Targeting of Smoothened for therapeutic gain Martial Ruat, Lucile Hoch, Hélène Faure, Didier Rognan  Trends in Pharmacological Sciences  Volume 35, Issue 5, Pages 237-246 (May 2014) DOI: 10.1016/j.tips.2014.03.002 Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 1 Chemical structures of Smo modulators. Trends in Pharmacological Sciences 2014 35, 237-246DOI: (10.1016/j.tips.2014.03.002) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 2 Hedgehog signaling pathway at the primary cilium. (A) In the absence of Hedgehog ligand (-Hh), the receptor Patched (Ptc), located in the cilium, inhibits Smoothened (Smo), a seven-transmembrane receptor mostly found outside the cilium, by an as yet unknown mechanism. Gli transcription factors (Gli) are in a complex with the anterograde kinesin-like motor protein Kif7. Repressor factors such as suppressor of fused (Sufu) and kinases including protein kinase A (PKA), casein kinase 1α and glycogen synthase 3β, promote Gli truncation and phosphorylation, respectively. The constitutive activity of GPR161, a member of the G-protein coupled receptor superfamily [94], induces an increase of cAMP at the primary cilia resulting in an activation of PKA. These events lead to the generation of Gli repressor forms (GliR) and inhibition of Hh target genes. Hip, a negative regulator of the pathway, binds Hh, and is found as both membrane-associated and soluble forms. Gas1, Cdo, and Boc are membrane proteins that bind Hh, and are considered positive regulators of the pathway. A proposed model is that Gas1, Cdo, and Boc form a physical complex with Ptc, and function as essential coreceptors mediating multiple cellular events in response to Hh [95,96]. In the presence of Hh ligand (+Hh), Smo inhibition is relieved, allowing its translocation and accumulation in the cilium, which involves interaction with β-arrestin (β-Arr) [97] and association with the Evc–Evc2 complex [98] to transduce the signal intracellularly. This leads to Sufu inhibition and Gli conversion into their activated forms (GliA). GliA enters the nucleus and activates transcription of Hh target genes, including Ptc and Gli1. Smo may move through a lateral transport pathway from the plasma membrane to the ciliary membrane [99]. (B) Schematic representation depicting Smo activation for the two-step model of Smo trafficking at the cilium [74]. Smo is translocated to the cilium (step 1), and is then activated (step 2). SANT-1, GDC-0449, and MRT-83 block an inactive form of the receptor (Smo1) and its translocation to the cilium. Cyclopamine promotes a Smo2 inactive form to the primary cilium. The Smo agonists 20(S)-hydroxycholesterol [20(S)-OHC] and SAG, a Smo-activating M2 mutation, or Hh ligands via Ptc activation induce an active form of the receptor (Smo3) leading to GliA. The Smo agonist GSA-10 activates the receptor through an active form (Smo4) that neither traffics to the cilium nor acts via the canonical pathway [61]. Trends in Pharmacological Sciences 2014 35, 237-246DOI: (10.1016/j.tips.2014.03.002) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 3 Modulation of canonical and noncanonical Hedgehog (Hh) signaling by Smoothened (Smo) modulators. Canonical Hh signaling. (A) When Hh ligand binds patched (Ptc), Smo is activated. Smo antagonists blocking the Hh response that have reached clinical trials are shown. (B) SAG, purmorphamine (Pur.), and 20(S)-hydroxycholesterol [20(S)-OHC] are Smo agonists that induce Gli1 and Ptc1 transcription via the canonical pathway, and promote Smo translocation to the primary cilium. Smo activation is inhibited by antagonists, thought to act competitively, at the agonist binding site, or modulated by positive (PAM) or negative (NAM) allosteric modulators [2,3,17,55,61,73]. Binding sites are indicated as ellipses and agonist activity is shown in green. Compounds are proposed to act at the level of Smo transmembrane domains or in its aminoterminal tail for 20(S)-OHC. (C) Noncanonical Hh and Smo signaling. Hh proteins and the Smo ligands SAG, Pur., GSA-10, 20(S)-OHC, GDC-0449 (GDC), and cyclopamine (Cyclo) induce multiple cell responses through noncanonical pathways. Stimulation of Src family kinase by Hh ligands is implicated in attraction of spinal cord commissural axons within minutes, suggesting a nontranscriptional mechanism [9]. Pur. promotes fibroblast migration via Rho small GTPases [12]. SAG stimulates Gαi, Ca2+ spikes, and inositol triphosphate transients at the primary cilium of embryonic spinal neurons [10]. GSA-10 stimulates Smo to induce cell differentiation by a mechanism that involves neither increased Gli1 or Ptc transcription nor Smo translocation to the primary cilium [61]. SAG acts via an AMP-activated protein kinase (AMPK), Ca2+-dependent, and Gαi pathway to promote rapid glucose uptake, which is dependent on the primary cilium [11]. 20(S)-OHC and two antagonists of canonical Hh signaling (GDC-0449 and cyclopamine) show agonist activities towards this response. Trends in Pharmacological Sciences 2014 35, 237-246DOI: (10.1016/j.tips.2014.03.002) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 4 Structure of the Smo receptor and structural insights from its ligand binding pockets. (A) X-ray structure of the human Smo transmembrane (TM) domain complexed with the antitumor agent LY-2940680 (PDB 4KJV) (adapted from [79]). The structure is composed of seven TM helices (tan ribbons) connected by three intracellular (red) and three extracellular (green) loops. The N-terminal part (orange) is truncated at S190 and fused to a thermostabilized apocytochrome b562RIL (not displayed) to favor crystallization. Likewise, the C terminus is truncated after helix 8 at Q555. (B) X-ray structure of zebrafish Smo cysteine-rich extracellular domain (PDB 2C79; adapted from [58]). The binding groove for oxysterols (red side chains) is structurally analogous to the palmitoleyl-binding pocket in Frizzled-8. 20-(S)-Hydroxycholesterol [20(S)-OHC, cyan sticks] is docked in the groove using standard AutoDock 4.2.5.1 settings [100]. (C) Human Smo TM binding site for two small-molecular-weight Smo antagonists (LY2940680, SANT-1). The rotameric state of a unique residue (Leu3253.36, TM3) controls the depth of the TM cavity to which the ligand binds. When bound to LY2940680 (tan sticks and transparent surface, PDB 4JKV), the Leu3253.36 side chain (orange) is perpendicular to the 7-TM bundle main axis and defines the bottom of the pocket. As a consequence, LY2940680 mainly interacts with the three extracellular loops of the receptor. When bound to another antagonist (SANT-1, green sticks and transparent surface, PDB 4NKW), the Leu3253.36 side chain flips to open the lower part of the TM cavity in which SANT-1 deeply penetrates. (D) Structure of the human Smo homodimer (monomer A, yellow ribbons; monomer B, cyan ribbons). The interface engages TM domains 4 and 5 of both monomers. The dimer exhibits four structurally druggable cavities identified by the VolSite program [101]. The canonical antagonist pocket (white surface, 1) is present in each monomer, but two additional cavities (2 and 3) are present at the dimer interface (green and orange surfaces) and constitute putative binding sites for a novel generation of Smo modulators. Trends in Pharmacological Sciences 2014 35, 237-246DOI: (10.1016/j.tips.2014.03.002) Copyright © 2014 Elsevier Ltd Terms and Conditions