Regulation of the oncoprotein Smoothened by small molecules

Sharpe, Hayley J.; Wang, Weiru; Hannoush, Rami N.; Sauvage, Frédéric J. de

doi:10.1038/nchembio.1776

Cited by 106 publications

(97 citation statements)

References 99 publications

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“…Of note is that Tyr106 is also conserved within smoothened (SMO; Tyr103 in SMO), a hedgehog pathway receptor that contains an extracellular FZD-like CRD and is distantly related to the FZD family (SI Appendix, Fig. S1A) (26). In the crystal structure of SMO CRD, Tyr103 forms a hydrogen bond with the hydroxyl group of 20 (S)-hydroxycholesterol (27).…”

Section: Resultsmentioning

confidence: 99%

Unsaturated fatty acyl recognition by Frizzled receptors mediates dimerization upon Wnt ligand binding

Nile¹,

Mukund²,

Stanger³

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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105

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Frizzled (FZD) receptors mediate Wnt signaling in diverse processes ranging from bone growth to stem cell activity. Moreover, high FZD receptor expression at the cell surface contributes to overactive Wnt signaling in subsets of pancreatic, ovarian, gastric, and colorectal tumors. Despite the progress in biochemical understanding of Wnt-FZD receptor interactions, the molecular basis for recognition of Wnt cis-unsaturated fatty acyl groups by the cysteine-rich domain (CRD) of FZD receptors remains elusive. Here, we determined a crystal structure of human FZD7 CRD unexpectedly bound to a 24-carbon fatty acid. We also report a crystal structure of human FZD5 CRD bound to C16:1 cis-Δ9 unsaturated fatty acid. Both structures reveal a dimeric arrangement of the CRD. The lipid-binding groove exhibits flexibility and spans both monomers, adopting a U-shaped geometry that accommodates the fatty acid. Re-evaluation of the published mouse FZD8 CRD structure reveals that it also shares the same architecture as FZD5 and FZD7 CRDs. Our results define a common molecular mechanism for recognition of the cis-unsaturated fatty acyl group, a necessary posttranslational modification of Wnts, by multiple FZD receptors. The fatty acid bridges two CRD monomers, implying that Wnt binding mediates FZD receptor dimerization. Our data uncover possibilities for the arrangement of Wnt-FZD CRD complexes and shed structural insights that could aide in the identification of pharmacological strategies to modulate FZD receptor function.nt signaling is evolutionarily conserved from metazoans to humans (1). It is critical for tissue homeostasis and during development (2, 3). On the other hand, derangements in Wnt signaling are associated with severe pathologies in mammals, including cancer (4-6). Signaling is initiated at the cell surface where the secreted, lipid-modified Wnt glycoprotein interacts with the extracellular N-terminal cysteine-rich domain (CRD) of the frizzled (FZD) receptor (7). This high-affinity interaction occurs at two distinct contact sites, one comprising a protein-fatty acyl interface and another a protein-protein interaction interface (8-10). Sequence analysis revealed that there are 10 human frizzled genes grouped into four clusters (SI Appendix, Fig. S1A).Among their multiple roles in physiology, FZD receptors have emerged as critical regulators of Wnt-dependent stem cell processes. For example, FZD7 is a critical component of the stem cell niche and was shown by genetic knockdown experiments to be essential for maintaining human embryonic and epithelial limbal stem cells in an undifferentiated state (11,12). Of the 10 mammalian frizzleds, FZDs 1, 2, and 7 are enriched at the base of mammalian adult intestinal crypts where the stem cells reside (13,14), and FZD7 is required for stem cell-mediated regeneration of the intestinal epithelium (15). Moreover, high FZD receptor expression at the cell surface contributes to overactive Wnt signaling in subsets of pancreatic, ovarian, gastric, and colorectal tumors (16)(17)(18)(19)...

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Section: Resultsmentioning

confidence: 99%

Unsaturated fatty acyl recognition by Frizzled receptors mediates dimerization upon Wnt ligand binding

Nile¹,

Mukund²,

Stanger³

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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105

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“…Neither of the two distinct sites on Smo that are known to bind modulatory lipids, however, is likely to respond to Patched regulation. The extracellular CRD domain of Smoothened is not required for Patched action (18), and mutations affecting the cyclopamine pocket generally fail to disrupt regulation by Patched (15), suggesting that the Smoothened site responding to Patched regulation remains unknown, as does the identity of the molecule(s) that mediate this regulation. We therefore undertook an unbiased search for possible mediators of Smoothened regulation, using a cultured cell reporter assay to identify activities from various lipid extracts.…”

