Article Figures & Data
Figures
- Fig. 1.
Multifilter virtual screening.
- Fig. 2.
(A) NMR structure of the N-terminal extracellular (EC) domain of PAC1 receptor in complex with PACAP 6–38 (green). (B) Interaction of Y22, V26, and R30 of PACAP 6–38 with PAC1 receptor. Hydrogen-bonding interactions are indicated by dashed yellow lines.
- Fig. 3.
Effects of 10 candidate compounds on the PACAP-induced phosphorylation of CREB in the CHO cells expressing PAC1 receptors. The cells were incubated with PACAP 6–38 (1 nM) or each compound (1 nM) for 30 minutes, then the cells were stimulated with PACAP (1 nM) for 30 minutes and lysed. The lysates containing equivalent amounts of protein (20 µg) were subjected to SDS-PAGE. Immunoblots were probed with specific antibodies that recognize phosphorylated form of CREB (pCREB) and total CREB. The data represent the mean ± S.E.M. (n = 4 to 5). *P < 0.05 vs. dimethylsulfoxide (DMSO) control.
- Fig. 4.
Effects of PA-8 or PA-9 on the PACAP-induced phosphorylation of CREB in the CHO cells expressing PAC1, VPAC1, or VPAC2 receptors. The cells were incubated with PA-8 or PA-9 compounds (10 pM to 10 nM) for 30 minutes, then the cells were stimulated with PACAP (1 nM). The data represent the mean ± S.E.M. (n = 3–5).
- Fig. 5.
Dose-dependent inhibitory effects of PA-8, PA-9, or PA-10 on the PACAP-induced cAMP elevation in the CHO cells expressing PAC1 receptors. The cells were incubated with PA-8, PA-9, or PA-10 (1 pM to 100 µM) for 30 minutes, then the cells were stimulated with PACAP (1 nM) for 1 hour and assessed the level of cAMP using cAMP-Glo Assay kit. The data represent the mean ± S.E.M. (n = 3–5).
- Fig. 6.
Blockade of PACAP-induced aversive responses and mechanical allodynia by the identified PA compounds. (A) A single intrathecal injection of PACAP (100 pmol) induced transient aversive responses, and the PACAP-induced behaviors were ameliorated by co-injection of PA-8, PA-9, or PA-10 (100 pmol each). (B) PA-8, PA-9, or PA-10 (100 pmol each intrathecally) in itself did not induce aversive responses. (C) A single intrathecal injection of PACAP (100 pmol) induced long-lasting mechanical allodynia, and the induction of allodynia was prevented by co-injection of PA-8, PA-9, or PA-10 (100 pmol each). (D) PA-8, PA-9, or PA-10 (100 pmol each intrathecally) per se did not induce mechanical allodynia. *P < 0.05 compared with vehicle. #P < 0.05 compared with PACAP.
- Fig. 7.
Docking models of PA-8 (A) or PA-9 (B) with PAC1 receptor. Hydrogen-bonding interactions are indicated by yellow dashed lines.
Additional Files
Data Supplement
- Supplemental Figure 1 -
3D pharmacophore models 1(A) and 2 (B) for first-stage screening.
- Supplemental Figure 2 -
Chemical structure of 10 compounds identified by in silico screening.
- Supplemental Figure 1 -
In this issue
Identification of Small-molecule Antagonist of PAC1
