Three-Dimensional Structure of the Smoothened Receptor: Implications for Drug Discovery

  • Didier RognanEmail author
  • Isabelle Mus-Veteau
Part of the Topics in Medicinal Chemistry book series (TMC, volume 16)


The recently described high resolution three-dimensional structures of the transmembrane and the extracellular domains of the human Smoothened (Smo) receptor higlight both conserved and unique structural features of this G protein-coupled receptor. It enables a better understanding of very subtle molecular mechanisms regulating Smo function and demonstrates the very plastic nature of this receptor which is able to accommodate a diverse array of small molecular weight ligands through several binding sites. This structural information should pave the way for designing small molecular weight modulators of Smo function targeting different binding sites and insensitive to clinically observed receptor mutations.


Binding mode Cystein-rich domain Drug design Transmembrane domain X-ray structure 



Basal cell carcinoma




Drosophila Smoothened


Extracellular domain


Cystein-rich domain


Extracellular loop


G protein-coupled receptor


Human Smoothened




Intracellular loop


Nuclear magnetic resonance






Zebrafish Smoothened


  1. 1.
    Ingham PW (2001) Hedgehog signaling: a tale of two lipids. Science 294:1879–1881CrossRefGoogle Scholar
  2. 2.
    Scales SJ, de Sauvage FJ (2009) Mechanisms of Hedgehog pathway activation in cancer and implications for therapy. Trends Pharmacol Sci 30:303–312CrossRefGoogle Scholar
  3. 3.
    Lagerstrom MC, Schioth HB (2008) Structural diversity of G protein-coupled receptors and significance for drug discovery. Nat Rev Drug Discov 7:339–357CrossRefGoogle Scholar
  4. 4.
    Rudin CM (2012) Vismodegib. Clin Cancer Res 18:3218–3222CrossRefGoogle Scholar
  5. 5.
    Amakye D, Jagani Z, Dorsch M (2013) Unraveling the therapeutic potential of the Hedgehog pathway in cancer. Nat Med 19:1410–1422CrossRefGoogle Scholar
  6. 6.
    Ruat M, Hoch L, Faure H, Rognan D (2014) Targeting of Smoothened for therapeutic gain. Trends Pharmacol Sci 35:237–246CrossRefGoogle Scholar
  7. 7.
    Yauch RL, Dijkgraaf GJ, Alicke B, Januario T, Ahn CP, Holcomb T, Pujara K, Stinson J, Callahan CA, Tang T, Bazan JF, Kan Z, Seshagiri S, Hann CL, Gould SE, Low JA, Rudin CM, de Sauvage FJ (2009) Smoothened mutation confers resistance to a Hedgehog pathway inhibitor in medulloblastoma. Science 326:572–574CrossRefGoogle Scholar
  8. 8.
    Wang C, Wu H, Katritch V, Han GW, Huang XP, Liu W, Siu FY, Roth BL, Cherezov V, Stevens RC (2013) Structure of the human smoothened receptor bound to an antitumour agent. Nature 497:338–343CrossRefGoogle Scholar
  9. 9.
    Rana R, Carroll CE, Lee HJ, Bao J, Marada S, Grace CR, Guibao CD, Ogden SK, Zheng JJ (2013) Structural insights into the role of the Smoothened cysteine-rich domain in Hedgehog signalling. Nat Commun 4:2965CrossRefGoogle Scholar
  10. 10.
    Nachtergaele S, Whalen DM, Mydock LK, Zhao Z, Malinauskas T, Krishnan K, Ingham PW, Covey DF, Siebold C, Rohatgi R (2013) Structure and function of the Smoothened extracellular domain in vertebrate Hedgehog signaling. eLife 2:e01340CrossRefGoogle Scholar
