Bio

Academic Appointments


Administrative Appointments


  • Executive Committee Member, Stanford University Chemical Biology Institute (2012 - Present)
  • Steering Committee Member, Stanford University School of Medicine Faculty Senate (2012 - Present)
  • Departmental Representative, Stanford Biosciences Committee on Graduate Admissions and Policy (2012 - Present)
  • Director, Advisory Committee for the Scholarly Concentrations Program in the Molecular Basis of Medicine (2012 - Present)
  • Alternate Member, Stanford University Administrative Panel on Laboratory Animal Care (2012 - Present)
  • Departmental Representative, Stanford University School of Medicine Faculty Senate (2009 - Present)
  • Member, Medical Scientist Training Program Admissions Committee (2004 - Present)
  • Executive Director, Stanford High-Throughput Bioscience Center (2003 - Present)

Honors & Awards


  • NSF INSPIRE Award, NSF (2013-2017)
  • Innovation Award, Alex's Lemonade Stand Foundation (2013-2015)
  • Nature SciCafe Award for Outstanding Research Achievement, Nature Biotechnology and Nature Medicine (2009)
  • NIH Director's Pioneer Award, NIH (2008-2013)
  • American Cancer Society Research Scholar Award, American Cancer Society (2008-2011)
  • Brain Tumor Society Award, Brain Tumor Society/Rachel Molly Markoff Foundation (2006-2008)
  • Astellas USA Foundation Award, Astella USA Foundation (2005-2006)
  • Terman Fellow, Stanford University (2005-2008)
  • Basil O'Connor Starter Scholar Research Award, March of Dimes (2005-2007)
  • Kimmel Scholar Award, Sidney Kimmel Foundation for Cancer Research (2004-2006)
  • W. Barry Wood, Jr. Research Award, Johns Hopkins School of Medicine (2003)
  • American Cancer Society Postdoctoral Fellowship, American Cancer Society (2002-2003)
  • Damon Runyon-Walter Winchell Postdoctoral Fellowship, Damon Runyon Cancer Research Foundation (1999-2002)

Professional Education


  • A.B., Harvard College, Chemistry (1991)
  • Ph.D., Harvard University, Chemistry and Chemical Biology (1999)
  • Postdoctoral Fellow, Johns Hopkins School of Medicine, Molecular Biology and Genetics (2003)

Research & Scholarship

Current Research and Scholarly Interests


The Chen laboratory integrates synthetic chemistry and developmental biology to interrogate the molecular mechanisms that control embryonic patterning, tissue regeneration, and oncogenesis. Our research group is currently focused on three major areas: the identification of small-molecule and genetic regulators of Hedgehog signaling, the development of chemical technologies for perturbing and observing the molecular programs of embryonic patterning and tumorigenesis, and the study of tissue regeneration using zebrafish as a model organism.

Our interest in the Hedgehog pathway arises from its critical role in the patterning of multiple tissues such as the neural tube, craniofacial structures, limbs, and somites. Aberrant Hedgehog pathway activation in children and adults is also linked to several cancers, including those of the skin, brain, and gut. Since the cellular events that transduce the Hedgehog signal from the cell surface to the nucleus are not well understood, we are pursuing genetic and small-molecule screens for new Hedgehog pathway modulators with novel modes of action. These studies will not only provide insights into the basic mechanisms of Hedgehog signal transduction but also provide chemical leads for the development of next-generation chemotherapies and reveal new “druggable” targets within this tumor-promoting pathway.

Our laboratory is also investigating how Hedgehog signaling and other developmental pathways regulate tissue formation and regeneration in vertebrates. We use the zebrafish as a model organism for these studies, exploiting its rapid ex utero development and amenability to real-time imaging. As part of these efforts, we have developed new strategies for activating and silencing gene expression in zebrafish embryos with unprecedented spatial and temporal precision. These methods utilize chemical probes developed by our research group, including caged reagents that allow light-controlled gene silencing in whole organisms. In conjunction with conventional genetic approaches, these chemical technologies will help us elucidate the genetic programs that control vertebrate development and physiology.

Understanding the molecular mechanisms that underlie tissue regeneration is an emerging interest in our laboratory. The zebrafish is an ideal system for studying these processes, since it has the unique ability to regenerate its heart, retina, spinal cord, fins, and other body parts. Our research group is working with Michael Longaker's laboratory to decipher the molecular and cellular events associated with larval tail regeneration, and we have identified several genes that are upregulated or downregulated in posterior cells after the tail is amputated. We are now using an interdisciplinary approach to determine the roles of these genes in the regenerative process.

Teaching

2013-14 Courses


Publications

Journal Articles


  • General method for regulating protein stability with light. ACS chemical biology Bonger, K. M., Rakhit, R., Payumo, A. Y., Chen, J. K., Wandless, T. J. 2014; 9 (1): 111-115

    Abstract

    Post-translational regulation of protein abundance in cells is a powerful tool for studying protein function. Here, we describe a novel genetically encoded protein domain that is degraded upon exposure to nontoxic blue light. We demonstrate that fusion proteins containing this domain are rapidly degraded in cultured cells and in zebrafish upon illumination.

