Knaus H G, Striessnig J, Hering S, Marrer S, Schwenner E, Höltje H D, Glossmann H
Institute for Biochemical Pharmacology, Innsbruck, Austria.
Mol Pharmacol. 1992 Feb;41(2):298-307.
A novel 35S-labeled dihydropyridine (DHP), 1,4-dihydro-2,6-dimethyl-4-(2-trifluoromethylphenyl)-pyridine-3,5-dic arboxyl-3- [2-(N-tert-butoxycarbonyl-L-[35S]methionyl)-aminoethyl]-ester-5-ethyl ester, ([35S]sadopine) (800-1400 Ci/mmol), the respective (+)- and (-)-diastereomers, and unlabeled (+/- )-, (-)-, and (+)-sadopine were synthesized. [35S]Sadopine is an excellent high affinity, high specific activity radioligand to label selectively the DHP receptor of L-type Ca2+ channels in tissue sections as well as in membrane fragments. Both diastereomers bind to the DHP receptors in a saturable and reversible manner, with equal, subnanomolar, dissociation constants. Despite their similar affinities, (+)- and (-)-sadopine differ with respect to their kinetic properties [the association and dissociation rate constants are 10-fold higher for (+)-[35S]sadopine at 22 degrees] and their allosteric modulation of the phenylaklylamine or benzothiazepine binding domain. (+)-Sadopine is a negative but (-)-sadopine a positive allosteric modulator of (-)-[N-methyl-3H]LU49888 or (+)-cis-[3H] diltiazem binding at 30 degrees. Both diastereomers act as L-type Ca2+ channel blockers in cardiac and smooth muscle cells. Computer-based analysis of the electrostatic potentials of the two diastereomers and calculation of the interaction energies with a hypothetical DHP receptor model predicted not only the similar affinities of (+)- and (-)-sadopine but also their Ca2+ channel-blocking effects. The temperature-dependent allosteric differences between the diastereomers suggest that two distinct conformational states of the DHP receptor are stabilized in vitro, both corresponding to a nonconducting state of the channel. Our data indicate that access to the DHP receptor site, but not binding affinity, is a function of the opposite stereochemistry of the sadopine diastereomers. Therefore, labeled and unlabeled (+)- and (-)-sadopine will be useful probes to further characterize the molecular basis of DHP-Ca2+ channel interaction and the pharmacological and physiological significance of the different allosteric conformations of the channel induced by Ca2+ channel-active drugs.
合成了一种新型的35S标记的二氢吡啶（DHP），即1,4-二氢-2,6-二甲基-4-(2-三氟甲基苯基)-吡啶-3,5-二羧酸-3-[2-(N-叔丁氧羰基-L-[35S]甲硫氨酰)-氨基乙基]-酯-5-乙酯，（[35S]萨多平）（800 - 1400 Ci/mmol），以及各自的（+）-和（-）-非对映异构体，还有未标记的（+/-）-、（-）-和（+）-萨多平。[35S]萨多平是一种出色的高亲和力、高比活性放射性配体，可选择性标记组织切片以及膜碎片中L型Ca2+通道的DHP受体。两种非对映异构体均以可饱和且可逆的方式与DHP受体结合，解离常数相等，均为亚纳摩尔级。尽管它们的亲和力相似，但（+）-和（-）-萨多平在动力学性质方面存在差异（在22℃时，（+）-[35S]萨多平的结合和解离速率常数高10倍），并且在苯烷基胺或苯并硫氮杂䓬结合域的变构调节方面也有所不同。在30℃时，（+）-萨多平是（-）-[N-甲基-3H]LU49888或（+）-顺式-[3H]地尔硫䓬结合的负性变构调节剂，而（-）-萨多平是正性变构调节剂。两种非对映异构体在心肌和平滑肌细胞中均作为L型Ca2+通道阻滞剂起作用。对两种非对映异构体的静电势进行基于计算机的分析，并与假设的DHP受体模型计算相互作用能，不仅预测了（+）-和（-）-萨多平的相似亲和力，还预测了它们的Ca2+通道阻断作用。非对映异构体之间温度依赖性的变构差异表明，在体外DHP受体的两种不同构象状态得以稳定，且均对应于通道的非导通状态。我们的数据表明，接近DHP受体位点，而非结合亲和力，是萨多平非对映异构体相反立体化学的函数。因此，标记和未标记的（+）-和（-）-萨多平将是有用的探针，可进一步表征DHP-Ca2+通道相互作用的分子基础以及Ca2+通道活性药物诱导的通道不同变构构象的药理和生理意义。
