Identification of a lacosamide binding protein using an affinity bait and chemical reporter strategy: 14-3-3 ζ.
Identification of a lacosamide binding protein using an affinity bait and chemical reporter strategy: 14-3-3 ζ.
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DOI：
10.1021/ja2034156
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发表时间：
2011-07-27
影响因子：
15
通讯作者：
Kohn, Harold
中科院分区：
文献类型：
--
作者：
Park, Ki Duk;Kim, Dongwook;Reamtong, Onrapak;Eyers, Claire;Gaskell, Simon J.;Liu, Rihe;Kohn, Harold
我们提出了一种有用的策略来阐明具有适度结合亲和力的配体（药物）的结合伙伴。该策略的关键是将亲和诱饵（AB）和化学报告基团（CR）连接到配体上，其中AB在结合时将配体不可逆地附着在受体上，CR组用于受体检测和分离。我们使用低分子量抗癫痫药物拉科沙胺（(R)-1）测试了这种AB&CR策略。我们证明，使用(R)-lacosamide AB&CR试剂（(R)-2） 14-3-3 ζ在啮齿类动物脑可溶性裂解物中优先内合，内合对AB&CR试剂具有立体特异性，内合取决于内源性小分子代谢产物黄嘌呤水平的存在。以(R)- 5取代(R)-2，质谱法确定14-3-3 ζ内合位点（K120）。在(R)-2存在的情况下增加(R)-1的竞争实验表明(R)-1结合在14-3-3 ζ上的(R)-2修饰位点或附近。黄嘌呤衍生物的结构-活性研究提供了有关该代谢物与重组14-3-3 ζ之间可能结合相互作用的信息。用黄嘌呤和黄嘌呤类似物1,7-二甲基黄嘌呤等温量热法获得了14-3-3 - ζ-黄嘌呤相互作用的记录。
We have advanced a useful strategy to elucidate binding partners of ligands (drugs) with modest binding affinity. Key to this strategy is attaching to the ligand an affinity bait (AB) and a chemical reporter (CR) group, where the AB irreversibly attaches the ligand to the receptor upon binding and the CR group is employed for receptor detection and isolation. We have tested this AB&CR strategy using lacosamide ((R)-1), a low-molecular-weight antiepileptic drug. We demonstrate that using a (R)-lacosamide AB&CR agent ((R)-2) 14-3-3 ζ in rodent brain soluble lysates is preferentially adducted, adduction is stereospecific with respect to the AB&CR agent, and adduction depends upon the presence of endogenous levels of the small molecule metabolite xanthine. Substitution of lacosamide AB agent ((R)- 5) for (R)-2 led to the identification of the 14-3-3 ζ adduction site (K120) by mass spectrometry. Competition experiments using increasing amounts of (R)-1 in the presence of (R)-2 demonstrated that (R)-1 binds at or near the (R)-2 modification site on 14-3-3 ζ. Structure-activity studies of xanthine derivatives provided information concerning the likely binding interaction between this metabolite and recombinant 14-3-3 ζ. Documentation of the 14-3-3 ζ-xanthine interaction was obtained with isothermal calorimetry using xanthine and the xanthine analogue 1,7-dimethylxanthine.
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DOI：
10.1126/stke.2962005re10
发表时间：
2005-08-09
期刊：
Science's STKE : signal transduction knowledge environment
影响因子：
--
作者：
Bridges, Dave;Moorhead, Greg B G
通讯作者：
Moorhead, Greg B G
影响因子：
2.9
作者：
de Felipe, KS;Carter, BT;Cornish, VW
通讯作者：
Cornish, VW
影响因子：
3.2
作者：
Ballell, Lluis;van Scherpenzeel, Monique;Pieters, Roland J.
通讯作者：
Pieters, Roland J.
影响因子：
4
作者：
Kekesi, Katalin A.;Kovacs, Zsolt;Palkovits, Miklos
通讯作者：
Palkovits, Miklos
影响因子：
20.1
作者：
Allouis, Marie;Le Bouffant, Francoise;Baro, Isabelle
通讯作者：
Baro, Isabelle