    View details for DOI 10.1021/cb400755b

    View details for PubMedID 24180414

  • Post-transcriptional mechanisms contribute to Etv2 repression during vascular development DEVELOPMENTAL BIOLOGY Moore, J. C., Sheppard-Tindell, S., Shestopalov, I. A., Yamazoe, S., Chen, J. K., Lawson, N. D. 2013; 384 (1): 128-140

    Abstract

    etv2 is an endothelial-specific ETS transcription factor that is essential for vascular differentiation and morphogenesis in vertebrates. While recent data suggest that Etv2 is dynamically regulated during vascular development, little is known about the mechanisms involved in this process. Here, we find that etv2 transcript and protein expression are highly dynamic during zebrafish vascular development, with both apparent during early somitogenesis and subsequently down-regulated as development proceeds. Inducible knockdown of Etv2 in zebrafish embryos prior to mid-somitogenesis stages, but not later, caused severe vascular defects, suggesting a specific role in early commitment of lateral mesoderm to the endothelial linage. Accordingly, Etv2-overexpressing cells showed an enhanced ability to commit to endothelial lineages in mosaic embryos. We further find that the etv2 3' untranslated region (UTR) is capable of repressing an endothelial autonomous transgene and contains binding sites for members of the let-7 family of microRNAs. Ectopic expression of let-7a could repress the etv2 3'UTR in sensor assays and was also able to block endogenous Etv2 protein expression, leading to concomitant reduction of endothelial genes. Finally, we observed that Etv2 protein levels persisted in maternal-zygotic dicer1 mutant embryos, suggesting that microRNAs contribute to its repression during vascular development. Taken together, our results suggest that etv2 acts during early development to specify endothelial lineages and is then down-regulated, in part through post-transcriptional repression by microRNAs, to allow normal vascular development.

    View details for DOI 10.1016/j.ydbio.2013.08.028

    View details for Web of Science ID 000326901100011

    View details for PubMedID 24036310

  • Centrosome repositioning in T cells is biphasic and driven by microtubule end-on capture-shrinkage JOURNAL OF CELL BIOLOGY Yi, J., Wu, X., Chung, A. H., Chen, J. K., Kapoor, T. M., Hammer, J. A. 2013; 202 (5): 779-792

    Abstract

    T cells rapidly reposition their centrosome to the center of the immunological synapse (IS) to drive polarized secretion in the direction of the bound target cell. Using an optical trap for spatial and temporal control over target presentation, we show that centrosome repositioning in Jurkat T cells exhibited kinetically distinct polarization and docking phases and required calcium flux and signaling through both the T cell receptor and integrin to be robust. In "frustrated" conjugates where the centrosome is stuck behind the nucleus, the center of the IS invaginated dramatically to approach the centrosome. Consistently, imaging of microtubules during normal repositioning revealed a microtubule end-on capture-shrinkage mechanism operating at the center of the IS. In agreement with this mechanism, centrosome repositioning was impaired by inhibiting microtubule depolymerization or dynein. We conclude that dynein drives centrosome repositioning in T cells via microtubule end-on capture-shrinkage operating at the center of the IS and not cortical sliding at the IS periphery, as previously thought.

    View details for DOI 10.1083/jcb.201301004

    View details for Web of Science ID 000323870500007

    View details for PubMedID 23979719

  • Diacylglycerol promotes centrosome polarization in T cells via reciprocal localization of dynein and myosin II PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Liu, X., Kapoor, T. M., Chen, J. K., Huse, M. 2013; 110 (29): 11976-11981

    Abstract

    Centrosome reorientation to the immunological synapse maintains the specificity of T-cell effector function by facilitating the directional release of cytokines and cytolytic factors toward the antigen-presenting cell. This polarization response is driven by the localized accumulation of diacylglycerol, which recruits multiple protein kinase (PK)C isozymes to the synaptic membrane. Here, we used T-cell receptor (TCR) photoactivation and imaging methodology to demonstrate that PKCs control centrosome dynamics through the reciprocal localization of two motor complexes, dynein and nonmuscle myosin (NM)II. Dynein accumulated in the region of TCR stimulation, whereas NMII clustered in the back of the cell, behind the polarizing centrosome. PKC activity, which shaped both dynein and NMII accumulation within this framework, controlled NMII localization directly by phosphorylating inhibitory sites within the myosin regulatory light chain, thereby suppressing NMII clustering in the region of TCR stimulation. Concurrently, phosphorylation of distinct sites within myosin regulatory light chain by Rho kinase drove NMII clustering in areas behind the centrosome. These results reveal a role for NMII in T-cell polarity and demonstrate how it is regulated by upstream signals.

