An In Vivo Chemical Genetic Screen Identifies Phosphodiesterase 4 as a Pharmacological Target for Hedgehog Signaling Inhibition

Abstract

SUMMARY Hedgehog (Hh) signaling plays an integral role in vertebrate development, and its dysregulation has been widely accepted as a driver of numerous malignancies. While a variety of small molecules target Smoothened (Smo) as a strategy for Hh inhibition, Smo gain of function mutations have limited their clinical implementation. Modulation of targets downstream of Smo could define a paradigm for treatment of Hh-dependent cancers. Here, we describe eggmanone, a small molecule identified from a chemical genetic zebrafish screen which induced a Hh-null phenotype. Eggmanone exerts its Hh-inhibitory effects through selective antagonism of phosphodiesterase (PDE) 4, leading to protein kinase A activation and subsequent Hh blockade. Our study implicates PDE4 as a target for Hh inhibition, suggests an improved strategy for Hh-dependent cancer therapy and identifies a unique probe of downstream-of-Smo Hh modulation.

Targeting GLI factors to inhibit the Hedgehog pathway

“…Eggmanone ( Figure 1) is a small molecule identified by an in vivo chemical genetic screening of 30 000 others compounds for their ability to reproduce the Hh-null phenotype in zebrafish embryo. This molecule affects some typical structural features such as ventral tail curvature, small eyes, loss of pectoral fins, enlarged rounded somites, loss of neurocranial chondrogenesis and impaired slow muscle formation [78]. Specifically, in vitro studies show that eggmanone antagonizes Hh signaling by inhibition of phosphodiesterase (PDE)4D3, thus determining an increase of cAMP levels ( Figure 2).…”

Synthetic Small Molecule Inhibitors of Hh Signaling As Anti-Cancer Chemotherapeutics

“…A screen of ~30,000 small molecules designed to identify compounds with the ability to alter embryonic zebrafish patterning was performed at Vanderbilt University [106]. This screen identified a scaffold termed eggmanone ( 68 ) that selectively reproduced the Hh-null phenotype [106].…”

“…This screen identified a scaffold termed eggmanone ( 68 ) that selectively reproduced the Hh-null phenotype [106]. Eggmanone inhibited Gli-luciferase activity in a dose-dependent manner when stimulated with recombinant SHh ligand (IC 50 ~ 1 μM); however, when Gli2 was transiently overexpressed, eggmanone treatment had no effect on Hh signaling, suggesting it targets a pathway component upstream of Gli.…”

“…PKA function in the basal body is activated in response to cyclic adenosine monophosphate (cAMP) and cAMP levels are tightly regulated through degradation by phosphodiesterase (PDEs) enzymes. Based on these control systems, eggmanone was evaluated for its ability to inhibit several PDEs and was identified as a potent, selective inhibitor of PDE4D3 (IC 50 = 72 nM) [106]. Additional studies in Hh-dependent cell culture following exogenous addition of PDE4D3 or the pan-PDE inhibitor rolipram demonstrated that inhibition of Hh signaling by eggmanone is through direct antagonism of PDE4, which ultimately results in PKA-mediated degradation of Gli [106].…”

Development of thieno- and benzopyrimidinone inhibitors of the Hedgehog signaling pathway reveals PDE4-dependent and PDE4-independent mechanisms of action

“…We previously reported the discovery of eggmanone (EGM1, 3 ) 25 from a high content screen for small molecule modulators of developmental patterning in embryonic zebrafish. Based on its recapitulation of the Hh-null phenotype, EGM1 was confirmed to inhibit Hh signaling in cell-based assays, functioning downstream of Smo and the negative regulator Sufu but upstream of Gli TFs.…”