Affinity-based chemical proteomic probe for dehydrogenases: fluorescence and visible binding assays in gels.
Affinity-based chemical proteomic probe for dehydrogenases: fluorescence and visible binding assays in gels.
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基于亲和力的脱氢酶化学蛋白质组探针：凝胶中的荧光和可见结合测定。
DOI：
10.1016/j.ab.2007.08.010
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发表时间：
2007
影响因子：
2.9
通讯作者：
Sem,DanielS
中科院分区：
文献类型：
--
作者：
Ge,Xia;Sem,DanielS
最近报道了一种针对脱氢酶的基于儿茶酚绕丹宁（CR）的特殊支架。该支架用作重点组合文库的模板，使用 NMR SOLVE 方法设计，为多种脱氢酶制备有效的 (50-200nM) 双配体抑制剂。据报道，这种 CR 支架也是一种用于溶液和凝胶内（天然）结合测定的荧光可见探针，使其成为脱氢酶的有用筛选试剂，并可用作基于亲和力的化学蛋白质组探针。这种荧光 CR 探针的最初应用是二氢吡啶二羧酸还原酶，一种抗感染药物靶标。该探针还显示出与两种乳酸脱氢酶同工酶和 1-脱氧-d-木酮糖-5-磷酸还原异构酶的凝胶内结合亲和力，使其成为多种脱氢酶普遍有用的凝胶内染色试剂。由于结合是非共价的，因此此类试剂可用于在天然凝胶中进行的置换测定，监测荧光带强度的降低。但是，由于该探针具有作为脱氢酶靶向组合文库的特殊支架的附加功能，因此它也可以用于直接结合测定，以筛选含有荧光 CR 的最高亲和力双配体抑制剂。最后，CR 探针可用作蛋白质组学研究中的染色剂，以分析在天然凝胶上运行的蛋白质混合物，以鉴定可能是脱氢酶的蛋白质。
A catechol rhodanine (CR)-based privileged scaffold, tailored to dehydrogenase enzymes, has recently been reported. This scaffold was used as a template in a focused combinatorial library, designed using the NMR SOLVE methodology, to prepare potent (50–200nM) biligand inhibitors for multiple dehydrogenases. It is reported here that this CR scaffold is also a fluorescent and visible probe for solution and in-gel (native) binding assays, making it a useful screening reagent for dehydrogenases, with applications as an affinity-based chemical proteomic probe. Initial application of this fluorescent CR probe was to dihydrodipicolinate reductase, an anti-infective drug target. The probe also shows in-gel binding affinity to two lactate dehydrogenase isozymes and to 1-deoxy-d-xylulose-5-phosphate reductoisomerase, making it a generally useful in-gel staining reagent for multiple dehydrogenases. Because binding is noncovalent, such a reagent could be used in a displacement assay performed in a native gel, monitoring decrease in fluorescent band intensity. But, because the probe has the added function of serving as a privileged scaffold for dehydrogenase-targeted combinatorial libraries, it could also be used in a direct binding assay to screen for the highest-affinity biligand inhibitors that contain the fluorescent CR. Finally, the CR probe could be used as a stain in proteomic studies, to profile mixtures of proteins run on a native gel, to identify proteins that are likely to be dehydrogenases.