    View details for DOI 10.1073/pnas.1306180110

    View details for Web of Science ID 000322086100071

    View details for PubMedID 23818610

  • Functional inhibition of UQCRB suppresses angiogenesis in zebrafish. Biochemical and biophysical research communications Cho, Y. S., Jung, H. J., Seok, S. H., Payumo, A. Y., Chen, J. K., Kwon, H. J. 2013; 433 (4): 396-400

    Abstract

    As a subunit of mitochondrial complex III, UQCRB plays an important role in complex III stability, electron transport, and cellular oxygen sensing. Herein, we report UQCRB function regarding angiogenesis in vivo with the zebrafish (Danio rerio). UQCRB knockdown inhibited angiogenesis in zebrafish leading to the suppression of VEGF expression. Moreover, the UQCRB-targeting small molecule terpestacin also inhibited angiogenesis and VEGF levels in zebrafish, supporting the role of UQCRB in angiogenesis. Collectively, UQCRB loss of function by either genetic and pharmacological means inhibited angiogenesis, indicating that UQCRB plays a key role in this process and can be a prognostic marker of angiogenesis- and mitochondria-related diseases.

    View details for DOI 10.1016/j.bbrc.2013.02.082

    View details for PubMedID 23454382

  • Emerging technologies in molecular imaging: new windows into biology. Current opinion in chemical biology Chen, J. K., Kikuchi, K. 2013

    View details for PubMedID 23886981

  • Small-molecule inhibitors of the AAA plus ATPase motor cytoplasmic dynein NATURE Firestone, A. J., Weinger, J. S., Maldonado, M., Barlan, K., Langston, L. D., O'Donnell, M., Gelfand, V. I., Kapoor, T. M., Chen, J. K. 2012; 484 (7392): 125-129

    Abstract

    The conversion of chemical energy into mechanical force by AAA+ (ATPases associated with diverse cellular activities) ATPases is integral to cellular processes, including DNA replication, protein unfolding, cargo transport and membrane fusion. The AAA+ ATPase motor cytoplasmic dynein regulates ciliary trafficking, mitotic spindle formation and organelle transport, and dissecting its precise functions has been challenging because of its rapid timescale of action and the lack of cell-permeable, chemical modulators. Here we describe the discovery of ciliobrevins, the first specific small-molecule antagonists of cytoplasmic dynein. Ciliobrevins perturb protein trafficking within the primary cilium, leading to their malformation and Hedgehog signalling blockade. Ciliobrevins also prevent spindle pole focusing, kinetochore-microtubule attachment, melanosome aggregation and peroxisome motility in cultured cells. We further demonstrate the ability of ciliobrevins to block dynein-dependent microtubule gliding and ATPase activity in vitro. Ciliobrevins therefore will be useful reagents for studying cellular processes that require this microtubule motor and may guide the development of additional AAA+ ATPase superfamily inhibitors.

    View details for DOI 10.1038/nature10936

    View details for Web of Science ID 000302343400047

    View details for PubMedID 22425997

  • The BAH domain of ORC1 links H4K20me2 to DNA replication licensing and Meier-Gorlin syndrome NATURE Kuo, A. J., Song, J., Cheung, P., Ishibe-Murakami, S., Yamazoe, S., Chen, J. K., Patel, D. J., Gozani, O. 2012; 484 (7392): 115-?

    Abstract

    The recognition of distinctly modified histones by specialized 'effector' proteins constitutes a key mechanism for transducing molecular events at chromatin to biological outcomes. Effector proteins influence DNA-templated processes, including transcription, DNA recombination and DNA repair; however, no effector functions have yet been identified within the mammalian machinery that regulate DNA replication. Here we show that ORC1--a component of ORC (origin of replication complex), which mediates pre-DNA replication licensing--contains a bromo adjacent homology (BAH) domain that specifically recognizes histone H4 dimethylated at lysine 20 (H4K20me2). Recognition of H4K20me2 is a property common to BAH domains present within diverse metazoan ORC1 proteins. Structural studies reveal that the specificity of the BAH domain for H4K20me2 is mediated by a dynamic aromatic dimethyl-lysine-binding cage and multiple intermolecular contacts involving the bound peptide. H4K20me2 is enriched at replication origins, and abrogating ORC1 recognition of H4K20me2 in cells impairs ORC1 occupancy at replication origins, ORC chromatin loading and cell-cycle progression. Mutation of the ORC1 BAH domain has been implicated in the aetiology of Meier-Gorlin syndrome (MGS), a form of primordial dwarfism, and ORC1 depletion in zebrafish results in an MGS-like phenotype. We find that wild-type human ORC1, but not ORC1-H4K20me2-binding mutants, rescues the growth retardation of orc1 morphants. Moreover, zebrafish depleted of H4K20me2 have diminished body size, mirroring the phenotype of orc1 morphants. Together, our results identify the BAH domain as a novel methyl-lysine-binding module, thereby establishing the first direct link between histone methylation and the metazoan DNA replication machinery, and defining a pivotal aetiological role for the canonical H4K20me2 mark, via ORC1, in primordial dwarfism.

