Designing inhibitors for glycosyltransferases

Details Designing inhibitors for glycosyltransferases Designing inhibitors for glycosyltransferases

1999-08-02

2002-07-02

Peselev, Elli (Department: 1623)

Data processing: measuring, calibrating, or testing

Measurement system in a specific environment

Chemical analysis

C536S018500, C536S127000

Reexamination Certificate

active

06415234

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to the design of carbohydrate processing inhibitors for glycosyltransferases based on the conformational properties of the sugar phosphate linkage and/or phosphate linkages in nucleotide-sugar donors for the glycosyltransferases.
BACKGROUND OF THE INVENTION
The oligosaccharide chains of N— and O-linked glycoproteins play a crucial role in a number of biological processes [1,2]. Their biosynthesis and degradation pathways are therefore areas of significant interest for biology, medicine, and biotechnology. The assembly of the various types of oligosaccharides involves several glycosidases and glycosyltransferases. In comparison with glycosidases, the mechanisms of which have been characterized in some detail [3-5] the catalytic mechanism of glycosyltransferases have not yet been investigated in detail, though some kinetic studies have been reported [6-18].
Glycosyltransferases are a diverse group of enzymes that catalyze the transfer of a single monosaccharide unit from a donor to the hydroxyl group of an acceptor saccharide. [19, 20] The acceptor can be either a free saccharide, glycoprotein, glycolipid, or polysaccharide. The donor can be a nucleotide-sugar, dolichol-phosphate-sugar or dolichol-pyrophosphate-oligosaccharide. Glycosyltransferases show a precise specificity for both the acceptor and sugar donor and generally require the presence of a metal cofactor, usually a divalent cation like manganese.
The knowledge of the structure of nucleotide-sugars is prerequisite for understanding the catalytic mechanism of glycosyltransferases and for developing inhibitors for these enzymes. The 3-D structure of nucleotide-sugars is determined to some extent by the conformation adopted by the phosphate linkage. However, despite the importance of the conformation adopted by the diphosphate linkages on the overall shape of nucleotide-sugars, the structure and the conformational properties of such linkage remains somewhat unspecified.
Few calculations have been performed on the diphosphate linkage. The structure of the lowest energy conformers of pyrophosphoric acid, its anions and alkali salts were studied to model the hydrolysis of pyrophosphate [2-5]. The presence of hydroxyl groups in pyrophosphates stabilizes their lowest energy conformers by intramolecular hydrogen bonds. However, such stabilizing interactions are not possible in nucleotide-sugars and the diphosphate linkage in nucleotide-sugars should exhibit a different conformational behavior.
SUMMARY OF THE INVENTION
The invention relates to the design of carbohydrate processing inhibitors for glycosyltransferases based on the conformational properties of the sugar-phosphate linkage and/or phosphate linkage in sugar nucleotide donors for the glycosyltransferases. The method permits the identification early in the drug development cycle of compounds which have advantageous properties.
In particular, the present inventors studied the conformational properties of the sugar-phosphate linkage with ab initio methods using the 2-O-methylphosphono-tetrahydropyran anion (1 in
FIG. 1
) and sodium 2-O-methylphosphono-tetrahydropyran (2 in
FIG. 1
) as models. The ab initio energy and geometry of the conformers around the C1—O1 and O—P bonds were determined at various levels of the self-consistent field (SCF) and adiabatic connection method of density functional theory. At all levels of ab initio theory, compound 1 preferred the trans to the gauche conformer around the C1—O1 bond. The presence of a sodium counter-ion completely reverses the relative energy of the conformers, such that in the ion-pair complex 2, the gauche conformer about the C1—O1 bond is favored
The present inventors also carried out an ab initio study of the sugar-diphosphate linkage. Ab initio molecular orbital calculations of the 2-O-methyldiphosphono-tetrahydropyran dianion and the magnesium 2-O-methyldiphosphono-tetrahydropyran were used to model the conformational behaviour of the sugar-diphosphate linkage in sugar-nucleotides. The geometry and energy of conformers were calculated at different basis set levels, from 6-31G* to cc-pVTZ(−f)++, using the SCF, DFT/B3LYP, and LMP2 methods. The vibrational frequencies were calculated at the HF/6-31G* level and the zero-point energy, thermal and entropy corrections were evaluated. The results of conformational analyses show that interactions of the diphosphate linkage with the Mg
2+
cation alter the conformational preferences about the anomeric and the diphosphate linkages. These changes influence the overall 3D-shape adopted by nucleotide-sugars.
The differences in structures with the ions indicates an important function of the metal cofactor in the catalytic mechanism of glycosyltransferases. Complexation of the phosphate with the metal ion changes the conformation about the phosphate linkages and more specifically about one of the P—O bonds going from gauche to trans orientation; it changes the conformation of the sugar-phosphate linkage from trans to gauche orientation; it influences the overall 3D-shape adopted by molecules containing phosphate linkages such as sugar donors in order to adopt a correct shape for optimal enzymatic recognition and to achieve maximal catalytic efficiency; it activates the sugar-oxygen glycosidic bond by elongating the sugar-oxygen bond; and/or, it changes the charge distribution to make the protonation of the glycosidic oxygen more favored.
Therefore broadly stated, the present invention relates to a method for preparing a potential inhibitor of a glycosyltransferase comprising:
(a) combining a first sugar, a phosphate group, and a second sugar that is transferred by the glycosyltransferase to an acceptor for the glycosyltransferase, under conditions appropriate for formation of a bond between a carbon atom of the first sugar and a first oxygen atom of the phosphate group, and formation of a linkage between a carbon atom of the second sugar and a second oxygen atom of the phosphate group, wherein the orientation of the linkage is antiperiplanar, and preferably the distance between the carbon atom linked to the first sugar and the carbon atom linked to the second sugar is in the range 3.7 Å to 4.2 Å;
(b) combining a first sugar, a phosphate group, an ion, and a second sugar that is transferred by the glycosyltransferase to an acceptor for the glycosyltransferase, under conditions appropriate for formation of a bond between a carbon atom of the first sugar and a first oxygen atom of the phosphate group, a linkage between a carbon atom of the second sugar and a second oxygen atom of the phosphate group, and an electrostatic interaction between free oxygen atoms of the phosphate group and the ion, and wherein the orientation of the linkage is synclinal, and, preferably the distance between the carbon atom linked to the first sugar and the carbon atom linked to the second sugar is in the range 3.7 Å to 4.5 Å;
(c) combining a first sugar, a diphosphate group, and a second sugar that is transferred by the glycosyltransferase to an acceptor for the glycosyltransferase, under conditions appropriate for formation of a bond between a carbon atom of the first sugar and an oxygen atom of a first phosphate of the diphosphate group, and formation of a linkage between a carbon atom of the second sugar and an oxygen atom of a second phosphate of the diphosphate group, wherein the orientation of the linkage is antiperiplanar, phosphorous-oxygen bonds linking the first phosphate to the second phosphate of the diphosphate group are in a synclinal or anticlinal orientation, and synclinal orientation, respectively, or symmetrically related orientation, and preferably the distance between the carbon atom linked to the first sugar and the carbon atom linked to the second sugar is in the range 4.9 Å to 5.3 Å; or
(d) combining a first sugar, a diphosphate group, an ion, and a second sugar that is transferred by the glycosyltransferase to an acceptor for the gly

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