Abstract
The neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) and their class II G protein-coupled receptors VPAC1, VPAC2, and PAC1 play important roles in human physiology. No small molecule modulator has ever been reported for the VIP/PACAP receptors, and there is a lack of specific VPAC2 antagonists. Via high-throughput screening of 1.67 million compounds, we discovered a single small molecule antagonist of human VPAC2, compound 1. Compound 1 inhibits VPAC2-mediated cAMP accumulation with an IC50 of 3.8 μM and the ligand-activated β-arrestin2 binding with an IC50 of 2.3 μM. Compound 1 acts noncompetitively in Schild analysis. It is a specific VPAC2 antagonist with no detectable agonist or antagonist activities on VPAC1 or PAC1. Compound 2, a close structural analog of compound 1, was also found to be weakly active. To our surprise, compound 1 is completely inactive on the closely related mouse VPAC2. Chimera experiments indicate that compounds 1 and 2 bind to the seven transmembrane (7TM) region of the receptor as opposed to the N-terminal extracellular domain, where the natural ligand binds. Compound 1, being the first small molecular antagonist that is specific for VPAC2, and the only VPAC2 antagonist molecule known to date that allosterically interacts with the 7TM region, will be a valuable tool in further study of VPAC2 and related receptors. This study also highlights the opportunities and challenges facing small molecule drug discovery for class II peptide G protein-coupled receptors.
Footnotes
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This work was supported by the Novartis Research Foundation.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.109.060137.
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ABBREVIATIONS:
- VIP
- vasoactive intestinal peptide
- PACAP
- pituitary adenylate cyclase-activating peptide
- GPCR
- G protein-coupled receptor
- ECD
- extracellular domain
- PG-99-465; HTRF
- homogeneous time-resolved fluorescence
- HEK
- human embryonic kidney
- IBMX
- 3-isobutyl-1-methylxanthine
- GLP
- glucagon-like peptide
- RLU
- relative luminescence unit
- aa
- amino acid(s)
- h
- human
- m
- mouse.
- Received August 6, 2009.
- Accepted October 23, 2009.
- © 2010 The American Society for Pharmacology and Experimental Therapeutics
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