    View details for DOI 10.1038/nature10956

    View details for Web of Science ID 000302343400045

    View details for PubMedID 22398447

  • Spatiotemporal resolution of the Ntla transcriptome in axial mesoderm development NATURE CHEMICAL BIOLOGY Shestopalov, I. A., Pitt, C. L., Chen, J. K. 2012; 8 (3): 270-276

    Abstract

    Transcription factors have diverse roles during embryonic development, combinatorially controlling cellular states in a spatially and temporally defined manner. Resolving the dynamic transcriptional profiles that underlie these patterning processes is essential for understanding embryogenesis at the molecular level. Here we show how temporal, tissue-specific changes in embryonic transcription factor function can be discerned by integrating caged morpholino oligonucleotides with photoactivatable fluorophores, fluorescence-activated cell sorting and microarray technologies. As a proof of principle, we have dynamically profiled No tail a (Ntla)-dependent genes at different stages of axial mesoderm development in zebrafish, discovering discrete sets of transcripts that are coincident with either notochord cell fate commitment or differentiation. Our studies reveal new regulators of notochord development and the sequential activation of distinct transcriptomes within a cell lineage by a single transcriptional factor and demonstrate how optically controlled chemical tools can dissect developmental processes with spatiotemporal precision.

    View details for DOI 10.1038/NCHEMBIO.772

    View details for Web of Science ID 000300600000012

    View details for PubMedID 22286130

  • Cyclic Caged Morpholinos: Conformationally Gated Probes of Embryonic Gene Function ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Yamazoe, S., Shestopalov, I. A., Provost, E., Leach, S. D., Chen, J. K. 2012; 51 (28): 6908-6911

    Abstract

    Feeling a bit cagey: morpholino-based antisense reagents have been caged through oligonucleotide cyclization, enabling photocontrol of gene expression in zebrafish embryos and larvae. Using these reagents, the timing of exocrine cell fate commitment in the developing pancreas has been examined.

    View details for DOI 10.1002/anie.201201690

    View details for Web of Science ID 000305990800017

    View details for PubMedID 22689470

  • Roles of Hedgehog pathway components and retinoic acid signalling in specifying zebrafish ventral spinal cord neurons DEVELOPMENT England, S., Batista, M. F., Mich, J. K., Chen, J. K., Lewis, K. E. 2011; 138 (23): 5121-5134

    Abstract

    In mouse, Hedgehog (Hh) signalling is required for most ventral spinal neurons to form. Here, we analyse the spinal cord phenotype of zebrafish maternal-zygotic smoothened (MZsmo) mutants that completely lack Hh signalling. We find that most V3 domain cells and motoneurons are lost, whereas medial floorplate still develops normally and V2, V1 and V0v cells form in normal numbers. This phenotype resembles that of mice that lack both Hh signalling and Gli repressor activity. Ventral spinal cord progenitor domain transcription factors are not expressed at 24 hpf in zebrafish MZsmo mutants. However, pMN, p2 and p1 domain markers are expressed at early somitogenesis stages in these mutants. This suggests that Gli repressor activity does not extend into zebrafish ventral spinal cord at these stages, even in the absence of Hh signalling. Consistent with this, ectopic expression of Gli3R represses ventral progenitor domain expression at these early stages and knocking down Gli repressor activity rescues later expression. We investigated whether retinoic acid (RA) signalling specifies ventral spinal neurons in the absence of Hh signalling. The results suggest that RA is required for the correct number of many different spinal neurons to form. This is probably mediated, in part, by an effect on cell proliferation. However, V0v, V1 and V2 cells are still present, even in the absence of both Hh and RA signalling. We demonstrate that Gli1 has a Hh-independent role in specifying most of the remaining motoneurons and V3 domain cells in embryos that lack Hh signalling, but removal of Gli1 activity does not affect more dorsal neurons.

    View details for DOI 10.1242/dev.066159

    View details for Web of Science ID 000296769200008

    View details for PubMedID 22069186

  • Hedgehog and retinoic acid signaling cooperate to promote motoneurogenesis in zebrafish DEVELOPMENT Mich, J. K., Chen, J. K. 2011; 138 (23): 5113-5119

    Abstract

    The precise requirements of Hedgehog (Hh) pathway activity in vertebrate central nervous system development remain unclear, particularly in organisms with both maternally and zygotically derived signaling. Here we describe the motoneural phenotype of zebrafish that lack maternal and zygotic contributions of the Hh signaling transducer Smoothened (MZsmo mutants) and therefore are completely devoid of ligand-dependent pathway activation. Some functional primary motoneurons (PMNs) persist in the absence of Hh signaling, and we find that their induction requires both basal Gli transcription factor activity and retinoic acid (RA) signaling. We also provide evidence that RA pathway activation can modulate Gli function in a Hh ligand-independent manner. These findings support a model in which Hh and RA signaling cooperate to promote PMN cell fates in zebrafish.

