Recent Developments of Transition-State Analogue Glycosidase Inhibitors of Non-Natural Product Origin

Fabry disease is a disorder of glycosphingolipid metabolism caused by deficiency of lysosomal alpha-galactosidase A (alpha-Gal A), resulting in renal failure along with premature myocardial infarction and strokes. No effective treatment of this disorder is available at present. Studies of residual activities of mutant enzymes in many Fabry patients showed that some of them had kinetic properties similar to those for normal alpha-Gal A, but were significantly less stable, especially in conditions of neutral pH (refs. 3-5). The biosynthetic processing was delayed in cultured fibroblasts of a Fabry patient, and the mutant protein formed an aggregate in endoplasmic reticulum, indicating that the enzyme deficiency in some mutants was mainly caused by abortive exit from the endoplasmic reticulum, leading to excessive degradation of the enzyme. We report here that 1-deoxy-galactonojirimycin (DGJ), a potent competitive inhibitor of alpha-Gal A, effectively enhanced alpha-Gal A activity in Fabry lymphoblasts, when administrated at concentrations lower than that usually required for intracellular inhibition of the enzyme. DGJ seemed to accelerate transport and maturation of the mutant enzyme. Oral administration of DGJ to transgenic mice overexpressing a mutant alpha-Gal A substantially elevated the enzyme activity in some organs. We propose a new molecular therapeutic strategy for genetic metabolic diseases of administering competitive inhibitors as 'chemical chaperons' at sub-inhibitory intracellular concentrations.

Accelerated transport and maturation of lysosomal alpha-galactosidase A in Fabry lymphoblasts by an enzyme inhibitor.

Publisher Summary This chapter discusses the results of the studies that permit some generalizations on the catalytic mechanism of glycoside hydrolases from widely differing sources and with different sugar and aglycon specificities and that have become available over the past 15 years. The strong inhibition of glycosidases by aldonolactones was first mentioned in 1940 by Japanese workers who studied β- D -glucosidases from Aspergillus (Taka-diastase) and almonds. Even though both of these groups of compounds are derived from normal substrates by only a minor modification of the glycon moiety, they are discussed together with pseudosubstrates because their reactions with glycosidases show, in many cases, unusual kinetic features. The information relevant to the mechanism of an enzyme-catalyzed reaction can, in general, only be obtained from irreversible inhibitors that react specifically at the active site, and thereby inactivate the enzyme. In many cases, inhibition studies were not carried out to obtain information on the reaction mechanism, but for other purposes. Thus, only inhibitors were tested that were considered suitable for the particular project, for example, studies on the biological function of the enzyme where glycosylamines and aldonolactones are unsuitable.

Glycoside Hydrolases: Mechanistic Information from Studies with Reversible and Irreversible Inhibitors

Binding Energy and Catalysis: The Implications for Transition-State Analogs and Catalytic Antibodies.

Enzyme-catalyzed organic synthesis: practical routes to aza sugars and their analogs for use as glycoprocessing inhibitors

A variety of reversible inhibitors of sweet almond beta-glucosidase were examined. These included simple sugars and sugar derivatives, amines and phenols. With respect to the sugar inhibitors and, indeed, the various glycoside substrates, the enzyme has what can be considered a "relaxed specificity". No single substituent on glucose, for example, is essential for binding. Replacement of a hydroxyl group with an anionic substituent reduces the affinity while substitution with a cationic (amine) substituent enhances the affinity. Amines, in general, are good inhibitors, binding more tightly than the corresponding alcohols: pKiRNH3+ = 0.645pKiROH + 1.77 (n = 9, r = 0.97). The affinity of a series of 10 primary amines was found to be strongly influenced by substituent hydrophobicity: pKi = 0.52 pi + 1.32 (r = 0.95). The major binding determinant of the glycoside substrates is the aglycon moiety. Thus, the Ki values of phenols are similar in magnitude to the Ks values of the corresponding aryl beta-glucoside. The pH dependence for the inhibition by various phenols indicates that it is the un-ionized phenol which binds to the enzyme when an enzymic group of pKa = 6.8 (+/- 0.1) is protonated. The affinity of the phenol inhibitor is dependent on its basicity with a Bronsted coefficient for binding of beta = -0.26 (n = 14, r = 0.98). The pH dependence of the binding of two particularly potent beta-glucosidase inhibitors was also examined. 1-Deoxynojirimycin (1,5-dideoxy-1,5-imino-D-glucitol) has a pH-corrected Ki = 6.5 microM, and D-glucono-1,5-lactam has a pH-corrected Ki = 29 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

K. A. R. Sastry

Papers

Reversible inhibitors of beta-glucosidase.