  11. 11.
    Wang C, Wu H, Han GW, Cherezov V, Stevens RC (2014) Structure of the human smoothened receptor in complex with SANT-1Google Scholar
  12. 12.
    Weierstall U, James D, Wang C, White TA, Wang D, Liu W, Spence JC, Bruce Doak R, Nelson G, Fromme P, Fromme R, Grotjohann I, Kupitz C, Zatsepin NA, Liu H, Basu S, Wacker D, Han GW, Katritch V, Boutet S, Messerschmidt M, Williams GJ, Koglin JE, Marvin Seibert M, Klinker M, Gati C, Shoeman RL, Barty A, Chapman HN, Kirian RA, Beyerlein KR, Stevens RC, Li D, Shah ST, Howe N, Caffrey M, Cherezov V (2014) Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography. Nat Commun 5:3309CrossRefGoogle Scholar
  13. 13.
    Riobo NA, Saucy B, Dilizio C, Manning DR (2006) Activation of heterotrimeric G proteins by Smoothened. Proc Natl Acad Sci U S A 103:12607–12612CrossRefGoogle Scholar
  14. 14.
    Fredriksson R, Lagerstrom MC, Lundin LG, Schioth HB (2003) The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol Pharmacol 63:1256–1272CrossRefGoogle Scholar
  15. 15.
    The GPCR Network Homepage (2014) Accessed Feb 2014
  16. 16.
    Dann CE, Hsieh JC, Rattner A, Sharma D, Nathans J, Leahy DJ (2001) Insights into Wnt binding and signalling from the structures of two Frizzled cysteine-rich domains. Nature 412:86–90CrossRefGoogle Scholar
  17. 17.
    Janda CY, Waghray D, Levin AM, Thomas C, Garcia KC (2012) Structural basis of Wnt recognition by Frizzled. Science 337:59–64CrossRefGoogle Scholar
  18. 18.
    Zhao Y, Tong C, Jiang J (2007) Hedgehog regulates smoothened activity by inducing a conformational switch. Nature 450:252–258CrossRefGoogle Scholar
  19. 19.
    Aanstad P, Santos N, Corbit KC, Scherz PJ, le Trinh A, Salvenmoser W, Huisken J, Reiter JF, Stainier DY (2009) The extracellular domain of Smoothened regulates ciliary localization and is required for high-level Hh signaling. Curr Biol 19:1034–1039CrossRefGoogle Scholar
  20. 20.
    Nedelcu D, Liu J, Xu Y, Jao C, Salic A (2013) Oxysterol binding to the extracellular domain of Smoothened in Hedgehog signaling. Nat Chem Biol 9:557–564CrossRefGoogle Scholar
  21. 21.
    Myers BR, Sever N, Chong YC, Kim J, Belani JD, Rychnovsky S, Bazan JF, Beachy PA (2013) Hedgehog pathway modulation by multiple lipid binding sites on the smoothened effector of signal response. Dev Cell 26:346–357CrossRefGoogle Scholar
  22. 22.
    Cooper MK, Wassif CA, Krakowiak PA, Taipale J, Gong R, Kelley RI, Porter FD, Beachy PA (2003) A defective response to Hedgehog signaling in disorders of cholesterol biosynthesis. Nat Genet 33:508–513CrossRefGoogle Scholar
  23. 23.
    Corcoran RB, Scott MP (2006) Oxysterols stimulate Sonic hedgehog signal transduction and proliferation of medulloblastoma cells. Proc Natl Acad Sci U S A 103:8408–8413CrossRefGoogle Scholar
  24. 24.
    Bidet M, Joubert O, Lacombe B, Ciantar M, Nehme R, Mollat P, Bretillon L, Faure H, Bittman R, Ruat M, Mus-Veteau I (2011) The hedgehog receptor patched is involved in cholesterol transport. PLoS One 6:e23834CrossRefGoogle Scholar
  25. 25.
