Target-assisted selection of galactosyltransferase binders from dynamic combinatorial libraries. An unexpected solution with restricted amounts of the enzyme
Target-assisted selection of galactosyltransferase binders from dynamic combinatorial libraries. An unexpected solution with restricted amounts of the enzyme
复制标题
DOI：
10.1002/cbic.200600022
复制
发表时间：
2006-07-01
期刊：
影响因子：
3.2
通讯作者：
Beau, Jean-Marie
中科院分区：
文献类型：
--
作者：
Valade, Anne;Urban, Dominique;Beau, Jean-Marie
在细胞表面附着于蛋白质和脂质的寡聚体参与主要的分子识别过程，包括细胞粘附、炎症和转移。[1]抑制负责这些碳水化合物基序合成的酶，特别是糖基转移酶（GT），对于更好地理解这些酶的作用机制及其产物的结构-功能关系特别有价值。这项研究也可能导致新的治疗干预剂的开发。遵循不同的ACHTUNGTRENNUNGferent策略来发现潜在的酶调节剂。[2]然而，由于几个原因，合理设计良好的GT粘合剂是困难的。首先，大多数GT的3D结构还不可用。[2]此外，这些酶的功能与复杂的四域催化位点（受体，核苷酸糖供体，金属）与其天然底物的弱结合。[2]在这种情况下，动态组合化学（DCC）[3]似乎是寻找GT结合剂的最佳选择，因为基本上不需要靶蛋白结构的先验知识。此外，这种ACHTUNGTRENNUNG方法能够产生有效的酶抑制剂，[4]即使系统表现出不良的结合特性。[5]该方法基于不同结构单元之间的可逆连接，以在热力学平衡下形成潜在配体库（动态组合库，DCL）。在存在模板（酶）的情况下，文库的分布可能会改变，最佳结合物的扩增可以通过适当的ACHTUNGTRENNUNG分析方法检测到。当扩增最多的成员与最佳结合剂的浓度与结构单元的总浓度相比保持较低时，遇到扩增最多的成员与最佳结合剂的直接相关性。[6]在这方面，亚胺已成功地用作水性介质中的DCL的活性成员，因为它们满足两个重要条件。首先，与起始构建单元相比，库的“亚胺”[7]仅以非常少量存在。[8]第二，组分的平衡混合物可以通过简单的还原步骤方便地关闭用于组成分析。[4a在所有报道的实施例中，由还原步骤产生的胺还原产物（RP）保持了母体亚胺的大部分结合性质。[4a实施这种DCC实验的一个缺点是该方法通常需要过量的酶，因此可能观察到再平衡。[4，5，10]不幸的是，GT通常只能获得少量。在没有其他选择的情况下，除了目标酶的有限浓度，简单的计算表明，在标准条件下，[4a，5]最好的结合剂将无法检测到，因为它们的浓度将保持远低于检测限。[11]唯一的解决方案将是相对于目标具有大量过量的起始结构单元，以增加亚胺粘合剂的浓度。在这些条件下，必须考虑与衍生RP的靶标的竞争性结合。当RP中亚胺的结合性质保持不变（解离常数KRP = KIm）时，如迄今为止报道的所有实施例所示，[4a，c，d，5]最佳结合剂的扩增[12]将随着时间的推移而趋于平稳，因为RP将有效地与DCL的活性成员竞争。另一方面，当结合性质丧失时（解离常数KRP！KIm），RP的放大将保持恒定...
Oligosaccharides attached to proteins and lipids at cell surfaces are involved in major molecular-recognition processes including cell adhesion, inflammation, and metastasis.[1] 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. Dif-ACHTUNGTRENNUNGferent strategies have been followed to discover potential enzyme modulators.[2] 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.[2] Moreover, these enzymes function with a complex four-domain catalytic site (acceptor, nucleotide sugar donor, metal) with a weak binding of their natural substrates.[2] Within this context, dynamic combinatorial chemistry (DCC)[3] 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,[4] even when the system exhibits poor binding properties.[5] 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.[6] 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”[7] of the library are present in only very small amounts compared to the starting building blocks.[8] Secondly, the equilibrating mixture of components can be conveniently turned off for composition analysis by a simple reductive step.[4a, c, d, 5] 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.[4a, c, d, 5, 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.[4, 5, 10] 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,[4a, 5] the best binders will go undetected because their concentration will remain far below the detection limits.[11] 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 [12] 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 …