells.

Chemical chaperone therapy: luciferase assay for screening of ß-galactosidase mutations.

ß-Galactosidosis is a group of disorder based on heterogeneous mutations of GLB1 gene coding for the lysosomal acid ß-galactosidase (ß-gal). A decrease of the ß-gal enzyme activity results in progressive accumulation of substrates in somatic cells, particularly in neurons, leading to severe neuronal dysfunction. We have previously reported that N-octyl-4-epi-ß-valienamine (NOEV), a chemical chaperone compound, stabilized various mutant human ß-gal proteins and increased residual enzyme activities in cultured fibroblasts from human patients. These data proved a potential therapeutic benefit of chemical chaperone therapy for patients with missense ß-gal. This effect is mutation specific. In this study, we have established a sensitive luciferase-based assay for measuring chaperone effect on mutant human ß-gal. A dinoflagellate luciferase (Dluc) cDNA was introduced to the C-terminus of human ß-gal. When COS7 cells expressing the Dluc-tagged human R201C ß-gal was treated with NOEV, there happened a remarkable increase of the mutant ß-gal activity. In the presence of NH(4)Cl, luciferase level in the medium increased in parallel with the enzyme activity in cell lysates. We also found that proteasome inhibitors enhance chaperone effect of NOEV. These results demonstrate that the luciferase-based assay is a reliable and convenient method for screening and evaluation of chaperone effects on human ß-gal mutants, and that it will be a useful tool for finding novel chaperone compounds in the future study.

The effect of N-octyl-ß-valienamine on ß-glucosidase activity in tissues of normal mice.

Gaucher disease (GD), mainly caused by a defect of acid ß-glucosidase (ß-Glu), is the most common sphingolipidosis. We have previously shown that a carbohydrate mimic N-octyl-ß-valienamine (NOV), an inhibitor of ß-Glu, could increase the protein level and enzyme activity of various mutant ß-Glu in cultured GD fibroblasts, suggesting that NOV acted as a pharmacological chaperone to accelerate transport and maturation of this mutant enzymes. In the present study, the NOV effect was evaluated for ß-Glu activity, tissue distribution and adverse effects in normal mice. We measured the ß-Glu activity in tissues of normal mice which received water containing increasing concentrations of NOV ad libitum for 1 week. Fluid intake and body weight were measured periodically throughout the study. Measurement of tissue NOV concentration, blood chemistry and urinalysis were performed at the end of the study. The results showed that NOV had no impact on the body weight but fluid intake in the 10mM NOV group mice decreased and there was a moderate increase in blood urea nitrogen (BUN). No other adverse effect was observed during this experiment. Tissue NOV concentration increased in all tissues examined with increasing NOV doses. No inhibitory effect of NOV on ß-Glu was observed. Furthermore, NOV increased the ß-Glu activity in the liver, spleen, muscle and cerebellum of the mice significantly. This study on NOV showed its oral availability and wide tissue distribution, including the brain and its lack of acute toxicity. These characteristics of NOV would make it a potential therapeutic chaperone in the treatment of GD with neurological manifestations and selected mutations.

The pharmacological chaperone effect of N-octyl-beta-valienamine on human mutant acid beta-glucosidases.

Gaucher's disease (GD), mainly caused by a defect of acid beta-glucosidase (beta-Glu), is the most common form of sphingolipidosis. We have previously shown that the carbohydrate mimic and inhibitor of beta-Glu, N-octyl-beta-valienamine (NOV), could increase the protein level and enzyme activity of various mutant beta-Glus in cultured GD fibroblasts and in COS cells, suggesting that NOV acts as a pharmacological chaperone to accelerate transport and maturation of these mutant enzymes. In present study, we continued to investigate the chaperone characteristics of NOV. More importantly, chaperone activities of NOV were evaluated in COS cells transiently expressing ten new, recombinant beta-Glu mutants with mutations located in domain I, II and III. NOV was only effective on the T369M mutation, located in domain III. As we suggested in a previous study, domain III may be a prerequisite for pharmacological rescue of the mutant beta-Glu by NOV. These characteristics of NOV could provide potential therapeutic chaperone properties that would be useful in the treatment of GD with neurological manifestations due to gene mutations in beta-Glu.

Chaperone therapy for neuronopathic lysosomal diseases: competitive inhibitors as chemical chaperones for enhancement of mutant enzyme activities.

