Báo cáo sinh học: "Research news Agonizing."

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Journal BioMed Central of Biology Research news Agonizing Hedgehog Jonathan B Weitzman Published: 6 November 2002 Journal of Biology 2002, 1:7 The electronic version of this article is the complete one and can be found online at http://jbiol.com/content/1/2/7 © 2002 BioMed Central Ltd ISSN 1475-4924 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 segmentation, and it was subsequently events in a range of developing tissues program teaches us something about found to control patterning of struc- (see the ‘Background’ box) [2,3]. the biology of the process that it tures such as the eye and the abdominal Recently, signaling by Hh has been attempts to modulate. The paper by cuticle. In mammals there are three hh shown to be important for patterning Maria Frank-Kamenetsky and col- homologs, called Sonic Hedgehog, of the cerebellum, where it promotes leagues in this issue of the Journal of Indian Hedgehog and Desert Hedgehog the proliferation of granule neuron Biology [1] presents a compelling (Shh, Ihh and Dhh, respectively), which precursors. A link between Hh proteins example of how the search for thera- have been implicated in patterning and stem-cell proliferation has raised peutics can provide powerful experi- mental tools and insights into fundamental biology, blurring the dis- The bottom line tinction between applied and basic research. By characterizing a small • Frank-Kamenetsky and colleagues designed a cell-based, high-throughput group of chemically similar agonists of assay to screen 140,000 compounds to find modulators of the Hedgehog the Hedgehog signaling pathway, signaling pathway. Frank-Kamenetsky et al. have been able to propose a new model for how the • They identified a small group of related synthetic non-peptidyl molecules Smoothened component of the that can act as agonists of Hedgehog signals at nanomolar concentra- Hedgehog-receptor complex works, tions, having previously identified small-molecule Hedgehog antagonists. and to hint at the existence of natural- ligand agonists of the Hedgehog • A range of in vitro and in vivo assays were used to show that the signaling pathway (see ‘The bottom agonists can be used as drugs to overcome Hedgehog-signaling defects line’ box for a summary of their work). and to promote cell proliferation. Hedgehog history • The action of the agonist compounds is independent of the Hedgehog Signaling by the Hedgehog (Hh) ligand and the inhibitory receptor Patched. Further characterization family of secreted proteins plays a revealed that the agonists bind directly to the receptor Smoothened. central role in regulating cell differenti- ation and tissue patterning during • These data give new insights into the nature of Hedgehog-Smoothened development [2]. The hedgehog gene signaling and raise the possibility of analogous endogenous modulators (hh) was first identified by virtue of its of Hh signaling. role in the specification of positional identity during Drosophila embryonic Journal of Biology 2002, 1:7
7.2 Journal of Biology 2002, Volume 1, Issue 2, Article 7 Weitzman http://jbiol.com/content/1/2/7 the enticing possibility that modulating a proto-oncogene and activates signal- (Johns Hopkins University School of Hh signaling might be relevant for the ing in response to Hh ligand. The Smo Medicine, Maryland, USA). “The previ- clinical management of certain degener- protein is a seven-transmembrane ous models are not tenable,” and he ative diseases. Indeed, it was recently receptor that resembles conventional G- suggests that alternative models of protein-coupled receptors (Figure 1a). demonstrated that Shh might be effec- receptor function must be considered. tive in treating peripheral nerve damage It appears that Ptc inhibits Smo, The power of chemical or degenerative brain disorders such as although the precise mechanisms are genetics Parkinson’s disease [4,5]. Perhaps not unclear. Hh stimulation relieves Smo surprisingly given its role in develop- from inhibition by Ptc, leading to the Frank-Kamenetsky et al. [1] chose to ment, misregulated Hh signaling has generation of an intracellular signal use chemical genetics in an attempt to also been implicated in cancer [3]. that culminates in a nuclear transcrip- identify compounds that could inter- Specifically, medulloblastoma and basal tional response (Figure 1b). When Ptc fere with the inhibition of Smo by Ptc, cell carcinoma (BCC) are associated is removed the pathway is constitu- or could activate Smo independent of with inappropriate activation of Hh sig- tively ‘on’, independent of the Hh Ptc, or that might act downstream of naling [6,7]. These observations moti- ligand, whereas certain mutations in Smo. In addition to making effective vated Frank-Kamenetsky and colleagues Smo can activate Hh signaling, bypass- drug candidates, these molecules might to search for small-molecule modula- ing Ptc regulation. A heteromeric also help to illuminate the complex tors of the Hh pathway, with the hope receptor model has been proposed, in mechanisms underlying signaling by that antagonists and agonists