N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A) interaction with LYS 3.28(192) is crucial for its inverse agonism at the cannabinoid CB1 receptor.
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.
Hurst
Dow P
DP
Department of Chemistry, Kennesaw State University, Kennesaw, Georgia 30144, USA.
Lynch
Diane L
DL
Barnett-Norris
Judy
J
Hyatt
Stephen M
SM
Seltzman
Herbert H
HH
Zhong
Miao
M
Song
Zhao-Hui
ZH
Nie
Jingjiang
J
Lewis
Deborah
D
Reggio
Patricia H
PH
D001665:Binding Sites; D002118:Calcium / Q000502:physiology; D002478:Cells, Cultured; D004594:Electrophysiology; D006801:Humans; D008024:Ligands; D008239:Lysine / Q000378:metabolism*; D008958:Models, Molecular; D009474:Neurons / Q000187:drug effects / Q000502:physiology; D010880:Piperidines / Q000494:pharmacology*; D011487:Protein Conformation; D011720:Pyrazoles / Q000494:pharmacology*; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug / Q000819:agonists* / Q000737:chemistry / Q000378:metabolism; D000077285:Rimonabant; D014162:Transfection
D008024:Ligands; D010880:Piperidines; D011720:Pyrazoles; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug; D008239:Lysine; D000077285:Rimonabant; D002118:Calcium