Roughing up Smoothened: chemical modulators of

Hedgehog signaling

Abstract
Thirty years before genetic experiments in Drosophila created the hedgehog mutant,
a naturally occurring 'chemical genetic' experiment had produced sheep with an
even more disturbing phenotype: cyclopia. It was discovered that mutation of the
Sonic hedgehog (Shh) gene in mice or humans could produce defects that
resembled those caused by administration of cyclopamine to animals.
Cyclopamine blocks the response of cells to Shh by activating a protein called
Smoothened. Smoothened is distantly related to G-protein coupled receptors, but no
G protein has yet been identified as essential for Hh-pathway signaling.
The Journal of Biology reports that Jeff Porter and colleagues identified a class of
synthetic small molecules that potently activate the Hh-signaling pathway by binding
to the Smo protein. These molecules are called leiosamines.
Cyclopamine and leiosamine were discovered by natural accidents, whereas
cyclopamine was discovered by systematic screening of chemical libraries using a
cell-based assay that monitored Hh-pathway activation. This approach is ambitious
and not without risk, but the recent success in identifying Smo as a target protein
suggests that the challenge can be overcome.
Cyclopamine blocks Hh signaling by binding to Smo, and this binding is specific to
the seven transmembrane-spanning domains of Smo. Cyclopamine also blocks
signaling by constitutively activated oncogenic mutant forms of Smo, although higher
concentrations were required.
The Curis group identified a family of compounds called leiosamines that can
activate the Hh pathway in the absence of the Shh ligand. These compounds can
also activate appropriate Hh-dependent biological responses, such as proliferation of
neonatal cerebellar granule neurons.
One leiosamine derivative was able to activate Ptc expression in mouse embryos
after oral administration to pregnant females, indicating that leiosamine can activate
the Hh pathway in the absence of Shh ligand.
Additional experiments in cell culture hinted that Smo might be a target of
leiosamine. When leiosamine was used to treat cells overexpressing Smo, it was
found to bind to the receptor with a dissociation constant of 0.37 nM, whereas the
binding of antagonists was substantially weakened.
The Beachy lab has recently characterized leiosamine and confirmed the Smo
protein as its target. Higher concentrations of leiosamine inhibit Hh signaling,
suggesting that it may also interact with a downstream effector protein.
The susceptibility of Smo to activation or inhibition by synthetic small molecules
suggests that endogenous small molecules may also regulate Smo activity. Ptc may
regulate Smo by transporting a small molecule that binds to Smo and regulates its
activity.