Small-molecule modulators of Hedgehog signaling: identification and characterization of Smoothened agonists and antagonists

Abstract

BackgroundThe Hedgehog (Hh) signaling pathway is vital to animal development as it mediates the differentiation of multiple cell types during embryogenesis. In adults, Hh signaling can be activated to facilitate tissue maintenance and repair. Moreover, stimulation of the Hh pathway has shown therapeutic efficacy in models of neuropathy. The underlying mechanisms of Hh signal transduction remain obscure, however: little is known about the communication between the pathway suppressor Patched (Ptc), a multipass transmembrane protein that directly binds Hh, and the pathway activator Smoothened (Smo), a protein that is related to G-protein-coupled receptors and is capable of constitutive activation in the absence of Ptc.ResultsWe have identified and characterized a synthetic non-peptidyl small molecule, Hh-Ag, that acts as an agonist of the Hh pathway. This Hh agonist promotes cell-type-specific proliferation and concentration-dependent differentiation in vitro, while in utero it rescues aspects of the Hh-signaling defect in Sonic hedgehog-null, but not Smo-null, mouse embryos. Biochemical studies with Hh-Ag, the Hh-signaling antagonist cyclopamine, and a novel Hh-signaling inhibitor Cur61414, reveal that the action of all these compounds is independent of Hh-protein ligand and of the Hh receptor Ptc, as each binds directly to Smo.ConclusionsSmo can have its activity modulated directly by synthetic small molecules. These studies raise the possibility that Hh signaling may be regulated by endogenous small molecules in vivo and provide potent compounds with which to test the therapeutic value of activating the Hh-signaling pathway in the treatment of traumatic and chronic degenerative conditions.

Defective cerebellar response to mitogenic Hedgehog signaling in Down's syndrome mice

“…A series of related small molecule agonists has been described that trigger Hh pathway activity by binding to and activating the Smoothened component of the Hh response pathway (28,29). These molecules cross the bloodbrain barrier and produce, either alone or in synergy with Hh ligands, the effects of normal pathway stimulation in vitro and in vivo, including activation of reporter constructs (28,29), induction of motor neuron differentiation (30), mitogenic stimulation of neural precursors in vivo (31), and even the substantial rescue of a deficit of Shh signal in utero (28).…”

Identification of a small molecule inhibitor of the hedgehog signaling pathway: Effects on basal cell carcinoma-like lesions

“…We identified several different chemical classes of inhibitors, each of which strongly inhibits Hh signaling in all test systems to which they have been applied to date. CUR61414 is a synthetic inhibitor that specifically binds to and antagonizes the activity of Smo in the Hh signaling pathway (24). Similarly, in a recent study by Chen et al (28) several other classes of small synthetic modulators of Hh signaling were presented that bind to Smo.…”

“…Intermediate (''naïve'') regions of embryonic chick neural tube (stage 10-11) were dissected, embedded in collagen, and cultured as described before (22)(23)(24). These explants (n ϭ 6) were then treated with vehicle alone, Shh OCT , or Shh OCT ϩ test compound.…”

Genomic characterization of Gli-activator targets in sonic hedgehog-mediated neural patterning

“…After 2 days, the media was changed to DFNB containing 500 nM retinoic acid and, when applicable, 1 M Hh agonist (HH-Ag) (Frank-Kamenetsky et al, 2002). EBs were grown for an additional 3 days to induce neural progenitor stages, at which point cultures were harvested for ChIP.…”

“…and W.H.W., unpublished) to identify and rank putative Hh targets. The EB transcriptional profiling screen was conducted by screening quadruplicate samples of neuralized EBs treated with Hh agonist (Frank-Kamenetsky et al, 2002) and 500 nM retinoic acid (under identical conditions to the ChIPs) versus EBs treated with 500 nM retinoic acid alone. Samples were profiled using the Affymetrix MOE4302.0 arrays and results were analyzed using dChip (GEO #GSE4936).…”