might be which Hh interacts directly with Ptc Hh, Ptc, and Smo (see the ‘Behind the used as drugs to treat proliferative or and thereby affects the interaction of scenes’ box for more of the background degenerative diseases, and that small Ptc with Smo [8]. “But things look to the work). molecules might prove more amenable much more complicated than we had The high-throughput screen was to pharmacological delivery than the earlier thought,” says Philip Beachy elegantly simple. First, Frank-Kamenetsky Hh-family proteins themselves. Sending the signal Background Hedgehog signaling challenges the way we normally think about signal • Mammals have three Hedgehog (Hh) proteins (Sonic Hedgehog, transduction pathways. Biology is full Indian Hedgehog and Desert Hedgehog) that are processed to of examples of extracellular ligands generate functional extracellular ligands. Two receptors are involved in that bind to specific cell-surface recep- Hh signaling: Patched (Ptc) is a negative regulator of the Hh-triggered tors, initiating a cascade of biochemi- signaling pathway and Smoothened (Smo) is a positive activator cal events (often involving protein (Hh, Ptc and Smo were all originally named for the cuticular pheno- kinases) that leads to the activation of types of mutant Drosophila larvae). When Hh ligands bind to Ptc they a transcription factor and the induc- relieve the negative inhibition and Smoothened initiates a signaling tion of a set of effector genes. But cascade that results in the activation of nuclear transcription factors of Hedgehog signaling is not so simple. the Gli family that regulate effector gene transcription (see Figure 1). Even the ligand is complicated: the Hh proteins undergo unusual process- • Hh signaling has been implicated in a wide range of developmental ing and cleavage to generate an extra- processes. Small-molecule modulators of Hh signaling are cellular cholesterol-linked peptide potential candidates as therapeutic drugs to treat human diseases – that serves as the signaling ligand [2]. molecules that mimic Hh signaling (agonists) might be used to treat And the receptor component is far degenerative disorders such as Parkinson’s disease, whereas from understood. blockers (antagonists) could be used as drugs against certain types The cellular response to Hh is con- of cancer. trolled by two transmembrane pro- teins, Patched (Ptc) and Smoothened • Chemical genetics uses synthetic small molecules to dissect cellular (Smo). The Ptc protein weaves across functions such as signal transduction pathways. By analogy with loss- the cell membrane twelve times and and gain-of-function mutations in genetics, the functional interactions resembles some transmembrane chan- between chemical inducers and inhibitors is used to define the nels. It acts as a negative regulator of hierarchical relationship between protein components of the pathway. the Hh signal and has been defined as a tumor-suppressor. In contrast, Smo is Journal of Biology 2002, 1:7
http://jbiol.com/content/1/2/7 Journal of Biology 2002, Volume 1, Issue 2, Article 7 Weitzman 7.3 researchers then turned to an in vivo (a) (b) model, feeding the compounds to Hedgehog ligands pregnant mice and following the Dhh Shh Ihh effects on the phenotypes of embryos lacking Shh or Smo. The treated embryos displayed activated Hh sig- naling, demonstrating that the com- pounds were not toxic and could cross the placental barrier. The devel- opmental defects of Shh-/- embryos Membrane were rescued by treatment with the Cytoplasm agonist but the compound had no effect on Hh signaling in the absence Patched Smoothened of Smo. “Once we knew that the agonists Nucleus were targeting Smo, we wanted to Gli investigate whether they bound to Smo directly and how they activated Effector gene Hh signaling,” says Porter. The cell line that was created for the screen served as a useful tool to test the effects of Figure 1 known antagonists on the function of The Hedgehog signaling pathway. (a) In the absence of Hedgehog ligands, Patched protein inhibits the agonist. An anti-Hh blocking anti- 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 body had no effect, so the agonist is freed to trigger an intracellular signaling cascade; this ultimately leads to transcriptional regulation must work downstream of the Hh-Ptc by transcription factors of the Gli family. interaction. But the agonist was blocked by antagonists that work at the level of Smo or further down- et al. identified a cell line that responds effective at eliciting a cellular response, stream. “We have similar conclusions,” well to Hh stimulation. The introduc- affecting cells when applied in the says Beachy, whose group used photo- tion of a reporter gene (encoding the nanomolar range. “Getting more affinity labeling and cross-linking firefly luciferase protein) that was potent compounds was essential if we experiments to show that small- turned on by Hh signaling permitted were to figure out where the agonists molecule agonists and antagonists screening for compounds that block or were acting” recalls Jefferey Porter who bind directly to Smo. induce signaling by monitoring the headed the