To identify small-molecule agonists of Hh signaling, we established a mammalian-cell-based assay. After testing several cell lines for Hh-dependent induction of the target genes Ptc1 and Gli12, we identified C3H10T1/2 and TM3 cells as optimal responders. We then introduced into each line a plasmid containing a luciferase reporter downstream of multimerized Gli binding sites and a minimal promoter 20. An isolated stable clone of the 10T1/2 cell transfectants (referred to as clone S12) gave a 10-20-fold up-regulation of luciferase activity (Figure 1a) when stimulated with Hh protein 21 for 24 hours. Using this assay system, we screened 140,000 synthetic compounds at a concentrations of 2-5 μM and isolated several putative agonists. One of these molecules - Hh-Ag 1.1 (Figure 1a,b) - was studied further. Hh-Ag 1.1 exhibited half-maximal stimulation (EC₅₀) at around 3 μM, and an activation maximum (A_max) of approximately 35% compared to the Hh protein control (Figure 1a). In the presence of sub-threshold signaling levels of Hh protein (0.3 nM), the EC₅₀ of Hh-Ag 1.1 was reduced to around 0.4 μM and the A_max approached 70% (Figure 1a).

We next tested whether expression of endogenous Hh-responsive genes was stimulated by the agonist. Using quantitative PCR, Hh-Ag 1.1 was shown clearly to elevate the expression of Gli1 and Ptc1 in a dose-dependent manner (Figure 1c).

In an effort to improve the potency of Hh-Ag 1.1, over 300 derivatives were synthesized and tested in the cell-based reporter assay. The relative potencies of the most active derivatives - 1.2, 1.3, 1.4 and 1.5 - are shown in Figure 1d. The most potent, Hh-Ag 1.5, had an EC₅₀ of approximately 1 nM. Thus, potency was increased over 1000-fold by chemical modification. The structures of compounds 1.2 and 1.3 are shown in Figure 1e. Hh-Ag 1.2 was the most stable derivative in vivo and in vitro (data not shown) and was used for most cell-based assays. Hh-Ag 1.3 showed lower toxicity in embryonic tissue cultures (data not shown) and was used for the neural plate explant assays described below. These experiments suggest that the agonist may have many of the properties of the Hh ligand. To specifically test this, we used two established in vitro assay systems that detect the effects of Hh on primary neuronal precursors.

To explore the site of action of the Hh agonist within the Hh pathway, we developed an in vivo assay for the agonist that would allow us to test its activity in Shh- and Smo-mutant mouse embryos in utero. First, we compared the expression of Ptc1 in vehicle- and agonist-treated Ptc1^lacZ/+ mouse embryos 25. The Ptc1^lacZ/+ mouse expresses β-galactosidase under control of Ptc1-regulatory elements and thus reports Hh-pathway activity in mouse tissues. Hh-Ag 1.2 (Figure 1e) was chosen for study on the basis of its relatively low toxicity, long serum half-life and ability to cross the placenta (data not shown). Hh-Ag 1.2 was delivered by oral gavage to pregnant mice at 7.5 and 8.5 days post coitum (7.5 and 8.5 dpc). Embryos were collected at embryonic day (E) 9.5 and analyzed by staining with the β-galactosidase chromogenic substrate X-gal. In vehicle-treated embryos, Ptc1 expression was confined primarily to the ventral neural tube (Figure 3a,c). In embryos treated with Hh-Ag 1.2, however, expression of Ptc1^lacZ was greatly extended dorsally in the neural tube and throughout the adjacent mesoderm (Figure 3b,d). These embryos also displayed open rostral neural tubes, similar to those of Ptc1^-/- embryos. These experiments demonstrate that the agonist compound effectively activates Hh signaling in vivo following oral administration.

Next, we sought to generate association, dissociation and saturation-binding curves, in order to derive affinity constants for the interaction of the Hh agonist and Smo. To control for nonspecific binding we used either Cur61414 or Hh-Ag 1.5 as unlabeled competitors. Similar results were generated if control membranes (from cells transfected with GFP, βAR, or Ptc) were used to define the non-specific level (data not shown).

