An approach using 'chemical genetics' has identified small-molecule agonists of the Hedgehog signaling pathway that may lead the way to drugs for chronic degenerative diseases.
It is a rare treat when a drug discovery program teaches us something about the biology of the process that it attempts to modulate. The paper by Maria Frank-Kamenetsky and colleagues in this issue of the Journal of Biology presents a compelling example of how the search for therapeutics can provide powerful experimental tools and insights into fundamental biology, blurring the distinction between applied and basic research. By characterizing a small group of chemically similar agonists of the Hedgehog signaling pathway, Frank-Kamenetsky et al. have been able to propose a new model for how the Smoothened component of the Hedgehog-receptor complex works, and to hint at the existence of natural-ligand agonists of the Hedgehog signaling pathway (see 'The bottom line' box for a summary of their work).
Signaling by the Hedgehog (Hh) family of secreted proteins plays a central role in regulating cell differentiation and tissue patterning during development . The hedgehog gene (hh) was first identified by virtue of its role in the specification of positional identity during Drosophila embryonic segmentation, and it was subsequently found to control patterning of structures such as the eye and the abdominal cuticle. In mammals there are three hh homologs, called Sonic Hedgehog, Indian Hedgehog and Desert Hedgehog (Shh, Ihh and Dhh, respectively), which have been implicated in patterning events in a range of developing tissues [2,3] (see the 'Background' box).
Recently, signaling by Hh has been shown to be important for patterning of the cerebellum, where it promotes the proliferation of granule neuron precursors. A link between Hh proteins and stem-cell proliferation has raised the enticing possibility that modulating Hh signaling might be relevant for the clinical management of certain degenerative diseases. Indeed, it was recently demonstrated that Shh might be effective in treating peripheral nerve damage or degenerative brain disorders such as Parkinson's disease[4,5]. Perhaps not surprisingly given its role in development, misregulated Hh signaling has also been implicated in cancer. Specifically, medulloblastoma and basal cell carcinoma (BCC) are associated with inappropriate activation of Hh signaling [6,7]. These observations motivated Frank-Kamenetsky and colleagues to search for small-molecule modulators of the Hh pathway, with the hope that antagonists and agonists might be used as drugs to treat proliferative or degenerative diseases, and that small molecules might prove more amenable to pharmacological delivery than the Hh-family proteins themselves.
Sending the signal
Hedgehog signaling challenges the way we normally think about signal transduction pathways. Biology is full of examples of extracellular ligands that bind to specific cell-surface receptors, initiating a cascade of biochemical events (often involving protein kinases) that leads to the activation of a transcription factor and the induction of a set of effector genes. But Hedgehog signaling is not so simple. Even the ligand is complicated: the Hh proteins undergo unusual processing and cleavage to generate an extracellular cholesterol-linked peptide that serves as the signaling ligand . And the receptor component is far from understood.
The cellular response to Hh is controlled by two transmembrane proteins, Patched (Ptc) and Smoothened (Smo). The Ptc protein weaves across the cell membrane twelve times and resembles some transmembrane channels. It acts as a negative regulator of the Hh signal and has been defined as a tumor-suppressor. In contrast, Smo is a proto-oncogene and activates signaling in response to Hh ligand. The Smo protein is a seven-transmembrane receptor that resembles conventional G-protein-coupled receptors (Figure 1a).
Figure 1. The Hedgehog signaling pathway. (a) In the absence of Hedgehog ligands, Patched protein inhibits the activity of Smoothened, which resembles G-protein-coupled receptors. (b) On activation by a ligand of the Hedgehog family, the inhibition of Smoothened by Patched is relieved and Smoothened is freed to trigger an intracellular signaling cascade; this ultimately leads to transcriptional regulation by transcription factors of the Gli family.
