[3H]Azidodantrolene:: Synthesis and use in identification of a putative skeletal muscle dantrolene binding site in sarcoplasmic reticulum
[3H]Azidodantrolene:: Synthesis and use in identification of a putative skeletal muscle dantrolene binding site in sarcoplasmic reticulum
复制标题
DOI：
10.1021/jm9805079
复制
发表时间：
1999-06-03
影响因子：
7.3
通讯作者：
Parness, J
中科院分区：
文献类型：
--
作者：
Palnitkar, SS;Bin, B;Parness, J
丹曲林钠是一种医学上重要的乙内酰脲衍生物，通过未知机制干扰骨骼肌细胞内储存的Ca 2+释放。丹曲林的分子靶点的鉴定将极大地有助于理解药物的作用机制和肌肉中细胞内Ca 2+释放的动力学。设计并合成了[H-3]叠氮丹曲林作为光亲和类似物，以鉴定骨骼肌中假定的丹曲林受体。在放射性配体合成过程中，需要将1摩尔原子的氚引入醛5 b中，以确保通过荧光方法检测光交联蛋白质的足够高的比活性。这是通过用定制合成的100%氚标记的三乙基硼三氢化锂还原酯3，然后用氧化锰（IV）氧化成5 b来完成的。通过NMR光谱法证明化合物6 b在亚胺位置处大于或等于95%的氚标记，并且通过HPLC和液体闪烁计数凭经验确定[H-3]叠氮丹曲林钠的比放射性为24.4Ci/mmol，类似于理论最大值的85%。[H-3]Azidodantrolene在与骨骼肌肌浆网膜的配体-受体结合研究中被发现具有抑制活性。通过SDS-PAGE和氚荧光分析的光交联实验已经鉴定出类似于160-kDa特异性标记的蛋白质作为推定的细胞内骨骼肌丹曲林受体。这种光标记的蛋白质comigrates与蛋白质在Western印迹免疫交叉反应的多克隆抗兔骨骼肌ryanodine受体抗体。因此，推定的丹曲林受体可能与骨骼肌兰尼碱受体有关。
Dantrolene sodium is a medically important hydantoin derivative that interferes with release of Ca2+ from intracellular stores of skeletal muscle by an unknown mechanism. Identification of the molecular target of dantrolene would greatly aid in understanding both the mechanism of action of the drug and the dynamics of intracellular Ca2+ release in muscle. [H-3]Azidodantrolene was designed and synthesized as a photoaffinity analogue in order to identify a putative dantrolene receptor in skeletal muscle. Introduction of 1 mole-atom of tritium into aldehyde 5b was required during radioligand synthesis in order to ensure high enough specific activity for detection of photo-cross-linked proteins by fluorographic methods. This was accomplished by reduction of ester 3 with custom synthesized, 100% tritium-labeled lithium triethylborotritide, followed by oxidation to 5b by manganese(IV) oxide. Compound 6b was demonstrated to be greater than or equal to 95% tritium-labeled at the imine position by NMR spectroscopy, and the specific radioactivity of [H-3]azidodantrolene sodium was empirically determined by HPLC and liquid scintillation counting to be 24.4 Ci/mmol, similar to 85% of theoretical maximum. [H-3]Azidodantrolene was found to be pharmacologically active in ligand-receptor binding studies with skeletal muscle sarcoplasmic reticulum membranes. Photo-cross-linking experiments analyzed by SDS-PAGE and tritium fluorography have identified a similar to 160-kDa specifically labeled protein as the putative, intracellular, skeletal muscle dantrolene receptor. This photolabeled protein comigrates with a protein in Western blots immunologically cross-reactive to a polyclonal anti-rabbit skeletal muscle ryanodine receptor antibody. Thus, the putative dantrolene receptor may be related to the skeletal muscle ryanodine receptor.