Current Topics in Medicinal Chemistry - Volume 9, Issue 1, 2009

Carbohydrates are used as nutrients, structural components and informational tags in life system and they are continuously synthesized, modified and degraded by numerous kinds of carbohydrate-related enzymes. The inhibitors of these enzymes, therefore, are commercial or potential medicines for diseases caused by upregulated or unwanted activities of these enzymes. Starch taken in the digestive system as a nutrient is degraded into glucose by amylase, maltase, etc., which is released into the blood circulatory system. In diabetics, the elevated glucose level promotes the glycation of proteins through Maillard reaction to help accumulation of advanced glycation end products (AGEs), which may fatally damage tiny blood vessels in the eyes, kidneys and so on. The inhibitors of amylase and maltase reduce blood glucose level and therefore suppress AGE accumulation. The effect of the inhibitors toward diabetes is exemplified by commercial antidiabetic drugs, acarbose (Glucobay) and voglibose (Basen). Another possible damage mechanism in diabetics includes an impaired blood stream caused by sorbitol-derived edema in cells. The elevated blood glucose level accelerates sorbitol synthesis by aldose reductase in the starting point of polyol pathway of carbohydrate metabolism, resulting in the accumulation of sorbitol in cells. In addition, fructose produced from sorbitol in the polyol pathway is more reactive toward the glycation of proteins than glucose. The aldose reductase inhibitor (epalrestat) therefore is used in symptomatic therapy of diabetes. Another potential diabetic drug is glycogen phosphorylase inhibitors, which would reduce the glucose reproduction through muscle glycogen degradation. As the outer shell of insects and crustaceans and the cell wall of Gram-positive bacteria are made of chitin and chitosan, the metabolism of these polysaccharides is a lifeline for these organisms. Thus polytoxins D, a chitin synthase inhibitor, and allosamidin, a chitinase inhibitor, are widely used as an agricultural fungicide and insecticide, respectively. On the other hand, validamycin A, a trehalase inhibitor, has antifungal activity. Trehalose is a storage carbohydrate for fungi and the trehalase inhibitor causes excess trehalose accumulation. Glycoproteins are synthesized in cells through correct folding of polypeptides and appropriate intracellular trafficking between compartments. In these biosynthetic processes, oligosaccharides are used as informative tags displaying the next actions to be conducted on the immature polypeptides or proteins. Glycoproteins with erroneously synthesized oligosaccharides are excluded from the synthetic process. Proper display of the oligosaccharides depends on timely trimming of intermediate oligosaccharides with glycosidases. Glycosidase inhibitors, such as N-butyl-1-deoxynojirimycin (NB-DNJ, Miglustat), can block the synthesis of glycoproteins required to reconstruct viruses in host cells. Therefore these inhibitors are potential antiviral agents and NB-DNJ has been subjected to clinical trials for human immunodeficiency virus (HIV). Oligosaccharides on glycoproteins can be knocked-down by a glycosyltransferase inhibitor, tunicamycin, which is used to study the functions of carbohydrates as informational tags.

α-Glucosidase inhibitors are marketed as therapeutic drugs for diabetes that act through the inhibition of carbohydrate metabolism. Inhibitors of the α-glucosidases that are involved in the biosynthesis of N-linked oligosaccharide chains have been reported to have antitumor, antiviral, and apoptosis-inducing activities, and some have been used clinically. α-Glucosidase inhibitors have interesting biological activities, and their design, synthesis, and screening are being actively performed. In quite a few reports, however, α-glucosidases with different origins than the target α-glucosidases, have been used to evaluate inhibitory activities. There might be confusion regarding the naming of α-glucosidases. For example, the term α-glucosidase is sometimes used as a generic name for α-glucoside hydrolases. Moreover, IUBMB recommends the use of “α-glucosidase” (EC 3.2.1.20) for exo-α-1,4-glucosidases, which are further classified into four families based on amino acid sequence similarities. Accordingly, substrate specificity and susceptibility to inhibitors varies markedly among enzymes in the IUBMB α-glucosidases. The design and screening of inhibitors without consideration of these differences is not efficient. For the development of a practical inhibitor that is operational in cells, HTS using the target α-glucosidase and the computer-aided design of inhibitors based on enzymatic information concerning the same α-glucosidase are essential.