Chaperone therapy is a newly developed molecular approach to lysosomal diseases, a group of human genetic diseases causing severe brain damage. We found two valienamine derivatives, N-octyl-4-epi-beta-valienamine (NOEV) and N-octyl-beta-valienamine (NOV), as promising therapeutic agents for human beta-galactosidase deficiency disorders (mainly G(M1)-gangliosidosis) and beta-glucosidase deficiency disorders (Gaucher disease), respectively. We briefly reviewed the historical background of research in carbasugar glycosidase inhibitors. Originally NOEV and NOV had been discovered as competitive inhibitors, and then their paradoxical bioactivities as chaperones were confirmed in cultured fibroblasts from patients with these disorders. Subsequently G(M1)-gangliosidosis model mice were developed and useful for experimental studies. Orally administered NOEV entered the brain through the blood-brain barrier, enhanced beta-galactosidase activity, reduced substrate storage, and improved neurological deterioration clinically. Furthermore, we executed computational analysis for prediction of molecular interactions between beta-galactosidase and NOEV. Some preliminary results of computational analysis of molecular interaction mechanism are presented in this article. NOV also showed the chaperone effect toward several beta-glucosidase gene mutations in Gaucher disease. We hope chaperone therapy will become available for some patients with G(M1)-gangliosidosis, Gaucher disease, and potentially other lysosomal storage diseases with central nervous system involvement.

5a-Carba-glycopyranoside primers: potential building blocks for biocombinatorial synthesis of glycosphingolipid analogues.

Three ether-linked alkyl 5a-carba-glycopyranosides 1b,d, and 5b, and 5a'-carba-lactoside 7b were examined as potent primers in mouse B16 melanoma cells for their feasibility as building blocks for oligosaccharide biosynthesis. Uptake by B16 cells was first observed for all carba-glycoside primers, and, especially, the 5a-carba-sugar analogues of N-acetyl-beta-d-glucosaminide 1b and beta-d-glucoside 1d were shown to produce two-to-four-fold larger amounts of glycosylated products than the corresponding true sugar primers 1a and 1c. The carba glycoside uptake by cells resulted in beta-galactosylation and subsequent sialylation of the incorporated galactose residues, giving rise to glycosylated products 3b and 3d having similar glycan structures as the ganglioside GM3. According to efficient uptake in cells, in addition to stability of the ether-linked pseudo-reducing ends of the oligosaccharides that formed, the carba glycoside primers have been demonstrated to be versatile building blocks for these biocombinatorial syntheses of glycolipid oligosaccharide mimetics. On the other hand, uptake for 5a-carba-galactopyranoside residue was found to be decreased by one-third for dodecyl 5a-carba-beta-d-galactopyranoside 5b. Observation of similar levels for 5a'-carba-beta-lactoside 7b under both cellular and cell-free conditions suggested that enzymes are likely to recognize the pyranose oxygen atom.

Design and synthesis of 5a-carbaglycopyranosylamime glycosidase inhibitors.

5a-Carba-alpha-D-glucopyranosylamine, validamine, and analogous compounds valienamine and valiolamine, have proved to be important lead compounds for development of clinically useful medicines, including the very strong alpha-glucosidase inhibitor, voglibose, N-(1,3-dihydroxyprop-2-yl)valiolamine, now used widely as a clinically important antidiabetic agent. In this review, we describe recent advances in development of glycosidase inhibitors on the basis of the ground-state mimics of the postulated glycopyranosyl cation, considered to be formed during hydrolysis of glycopyranosides, and introduce a new type of highly potent alpha-fucosidase inhibitor, 5a-carba-alpha-L-fucopyranosylamine, alpha-fuco validamine. Interestingly, the corresponding beta-anomer, and in particular its D-enantiomer, has been shown to possess very strong cross-inhibitory activity toward beta-galactosidase and beta-glucosidase. Structure and inhibitory activity relationships concerning these alpha,beta-fuco derivatives, as well as parent alpha,beta-galacto validamines are discussed here with reference to our results.

Chemical chaperone therapy: clinical effect in murine G(M1)-gangliosidosis.

Certain low-molecular-weight substrate analogs act both as in vitro competitive inhibitors of lysosomal hydrolases and as intracellular enhancers (chemical chaperones) by stabilization of mutant proteins. In this study, we performed oral administration of a chaperone compound N-octyl-4-epi-beta-valienamine to G(M1)-gangliosidosis model mice expressing R201C mutant human beta-galactosidase. A newly developed neurological scoring system was used for clinical assessment. N-Octyl-4-epi-beta-valienamine was delivered rapidly to the brain, increased beta-galactosidase activity, decreased ganglioside G(M1), and prevented neurological deterioration within a few months. No adverse effect was observed during this experiment. N-Octyl-4-epi-beta-valienamine will be useful for chemical chaperone therapy of human G(M1)-gangliosidosis.

Development and medical application of unsaturated carbaglycosylamine glycosidase inhibitors.