First, we performed a kinetic analysis to establish the reversibility of the binding reaction and the approximate incubation time required for equilibrium binding studies. Association assays were performed at 37°C by combining 2 nM [³H]-Hh-Ag 1.5 with Smo-containing membranes for various times prior to harvesting and counting. Dissociation studies were initiated by adding 2 μM unlabeled Hh-Ag 1.5 after 2 hours of association. Samples were then incubated for 1-26 hours prior to harvesting and counting. Figure 7a shows the association and dissociation phases of agonist binding to Smo-containing membranes. Using the Prism GraphPad software, these data were fit to one-phase exponential association and decay curves, respectively, and gave an association t½ of approximately 1 hour and a dissociation t½ of approximately 10 hours. These results demonstrate that the binding of the agonist to Smo is reversible and that equilibrium binding will require binding reaction times of approximately 50 hours (five times the t½ of dissociation).

Next, we performed a saturation binding experiment. To establish total, nonspecific, and specific binding curves (Figure 7b), we added a range of [³H]-Hh-Ag 1.5 concentrations (0.01-3 nM) in the presence or absence of unlabeled Hh-Ag-1.5 at 2 μM. Identical results were seen if Cur61414 at 10 μM was used as the competitor (data not shown). On the basis of the binding kinetics, incubations were carried out for approximately 45 hours at 37°C, to allow for equilibrium to be reached. Using the Prism GraphPad software to perform non-linear regression analysis and curve fitting, we concluded that the data best fit a simple one-site binding model with a predicted K_d of 0.37 nM for Hh-Ag 1.5. This K_d is in general agreement with the EC₅₀ values observed in the cell-based assay (0.37 nM as compared to 1 nM).

To further validate our binding results, we performed a competition assay using several agonist derivatives across a range of concentrations (0.01 nM to 1 μM). Figure 7c shows the competition curves for Hh-Ag 1.5, Hh-Ag 1.3, Hh-Ag 1.2, Hh-Ag 1.1, and the signaling-inactive Hh-Ag 1.1 derivative described above. With the exception of the inactive derivative, these compounds all compete out the binding of [³H]-Hh-Ag 1.5 (0.4 nM) to the Smo-containing membranes. These data are best fit to a single-binding-site competition model that predicts the following K_i values: Hh-Ag 1.5, 0.52 nM; Hh-Ag 1.3, 8.4 nM; Hh-Ag 1.2, 22 nM; and Hh-Ag 1.1, 96 nM. These K_i values are in general agreement with the agonist EC₅₀ values in cell culture for these compounds, with the exception that the Hh-Ag 1.1 compound is not as potent in signaling assays (EC₅₀ 2 μM) as its K_i (96 nM) might predict. This suggests an uncoupling of binding and signaling for certain agonists. Although binding affinities and signaling efficacy can correspond for certain ligand/receptor complexes, exceptions often arise 28 because binding affinity does not necessarily measure the ability of a compound to induce an active receptor conformation. As a control for these binding studies, identical competition experiments were performed with membranes from cells transfected with GFP, or with the β-adrenergic receptor. No specific binding or apparent competition was seen under these conditions (data not shown).

We next compared binding of KAAD-cyclopamine, Cur61414 and Hh-Ag 1.5 to a constitutively active mutant of Smo (Smo^act) or to wild-type Smo (Smo^wt). The two Hh-signaling antagonists, KAAD-cyclopamine and Cur61414, have shown decreased potency on Smo^act-expressing cells, leading to the speculation that they may bind this mutant form of Smo less well than the wild-type form 1117. We sought to determine whether the Hh agonist binds Smo^act with a higher affinity, an observation seen with certain ligands and constitutively active mutants of GPCRs 29. To perform this experiment, we isolated membranes from cells transfected with a cDNA construct encoding a tryptophan-to-leucine mutation at residue 539 (W539L) of murine Smo. This oncogenic mutation has been found in human basal cell carcinoma 3 and the correspondingly mutated protein is capable of ligand-independent activation of the Hh pathway in cell-culture assays 11. A kinetic and saturation binding assay with Smo^act-containing membranes showed that this mutant protein binds the Hh agonist with an affinity identical to that of Smo^wt (data not shown).

