肝醣合成酶激酶 3 (glycogen synthase kinase 3, GSK-3)，是一種絲胺酸/蘇胺酸蛋白質激酶，在阿茲海默症 (AD) 的發病機制上扮演著重要的角色。根據推測，當GSK-3β活性太高會導致tau 蛋白過度磷酸化因而產生AD症狀，如果能設計有效的抑制劑分子去抑制GSK-3β的活性，則可能達到減輕或治療AD疾病的效果。因此，我們使用熱力學積分ΔΔG的模擬方法去計算蛋白質與小分子間的相對結合自由能，藉此幫助我們去設計小分子抑制劑。首先，我們先計算已知實驗值的分子的結合親和力，所計算結果與實驗值有不錯的吻合。之後，我們利用相同計算程序去預測類似物分子的結合力，並且將這些分子推薦去做酵素實驗。我們總共計算了11個分子的相對自由能ΔΔG (kcal/mol)。有三個類似物分子 PP1、PP2和LL4 在酵素實驗上發現到有效的抑制能力。此外，我們對這三個類似物分別去探討不同的結合位向的關係。在不同結合位向計算上，我們發現計算LL4是以和ZRM相同的結合位向結合蛋白質；而PP1和PP2則是以不相同結合位向結合蛋白質。PP1、PP2和LL4的ΔΔG計算結果分別為5.1, 2.7和1.2 kcal/mol，實驗結果分別為3.7, 0.7和-0.4 kcal/mol，計算值和實驗值有一定的相關性，相關係數為0.83，在考慮不同位向計算上，docking結合TI方法的計算可以更準確預測到實驗結果。此外，在探討小分子與蛋白質的結合親和力關係上，我們建議在指向水的分子結構位置上加上親水性的官能基團，預測可能有效增進蛋白質與小分子間的結合親力。最後，我們希望本篇模擬結果提供的資訊，能在未來上更進一步幫助GSK-3β去設計有效的小分子抑制劑。

Glycogen synthase kinase 3 (GSK-3) is a serine/threonine kinase that plays an important role in the pathogenesis of Alzheimer's disease (AD). It was postulated that when GSK-3β kinase’s activity is too high, it over-phosphorylates tau protein and causes AD.Inhibition of GSK-3βkinase would provide therapeutic effect in alleviated and/or cure AD.In the present study, we used thermodynamic integration molecular dynamics (TI-MD) simulation to calculate protein-ligand binding free energy to aid in design of GSK-3β inhibitors. First, we computed the affinity of analogous GSK-3β kinase inhibitors of available experimental data and the computed results were in reasonably good agreement with the experimental value.Subsequently, we employed the same protocol in order to identify new analogous inhibitors which would be recommended for kinase experiment. We presented predicted results of relative binding free energy ΔΔG (kcal/mol) for 11 compounds. Three analogs PP1, PP2 and LL4 were found to be effective inhibitors in kinase assay experiment.In addition, the binding modes of these 3 compounds were investigated. It was found that the LL4 adopted binding mode similar to ZRM's but PP1 and PP2 is adopted different binding modes. The computed ΔΔG values of PP1,PP2 and LL4 were 5.1, 2.7, and 1.2 kcal/mol , correlating well with experiment data of 3.7, 0.7, and -0.4 kcal/mol, respectively. The correlation coefficient (R2) was 0.83. Analysis of interactions between ligands and GSK-3β kinase suggested that addition the hydrophilic group at the site of the functional group pointing towards water can enhance the binding affinity of lignad-protein binding. These results provide useful insight for further design of GSK-3β kinase inhibitors in the future.

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