    Dwyer JR, Sever N, Carlson M, Nelson SF, Beachy PA, Parhami F (2007) Oxysterols are novel activators of the hedgehog signaling pathway in pluripotent mesenchymal cells. J Biol Chem 282:8959–8968CrossRefGoogle Scholar
  26. 26.
    Nachtergaele S, Mydock LK, Krishnan K, Rammohan J, Schlesinger PH, Covey DF, Rohatgi R (2012) Oxysterols are allosteric activators of the oncoprotein Smoothened. Nat Chem Biol 8:211–220CrossRefGoogle Scholar
  27. 27.
    Wang Y, Davidow L, Arvanites AC, Blanchard J, Lam K, Xu K, Oza V, Yoo JW, Ng JM, Curran T, Rubin LL, McMahon AP (2012) Glucocorticoid compounds modify smoothened localization and hedgehog pathway activity. Chem Biol 19:972–982CrossRefGoogle Scholar
  28. 28.
    Taipale J, Cooper MK, Maiti T, Beachy PA (2002) Patched acts catalytically to suppress the activity of Smoothened. Nature 418:892–897CrossRefGoogle Scholar
  29. 29.
    Eaton S (2008) Multiple roles for lipids in the Hedgehog signalling pathway. Nat Rev 9:437–445CrossRefGoogle Scholar
  30. 30.
    Roberg-Larsen H, Strand MF, Grimsmo A, Olsen PA, Dembinski JL, Rise F, Lundanes E, Greibrokk T, Krauss S, Wilson SR (2012) High sensitivity measurements of active oxysterols with automated filtration/filter backflush-solid phase extraction-liquid chromatography-mass spectrometry. J Chromatogr A 1255:291–297CrossRefGoogle Scholar
  31. 31.
    Liu W, Wacker D, Gati C, Han GW, James D, Wang D, Nelson G, Weierstall U, Katritch V, Barty A, Zatsepin NA, Li D, Messerschmidt M, Boutet S, Williams GJ, Koglin JE, Seibert MM, Wang C, Shah ST, Basu S, Fromme R, Kupitz C, Rendek KN, Grotjohann I, Fromme P, Kirian RA, Beyerlein KR, White TA, Chapman HN, Caffrey M, Spence JC, Stevens RC, Cherezov V (2013) Serial femtosecond crystallography of G protein-coupled receptors. Science 342:1521–1524CrossRefGoogle Scholar
  32. 32.
    Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, Le Trong I, Teller DC, Okada T, Stenkamp RE, Yamamoto M, Miyano M (2000) Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289:739–745CrossRefGoogle Scholar
  33. 33.
    The GPCR Structural Database (2014) Accessed Jan 2014
  34. 34.
    Mus-Veteau I, Zito F, Demange P (2014) Membrane protein production for structural analysis. In: Mus-Veteau I (eds) Membrane protein production for structural analysis. Springer, New YorkGoogle Scholar
  35. 35.
    Ahn KH, Nishiyama A, Mierke DF, Kendall DA (2010) Hydrophobic residues in helix 8 of cannabinoid receptor 1 are critical for structural and functional properties. Biochemistry 49:502–511CrossRefGoogle Scholar
  36. 36.
    Delos Santos NM, Gardner LA, White SW, Bahouth SW (2006) Characterization of the residues in helix 8 of the human beta1-adrenergic receptor that are involved in coupling the receptor to G proteins. J Biol Chem 281:12896–12907CrossRefGoogle Scholar
  37. 37.
    Awwad HO, Millman EE, Alpizar-Foster E, Moore RH, Knoll BJ (2010) Mutating the dileucine motif of the human beta(2)-adrenoceptor reduces the high initial rate of receptor phosphorylation by GRK without affecting postendocytic sorting. Eur J Pharmacol 635:9–15CrossRefGoogle Scholar
  38. 38.