Glycosidases mediate the digestion of oligosaccharides in the small intestine as well as the processing of cell surface oligosaccharides, which play important roles in cell to cell recognition during infections, metastasis, and immune responses. Thus, agents that control the activities of glycosidases could have therapeutic effects against many carbohydrate-related diseases. In fact, many fruitful efforts have been made to design and synthesize glycosidase inhibitors as clinically practical medicines. This review summarizes the current stage of development of such inhibitors to treat diabetes, influenza, virus infection of HIV, tumor metastasis, and glycosyl sphingo lipid (GSL) storage diseases.

Glycosidases are involved in various important biological processes including digestion of starch in the intestine, oligosaccharide processing inside rough ER and Golgi apparatus, and degradation of glycoconjugates in lysosomes. It is apparent that inhibitors of this class of enzymes are useful in the investigation of biological functions of glycoconjugates. Furthermore, it is believed that these compounds are important as pharmaceuticals. The structures of glycosidase inhibitors can be categorized into two major classes; ground-state mimetics and transition-state mimetics. The former has a chair-shaped six-membered structure that mimics monosaccharides where ring oxygens are often replaced with other elements for an improved binding affinity. On the other hand, the latter possesses a somewhat distorted shape compared with the chair conformation of carbohydrates. One of the ways to derive such distortion is by the introduction of sp2 character into the six-membered ring, and another is by ring contraction to form a five-membered system for the transition-state mimetics. The functions of these transition-state mimetics are often unpredictable regarding inhibitory activity and enzyme specificity. This review focuses on such “difficult to predict” species in an attempt to extract information or common aspects for the future development of inhibitors of glycosidases based on transition-state mimetics.

5a-Carba-α-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 α- 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 α-fucosidase inhibitor, 5a-carba-α-L-fucopyranosylamine, α- fuco validamine. Interestingly, the corresponding β-anomer, and in particular its D-enantiomer, has been shown to possess very strong cross-inhibitory activity toward β-galactosidase and β-glucosidase. Structure and inhibitory activity relationships concerning these α,β-fuco derivatives, as well as parent α,β-galacto validamines are discussed here with reference to our results.

The inhibitory activities against glycosidases of synthetic thiasugars, the ring sulfur analogs of carbohydrate, were surveyed with a special emphasis on our own studies. 5-Thio-L-fucose, the ring sulfur analog of L-fucose, was the first thiopyran that shows a Ki value in the micromolar range against a glycosidase. The structure-activity relationship studies for the fucosidase inhibition by 5-thio-L-fucose disclosed that a hydrophobic interaction between the ring sulfur atom and the enzyme is responsible for the strong binding. The syntheses and activities of di- and trisaccharide analogs incorporating thiasugars are also outlined. These oligosaccharide analogs are glycosidase-resistant and some of them show strong binding to antibodies or lectins. We created a sulfylimine compound having a thiafuran structure as a transitionstate analog inhibitor of glucosidases and found that it has a weak inhibition against a glucosidase. The same thiafuran structure could be found in a natural product, salacinol, which was isolated and elucidated to be a strong glucosidase inhibitor by another group. A study of the structure-activity relationship using the synthetic analogs of salacinol indicated the relevance of both the sulfonium ion in the thiafuran ring and its intramolecular counter anion, sulfate, to the inhibition activity.

Oligosaccharides in glycoconjugates such as glycoproteins and glycolipids play important roles in a variety of biological functions. Since glycosyltransferases are responsible for the biosynthesis of these oligosaccharides, inhibitors of glycosyltransferases are targets for drug discovery. Bisubstrate analogues, in which donor and acceptor analogue are covalently attached to each other, offer donor's high affinity and acceptor's high selectivity. In this review, we describe the design and synthesis of bisubstrate analogues of glycosyltransferases as well as their inhibitory potency hoping to inform the development of potent and selective inhibitors.

Recent structural and kinetic studies indicated that enzymes shift their peripheral structures of the catalytic sites dynamically to modify the substrate structures. Molecules which disturb such mechanism of specific enzymes may become potent candidates for therapeutic reagent. This article describes a versatile strategy to synthesize new class of mechanism-based inhibitors for glycosyltransferases and glycoside hydrolases. Combination of irreversible tagging and proteomic analysis of crucial amino acid residues using MALDI-TOF/TOF mass spectrometry allowed a promising method to probe such invisible transitional state in the enzymatic reactions. Feasibility of the fluorescence energy resonance transfer (FRET) is also documented as novel protocol for the real-time and continuous monitoring of glycosyltransferase catalyzed reactions. It was demonstrated that FRET method greatly facilitates discovery research of selective inhibitors in combination with click chemistry.

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