Hurst Dow P, Lynch Diane L, Barnett-Norris Judy, Hyatt Stephen M, Seltzman Herbert H, Zhong Miao, Song Zhao-Hui, Nie Jingjiang, Lewis Deborah, Reggio Patricia H
Department of Chemistry, Kennesaw State University, Kennesaw, Georgia 30144, USA.
Mol Pharmacol. 2002 Dec;62(6):1274-87. doi: 10.1124/mol.62.6.1274.
In superior cervical ganglion neurons, N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A) competitively antagonizes the Ca(2+) current effect of the cannabinoid (CB) agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55212-2), and behaves as an inverse agonist by producing opposite current effects when applied alone. In contrast, in neurons expressing CB1 with a K-->A mutation at residue 3.28(192) (i.e., K3.28A), SR141716A competitively antagonizes the effects of WIN55212-2, but behaves as a neutral antagonist by producing no current effects itself. Receptor modeling studies suggested that in the CB1 inactive (R) state, SR1417A16A stabilizes transmembrane helix 6 in its inactive conformation via aromatic stacking with F3.36/W6.48. In this binding site, SR141716A would exhibit higher affinity for CB1 R due to a hydrogen bond between the SR141716A C3 substituent and K3.28(192), a residue available to SR141716A only in R. To test this hypothesis, a "mutant thermodynamic cycle" was constructed that combined the evaluation of SR141716A affinity at WT CB1 and K3.28A with an evaluation of the wild-type CB1 and K3.28A affinities of an SR141716A analog, 5-(4-chlorophenyl)-3-[(E)-2-cyclohexylethenyl]-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole (VCHSR), that lacks hydrogen bonding potential at C3. Binding affinities suggested that K3.28 is involved in a strong interaction with SR141716A in WT CB1, but does not interact with VCHSR. Thermodynamic cycle calculations indicated that a direct interaction occurs between the C3 substituent of SR141716A and K3.28 in WT CB1. Consistent with these results, VCHSR acted as a neutral antagonist at WT CB1. These results support the hypothesis that hydrogen bonding of the SR141716A C3 substituent with K3.28 is responsible for its higher affinity for the inactive R state, leading to its inverse agonism.
在颈上神经节神经元中，N-(哌啶-1-基)-5-(4-氯苯基)-1-(2,4-二氯苯基)-4-甲基-1H-吡唑-3-甲酰胺（SR141716A）竞争性拮抗大麻素（CB）激动剂(R)-(+)-[2,3-二氢-5-甲基-3-(4-吗啉基甲基)吡咯并[1,2,3-de]-1,4-苯并恶嗪-6-基]-1-萘基甲酮（WIN55212-2）的钙电流效应，并且单独应用时通过产生相反的电流效应而表现为反向激动剂。相比之下，在3.28(192)位残基发生K→A突变（即K3.28A）的表达CB1的神经元中，SR141716A竞争性拮抗WIN55212-2的效应，但自身不产生电流效应，表现为中性拮抗剂。受体模型研究表明，在CB1的无活性（R）状态下，SR1417A16A通过与F3.36/W6.48的芳香堆积作用，使跨膜螺旋6稳定在其无活性构象。在这个结合位点，由于SR141716A的C3取代基与K3.28(192)之间存在氢键，SR141716A对CB1 R的亲和力会更高，而K3.28(192)是SR141716A仅在R状态下可作用的一个残基。为了验证这一假设，构建了一个“突变体热力学循环”，该循环结合了对SR141716A在野生型CB1和K3.28A上的亲和力评估，以及对SR141716A类似物5-(4-氯苯基)-3-[(E)-2-环己烯基乙烯基]-1-(2,4-二氯苯基)-4-甲基-1H-吡唑（VCHSR）在野生型CB1和K3.28A上的亲和力评估，VCHSR在C3处缺乏氢键结合潜力。结合亲和力表明，K3.28在野生型CB1中与SR141716A存在强相互作用，但不与VCHSR相互作用。热力学循环计算表明，在野生型CB1中，SR141716A的C3取代基与K3.28之间存在直接相互作用。与这些结果一致，VCHSR在野生型CB1上表现为中性拮抗剂。这些结果支持了以下假设：SR141716A的C3取代基与K3.