    Kirchberg K, Kim TY, Moller M, Skegro D, Dasara Raju G, Granzin J, Buldt G, Schlesinger R, Alexiev U (2011) Conformational dynamics of helix 8 in the GPCR rhodopsin controls arrestin activation in the desensitization process. Proc Natl Acad Sci U S A 108:18690–18695CrossRefGoogle Scholar
  39. 39.
    Aratake Y, Okuno T, Matsunobu T, Saeki K, Takayanagi R, Furuya S, Yokomizo T (2012) Helix 8 of leukotriene B4 receptor 1 inhibits ligand-induced internalization. FASEB J 26:4068–4078CrossRefGoogle Scholar
  40. 40.
    Venkatakrishnan AJ, Deupi X, Lebon G, Tate CG, Schertler GF, Babu MM (2013) Molecular signatures of G-protein-coupled receptors. Nature 494:185–194CrossRefGoogle Scholar
  41. 41.
    Xie J, Murone M, Luoh SM, Ryan A, Gu Q, Zhang C, Bonifas JM, Lam CW, Hynes M, Goddard A, Rosenthal A, Epstein EH Jr, de Sauvage FJ (1998) Activating Smoothened mutations in sporadic basal-cell carcinoma. Nature 391:90–92CrossRefGoogle Scholar
  42. 42.
    Umbhauer M, Djiane A, Goisset C, Penzo-Mendez A, Riou JF, Boucaut JC, Shi DL (2000) The C-terminal cytoplasmic Lys-thr-X-X-X-Trp motif in frizzled receptors mediates Wnt/beta-catenin signalling. EMBO J 19:4944–4954CrossRefGoogle Scholar
  43. 43.
    Chen Y, Li S, Tong C, Zhao Y, Wang B, Liu Y, Jia J, Jiang J (2010) G protein-coupled receptor kinase 2 promotes high-level Hedgehog signaling by regulating the active state of Smo through kinase-dependent and kinase-independent mechanisms in Drosophila. Genes Dev 24:2054–2067CrossRefGoogle Scholar
  44. 44.
    Chen JK, Taipale J, Cooper MK, Beachy PA (2002) Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened. Genes Dev 16:2743–2748CrossRefGoogle Scholar
  45. 45.
    Buonamici S, Williams J, Morrissey M, Wang A, Guo R, Vattay A, Hsiao K, Yuan J, Green J, Ospina B, Yu Q, Ostrom L, Fordjour P, Anderson DL, Monahan JE, Kelleher JF, Peukert S, Pan S, Wu X, Maira SM, Garcia-Echeverria C, Briggs KJ, Watkins DN, Yao YM, Lengauer C, Warmuth M, Sellers WR, Dorsch M (2010) Interfering with resistance to smoothened antagonists by inhibition of the PI3K pathway in medulloblastoma. Sci Transl Med 2:51ra70CrossRefGoogle Scholar
  46. 46.
    Roberg-Larsen H, Strand MF, Krauss S, Wilson SR (2014) Metabolites in vertebrate Hedgehog signaling. Biochem Biophys Res Commun. doi: 10.1016/j.bbrc.2014.01.087, Scholar
  47. 47.
    Dijkgraaf GJ, Alicke B, Weinmann L, Januario T, West K, Modrusan Z, Burdick D, Goldsmith R, Robarge K, Sutherlin D, Scales SJ, Gould SE, Yauch RL, de Sauvage FJ (2011) Small molecule inhibition of GDC-0449 refractory smoothened mutants and downstream mechanisms of drug resistance. Cancer Res 71:435–444CrossRefGoogle Scholar
  48. 48.
    Rasmussen SG, DeVree BT, Zou Y, Kruse AC, Chung KY, Kobilka TS, Thian FS, Chae PS, Pardon E, Calinski D, Mathiesen JM, Shah ST, Lyons JA, Caffrey M, Gellman SH, Steyaert J, Skiniotis G, Weis WI, Sunahara RK, Kobilka BK (2011) Crystal structure of the beta2 adrenergic receptor-Gs protein complex. Nature 477:549–555CrossRefGoogle Scholar
  49. 49.
    Chen JK, Taipale J, Young KE, Maiti T, Beachy PA (2002) Small molecule modulation of Smoothened activity. Proc Natl Acad Sci U S A 99:14071–14076CrossRefGoogle Scholar
  50. 50.