Using Smo^act- and Smo^wt-containing membranes, we then performed competition binding studies by adding increasing concentrations of unlabeled Hh-Ag 1.5, KAAD-cyclopamine or Cur61414 in the presence of [³H]-Hh-Ag 1.5 (0.4 nM). These binding curves (Figure 7d) can be fit to a single-site competition model. Although the K_i for Hh-Ag 1.5 on Smo^act-containing membranes was essentially identical to that observed for Smo^wt-containing membranes (approximately 0.5 nM), the K_i values of the Hh antagonists were seven-fold higher on the Smo^act- compared to the Smo^wt-containing membranes for both KAAD-cyclopamine (38.3 nM versus 5.8 nM) and Cur61414 (309 nM versus 44 nM). These results strongly support the model, initially hypothesized for cyclopamine 11, that the reduced potency observed for Hh antagonists on Smo^act-expressing cells is directly due to a reduced affinity of the antagonist for the mutated Smo protein. The agonist, on the other hand, would be predicted to bind to a site on Smo that is not affected by this gain-of-function Smo^act mutation.

To interpret the results of the competition binding studies, we assume that the mutation in Smo^act, like those in constitutively activate mutants of other GPCRs 29, indirectly influences ligand binding by creating a change in the normal equilibrium between the different conformations of Smo. Thus, the mutation would not directly influence the binding pocket for either ligand. A simple two-state model (Figure 8a) predicts that the agonist (Ag, green square) and the antagonist (Ant, pink circle) compete for the same site on Smo to activate or inactivate Hh-pathway signaling. It also suggests that antagonists should bind Smo^act with a lower affinity than they bind Smo^wt, while the agonist should bind with a higher affinity, as it prefers the active conformation. Such a model cannot accommodate our observations with the gain-of-function Smo mutant. Thus we introduce a ternary complex model (Figure 8b), used traditionally to describe the behavior of GPCRs in binding studies with agonist and antagonists 31, as well as constitutively active receptors 29. The ternary complex model for Hh signaling suggests that there are two independent binding sites on Smo^wt, one specific for the agonist and another specific for antagonists. Binding at either site would decrease the affinity for interactions at the other site (allosteric binding with high negative cooperativity). The agonist-bound form represents the normal activated state, while the antagonist-bound form is considered the inactive conformation. There are also other conformations that would not be bound, or would be transiently bound, by both ligands. A signaling pathway coupler, or effector (in blue), is proposed to bind the activated state of Smo^wt so as to generate a complex competent to initiate Hh signaling. Throughout the discussion the term 'coupler/effector' is used to describe an unknown molecule that binds activated Smo in such a way as to trigger signal transduction. The model further suggests that the Smo^act protein resides in a stable conformation in the absence of agonist that is capable of forming an active coupler/effector complex resistant to antagonist, but not agonist, binding.

Specifically, our data suggest that the agonist binds and stabilizes (or induces) an active signaling state of Smo while the antagonists bind and stabilize (or induce) an inactive form. Furthermore, the gain-of-function Smo mutation renders the protein less sensitive to the inhibitors, presumably because the amino-acid substitution directly stabilizes or induces an active conformation. On the basis of a simple two-state model, one might predict an increased affinity of the agonist for the mutant form, but in our studies binding of the agonist, unlike the antagonist, is not affected by the activating mutation, suggesting that a more complex model requiring two binding sites and perhaps multiple active conformations is needed to account for the observations. Thus, we propose a variation of the classic 'ternary complex model' (Figure 8), a decades-old paradigm that has provided the foundation for describing ligand induced conformational changes of GPCRs 31.