    View details for DOI 10.1242/dev.066225

    View details for Web of Science ID 000296769200007

    View details for PubMedID 22069185

  • Neuropilins are positive regulators of Hedgehog signal transduction GENES & DEVELOPMENT Hillman, R. T., Feng, B. Y., Ni, J., Woo, W., Milenkovic, L., Gephart, M. G., Teruel, M. N., Oro, A. E., Chen, J. K., Scott, M. P. 2011; 25 (22): 2333-2346

    Abstract

    The Hedgehog (Hh) pathway is essential for vertebrate embryogenesis, and excessive Hh target gene activation can cause cancer in humans. Here we show that Neuropilin 1 (Nrp1) and Nrp2, transmembrane proteins with roles in axon guidance and vascular endothelial growth factor (VEGF) signaling, are important positive regulators of Hh signal transduction. Nrps are expressed at times and locations of active Hh signal transduction during mouse development. Using cell lines lacking key Hh pathway components, we show that Nrps mediate Hh transduction between activated Smoothened (Smo) protein and the negative regulator Suppressor of Fused (SuFu). Nrp1 transcription is induced by Hh signaling, and Nrp1 overexpression increases maximal Hh target gene activation, indicating the existence of a positive feedback circuit. The regulation of Hh signal transduction by Nrps is conserved between mammals and bony fish, as we show that morpholinos targeting the Nrp zebrafish ortholog nrp1a produce a specific and highly penetrant Hh pathway loss-of-function phenotype. These findings enhance our knowledge of Hh pathway regulation and provide evidence for a conserved nexus between Nrps and this important developmental signaling system.

    View details for DOI 10.1101/gad.173054.111

    View details for Web of Science ID 000297154700003

    View details for PubMedID 22051878

  • A crucial requirement for Hedgehog signaling in small cell lung cancer NATURE MEDICINE Park, K., Martelotto, L. G., Peifer, M., Sos, M. L., Karnezis, A. N., Mahjoub, M. R., Bernard, K., Conklin, J. F., Szczepny, A., Yuan, J., Guo, R., Ospina, B., Falzon, J., Bennett, S., Brown, T. J., Markovic, A., Devereux, W. L., Ocasio, C. A., Chen, J. K., Stearns, T., Thomas, R. K., Dorsch, M., Buonamici, S., Watkins, D. N., Peacock, C. D., Sage, J. 2011; 17 (11): 1504-U1506

    Abstract

    Small-cell lung cancer (SCLC) is an aggressive neuroendocrine subtype of lung cancer for which there is no effective treatment. Using a mouse model in which deletion of Rb1 and Trp53 in the lung epithelium of adult mice induces SCLC, we found that the Hedgehog signaling pathway is activated in SCLC cells independently of the lung microenvironment. Constitutive activation of the Hedgehog signaling molecule Smoothened (Smo) promoted the clonogenicity of human SCLC in vitro and the initiation and progression of mouse SCLC in vivo. Reciprocally, deletion of Smo in Rb1 and Trp53-mutant lung epithelial cells strongly suppressed SCLC initiation and progression in mice. Furthermore, pharmacological blockade of Hedgehog signaling inhibited the growth of mouse and human SCLC, most notably following chemotherapy. These findings show a crucial cell-intrinsic role for Hedgehog signaling in the development and maintenance of SCLC and identify Hedgehog pathway inhibition as a therapeutic strategy to slow the progression of disease and delay cancer recurrence in individuals with SCLC.

    View details for DOI 10.1038/nm.2473

    View details for Web of Science ID 000296779300043

    View details for PubMedID 21983857

  • A Small-Molecule Smoothened Agonist Prevents Glucocorticoid-Induced Neonatal Cerebellar Injury SCIENCE TRANSLATIONAL MEDICINE Heine, V. M., Griveau, A., Chapin, C., Ballard, P. L., Chen, J. K., Rowitch, D. H. 2011; 3 (105)

    Abstract

    Glucocorticoids are used for treating preterm neonatal infants suffering from life-threatening lung, airway, and cardiovascular conditions. However, several studies have raised concerns about detrimental effects of postnatal glucocorticoid administration on the developing brain leading to cognitive impairment, cerebral palsy, and hypoplasia of the cerebellum, a brain region critical for coordination of movement and higher-order neurological functions. Previously, we showed that glucocorticoids inhibit Sonic hedgehog-Smoothened (Shh-Smo) signaling, the major mitogenic pathway for cerebellar granule neuron precursors. Conversely, activation of Shh-Smo in transgenic mice protects against glucocorticoid-induced neurotoxic effects through induction of the 11?-hydroxysteroid dehydrogenase type 2 (11?-HSD2) pathway. Here, we show that systemic administration of a small-molecule agonist of the Shh-Smo pathway (SAG) prevented the neurotoxic effects of glucocorticoids. SAG did not interfere with the beneficial effects of glucocorticoids on lung maturation, and despite the known associations of the Shh pathway with neoplasia, we found that transient (1-week-long) SAG treatment of neonatal animals was well tolerated and did not promote tumor formation. These findings suggest that a small-molecule agonist of Smo has potential as a neuroprotective agent in neonates at risk for glucocorticoid-induced neonatal cerebellar injury.

    View details for DOI 10.1126/scitranslmed.3002731

    View details for Web of Science ID 000296565100006

    View details for PubMedID 22013124

  • Chemical 'Jekyll and Hyde's: small-molecule inhibitors of developmental signaling pathways CHEMICAL SOCIETY REVIEWS Sakata, T., Chen, J. K. 2011; 40 (8): 4318-4331

    Abstract

    Small molecules that perturb developmental signaling pathways can have devastating effects on embryonic patterning, as evidenced by the chemically induced onset of cyclopic lambs and children with severely shortened limbs during the 1950s. Recent studies, however, have revealed critical roles for these pathways in human disorders and diseases, spurring the re-examination of these compounds as new targeted therapies. In this tutorial review, we describe four case studies of teratogenic compounds, including inhibitors of the Hedgehog (Hh), Wnt, and bone morphogenetic protein (BMP) pathways. We discuss how these teratogens were discovered, their mechanisms of action, their utility as molecular probes, and their potential as therapeutic agents. We also consider current challenges in the field and possible directions for future research.