    Gorojankina T, Hoch L, Faure H, Roudaut H, Traiffort E, Schoenfelder A, Girard N, Mann A, Manetti F, Solinas A, Petricci E, Taddei M, Ruat M (2013) Discovery, molecular and pharmacological characterization of GSA-10, a novel small-molecule positive modulator of Smoothened. Mol Pharmacol 83:1020–1029CrossRefGoogle Scholar
  51. 51.
    Yang H, Xiang J, Wang N, Zhao Y, Hyman J, Li S, Jiang J, Chen JK, Yang Z, Lin S (2009) Converse conformational control of smoothened activity by structurally related small molecules. J Biol Chem 284:20876–20884CrossRefGoogle Scholar
  52. 52.
    Duarte JM, Biyani N, Baskaran K, Capitani G (2013) An analysis of oligomerization interfaces in transmembrane proteins. BMC Struct Biol 13:21CrossRefGoogle Scholar
  53. 53.
    Shi D, Lv X, Zhang Z, Yang X, Zhou Z, Zhang L, Zhao Y (2013) Smoothened oligomerization/higher order clustering in lipid rafts is essential for high Hedgehog activity transduction. J Biol Chem 288:12605–12614CrossRefGoogle Scholar
  54. 54.
    Gomes I, Fujita W, Gupta A, Saldanha SA, Negri A, Pinello CE, Eberhart C, Roberts E, Filizola M, Hodder P, Devi LA (2013) Identification of a mu-delta opioid receptor heteromer-biased agonist with antinociceptive activity. Proc Natl Acad Sci U S A 110:12072–12077CrossRefGoogle Scholar
  55. 55.
    Hiller C, Kuhhorn J, Gmeiner P (2013) Class A G-protein-coupled receptor (GPCR) dimers and bivalent ligands. J Med Chem 56:6542–6559CrossRefGoogle Scholar
  56. 56.
    Desaphy J, Azdimousa K, Kellenberger E, Rognan D (2012) Comparison and druggability prediction of protein-ligand binding sites from pharmacophore-annotated cavity shapes. J Chem Inf Model 52:2287–2299CrossRefGoogle Scholar
  57. 57.
    Kim J, Tang JY, Gong R, Kim J, Lee JJ, Clemons KV, Chong CR, Chang KS, Fereshteh M, Gardner D, Reya T, Liu JO, Epstein EH, Stevens DA, Beachy PA (2010) Itraconazole, a commonly used antifungal that inhibits Hedgehog pathway activity and cancer growth. Cancer Cell 17:388–399CrossRefGoogle Scholar
  58. 58.
    Kwon HJ, Abi-Mosleh L, Wang ML, Deisenhofer J, Goldstein JL, Brown MS, Infante RE (2009) Structure of N-terminal domain of NPC1 reveals distinct subdomains for binding and transfer of cholesterol. Cell 137:1213–1224CrossRefGoogle Scholar
  59. 59.
    Wang ML, Motamed M, Infante RE, Abi-Mosleh L, Kwon HJ, Brown MS, Goldstein JL (2010) Identification of surface residues on Niemann-Pick C2 essential for hydrophobic handoff of cholesterol to NPC1 in lysosomes. Cell Metab 12:166–173CrossRefGoogle Scholar
  60. 60.
    Pamplona FA, Ferreira J, de Lima M, Jr O, Duarte FS, Bento AF, Forner S, Villarinho JG, Bellocchio L, Wotjak CT, Lerner R, Monory K, Lutz B, Canetti C, Matias I, Calixto JB, Marsicano G, Guimaraes MZ, Takahashi RN (2012) Anti-inflammatory lipoxin A4 is an endogenous allosteric enhancer of CB1 cannabinoid receptor. Proc Natl Acad Sci U S A 109:21134–21139CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  1. 1.Laboratoire d’Innovation ThérapeutiqueUMR 7200 CNRS-Université de StrasbourgIllkirchFrance
  2. 2.Institut de Pharmacologie Moléculaire et CellulaireUMR 7275 CNRS-Université de Nice-Sophia AntipolisIllkirchFrance

Personalised recommendations