It appears that Ptc inhibits Smo, although the precise mechanisms are unclear. Hh stimulation relieves Smo from inhibition by Ptc, leading to the generation of an intracellular signal that culminates in a nuclear transcriptional response (Figure 1b). When Ptc is removed the pathway is constitutively 'on', independent of the Hh ligand, whereas certain mutations in Smo can activate Hh signaling, bypassing Ptc regulation. A heteromeric receptor model has been proposed, in which Hh interacts directly with Ptc and thereby affects the interaction of Ptc with Smo . "But things look much more complicated than we had earlier thought," says Philip Beachy (Johns Hopkins University School of Medicine, Maryland, USA). "The previous models are not tenable," and he suggests that alternative models of receptor function must be considered.
The power of chemical genetics
Frank-Kamenetsky et al. chose to use chemical genetics in an attempt to identify compounds that could interfere with the inhibition of Smo by Ptc, or could activate Smo independent of Ptc, or that might act downstream of Smo. In addition to making effective drug candidates, these molecules might also help to illuminate the complex mechanisms underlying signaling by Hh, Ptc, and Smo (see the 'Behind the scenes' box for more of the background to the work).
The high-throughput screen was elegantly simple. First, Frank-Kamenetsky et al. identified a cell line that responds well to Hh stimulation. The introduction of a reporter gene (encoding the firefly luciferase protein) that was turned on by Hh signaling permitted screening for compounds that block or induce signaling by monitoring the expression of the luciferase protein (using a simple luminescence test). They had previously used such a screen to isolate antagonists of Hh signaling, and had demonstrated the effectiveness of some antagonists as potential anti-tumor drugs to treat BCC . Their new screen of 140,000 synthetic compounds led to the discovery of a few candidate agonists that could stimulate the reporter gene and mimic Hh activity.
Once the cell-based screen was completed, the chemists took over, synthesizing 300 derivative molecules until they found a few compounds that were related to the previous 'best' agonist but that were a thousand times more effective at eliciting a cellular response, affecting cells when applied in the nanomolar range. "Getting more potent compounds was essential if we were to figure out where the agonists were acting" recalls Jefferey Porter who headed the team at Curis, Inc.
Then the cell biologists began again, studying the effects of the agonists on the proliferation of primary neonatal cerebellar granule neuron precursors. They monitored the incorporation of tritiated thymidine into cultured rat neurons (as a marker of DNA synthesis and hence proliferation) and were pleased to see that the agonists were as effective in this assay as the Hh protein itself. An assay using an explant of embryonic neural plate was used to confirm that the agonists could induce dose-dependent gene expression in neural precursors, just as the Shh protein does.
Having established the effects of the agonists in culture assays, the researchers then turned to an in vivo model, feeding the compounds to pregnant mice and following the effects on the phenotypes of embryos lacking Shh or Smo. The treated embryos displayed activated Hh signaling, demonstrating that the compounds were not toxic and could cross the placental barrier. The developmental defects of Shh-/- embryos were rescued by treatment with the agonist but the compound had no effect on Hh signaling in the absence of Smo.
"Once we knew that the agonists were targeting Smo, we wanted to investigate whether they bound to Smo directly and how they activated Hh signaling," says Porter. The cell line that was created for the screen served as a useful tool to test the effects of known antagonists on the function of the agonist. An anti-Hh blocking antibody had no effect, so the agonist must work downstream of the Hh-Ptc interaction. But the agonist was blocked by antagonists that work at the level of Smo or further downstream. "We have similar conclusions," says Beachy, whose group used photo-affinity labeling and cross-linking experiments to show that small-molecule agonists and antagonists bind directly to Smo.
