Institute for Glyco-core Research (iGCORE), Gifu University, Gifu 501-1193, Japan.
Institute for Glyco-core Research (iGCORE), Gifu University, Gifu 501-1193, Japan.
Biochim Biophys Acta Gen Subj. 2022 Jun;1866(6):130118. doi: 10.1016/j.bbagen.2022.130118. Epub 2022 Mar 4.
N-Glycan branching regulates various functions of glycoproteins. N-Acetylglucosaminyltransferase V (GnT-V) is a GlcNAc transferase that acts on N-glycans and the GnT-V-producing branch is highly related to cancer progression. This indicates that specific GnT-V inhibitors may be drug candidates for cancer treatment. To design novel GnT-V inhibitors, we focused on the unique and weak recognition of the donor substrate UDP-GlcNAc by GnT-V. On the basis of the catalytic pocket structure, we hypothesized that UDP-GlcNAc analogs with increasing hydrophobicity may be GnT-V inhibitors.
We chemically synthesized 10 UDP-GlcNAc analogs in which one or two phosphate groups were replaced with hydrophobic groups. To test these compounds, we set up an HPLC-based enzyme assay system for all N-glycan-branching GlcNAc transferases in which GnT-I-V activity was measured using purified truncated enzymes. Using this system, we assessed the inhibitory effects of the synthesized compounds on GnT-V and their specificity.
Several UDP-GlcNAc analogs inhibited GnT-V activity, although the inhibition potency was modest. Compared with other GnTs, these compounds showed a preference for GnT-V, which suggested that GnT-V was relatively tolerant of hydrophobicity in the donor substrate. Docking models of the inhibitory compounds with GnT-V suggested the mechanisms of how these compounds interacted with GnT-V and inhibited its action.
Chemical modification of the donor substrate may be a promising strategy to develop selective inhibitors of GnT-V.
Our findings provide new insights into the design of GnT inhibitors and how GnTs recognize the donor substrate.
N-糖链分支调节糖蛋白的各种功能。N-乙酰氨基葡萄糖转移酶 V（GnT-V）是一种作用于 N-糖链的葡萄糖胺转移酶，其产生的分支与癌症进展高度相关。这表明特定的 GnT-V 抑制剂可能是癌症治疗的候选药物。为了设计新型 GnT-V 抑制剂，我们专注于 GnT-V 对供体底物 UDP-GlcNAc 的独特且较弱的识别。基于催化口袋结构，我们假设 UDP-GlcNAc 类似物的疏水性增加可能是 GnT-V 抑制剂。
我们通过化学方法合成了 10 种 UDP-GlcNAc 类似物，其中一个或两个磷酸基团被疏水性基团取代。为了测试这些化合物，我们建立了一个基于 HPLC 的酶测定系统，用于所有 N-糖链分支 GlcNAc 转移酶，其中使用纯化的截断酶测量 GnT-I-V 活性。使用该系统，我们评估了合成化合物对 GnT-V 的抑制作用及其特异性。
几种 UDP-GlcNAc 类似物抑制 GnT-V 活性，尽管抑制效力适中。与其他 GnTs 相比，这些化合物对 GnT-V 具有偏好性，这表明 GnT-V 对供体底物的疏水性相对耐受。与 GnT-V 的抑制性化合物的对接模型表明了这些化合物与 GnT-V 相互作用并抑制其作用的机制。
供体底物的化学修饰可能是开发 GnT-V 选择性抑制剂的一种有前途的策略。
我们的发现为 GnT 抑制剂的设计以及 GnTs 如何识别供体底物提供了新的见解。