team at Curis, Inc. Next, for Frank-Kamenetsky et al., it expression of the luciferase protein Then the cell biologists began was time for some careful biochemistry. (using a simple luminescence test). again, studying the effects of the ago- Analysis of the expression of fusion They had previously used such a screen nists on the proliferation of primary proteins of Ptc or Smo showed that, to isolate antagonists of Hh signaling, neonatal cerebellar granule neuron unlike the Hh ligand itself, the agonist and had demonstrated the effective- precursors. They monitored the incor- had no effect on the stability of the Ptc ness of some antagonists as potential poration of tritiated thymidine into protein. In contrast, both Hh and the anti-tumor drugs to treat BCC [7]. cultured rat neurons (as a marker of agonist could increase the stability of Their new screen of 140,000 synthetic DNA synthesis and hence prolifera- the Smo receptor. Immunoprecipita- compounds led to the discovery of a tion) and were pleased to see that the tion experiments with radiolabeled few candidate agonists that could stim- agonists were as effective in this assay agonist showed that the agonist must ulate the reporter gene and mimic as the Hh protein itself. An assay using bind directly to Smo receptors, and Hh activity. an explant of embryonic neural plate that Hh-signaling inhibitors compete Once the cell-based screen was com- was used to confirm that the agonists with the agonist for binding. Pharmo- pleted, the chemists took over, synthe- could induce dose-dependent gene kinetic analysis provided evidence for sizing 300 derivative molecules until expression in neural precursors, just as a single binding site competition they found a few compounds that were the Shh protein does. model. Finally, Frank-Kamenetsky et related to the previous ‘best’ agonist Having established the effects of al. exploited an oncogenic, constitu- but that were a thousand times more the agonists in culture assays, the tively active mutant form of Smo Journal of Biology 2002, 1:7
7.4 Journal of Biology 2002, Volume 1, Issue 2, Article 7 Weitzman http://jbiol.com/content/1/2/7 (Smoact); the agonists bound equally According to this model, active and or antagonists at independent sites. well to mutant and wild-type forms, inactive conformations of Smo are Furthermore, the model predicts that whereas the antagonist bound less well selected by the binding of agonists Smo binds to a novel effector molecule to the mutant form. Learning lessons from drugs Behind the scenes Frank-Kamenetsky et al. have demon- strated convincingly that high-through- Journal of Biology asked Jeff Porter, group leader at Curis Inc., to comment put chemical genetics can be used to on the background to the project to search for small-molecule modulators isolate modulators of a developmental of Hedgehog signaling. signaling pathway. The agonists that they have generated are efficient and What prompted you to set up a screen for agonists of the apparently non-toxic mimics of Hh Hedgehog pathway? signals and are promising candidates There was evidence that manipulating Hedgehog signaling might be useful for drugs for regenerative medicine. in a therapeutic context to treat degenerative neurological diseases. There The authors work at the biotechnology were promising data using modified Hedgehog ligands in animal disease company Curis Inc. (Cambridge, models, but we felt that a small molecule would be a more effective thera- USA), so finding new drugs is obvi- peutic. We had previously set up the cell-based assay to screen for antago- ously their primary objective. But the nists of Hedgehog signaling and successfully isolated inhibitors that could agonists also provide useful tools for inhibit tumor growth. So we adapted the assay to find Hedgehog agonists. probing the complex Hh-Ptc-Smo signaling pathway. What was your initial reaction to the results, and how were they “A lot of our biological insight is received by others? driven by having specific chemical This was a kind of ‘black box’ screen - we were looking for a change in a inhibitors, and many drugs are used biological readout, in contrast to traditional pharmacological screens that as tools to dissect signaling systems as focus on a particular target. So, we couldn’t be sure what type of molecule a substitute for genetic studies,” says we would get; we could have predicted that we’d find inhibitors of Arnon Rosenthal (Rinat Neuroscience Patched or stabilizers of Gli. I was surprised and excited when we realized Corp., Palo Alto, USA). Beachy agrees: that our agonists were targeting Smoothened. Once we had nailed down “these compounds, first the plant- the target, there was a lot of interest in these compounds and what they derived inhibitor cyclopamine and can teach us about Hedgehog signaling. more recently the agonists, have really helped us to understand Smoothened How long did the project take? function.” Recent work from Beachy’s We began the screen in late 1999. It took a few months to get the initial lab [9,10], demonstrating that Ptc compounds, and then we began the process of making slow improve- suppresses Smo in catalytic