Briefly, if this model is applied to Hh signaling, it proposes that, firstly, agonist and antagonists act at independent sites to select active and inactive conformations of Smo; secondly, that Smo engages an undefined coupler/effector, when it is in its signaling state; and finally, that the gain-of-function mutant form of Smo, Smo^act, adopts an abnormal conformation that resembles the coupler/effector-bound signaling state of Smo with low affinity for antagonists but normal affinity for the agonist.

As a receptor class, GPCRs are considered excellent drug targets because they are often regulated through interactions with small natural ligands 3233. Specifically, studies of classic GPCRs, such as the β-adrenergic receptors, show that in the absence of endogenous ligand (agonists) these receptors exist in multiple interconvertible conformations that are predominately inactive 34. Upon exposure to their natural ligands, however, the active receptor forms are preferentially stabilized, allowing them to readily engage G-protein couplers and to create signaling-competent complexes. Multiple compound classes have been isolated on the basis of their ability to compete for the binding of β-adrenergic receptors by their natural ligands. These competitors can mimic the natural ligand activity (agonists) or interfere with it (antagonists).

In addition to the binding sites for natural or endogenous agonists (orthosteric sites), many GPCRs have also been found to have allosteric sites 35. These sites can bind natural ligands, as in the case of Zn ions and heparin for the dopamine and neurokinin receptors, respectively, or bind synthetic drugs such gallamine, in the case of the muscarinic receptors 35. Binding of small molecules to these allosteric sites can modulate activity of a receptor without directly mimicking or competing out the interaction of ligands to the orthosteric sites. In summary, GPCRs have an array of potential regulatory binding sites, or potential drug targets.

How does Smo compare with other GPCRs with regard to the properties described above? Although there is clear structural homology between Smo and other GPCRs, endogenous ligands have yet to be discovered. Early models of Hh signaling proposed a Hh-regulated Ptc-Smo complex that directly controlled the conformation of Smo, making endogenous ligands unnecessary. But recent studies argue against this stoichiometric model 536, indicating that perhaps natural ligands should be considered. Furthermore, regarding G-protein-effector coupling for Smo, the results are also equivocal. Although no compelling data have been presented that directly link classic G-protein activation 37 to the canonical Hh pathway involving Ci/Gli stimulation 2, recent studies show that under certain conditions Smo can engage G-protein subunits in a Ptc-dependent manner 38 and that G-protein-mediated cAMP modulation may underlie certain effects of Hh on neuronal tissue 39.

Finally, with respect to pharmacological properties, our studies indicate that Smo behaves like a classic GPCR in many regards and that the models used to describe this large family of receptors can be applied to Hh signaling (Figure 8). Two relatively novel concepts for Hh signaling are raised by these GPCR models: firstly, the importance of considering the active Smo-coupler/effector complexes when modeling pathway regulation, and secondly the potential for endogenous ligands in the regulation of Smo activity.

A recent study in Drosophila suggested that Hh stimulates the pathway via Ptc degradation and, as a result, Smo is stabilized through extensive phosphorylation at the plasma membrane where it initiates signaling 5. This led to speculation that a Ptc-regulated phosphatase may control the subcellular distribution and stability of Smo 5. Although suggestive, the studies in Drosophila did not establish that phosphorylation, translocation or stabilization of Smo is required for pathway stimulation. It is plausible that some or all of these effects on Smo result from a feedback inhibition mechanism that targets the activated Smo receptor. Interestingly, while we did observe stabilization of Smo by Hh in our mammalian cell experiments (Figure 5), we did not detect Smo phosphorylation or translocation to the plasma membrane (data not shown). Perhaps the cellular (or species) context dictates the degree to which the active conformations of Smo are associated with such changes. Thus, in considering models of Ptc function based on Hh-stimulated effects it is important to consider whether the form of Smo that is being observed is the active (coupler/effector bound) signaling state, or perhaps a downregulated and inactive form.

