Probing the environment of tubulin-bound paclitaxel using fluorescent paclitaxel analogues.
Probing the environment of tubulin-bound paclitaxel using fluorescent paclitaxel analogues.
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使用荧光紫杉醇类似物探测微管蛋白结合紫杉醇的环境。
DOI：
10.1021/bi962891m
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Himes,RH
中科院分区：
文献类型：
--
作者：
Sengupta,S;Boge,TC;Liu,Y;Hepperle,M;Georg,GI;Himes,RH
为了确定紫杉醇分子与微管蛋白结合时不同位置的环境，我们合成了六种荧光类似物，其中在7-和10-位引入了（二甲氨基）苯甲酰基，并且在2-和N-以及3 '-苯环上的苯甲酰基已经用二甲氨基官能团修饰。在微管蛋白组装试验中，N-m-和N-p-（二甲氨基）苯甲酰基衍生物具有与紫杉醇相当的活性。2-、3 '-和10-类似物的活性略有降低，7-衍生物的活性约为紫杉醇的5%。根据溶剂对荧光发射光谱影响的研究结果，提出未结合的类似物与质子溶剂形成氢键。但7-和10-取代的类似物似乎比其他类似物更受质子溶剂的影响。先前，我们研究了N-元衍生物与微管蛋白和微管的结合[Sengupta，S.，等人（1995）Biochemistry 34，11889 - 11894]。在这项研究中，我们将研究扩展到包括2-，7-和10-衍生物。与N-取代类似物相似，2-衍生物与微管蛋白的结合伴随着大的蓝移，而7-和10-取代衍生物结合时发生非常小的蓝移。2-andN-取代的类似物结合到微管与增加的荧光强度，这是观察到与微管蛋白，而结合的7-和10-取代的类似物是伴随着一个大的荧光淬灭。这种猝灭可能是由于蛋白质中7-和10-（二甲氨基）苯甲酰基附近存在带电残基，或由于这些基团与芳香族侧链的π堆积。紫杉醇与微管的存在下，防止荧光增加的2-和N-衍生物和淬灭的7-和10-衍生物。结合到聚合微管蛋白上的荧光类似物的行为差异，加上对游离药物的溶剂研究，表明紫杉醇的2-和N-苯甲酰基结合在微管蛋白的疏水口袋中，但可以参与氢键，7-和10-位处于更亲水的环境中。
To determine the environment of different positions in the paclitaxel molecule when bound to tubulin, we have synthesized six fluorescent analogues in which a (dimethylamino)benzoyl group has been introduced into the 7- and 10-positions, and the benzoyl groups at the 2- andN- as well as the 3‘-phenyl ring have been modified with dimethylamino functions. In a tubulin assembly assay, theN-m-andN-p-(dimethylamino)benzoyl derivatives had activities comparable to the activity of paclitaxel. The 2-, 3‘-, and 10-analogues had slightly reduced activity, and the 7-derivative was about 5% as active as paclitaxel. On the basis of the results of studies of the effect of solvents on the fluorescence emission spectra, it is proposed that the unbound analogues form hydrogen bonds with protic solvents. But the 7- and 10-substituted analogues appear to be more affected by protic solvents than the other analogues. Previously, we studied the binding of theN-metaderivative to tubulin and microtubules [Sengupta, S., et al. (1995)Biochemistry 34, 11889−11894]. In this study, we extended the studies to include the 2-, 7-, and 10-derivatives. Similar to theN-substituted analogue, binding of the 2-derivative to tubulin was accompanied by a large blue shift, whereas a very small shift occurred when the 7- and 10-substituted derivatives bound. The 2- andN-substituted analogues bind to microtubules with an increase in fluorescence intensity over that which was observed with tubulin, whereas binding of the 7- and 10-substituted analogues was accompanied by a large quenching in fluorescence. This quenching may be due to the presence of charged residues in the protein near the 7- and 10-(dimethylamino)benzoyl groups or to π stacking of the groups with an aromatic side chain. The presence of paclitaxel with microtubules prevented the fluorescence increase of the 2- andN-derivatives and quenching of the 7- and 10-derivatives. The difference in behavior of the fluorescent analogues upon binding to polymerized tubulin, coupled with the solvent studies on the free drugs, suggests that the 2- andN-benzoyl groups of paclitaxel bind in a hydrophobic pocket of tubulin but could participate in hydrogen bonding, and the 7- and 10-positions are in a more hydrophilic environment.