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.

Fig 1.

SAG acts downstream of Ptch1 in the Hh pathway and counteracts cyclopamine inhibition of Smo. (A) Chemical structure of SAG and its activity in Shh-LIGHT2 cells. (B) SAG induces firefly luciferase expression in Shh-LIGHT2 cells with an EC₅₀ of 3 nM and then inhibits expression at higher concentrations. For comparison, the luciferase activity induced by 2 nM ShhNp is indicated by the green line. (C) SAG induces β-galactosidase expression in P2^Ptch1−/− cells treated with 100 nM KAAD-cyclopamine. Hh pathway activation in these cells is indicated by β-galactosidase activity, because expression of this reporter enzyme is under the control of the Ptch1 promoter, and Ptch1 itself is a transcriptional target of Hh signaling. Observed β-galactosidase activities in the absence of pharmacological modulation and with 100 nM KAAD-cyclopamine alone are indicated by the green and red lines, respectively. (D) SAG induces firefly luciferase expression in SmoA1-LIGHT2 cells treated with 1.5 μM KAAD-cyclopamine. Luciferase activities in the absence of small molecules and in the presence of 1.5 μM KAAD-cyclopamine alone are depicted by the green and red lines, respectively. (E) Cyclopamine and SAG have antagonistic effects on Hh pathway activation in Shh-LIGHT2 cells. Fifteenfold higher concentrations of KAAD-cyclopamine are required to inhibit luciferase expression induced by 100 nM SAG (red trace) than are necessary to block luciferase expression induced by 4 nM ShhNp (black trace). Relative luciferase activities are normalized with respect to maximum activity levels. (F) Similarly, 20 times more SAG is required to activate the Hh pathway in cells treated with 200 nM KAAD-cyclopamine (red trace) than is necessary to activate the pathway to comparable levels in untreated Shh-LIGHT2 cells (black trace). (G) Cross-linking of ER-localized (white arrowhead; see text and ref. 25) and post-ER (black arrowhead) forms of Smo-Myc₃ in Cos-1 cells with ¹²⁵I-labeled PA-cyclopamine is inhibited by SAG in a dose-dependent manner (Top). Cellular levels of Smo-Myc₃ are not affected by agonist treatment (Bottom). (H) Ptch1 inhibits SAG-induced pathway activation in a dose-dependent manner. NIH 3T3 cells were transiently transfected with the Gli-dependent firefly luciferase reporter and varying amounts of a Ptch1 expression construct. Transfected cells then were treated with a range of SAG concentrations, and the maximum luciferase activities observed for each amount of transfected Ptch1 cDNA are shown. All firefly luciferase and β-galactosidase activities are the average of three experiments and are normalized relative to a control reporter.

Fig 2.

SAG binds directly to Smo heptahelical bundle. (A) Chemical structure of the photoaffinity reagent PA-SAG and its activity in Shh-LIGHT2 cells. (B) ¹²⁵I-labeled PA-SAG cross-links the post-ER form of Smo-Myc₃ (black arrowhead) expressed in Cos-1 cells upon photoactivation, and this reaction is inhibited by 150 nM SAG (Left). The ER-localized form of Smo-Myc₃ (white arrowhead) is not detectably cross-linked, and cells expressing GFP as a control or SmoA1-Myc₃ do not yield specifically cross-linked products. An endogenous Cos-1 protein that is nonspecifically labeled by PA-SAG is denoted by the asterisk. Expression levels of Smo-Myc₃ and SmoA1-Myc₃ as determined by Western analysis are shown for comparison (Right). (C) SAG competes for PA-SAG cross-linking of post-ER Smo-Myc₃ (Left) in a manner similar to its ability to inhibit PA-cyclopamine cross-linking of Smo-Myc₃ (see Fig. 1G). Cellular levels of post-ER Smo-Myc₃ are not affected by SAG (Right). (D) KAAD-cyclopamine inhibits PA-SAG cross-linking of post-ER Smo-Myc₃, but concentrations greater than its apparent K_D for Smo (23 nM; ref. 25) are required (Left). Expression levels of post-ER Smo-Myc₃ are shown for comparison (Right). (E) SAG competes for the binding of BODIPY-cyclopamine to Smo-expressing cells, yielding an apparent dissociation constant of 59 nM for the SAG/Smo complex. (F) The binding of BODIPY-cyclopamine to Cos-1 cells expressing Smo, SmoΔCRD, or SmoΔCT is inhibited by 150 nM SAG with similar potencies, demonstrating that the SAG-binding site is localized to the Smo heptahelical bundle.

Fig 3.

Smo antagonists identified from a screen of 10,000 small molecules. (A) Chemical structures of SANTs and their activities in the Shh-LIGHT2 assay. The color-coding scheme used in the graphs is depicted by the blue, green, orange, and red lines. (B) The SANT compounds inhibit ShhNp-induced firefly luciferase expression in Shh-LIGHT2 cells. (C) The SANT molecules block the binding of BODIPY-cyclopamine to Smo-expressing Cos-1 cells, but SANT-1 and SANT-3 are unable to reduce BODIPY-cyclopamine binding to nonspecific levels. Nonspecific binding as defined by cellular BODIPY-cyclopamine levels in the presence of 500 nM KAAD-cyclopamine is indicated by the gray line. (D) Higher SAG concentrations are required to induce luciferase expression in Shh-LIGHT2 cells treated with 100 nM SANT-1 (blue trace), 150 nM SANT-2 (green trace), 500 nM SANT-3 (orange trace), or 1 μM SANT-4 (red trace) than are necessary to induce comparable luciferase activities in untreated Shh-LIGHT2 cells (black trace). (E) Unlike cyclopamine and its derivatives, the SANT compounds inhibit constitutive firefly luciferase expression in SmoA1-LIGHT2 cells with potencies that are similar to those required to inhibit Shh signaling in the Shh-LIGHT2 cells. Note that SANT-3 cannot completely inhibit luciferase expression in the SmoA1-LIGHT2 cells. Luciferase activity in SmoA1-LIGHT2 cells treated with 15 μM KAAD-cyclopamine is indicated by the gray line. All firefly luciferase activities are the average of three experiments and are normalized relative to a control reporter and to maximum activity levels.

Fig 4.

A bivalent model of SAG action. Hh pathway stimulation or inhibition by SAG at low or high concentrations, respectively, can be accounted for by bivalent binding of SAG to Smo and to a downstream effector. In this model, Hh pathway activation would normally involve the recruitment of a downstream effector (green) by a subpopulation of Smo molecules (blue). At subsaturating concentrations, SAG (red) can bind both Smo and the effector, thereby promoting Smo/effector association and increasing pathway activity levels. Higher concentrations of SAG, however, can inhibit the formation of this ternary complex by independently binding both proteins.