Site-directed ligand discovery
Site-directed ligand discovery
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DOI：
10.1073/pnas.97.17.9367
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发表时间：
2000-08-15
影响因子：
11.1
通讯作者：
Wells, JA
中科院分区：
文献类型：
--
作者：
Erlanson, DA;Braisted, AC;Wells, JA
我们报告了一种策略（称为“栓系”），以发现低分子量配体（约50 Da），通过中间二硫系链弱结合到蛋白质上的目标位点。蛋白质中的天然半胱氨酸或工程半胱氨酸可与少量含二硫分子库（约1200种化合物）发生可逆反应，其浓度通常用于药物筛选（10至200 μ M）。半胱氨酸捕获的配体，很容易被质谱识别，是最稳定的配合物之一，即使在没有共价系链的情况下，配体的结合可能非常弱。这种方法被应用于产生胸苷酸合成酶的有效抑制剂，胸苷酸合成酶是嘧啶代谢的必需酶，在癌症和传染病的治疗中有应用。通过利用拴链配合物的晶体结构合成一组类似物，将非拴链配体的亲和力（K(i)接近1 mM）提高了3000倍。这种位点导向配体发现允许人们从空间靶向铅片段进行药物设计。
We report a strategy (catled "tethering") to discover low molecular weight ligands (approximate to 50 Da) that bind weakly to targeted sites on proteins through an intermediary disulfide tether. A native or engineered cysteine in a protein is allowed to react reversibly with a small library of disulfide-containing molecules (approximate to 1,200 compounds) at concentrations typically used in drug screening (10 to 200 mu M). The cysteine-captured ligands, which are readily identified by MS, are among the most stable complexes, even though in the absence of the covalent tether the ligands may bind very weakly. This method was applied to generate a potent inhibitor for thymidylate synthase, an essential enzyme in pyrimidine metabolism with therapeutic applications in cancer and infectious diseases. The affinity of the untethered ligand (K(i)approximate to 1 mM) was improved 3,000-fold by synthesis of a small set of analogs with the aid of crystallographic strucutres of the tethered complex. Such site-directed ligand discovery allows one to nucleate drug design from a spatially targeted lead fragment.