    View details for DOI 10.1039/c1cs15019g

    View details for Web of Science ID 000292984900007

    View details for PubMedID 21505654

  • Spatiotemporal Control of Embryonic Gene Expression Using Caged Morpholinos ZEBRAFISH: GENETICS, GENOMICS AND INFORMATICS, 3RD EDITION Shestopalov, I. A., Chen, J. K. 2011; 104: 151-172

    Abstract

    Embryonic development depends on spatial and temporal control of gene function, and deciphering the molecular mechanisms that underlie pattern formation requires methods for perturbing gene expression with similar precision. Emerging chemical technologies can enable such perturbations, as exemplified by the use of caged morpholino (cMO) oligonucleotides to photo-inactivate genes in zebrafish embryos with spatiotemporal control. This chapter describes general principles for cMO design and methods for cMO assembly in three steps from commercially available reagents. Experimental techniques for the microinjection and photoactivation of these reagents are described in detail, as well as the preparation and application of caged fluorescein dextran (cFD) for labeling irradiated cells. Using these protocols, cMOs can be effective tools for functional genomic studies in zebrafish and other model organisms.

    View details for DOI 10.1016/B978-0-12-374814-0.00009-4

    View details for Web of Science ID 000295551800009

    View details for PubMedID 21924162

  • Lineage labeling of zebrafish cells with laser uncagable fluorescein dextran. Journal of visualized experiments : JoVE Clanton, J. A., Shestopalov, I., Chen, J. K., Gamse, J. T. 2011

    Abstract

    A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling and pressure injection of traceable enzymes to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularity attractive tools for lineage labeling and to observe the migration of cells in vivo. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequently, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

    View details for DOI 10.3791/2672

    View details for PubMedID 21559005

  • Synthetic Strategies for Studying Embryonic Development CHEMISTRY & BIOLOGY Ouyang, X., Chen, J. K. 2010; 17 (6): 590-606

    Abstract

    Developmental biology has evolved from a descriptive science to one based on genetic principles and molecular mechanisms. Although molecular biology and genetic technologies have been the primary drivers of this transformation, synthetic strategies have been increasingly utilized to interrogate the mechanisms of embryonic patterning with spatial and temporal precision. In this review, we survey how chemical tools and engineered proteins have been used to perturb developmental processes at the DNA, RNA, protein, and cellular levels. We discuss the design principles, experimental capabilities, and limitations of each method, as well as future challenges for the chemical and developmental biology communities.

    View details for DOI 10.1016/j.chembiol.2010.04.013

    View details for Web of Science ID 000279616700007

    View details for PubMedID 20609409

  • Oligonucleotide-Based Tools for Studying Zebrafish Development ZEBRAFISH Shestopalov, I. A., Chen, J. K. 2010; 7 (1): 31-40

    Abstract

    Synthetic and nonnatural oligonucleotides have been used extensively to interrogate gene function in zebrafish. In this review, we survey the capabilities and limitations of various oligonucleotide-based technologies for perturbing RNA function and tracking RNA expression. We also examine recent strategies for achieving spatiotemporal control of oligonucleotide function, particularly light-gated technologies that exploit the optical transparency of zebrafish embryos.

    View details for DOI 10.1089/zeb.2010.0650

    View details for Web of Science ID 000277029700005

    View details for PubMedID 20392138

  • Controlling Destiny through Chemistry: Small-Molecule Regulators of Cell Fate ACS CHEMICAL BIOLOGY Firestone, A. J., Chen, J. K. 2010; 5 (1): 15-34

    Abstract

    Controlling cell fate is essential for embryonic development, tissue regeneration, and the prevention of human disease. With each cell in the human body sharing a common genome, achieving the appropriate spectrum of stem cells and their differentiated lineages requires the selective activation of developmental signaling pathways, the expression of specific target genes, and the maintenance of these cellular states through epigenetic mechanisms. Small molecules that target these regulatory processes are therefore valuable tools for probing and manipulating the molecular mechanisms by which stem cells self-renew, differentiate, and arise from somatic cell reprogramming. Pharmacological modulators of cell fate could also help remediate human diseases caused by dysregulated cell proliferation or differentiation, heralding a new era in molecular therapeutics.