Several potential points for regulation by Ptc during the formation of a Smo-coupler/effector complex are apparent in a ternary complex model. The ideas proposed for the Drosophila system, in which Ptc may affect the levels of Smo or its subcellular localization, are easily accommodated. Ptc-induced instability of Smo protein would indirectly reduce the concentration of an active Smo-coupler/effector complex. Furthermore, limiting access of Smo to its effector through targeted vesicle trafficking would prevent a signaling-competent complex from forming. Alternatively, Ptc could more directly maintain Smo in an inactive state. On the basis of our studies it is tempting to speculate that native small molecules with properties similar to our agonist and antagonists act directly on Smo in a Ptc-dependent manner. These putative endogenous Smo modulators could represent orthosteric or allosteric ligands.

The simplest model would have Ptc acting catalytically to dock a natural antagonist directly onto (or remove an agonist from) Smo. A more complex model would involve Ptc restricting the distribution of Smo such that it is forced into compartments containing the natural antagonists or lacking the natural agonists. Finally, Ptc could control the distribution of the endogenous small-molecule modulators themselves. Ptc shares sequence homology with molecules associated with vesicle trafficking and transporter activity, namely SCAP and NPC1 36404142; if it also shares the activities of these molecules, as suggested by a recent study 36, then the possibility of the existence of endogenous Smo ligands that are docked via Ptc should be explored.

Various studies in mammals have shown that Hh genes are expressed in discrete areas of the adult organism and may function in the normal maintenance of mature organ systems 43444546. In addition, the regenerative healing of vascular and skeletal tissues following acute injuries appears to be aided by re-activating the Hh-signaling cascade 4748. Taken together, these observations suggest that the Hh pathway may represent a point of intervention for treating certain degenerative disorders. Two recent studies in models of Parkinson's disease and peripheral nerve damage support this claim, by demonstrating that pathway activation with a Hh-protein ligand has therapeutic value 4950. On the basis of our current understanding of these models and the specific mechanism of action of the Hh agonists, we predict that an agonist-derivative with low toxicity and favorable pharmacokinetics would replicate these positive results. As a drug, a Hh agonist would represent an attractive alternative to an expensive Hh-protein therapeutic. Beyond the economics, for disorders of the central nervous system a small molecule with the potential to cross the blood-brain barrier would eliminate the need for injections directly into the brain, the current delivery mode for central nervous system protein therapies.

The compound libraries used in our screens were purchased from a number of commercial vendors and were primarily generated by combinatorial chemistry approaches. The Hh-agonist class was isolated from a library synthesized by Oxford Asymmetry International, now EvotecOAI. The derivatization of this compound class utilized standard procedures, the details of which will be published elsewhere.

TM3 and C3H10T1/2 cells (ATCC; Manassas, USA) were maintained according to the instructions of ATCC. Stable Hh-signaling reporter cell lines were established by G418 selection following transfection with a luciferase reporter plasmid 20 containing the neomycin-resistance gene. Hh signaling was monitored by plating cells at 70% confluence in growth medium. After 24 hours the cells were changed to 0.5% serum-containing medium, and Hh protein or compounds were added; 24 hours later the cells were either monitored for luciferase activity using the Luc-lite assay kit (Packard Instrument Company, Meriden, USA) or harvested for RNA isolation using an RNA isolation kit (Qiagen; Valencia, USA). RNA was subjected to quantitative RT-PCR analysis (Taqman; Applied Biosystems, Foster City, USA) utilizing Gli1, Ptc1 and GAPDH primers and probes. Assays were run on a Prism 7700 instrument (ABI; Applied Biosystems).

The Hh protein used in the studies described here was bacterially overexpressed amino-terminal human Shh modified at its amino-terminal cysteine by an octyl maleimide moiety 21. This lipophilic Shh form showed comparable potency to native Shh in the cell-based reporter assay (data not shown).

