Lu Y F, Xu H, Liu-Chen L Y, Chen C, Partilla J S, Brine G A, Carroll F I, Rice K C, Lai J, Porreca F, Sadee W, Rothman R B
Division of Intramural Research, NIDA, NIH, Baltimore, Maryland 21224, USA.
Synapse. 1998 Feb;28(2):117-24. doi: 10.1002/(SICI)1098-2396(199802)28:2<117::AID-SYN2>3.0.CO;2-E.
Mutational analysis of opioid receptors supports the hypothesis that dissimilar receptor domains contribute to the binding affinity of different ligands. To determine whether enantiomeric ligands can serve to distinguish between different binding pockets (which focuses the analysis on asymmetric structural factors while avoiding confounding changes in physiochemical characteristics), we analyzed the binding of the 3-methylfentanyl congeners RTI-4614-4 [(+/-)-cis-N-[1-(2-hydroxy-2-phenylethyl)-3-methyl-4-piperidyl]-N- phenylpropanamide HCl)], its four stereoisomers [(2S,3R,4S)-1a, (2R,3R,4S)-1b, (2R,3S,4R)-1c, and (2S,3S,4R)-1d], and other mu agonists with cloned rat mu opioid receptors stably expressed in HEK-293 cells and mu/kappa receptor chimeras. Chimera III (kappa[aminoacids 1-141]/mu[aminoacids 151-398]), chimera IV (mu[aminoacids 1-150]/kappa[aminoacids 142-380]), and chimera XII (kappa[aminoacids 1-262]/mu[aminoacids 269-398]) bound [(125)I]IOXY (6beta-iodo-3,14-dihydoxy-17-cyclopropylmethyl-4,5alpha++ +-epoxymorphinan) with high affinities. The Ki values of 1a, 1b, 1c, and 1d at the wild-type mu receptor were 0.55 nM, 0.66 nM, 124 nM, and 59.2 nM, respectively. When the region from the N terminal to the start of the transmembrane helix 3 (TMH3) of the mu receptor was substituted by that of the kappa receptor (chimera III), the Ki value of 1b was increased (relative to the mu receptor) 590-fold compared to a 73-fold increase for 1a. When this portion of the kappa receptor was replaced by that of the mu receptor (chimera IV), the loss of affinity was not as great: 11.7-fold for 1a and 58.5-fold for 1b. Replacement of the middle of the third intracellular loop and third extracellular loop (e3) of the kappa receptor with that of the mu receptor (chimera XII) lowered (relative to their Ki values at the kappa receptor) the Ki values of [D-Ala2,D-Leu5]enkephalin and [D-Ala2-MePhe4,Gly-ol5]enkephalin to a much greater extent than the Ki values of the isomers. The kappa/chimera XII shift was greater for isomers 1c and 1d than for 1b and 1a. Viewed collectively, these data suggest that the region from the N terminal to the start of the TMH3 of the mu opioid receptor determines the binding affinity of RTI-4614-4 and its isomers and that the e3 loop also plays a major role in determining the binding affinity of mu agonist peptides. These data also show that the stereoisomers of RTI-4614-4 probably bind to different domains of the mu receptor and suggest that manipulation of stereochemistry may be a useful tool for designing domain-specific ligands.
阿片受体的突变分析支持这样一种假说，即不同的受体结构域对不同配体的结合亲和力有贡献。为了确定对映体配体是否可用于区分不同的结合口袋（这将分析聚焦于不对称结构因素，同时避免物理化学特性的混淆变化），我们分析了3 - 甲基芬太尼同系物RTI - 4614 - 4 [（±）-顺式 - N - [1 - （2 - 羟基 - 2 - 苯乙基）- 3 - 甲基 - 4 - 哌啶基] - N - 苯基丙酰胺盐酸盐]、其四种立体异构体[（2S，3R，4S）- 1a，（2R，3R，4S）- 1b，（2R，3S，4R）- 1c和（2S，3S，4R）- 1d]以及其他μ激动剂与稳定表达于HEK - 293细胞中的克隆大鼠μ阿片受体和μ/κ受体嵌合体的结合情况。嵌合体III（κ[氨基酸1 - 141]/μ[氨基酸151 - 398]）、嵌合体IV（μ[氨基酸1 - 150]/κ[氨基酸142 - 380]）和嵌合体XII（κ[氨基酸1 - 262]/μ[氨基酸269 - 398]）对[125I]IOXY（6β - 碘 - 3,14 - 二羟基 - 17 - 环丙基甲基 - 4,5α - 环氧吗啡喃）具有高亲和力。1a、1b、1c和1d在野生型μ受体处的Ki值分别为0.55 nM、0.66 nM、124 nM和59.2 nM。当μ受体从N末端到跨膜螺旋3（TMH3）起始处的区域被κ受体的相应区域取代时（嵌合体III），1b的Ki值（相对于μ受体）增加了590倍，而1a增加了73倍。当κ受体的这部分被μ受体的相应部分取代时（嵌合体IV），亲和力的丧失没那么大：1a为11.7倍，1b为58.5倍。用μ受体的相应部分替换κ受体的第三个细胞内环和第三个细胞外环（e3）的中间部分（嵌合体XII），相对于它们在κ受体处的Ki值，[D - Ala2，D - Leu5]脑啡肽和[D - Ala2 - MePhe4，Gly - ol5]脑啡肽的Ki值降低的程度比异构体的Ki值大得多。异构体1c和1d的κ/嵌合体XII的变化比1b和1a更大。总体来看，这些数据表明μ阿片受体从N末端到TMH3起始处的区域决定了RTI - 4614 - 4及其异构体的结合亲和力，并且e3环在决定μ激动剂肽的结合亲和力方面也起主要作用。这些数据还表明RTI - 4614 - 4的立体异构体可能与μ受体的不同结构域结合，并表明立体化学的操控可能是设计结构域特异性配体的有用工具。
