Lape Michael, Elam Christopher, Versluis Maria, Kempton Robert, Paula Stefan
Department of Chemistry, Northern Kentucky University, Highland Heights, Kentucky 41099-1905, USA.
Proteins. 2008 Feb 15;70(3):639-49. doi: 10.1002/prot.21542.
The ion transport activity of the sarco/endoplasmic reticulum calcium ATPase (SERCA) is specifically and potently inhibited by the small molecule 2,5-di-tert-butylhydroquinone (BHQ). In this study, we investigated the relative importance of the nature and position of BHQ's four substituents for enzyme inhibition by employing a combination of experimental and computational techniques. The inhibitory potencies of 21 commercially available or synthesized BHQ derivatives were determined in ATPase activity assays, and 11 compounds were found to be active. Maximum inhibitory potency was observed in compounds with two para hydroxyl groups, whereas BHQ analogues with only one hydroxyl group were still active, albeit with a reduced potency. The results also demonstrated that two alkyl groups were an absolute requirement for activity, with the most potent compounds having 2,5-substituents with four or five carbon atoms at each position. Using the program GOLD in conjunction with the ChemScore scoring function, the structures of the BHQ analogues were docked into the crystal structure of SERCA mimicking the enzyme's E(2) conformation. Analysis of the docking results indicated that inhibitor binding to SERCA was primarily mediated by a hydrogen bond between a hydroxyl group and Asp-59 and by hydrophobic interactions involving the bulky inhibitor alkyl groups. Attempts to dock BHQ into crystal structures corresponding to the E(1) conformation of the enzyme failed, because the conformational changes accompanying the E(2)/E(1) transition severely restricted the size of the binding site, suggesting that BHQ stabilizes the enzyme in its E(2) form. The potential role of Glu309 in enzyme inhibition is discussed in the context of the computational results. The docking scores correlated reasonably well with the measured inhibitory potencies and allowed the distinction between active and inactive compounds, which is a key requirement for future virtual screening of large compound databases for novel SERCA inhibitors.
小分子2,5 - 二叔丁基对苯二酚（BHQ）可特异性且强效地抑制肌浆网/内质网钙ATP酶（SERCA）的离子转运活性。在本研究中，我们通过结合实验和计算技术，研究了BHQ四个取代基的性质和位置对酶抑制作用的相对重要性。在ATP酶活性测定中确定了21种市售或合成的BHQ衍生物的抑制效力，发现其中11种化合物具有活性。在具有两个对羟基的化合物中观察到最大抑制效力，而仅具有一个羟基的BHQ类似物仍具有活性，尽管效力有所降低。结果还表明，两个烷基是活性的绝对必要条件，最有效的化合物在2,5位各有四个或五个碳原子的取代基。使用GOLD程序结合ChemScore评分函数，将BHQ类似物的结构对接至模拟酶E(2)构象的SERCA晶体结构中。对接结果分析表明，抑制剂与SERCA的结合主要由羟基与Asp - 59之间的氢键以及涉及庞大抑制剂烷基的疏水相互作用介导。将BHQ对接至与酶E(1)构象对应的晶体结构的尝试失败了，因为伴随E(2)/E(1)转变的构象变化严重限制了结合位点的大小，这表明BHQ使酶稳定在其E(2)形式。结合计算结果讨论了Glu309在酶抑制中的潜在作用。对接分数与测得的抑制效力合理相关，并能够区分活性和非活性化合物，这是未来对大型化合物数据库进行新型SERCA抑制剂虚拟筛选的关键要求。