Mouse Smo and Ptc1 genes were introduced by a retroviral approach utilizing the pLPCX vector (Clontech; Palo Alto, USA) to limit the copy number per cell. Stable HA-Smo and Ptc-GFP lines were established by puromycin selection following infection of TM3 cells with the respective retroviruses. TM3 cells expressing both epitope-tagged Ptc and Smo were derived by infecting first with an HA-Smo construct and subsequently with a Ptc-GFP construct. The levels of Ptc were relatively low in these lines and a standard immunoprecipitation procedure followed by western blotting was required to detect the Ptc-GFP protein. The HA-Smo protein was highly expressed and was easily detected in western blots of whole cell extracts. The HA tag was sub-cloned into the Smo gene so that it would reside immediately after the Smo signal sequence. The GFP tag was inserted before the stop codon of the Ptc open reading frame.

Generation of Ptc1^lacZ, Shh and Smo mutant mice has been described previously 2526. Ptc1^lacZ/+ mice were kindly provided by Matthew Scott. Shh^+/- and Smo^+/- mice were kindly provided by Andrew McMahon. Agonist solution was prepared in fine suspension in 0.5% methylcellulose/0.2% Tween 80 at 1.5 mg/ml. Compound was administered by oral gavage to pregnant mice once a day for two days at 100 μl per 10 g body weight. Embryos were collected 24 hours later. Whole-mount in situ hybridization with Ptc1 probe and X-gal staining for whole-mount β-galactosidase detection were performed as described 26. For histology, embryos stained with X-gal were post-fixed in 4% paraformaldehyde, wax-embedded, and 20 μm sections were prepared.

Cerebellar neurons were dissected out of postnatal (one week) rat brains, and placed into primary cell culture. Briefly, cells were placed in 96-well plates at a density of approximately 150,000 cells per well in basal medium of Eagle (Gibco; Carlsbad, USA) supplemented with 26 mM KCl, 2 mM glutamine and 10% calf serum. Treatment agents were added once, on the first day of culture (0 DIV). Cells were left in culture until 2 DIV, when [³H]-thymidine was added for 5 hours. Cells were then lysed, and the incorporation of [³H]-thymidine was determined by scintillation counting.

Intermediate regions of the open neural tube (i-explants) were dissected from stage 10-11 chick embryos and embedded in collagen gel 23. Explants were cultured in Ham-F12 supplemented with 3 g/l D-glucose, Mito Serum Extender (Collaborative Research; Bedford, USA), penicillin/streptomycin (Gibco), 2 mM L-glutamine (Gibco) and Hh-Ag 1.3 (0.1, 1, 10, 20, 200 and 1000 nM prepared as 1000X stocks in DMSO; n = 6 explants). As a control, some explants were cultured with vehicle alone or with octylated Hh-N recombinant protein. Cultures were fixed after 22 hours, stained with mouse monoclonal antibodies against Pax7, MNR2 or rabbit polyclonal antibodies against Nkx2.2 and the number of immunoreactive cells per explant counted.

Cultured cells - 70% confluent 293T cells in 6-well plates - were either left untransfected or transfected using Fugene6 with a pCDNA3.1 construct containing HA-tagged Smo or v5-epitope tagged β2AR (Invitrogen; Carlsbad, USA). After 48 hours cells were switched from 10% fetal bovine serum containing DME media to 0.5% FBS containing media supplemented with either 5 nM [³H]-Hh-Ag 1.5 alone or 5 nM [³H]-agonist plus 5 μM of various competitors (see Results). After 2 hours of incubation at 37°C, cells were washed one time with PBS and subsequently lysed in 0.5 ml of lysis/wash buffer containing 1% NP40 in Tris-buffered saline plus an EDTA-free protease inhibitor cocktail (Roche; Indianapolis, USA) for 20 minutes at 4°C. Cell extracts were spun at 14,000 rpm in a microcentrifuge and supernatants were incubated for 40-60 minutes with either anti-HA beads (Roche) or anti-v5 antibody (Invitrogen) and Protein A beads (Pierce; Rockford, USA) to form immunocomplexes. Immunobeads were then spun down and washed three times with 0.5 ml lysis/wash buffer per wash. The washed pellets were then resuspended in SDS sample buffer and combined with scintillation fluid. Counts per minute (cpm) for each sample were then determined in a scintillation counter (Packard topcount).