manner, ments by chemical modification. Without the improvements we couldn’t has led to speculation that Ptc may have figured out what was going on. We had to establish the techniques function as a transporter protein and acquire the reagents necessary to characterize the agonists in detail. pumping natural small-molecule Smo The potent compound derivatives were also key to the success of the modulators across the cell membrane. cell-free binding assays - they look simple but the binding experiments Rosenthal adds that “good basic were very tricky. research always leads to better medi- cines, since the more we understand What are the next steps? about the mechanisms operating in We want to figure out the mechanisms of Smo regulation and signal trans- the body, the better able we are to duction - how Ptc talks to Smo and how Smo talks to Gli. And, of course, modulate them rationally.” we are also testing these molecules as potential therapeutics in animal The data presented by Frank- models of disorders of the central nervous system. The preclinical results Kamenetsky et al. [1] led them to are promising: the compounds show low toxicity, they can be administered propose a new model for Hh signaling orally and cross the blood-brain barrier. These compounds might also be based on the classic ‘ternary complex useful in ex vivo therapies to stimulate stem cell proliferation. I am optimistic model’ that was developed to describe that Hedgehog agonists will be tested in human trials in the future. ligand-induced conformational changes of G-protein-coupled receptors [11]. Journal of Biology 2002, 1:7
http://jbiol.com/content/1/2/7 Journal of Biology 2002, Volume 1, Issue 2, Article 7 Weitzman 7.5 Chen JK, Cooper MK, Taipale J, Olson and it raises the possibility that excitement of ‘real’ scientific discovery JM, Beachy PA: Medulloblastoma endogenous ligands, analogous to the with the satisfaction of isolating growth inhibition by Hedgehog newly found agonist, may naturally compounds that might be used for pathway blockade. Science 2002, 297:1559-1561. regulate Smo activity. tomorrow’s therapies. 7. Williams JA, Guicherit OM, Zaharian BI, Cheryll Tickle (University of Dundee, Xu Y, Chai L, Gatchalian C, Porter JA, References UK) studies the role of Hh in develop- Rubin LL, Wang FY: Identification of 1. Frank-Kamenetsky M, Zhang XM, novel inhibitors of the hedgehog sig- ment and finds the possibility that there Bottega S, Guicherit O, Wichterle H, naling pathway: effects on basal cell are endogenous small-molecule agonists Dudek H, Bumcrot D, Wang FY, Jones F, carcinoma-like lesions. Proc Natl Acad that interact with Smoothened particu- Shulok J, Rubin LL Porter JA: Small Sci USA, in press. molecule modulators of Hedgehog 8. Stone DM, Hynes M, Armanini M, larly intriguing. “This would mean that signaling: identification and charac- Swanson TA, Gu Q, Johnson RL, Scott we are missing a whole layer of control terization of Smoothened agonists MP, Pennica D, Goddard A, Phillips H, et and antagonists. J Biol 2002, 1:10. al.: The tumor-suppressor gene of Hedgehog signaling in our current 2. Ingham PW, McMahon AP: Hedgehog patched encodes a candidate recep- models,” she notes. Porter adds, “It is signaling in animal development; tor for Sonic Hedgehog. Nature 1996, tempting to speculate that endogenous paradigms and principles. Genes Dev 384:129-134. 2001, 15:3059-3087. 9. Chen JK, Taipale J, Young KE, Maiti T, molecules act directly on Smo, bypass- 3. Taipale J, Beachy PA: The Hedgehog Beachy PA: Small molecule modula- ing Ptc. That is consistent with what we and Wnt signaling pathways in tion of Smoothened activity. Proc know about how G-protein-coupled cancer. Nature 2001, 411:349-354. Natl Acad Sci USA 2002, 99:14071-14076. 4. Pepinsky RB, Shapiro RI, Wang S, 10. Taipale J, Cooper MK, Maiti T, Beachy receptors work. But at the moment, it’s Chakraborty A, Gill A, Lepage DJ, Wen PA: Patched acts catalytically to sup- pure speculation.” D, Rayhorn P, Horan GS, Taylor FR, et al.: press the activity of Smoothened. The study by Frank-Kamenetsky et Long-acting forms of Sonic hedge- Nature 2002, 418:892-896. hog with improved pharmocokinetic 11. De Lean A, Stadel JM, Lefkowitz RJ: A al. [1] exploits a dazzling variety of and pharmacodynamic properties ternary complex model explains the experimental techniques to illustrate are efficacious in a nerve injury agonist-specific binding properties the path from high-throughput screen- model. J Pharm Sci 2002, 91:371-387. of the adenylate cyclase-coupled 5. Tsuboi K, Shults CW: Intrastriatal beta-adrenergic receptor. J Biol Chem ing to compound characterization. injection of sonic hedgehog reduces 1980, 255:7108-7117. Biochemists, cell biologists, pharm- behavioural impairment in a rat model of Parkinson’s disease. Exp acologists and chemists have come Neurol 2002, 173:95-104. Jonathan B Weitzman is a scientist and science together to demonstrate effectively that 6. Berman DM, Karhadkar SS, Hallahan AR, writer based in Paris, France. ‘drug discovery’ can combine all the Pritchard JI, Eberhart CG, Watkins DN, E-mail: jonathan.weitzman@hotmail.com Journal of Biology 2002, 1:7