This article reviews synthesis and structures of carbaglycosylamines, a group of carbocyclic sugar analogues. Some unsaturated derivatives are known to be potent glycosidase inhibitors. Among them, N-octyl-4-epi-beta-valienamine as a lysosomal beta-galactosidase inhibitor is currently undergoing a new molecular therapeutic trial (chemical chaperone therapy) for control of the human beta-galactosidase deficiency disorder, G(M1)-gangliosidosis.

Enzyme enhancement activity of N-octyl-beta-valienamine on beta-glucosidase mutants associated with Gaucher disease.

Gaucher disease (GD), caused by a defect of beta-glucosidase (beta-Glu), is the most common form of sphingolipidosis. We have previously shown that a carbohydrate mimic N-octyl-beta-valienamine (NOV), an inhibitor of beta-Glu, could increase the protein level and enzyme activity of F213I mutant beta-Glu in cultured GD fibroblasts, suggesting that NOV acted as a pharmacological chaperone to accelerate transport and maturation of this mutant enzyme. In the current study, NOV effects were evaluated in GD fibroblasts with various beta-Glu mutations and in COS cells transiently expressing recombinant mutant proteins. In addition to F213I, NOV was effective on N188S, G202R and N370S mutant forms of beta-Glu, whereas it was ineffective on G193W, D409H and L444P mutants. When expressed in COS cells, the mutant proteins as well as the wild-type protein were localized predominantly in the endoplasmic reticulum and were sensitive to Endo-H treatment. NOV did not alter this localization or Endo-H sensitivity, suggesting that it acted in the endoplasmic reticulum. Profiling of N-alkyl-beta-valienamines with various lengths of the acyl chain showed that N-dodecyl-beta-valienamine was as effective as NOV. These results suggest a potential therapeutic value of NOV and related compounds for GD with a broad range of beta-Glu mutations.

Synthesis of valiolamine and some precursors for bioactive carbaglycosylamines from (-)-vibo-quercitol produced by biogenesis of myo-inositol.

A convenient and practical synthesis of valiolamine (4) and its related carbaglycosylamine glycosidase inhibitors from (-)-vibo-quercitol (13), a compound readily produced by biogenesis of myo-inositol (9), is described.

Convenient synthesis of 3- and 6-deoxy-D-myo-inositol phosphate analogues from (+)-epi- and (-)-vibo-quercitols.

Starting from (+)-epi- and (-)-vibo-quercitols readily produced by bioconversion of myo-inositol, some biologically interesting phosphate and polyphosphate analogues, including the Ins(1,4,5)P(3) derivatives of 3-deoxy- and 6-deoxy-D-myo-inositol, could be readily prepared in a conventional manner. In addition, chemical modification at C-2 of the 3-deoxy Ins(1,4,5)P(3) provided 2-epimer, and 2-deoxy and 2-deoxy-2-fluoro forms. Eight polyphosphate analogues obtained were assayed for biological activity against PDH-Pase and PDH-K, and G6Pase, but none proved positive.

Fibroblast screening for chaperone therapy in beta-galactosidosis.

We performed screening of beta-galactosidase-deficient fibroblasts for possible chemical chaperone therapy using N-octyl-4-epi-beta-valienamine (NOEV) in patients with GM1-gangliosidosis and Morquio B disease (beta-galactosidosis). Fibroblasts were cultured with NOEV for 4 days and beta-galactosidase activity was measured. Mutation analysis was performed simultaneously. Two separate criteria were set for evaluation of the chaperone effect: a relative increase of enzyme activity (more than 3-fold), and an increase up to more than 10% normal enzyme activity. Among the 50 fibroblast strains tested, more than 3-fold increase was achieved in 17 cell strains (34%), and more than 10% normal activity in 10 (20%). Both criteria were satisfied in 6 (12%), and either of them in 21 (42%). Juvenile GM1-gangliosidosis was most responsive, and then infantile GM1-gangliosidosis. This enhancement was mutation-specific. We estimate that the NOEV chaperone therapy will be effective in 20-40% of the patients, mainly in juvenile and infantile GM1-gangliosidosis patients. A molecular design may produce mutation-specific chaperone compounds for the other disease phenotypes. This cellular screening will be useful for identification of human patients with beta-galactosidase deficiency for chaperone therapy to be started in the near future.

Design and synthesis of glycosidase inhibitor 5-amino-1,2,3,4-cyclohexanetetrol derivatives from (-)-vibo-quercitol.

In continuation of development of bioactive inositol derivatives, a 1-O-methyl derivative of 5-amino-5-deoxy-L-talo-quercitol was designed and synthesized as an analogue of the strong alpha-fucosidase inhibitor, 5a-carba-alpha-L-fucopyranosylamine, the methyl branch being replaced with methoxyl, and demonstrated to be a moderate alpha-fucosidase inhibitor. The present approach provides a possible route to apply alkyl ethers of aminodeoxyinositols as hexopyranose mimics of biological interest.