Small molecule modulation of Smoothened activity

Abstract

Smoothened (Smo), a distant relative of G protein-coupled receptors, mediates Hedgehog (Hh) signaling during embryonic development and can initiate or transmit ligand-independent pathway activation in tumorigenesis. Although the cellular mechanisms that regulate Smo function remain unclear, the direct inhibition of Smo by cyclopamine, a plant-derived steroidal alkaloid, suggests that endogenous small molecules may be involved. Here we demonstrate that SAG, a chlorobenzothiophene-containing Hh pathway agonist, binds to the Smo heptahelical bundle in a manner that antagonizes cyclopamine action. In addition, we have identified four small molecules that directly inhibit Smo activity but are structurally distinct from cyclopamine. Functional and biochemical studies of these compounds provide evidence for the small molecule modulation of Smo through multiple mechanisms and yield insights into the physiological regulation of Smo activity. The mechanistic differences between the Smo antagonists may be useful in the therapeutic manipulation of Hh signaling. H edgehog (Hh) signaling normally functions to specify embryonic pattern by directing cellular differentiation and proliferation (1), whereas aberrant Hh pathway activation is associated with the formation of tumors such as basal cell carcinoma and medulloblastoma (2-4). Cellular responses to the secreted Hh polypeptide are mediated by two integral membrane proteins, Patched (Ptc) and Smoothened (Smo), which were first identified by genetic screens in Drosophila (5-9). Hh binds to the twelve-pass transmembrane protein Ptc (8, 10, 11), thereby alleviating Ptc-mediated suppression of Smo (12), a distant relative of G protein coupled receptors. Smo activation then triggers a series of intracellular events, culminating in the stabilization of the transcription factor Cubitus interruptus (Ci) and the expression of Ci-dependent genes (13,14). These events are recapitulated during mammalian development and tumorigenesis through multiple protein homologues, including three distinct Hh family members [Sonic (Shh), Indian (Ihh), and Desert (Dhh)], two Ptc proteins (Ptch1 and Ptch2), and three Ci-like transcription factors (Gli1, Gli2, and Gli3; ref. (25), and Ptc is structurally related to the resistance-nodulation-cell division (RND) family of prokaryotic permeases and to the Niemann-Pick C1 (NPC1) protein, both of which are capable of transporting hydrophobic compounds (26, 27). Thus, Ptc might control Smo function by influencing its interactions with cellular small molecules.To study the biochemical basis of Smo activation further, we set out to identify and characterize other small molecules that modulate Smo function. We report here that a family of chlorobenzothiophene molecules identified as Hh pathway agonists (28) act by binding to the Smo heptahelical bundle. We also describe four previously uncharacterized Smo antagonists discovered through small molecule screens for Hh pathway inhibitors. In addition to providing mechanistic insights, such modulators may have ther...

Regulation of Hedgehog signaling: a complex story

“…Ptc appears to function catalytically in repression and destabilization of Smo, as Ptc can block Smo signaling at substochiometric levels [45]. As mentioned above, Smo repression has been suggested to occur via Ptc-mediated transport of an as yet unidentified small-molecule inhibitor [45,46]. This hypothesis is supported by recent work describing small molecule inhibitors of Smo that functionally mimic Ptc over-expression [36,46].…”

“…As mentioned above, Smo repression has been suggested to occur via Ptc-mediated transport of an as yet unidentified small-molecule inhibitor [45,46]. This hypothesis is supported by recent work describing small molecule inhibitors of Smo that functionally mimic Ptc over-expression [36,46]. These antagonists appear to target the hepta-helical bundle of Smo, the domain demonstrated to be affected by Ptc [46].…”

Expression patterns of hedgehog signaling peptides in naturally acquired equine osteochondrosis

“…Also, the apparent overlap of Ptcl and Ptc2 and their expression in articular cartilage have not been previously determined. The association between Ptc and Smo remains largely uncharacterized, although recent research indicates that Ptc may act catalytically to suppress Smo activity, possibly by small molecule translocation [7,28]. Binding of Ihh to Ptc inhibits this catalytic activity and allows Smo activation.…”

Targeting the CB2 cannabinoid receptor in osteoporosis

“…The first step involves the movement of Smo proteins from the plasma membrane and endoplasmic vesicles into primary cilium and here unliganded Ptch acts as a gatekeeper that restricts access of Smo to the primary cilium. Ptch action in this regard is mimicked by the drug, SANT-1, which similarly suppresses ciliary accumulation of Smo, even in the presence of ligand [35,36]. Once in the primary cilia, however, Smo activation requires a secondary step that is also regulated by Ptch, and this activation step is operationally defined by inhibition with cyclopamine or derivatives that allow Smo ciliary accumulation but prevent any further downstream signaling activities.…”

“…This is mainly attributable to the unique nature of the Smo molecule, whose activity is strongly influenced by its association with sterols or other low-molecular-weight compounds. Sterol-like compounds, such as Smo agonist [35] or purmorphomine [56], promote the activated Smo state and these molecules provide an alternative means of antagonizing hedgehog for experimental purposes. By contrast, sterols modeled after the phyto-derived jerveratrum alkaloid, cyclopamine, strongly inhibit Smo activation and these drugs are frequently used experimentally to antagonize hedgehog signaling [57].…”