Target identification for a Hedgehog pathway inhibitor reveals the receptor GPR39

Abstract

Hedgehog (Hh) signaling determines cell fate during development and can drive tumorigenesis. We performed a screen for new compounds that can impinge on Hh signaling downstream of Smoothened (Smo). A series of cyclohexyl-methyl aminopyrimidine chemotype compounds ('CMAPs') were identified that could block pathway signaling in a Smo-independent manner. In addition to inhibiting Hh signaling, the compounds generated inositol phosphates through an unknown GPCR. Correlation of GPCR mRNA expression levels with compound activity across cell lines suggested the target to be the orphan receptor GPR39. RNA interference or cDNA overexpression of GPR39 demonstrated that the receptor is necessary for compound activity. We propose a model in which CMAPs activate GPR39, which signals to the Gli transcription factors and blocks signaling. In addition to the discovery of GPR39 as a new target that impinges on Hh signaling, we report on small-molecule modulators of the receptor that will enable in vitro interrogation of GPR39 signaling in different cellular contexts.

Targeting GLI factors to inhibit the Hedgehog pathway

“…Bassilana and coworkers have discovered a CMAP chemotype able to block GLI functions and, through a chemoproteomics strategy, they also have identified the orphan GPCR GPR39 as the specific target of this molecule [63]. Although the authors failed to demonstrate the direct binding of CMAP to GPR39, data document that CMAP is a specific agonist of this receptor.…”

Discovery of new GPCR ligands to illuminate new biology

“…Gene expression analysis suggested that GPR39 might be the target, consistent with specific gene knockdown and ultimately on-target dose–response studies with optimized compounds. A role for this oGPCR in modulation of the hedgehog pathway protein Gli was suggested 66 .…”

Identifying compound efficacy targets in phenotypic drug discovery

“…Noncovalent, lysate-based approaches have been successfully applied to the identification of a wide range of targets, including soluble enzymes such as protein kinases [5], the lipid kinase PIKFyve [11] and many others. However, a number of therapeutically relevant target families, in particular integral membrane proteins such as ion channels and G-protein-coupled receptors, are notoriously incompatible owing to loss of their binding-competent conformation during the experimental workflow [12,13].…”