Membranes were prepared as follows. Briefly, approximately 10⁸ cells were transfected with pcDNA 3.1 constructs (Invitrogen) bearing either murine Smo (wild-type or W539L mutant), GFP, rat β2AR (Invitrogen), or murine Ptc cDNAs using Fugene 6 (Roche). After 48 hours cells were harvested by scraping in PBS, centrifuged at 1,000 × g for 10 minutes, and gently resuspended in around 10 ml of a 50 mM Tris pH 7.5, 250 mM sucrose buffer containing an EDTA-free protease inhibitor cocktail (Roche). This cell suspension was then placed in a nitrogen cavitation device (Parr Instrument Co, Moline, USA) and exposed to nitrogen gas (230 psi) for 10 minutes. Lysed cells were released from the device and centrifuged at 20,000 rpm in an SS34 rotor for 20 minutes at 4°C. Supernatants were discarded and the pellets were resuspended in 10% sucrose, 50 mM Tris pH 7.5, 5 mM MgCl, 1 mM EDTA solution using three 10-second pulses with a Polytron (Brinkman; Westbury, USA) at a power setting of 12. Using these membranes, filtration binding assays were performed according to previously described protocols 28. To reduce nonspecific binding, 96-well filtration plates (fiberglass FB filters; Millipore, Bedford, USA) were pre-coated as suggested by the manufacturer with 0.5% polyethyleneimine + 0.1% BSA and then washed four times with 0.1% BSA.

For association and dissociation studies, membranes (1.5 μg total protein) were incubated in polypropylene tubes with 2 nM [³H]-Hh-Ag 1.5 in the presence or absence of 2 μM competitor in binding buffer (50 mM Tris pH 7.5, 5 mM MgCl, 1 mM EDTA, 0.1 % bovine serum albumin) plus EDTA-free protease inhibitor cocktail (Roche) in a final volume of 250 μl for 1-26 hours at 37°C. For saturation and competition binding analysis, membranes (1.5 μg total protein) were incubated on the plates with various concentrations of the [³H]-Hh-Ag 1.5 (plus and minus competitors) in binding buffer plus EDTA-free protease inhibitor cocktail (Roche) at a final volume of 1 ml for approximately 45 hours at 37°C to allow binding to reach apparent equilibrium. Binding reaction mixtures (0.2 ml for association/dissociation studies and 0.75 ml in saturation and competition experiments) were then transferred to the pre-coated 96-well filtration plates (Millipore fiberglass FB filters), filtered and washed over a vacuum manifold with six 300 μl per well washes of binding buffer supplemented with 2% hydoxypropyl cyclodextrin (HPCD; Sigma; ST Louis, USA) + 0.1% BSA to decrease non-specific binding. Identical results were obtained if incubations were done in borosilicate glass or siliconized plastic tubes. Centrifugation assays were also performed that replicate the filtration assay results (data not shown). Additionally, these experiments showed that the extent of ligand depletion was less than 10% in these studies. Binding-kinetics experiments were performed similarly to the saturation and competition studies. All binding data were evaluated using a nonlinear regression analysis program (Prism; GraphPad; San Diego, USA). K_i values were calculated using the Cheng-Prusoff correction equation 28, where K_i = IC₅₀/1+ [L]/K_d, and K_d for Hh-Ag 1.5 was determined to be 0.37 nM by the saturation analysis.

Related results demonstrating the action of cyclopamine on Smo have been reported by Beachy and colleagues 5152.