    View details for DOI 10.1021/cb900249y

    View details for Web of Science ID 000273653800002

    View details for PubMedID 20000447

  • Versatile Synthesis and Rational Design of Caged Morpholinos JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Ouyang, X., Shestopalov, I. A., Sinha, S., Zheng, G., Pitt, C. L., Li, W., Olson, A. J., Chen, J. K. 2009; 131 (37): 13255-13269

    Abstract

    Embryogenesis is regulated by genetic programs that are dynamically executed in a stereotypic manner, and deciphering these molecular mechanisms requires the ability to control embryonic gene function with similar spatial and temporal precision. Chemical technologies can enable such genetic manipulations, as exemplified by the use of caged morpholino (cMO) oligonucleotides to inactivate genes in zebrafish and other optically transparent organisms with spatiotemporal control. Here we report optimized methods for the design and synthesis of hairpin cMOs incorporating a dimethoxynitrobenzyl (DMNB)-based bifunctional linker that permits cMO assembly in only three steps from commercially available reagents. Using this simplified procedure, we have systematically prepared cMOs with differing structural configurations and investigated how the in vitro thermodynamic properties of these reagents correlate with their in vivo activities. Through these studies, we have established general principles for cMO design and successfully applied them to several developmental genes. Our optimized synthetic and design methodologies have also enabled us to prepare a next-generation cMO that contains a bromohydroxyquinoline (BHQ)-based linker for two-photon uncaging. Collectively, these advances establish the generality of cMO technologies and will facilitate the application of these chemical probes in vivo for functional genomic studies.

    View details for DOI 10.1021/ja809933h

    View details for Web of Science ID 000270186500036

    View details for PubMedID 19708646

  • Small-molecule inhibitors reveal multiple strategies for Hedgehog pathway blockade PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hyman, J. M., Firestone, A. J., Heine, V. M., Zhao, Y., Ocasio, C. A., Han, K., Sun, M., Rack, P. G., Sinha, S., Wu, J. J., Solow-Cordero, D. E., Jiang, J., Rowitch, D. H., Chen, J. K. 2009; 106 (33): 14132-14137

    Abstract

    Inappropriate activation of the Hedgehog (Hh) signaling pathway has been implicated in a diverse spectrum of cancers, and its pharmacological blockade has emerged as an anti-tumor strategy. While nearly all known Hh pathway antagonists target the transmembrane protein Smoothened (Smo), small molecules that suppress downstream effectors could more comprehensively remediate Hh pathway-dependent tumors. We report here four Hh pathway antagonists that are epistatic to the nucleocytoplasmic regulator Suppressor of Fused [Su(fu)], including two that can inhibit Hh target gene expression induced by overexpression of the Gli transcription factors. Each inhibitor has a unique mechanism of action, and their phenotypes reveal that Gli processing, Gli activation, and primary cilia formation are pharmacologically targetable. We further establish the ability of certain compounds to block the proliferation of cerebellar granule neuron precursors expressing an oncogenic form of Smo, and we demonstrate that Hh pathway inhibitors can have tissue-specific activities. These antagonists therefore constitute a valuable set of chemical tools for interrogating downstream Hh signaling mechanisms and for developing chemotherapies against Hh pathway-related cancers.

    View details for DOI 10.1073/pnas.0907134106

    View details for Web of Science ID 000269078700091

    View details for PubMedID 19666565

  • Converse Conformational Control of Smoothened Activity by Structurally Related Small Molecules JOURNAL OF BIOLOGICAL CHEMISTRY Yang, H., Xiang, J., Wang, N., Zhao, Y., Hyman, J., Li, S., Jiang, J., Chen, J. K., Yang, Z., Lin, S. 2009; 284 (31): 20876-20884

    Abstract

    The seven-pass transmembrane protein Smoothened (Smo) is an essential component of the Hedgehog (Hh) signaling pathway that is critically involved in normal animal development as well as pathological malignancies. In studying Hh-related biological processes, it would be highly desirable if Smo activity could be instantly switched between activation and inhibition. Using Gli1-dependent GFP transgenic zebrafish and in vitro biochemical assays, we identified and characterized two potent Smo inhibitors, SANT74 and 75 (Smoothened antagonist 74 and 75), by screening a small molecule library designed based on the scaffold of Smo agonist SAG. These compounds are structural analogs of SAG with the methyl group substituted by a propyl or allyl group in SANTs. We show that SANTs and SAG exert opposite effects on Smo activity by regulating protein conformation. Our study represents the first demonstration of conformational regulation of Smo by small molecule analogs, and the combinational use of these Smo modulators in a temporal controlled fashion should be useful for studying Hh biology.

    View details for DOI 10.1074/jbc.M807648200

    View details for Web of Science ID 000268316100051

    View details for PubMedID 19366682

  • Germ cell migration in zebrafish is cyclopamine-sensitive but Smoothened-independent DEVELOPMENTAL BIOLOGY Mich, J. K., Blaser, H., Thomas, N. A., Firestone, A. J., Yelon, D., Raz, E., Chen, J. K. 2009; 328 (2): 342-354

    Abstract

    Primordial germ cells (PGCs) are the progenitors of reproductive cells in metazoans and are an important model for the study of cell migration in vivo. Previous reports have suggested that Hedgehog (Hh) protein acts as a chemoattractant for PGC migration in the Drosophila embryo and that downstream signaling proteins such as Patched (Ptc) and Smoothened (Smo) are required for PGC localization to somatic gonadal precursors. Here we interrogate whether Hh signaling is required for PGC migration in vertebrates, using the zebrafish as a model system. We find that cyclopamine, an inhibitor of Hh signaling, causes strong defects in the migration of PGCs in the zebrafish embryo. However, these defects are not due to inhibition of Smoothened (Smo) by cyclopamine; rather, we find that neither maternal nor zygotic Smo is required for PGC migration in the zebrafish embryo. Cyclopamine instead acts independently of Smo to decrease the motility of zebrafish PGCs, in part by dysregulating cell adhesion and uncoupling cell polarization and translocation. These results demonstrate that Hh signaling is not required for zebrafish PGC migration, and underscore the importance of regulated cell-cell adhesion for cell migration in vivo.

