Target‐Assisted Selection of Galactosyltransferase Binders from Dynamic Combinatorial Libraries. An Unexpected Solution with Restricted Amounts of the Enzyme

Oligosaccharides attached to proteins and lipids at cell surfaces are involved in major molecular-recognition processes including cell adhesion, inflammation, and metastasis. Inhibition of the enzymes responsible for the synthesis of these carbohydrate motifs, especially the glycosyltransferases (GT), is particularly valuable for a better understanding of the mechanism of action of these enzymes and the structure–function relationship of their products. This research might also lead to the ACHTUNGTRENNUNGdevelopment of new agents for therapeutic intervention. DifACHTUNGTRENNUNGferent strategies have been followed to discover potential enzyme modulators. The rational design of good GT binders is, however, difficult for several reasons. First, most of the 3D structures of GTs are not yet available. Moreover, these enzymes function with a complex four-domain catalytic site (acceptor, nucleotide sugar donor, metal) with a weak binding of their natural substrates. Within this context, dynamic combinatorial chemistry (DCC) appears to be the best choice in the search for GT binders because, basically, no prior knowledge of the target protein structure is required. Furthermore, this ACHTUNGTRENNUNGapproach is able to generate potent enzyme inhibitors, even when the system exhibits poor binding properties. This approach is based on the reversible connection between different building blocks to form a library of potential ligands under thermodynamic equilibrium (dynamic combinatorial library, DCL). In the presence of a template (enzyme), the distribution of the library may be altered, with an amplification of the best binders, which can be detected by an adequate ACHTUNGTRENNUNGanalytical method. A direct correlation of the most amplified members with the best binders is encountered when their concentration is kept low compared to the total concentration of the building blocks. In this respect, imines have been successfully used as active members of the DCL in an aqueous medium because they fulfill two important conditions. First, the “imines” of the library are present in only very small amounts compared to the starting building blocks. Secondly, the equilibrating mixture of components can be conveniently turned off for composition analysis by a simple reductive step. In all the examples reported, the amine reduction products (RP) resulting from the reductive step maintain most of the binding properties of the parent imines. 9] One drawback for the implementation of such DCC experiments is that the method usually requires excess quantities of the enzyme, so re-equilibration might be observed. Unfortunately, GTs are often only accessible in tiny amounts. With no other choice than a restricted concentration of the target enzyme, simple calculations show that, under standard conditions, the best binders will go undetected because their concentration will remain far below the detection limits. The only solution would be to have a large excess of the starting building blocks with respect to the target in order to increase the concentration of the imine binders. Under these conditions, competitive binding to the target of the derived RP must be considered. When the binding properties of the imines are maintained in the RP (dissociation constants KRP~ KIm), as seen in all examples reported thus far, [4a,c,d, 5] the amplification of the best binders will then level off over time, because the RP will efficiently compete with the active members of the DCL. On the other hand, when the binding properties are lost (dissociation constants KRP !KIm), the amplification of the RP will stay constant over time so that they will continually accumulate in the system thus becoming detectable. Scheme 1 illustrates this qualitative prediction.