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 appears to be non-competitive in Schild analysis. It is a specific VPAC₂ antagonist with no detectable agonist or antagonist activities on VPAC₁ or PAC₁. A close structural analogue of Compound 1, Compound 2, 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 Compound 1 and Compound 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 GPCRs.