Section: Discussionmentioning

confidence: 99%

“…Several small molecules including cyclopamine derivatives and other synthetic compounds that have been characterized as inhibiting Smoothened by binding to the "cyclopamine pocket" are in clinical trials or have been approved for use as anticancer drugs (17). Recent structural studies define the cyclopamine pocket in atomic detail, but mutations affecting this pocket generally fail to disrupt regulation by Patched (15), suggesting a distinct site of action for an endogenous mediator of Patched regulation.…”

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confidence: 99%

“…Various small-molecule agonists and antagonists that bind to the Smoothened transmembrane domain have been discovered, including the steroidal plant compounds cyclopamine and jervine and synthetic Smoothened antagonists and agonists (SAGs) (15,16). Several small molecules including cyclopamine derivatives and other synthetic compounds that have been characterized as inhibiting Smoothened by binding to the "cyclopamine pocket" are in clinical trials or have been approved for use as anticancer drugs (17).…”

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confidence: 99%

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Endogenous B-ring oxysterols inhibit the Hedgehog component Smoothened in a manner distinct from cyclopamine or side-chain oxysterols

Sever

Mann

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Cellular lipids are speculated to act as key intermediates in Hedgehog signal transduction, but their precise identity and function remain enigmatic. In an effort to identify such lipids, we pursued a Hedgehog pathway inhibitory activity that is particularly abundant in flagellar lipids of Chlamydomonas reinhardtii, resulting in the purification and identification of ergosterol endoperoxide, a B-ring oxysterol. A mammalian analog of ergosterol, 7-dehydrocholesterol (7-DHC), accumulates in Smith–Lemli–Opitz syndrome, a human genetic disease that phenocopies deficient Hedgehog signaling and is caused by genetic loss of 7-DHC reductase. We found that depleting endogenous 7-DHC with methyl-β-cyclodextrin treatment enhances Hedgehog activation by a pathway agonist. Conversely, exogenous addition of 3β,5α-dihydroxycholest-7-en-6-one, a naturally occurring B-ring oxysterol derived from 7-DHC that also accumulates in Smith–Lemli–Opitz syndrome, blocked Hedgehog signaling by inhibiting activation of the essential transduction component Smoothened, through a mechanism distinct from Smoothened modulation by other lipids.

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“…14 These efforts have led to the identification of several potent inhibitors of GPCR-like Smoothened (Smo) receptor. 14 These include cyclopamine 15 and vismodegib 16 , which correspond to the archetypal member and the first FDA-approved drug, respectively, belonging to this class of Hh pathway antagonists. Compounds that target downstream components of this pathway 14 or processes involved in Shh maturation 17 have also been reported.…”

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confidence: 99%

Design and Evolution of a Macrocyclic Peptide Inhibitor of the Sonic Hedgehog/Patched Interaction

et al. 2017

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The Hedgehog (Hh) signaling pathway plays a central role during embryonic development and its aberrant activation has been implicated in the development and progression of several human cancers. Major efforts toward the identification of chemical modulators of the Hedgehog pathway has yielded several antagonists of the GPCR-like Smoothened receptor. In contrast, potent inhibitors of the Sonic Hedgehog/Patched interaction, the most upstream event in ligand-induced activation of this signaling pathway, have been elusive. To address this gap, a genetically encoded cyclic peptide was designed based on the Shh-binding loop of Hedgehog-Interacting Protein (HHIP) and subjected to multiple rounds of affinity maturation through the screening of macrocyclic peptide libraries produced in E. coli cells. Using this approach, an optimized macrocyclic peptide inhibitor (HL2-m5) was obtained that binds Shh with a KD of 170 nM, which corresponds to a 120-fold affinity improvement compared to the parent molecule. Importantly, HL2-m5 is able to effectively suppress Shh-mediated Hedgehog signaling and Gli-controlled gene transcription in living cells (IC50 = 250 nM), providing the most potent inhibitor of the Sonic Hedgehog/Patched interaction reported to date. This first-in-class macrocyclic peptide modulator of the Hedgehog pathway is expected to provide a valuable probe for investigating and targeting ligand-dependent Hedgehog pathway activation in cancer and other pathologies. This work also introduces a general strategy for the development of cyclopeptide inhibitors of protein-protein interactions.

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Regulation of the oncoprotein Smoothened by small molecules

Cited by 106 publications

References 99 publications

Unsaturated fatty acyl recognition by Frizzled receptors mediates dimerization upon Wnt ligand binding

Unsaturated fatty acyl recognition by Frizzled receptors mediates dimerization upon Wnt ligand binding

Endogenous B-ring oxysterols inhibit the Hedgehog component Smoothened in a manner distinct from cyclopamine or side-chain oxysterols

Design and Evolution of a Macrocyclic Peptide Inhibitor of the Sonic Hedgehog/Patched Interaction

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