Next, for Frank-Kamenetsky et al., it was time for some careful biochemistry. Analysis of the expression of fusion proteins of Ptc or Smo showed that, unlike the Hh ligand itself, the agonist had no effect on the stability of the Ptc protein. In contrast, both Hh and the agonist could increase the stability of the Smo receptor. Immunoprecipitation experiments with radiolabeled agonist showed that the agonist must bind directly to Smo receptors, and that Hh-signaling inhibitors compete with the agonist for binding. Pharmokinetic analysis provided evidence for a single binding site competition model. Finally, Frank-Kamenetsky et al. exploited an oncogenic, constitutively active mutant form of Smo (Smoact); the agonists bound equally well to mutant and wild-type forms, whereas the antagonist bound less well to the mutant form.
Learning lessons from drugs
Frank-Kamenetsky et al. have demonstrated convincingly that high-throughput chemical genetics can be used to isolate modulators of a developmental signaling pathway. The agonists that they have generated are efficient and apparently non-toxic mimics of Hh signals and are promising candidates for drugs for regenerative medicine. The authors work at the biotechnology company Curis Inc. (Cambridge, USA), so finding new drugs is obviously their primary objective. But the agonists also provide useful tools for probing the complex Hh-Ptc-Smo signaling pathway.
"A lot of our biological insight is driven by having specific chemical inhibitors, and many drugs are used as tools to dissect signaling systems as a substitute for genetic studies," says Arnon Rosenthal (Rinat Neuroscience Corp., Palo Alto, USA). Beachy agrees: "these compounds, first the plant-derived inhibitor cyclopamine and more recently the agonists, have really helped us to understand Smoothened function." Recent work from Beachy's lab [9,10], demonstrating that Ptc suppresses Smo in catalytic manner, has led to speculation that Ptc may function as a transporter protein pumping natural small-molecule Smo modulators across the cell membrane. Rosenthal adds that "good basic research always leads to better medicines, since the more we understand about the mechanisms operating in the body, the better able we are to modulate them rationally."
The data presented by Frank-Kamenetsky et al.  led them to propose a new model for Hh signaling based on the classic 'ternary complex model' that was developed to describe ligand-induced conformational changes of G-protein-coupled receptors . According to this model, active and inactive conformations of Smo are selected by the binding of agonists or antagonists at independent sites. Furthermore, the model predicts that Smo binds to a novel effector molecule and it raises the possibility that endogenous ligands, analogous to the newly found agonist, may naturally regulate Smo activity.
Cheryll Tickle (University of Dundee, UK) studies the role of Hh in development and finds the possibility that there are endogenous small-molecule agonists that interact with Smoothened particularly intriguing. "This would mean that we are missing a whole layer of control of Hedgehog signaling in our current models," she notes. Porter adds, "It is tempting to speculate that endogenous molecules act directly on Smo, bypassing Ptc. That is consistent with what we know about how G-protein-coupled receptors work. But at the moment, it's pure speculation."
The study by Frank-Kamenetsky et al. exploits a dazzling variety of experimental techniques to illustrate the path from high-throughput screening to compound characterization. Biochemists, cell biologists, pharmacologists and chemists have come together to demonstrate effectively that 'drug discovery' can combine all the excitement of 'real' scientific discovery with the satisfaction of isolating compounds that might be used for tomorrow's therapies.
Frank-Kamenetsky M, Zhang XM, Bottega S, Guicherit O, Wichterle H, Dudek H, Bumcrot D, Wang FY, Jones F, Shulok J, Rubin LL, Porter JA: Small molecule modulators of Hedgehog signaling: identification and characterization of Smoothened agonists and antagonists.
Pepinsky RB, Shapiro RI, Wang S, Chakraborty A, Gill A, Lepage DJ, Wen D, Rayhorn P, Horan GS, Taylor FR, et al.: Long-acting forms of Sonic hedgehog with improved pharmocokinetic and pharmacodynamic properties are efficacious in a nerve injury model.
Williams JA, Guicherit OM, Zaharian BI, Xu Y, Chai L, Gatchalian C, Porter JA, Rubin LL, Wang FY: Identification of novel inhibitors of the hedgehog signaling pathway: effects on basal cell carcinoma-like lesions.