    View details for DOI 10.1016/j.ydbio.2009.01.036

    View details for Web of Science ID 000265239100015

    View details for PubMedID 19389352

  • A small molecule that binds Hedgehog and blocks its signaling in human cells NATURE CHEMICAL BIOLOGY Stanton, B. Z., Peng, L. F., Maloof, N., Nakai, K., Wang, X., Duffner, J. L., Taveras, K. M., Hyman, J. M., Lee, S. W., Koehler, A. N., Chen, J. K., Fox, J. L., Mandinova, A., Schreiber, S. L. 2009; 5 (3): 154-156

    Abstract

    Small-molecule inhibition of extracellular proteins that activate membrane receptors has proven to be extremely challenging. Diversity-oriented synthesis and small-molecule microarrays enabled the discovery of robotnikinin, a small molecule that binds the extracellular Sonic hedgehog (Shh) protein and blocks Shh signaling in cell lines, human primary keratinocytes and a synthetic model of human skin. Shh pathway activity is rescued by small-molecule agonists of Smoothened, which functions immediately downstream of the Shh receptor Patched.

    View details for DOI 10.1038/nchembio.142

    View details for Web of Science ID 000263556100012

    View details for PubMedID 19151731

  • The Imidazopyridine Derivative JK184 Reveals Dual Roles for Microtubules in Hedgehog Signaling ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Cupido, T., Rack, P. G., Firestone, A. J., Hyman, J. M., Han, K., Sinha, S., Ocasio, C. A., Chen, J. K. 2009; 48 (13): 2321-2324

    Abstract

    Eradicating hedgehogs: The title molecule has been previously identified as a potent inhibitor of the Hedgehog signaling pathway, which gives embryonic cells information needed to develop properly. This molecule is shown to modulate Hedgehog target gene expression by depolymerizing microtubules, thus revealing dual roles of the cytoskeleton in pathway regulation (see figure).

    View details for DOI 10.1002/anie.200805666

    View details for Web of Science ID 000264784400011

    View details for PubMedID 19222062

  • The decoupling of Smoothened from G alpha(i) proteins has little effect on Gli3 protein processing and Hedgehog-regulated chick neural tube patterning DEVELOPMENTAL BIOLOGY Low, W., Wang, C., Pan, Y., Huang, X., Chen, J. K., Wang, B. 2008; 321 (1): 188-196

    Abstract

    The Hedgehog (Hh) signal is transmitted by two receptor molecules, Patched (Ptc) and Smoothened (Smo). Ptc suppresses Smo activity, while Hh binds Ptc and alleviates the suppression, which results in activation of Hh targets. Smo is a seven-transmembrane protein with a long carboxyl terminal tail. Vertebrate Smo has been previously shown to be coupled to Galpha(i) proteins, but the biological significance of the coupling in Hh signal transduction is not clear. Here we show that although inhibition of Galpha(i) protein activity appears to significantly reduce Hh pathway activity in Ptc(-/-) mouse embryonic fibroblasts and the NIH3T3-based Shh-light cells, it fails to derepress Shh- or a Smo-agonist-induced inhibition of Gli3 protein processing, a known in vivo indicator of Hh signaling activity. The inhibition of Galpha(i) protein activity also cannot block the Sonic Hedgehog (Shh)-dependent specification of neural progenitor cells in the neural tube. Consistent with these results, overexpression of a constitutively active Galpha(i) protein, Galpha(i2)QL, cannot ectopically specify the neural cell types in the spinal cord, whereas an active Smo, SmoM2, can. Thus, our results indicate that the Smo-induced Galpha(i) activity plays an insignificant role in the regulation of Gli3 processing and Shh-regulated neural tube patterning.

    View details for DOI 10.1016/j.ydbio.2008.06.014

    View details for Web of Science ID 000258710800016

    View details for PubMedID 18590719

  • Targeted and Conditional Gene Expression Workshop, 8th International Conference on Zebrafish Development and Genetics. Zebrafish Chen, J. K., Hurlstone, A. F. 2008; 5 (3): 193-195

    View details for PubMedID 20798783

  • Chemical technologies for probing embryonic development CHEMICAL SOCIETY REVIEWS Shestopalov, I. A., Chen, J. K. 2008; 37 (7): 1294-1307

    Abstract

    Embryogenesis is a remarkable program of cell proliferation, migration, and differentiation that transforms a single fertilized egg into a complex multicellular organism. Understanding this process at the molecular and systems levels will require an interdisciplinary approach, including the concepts and technologies of chemical biology. This tutorial review provides an overview of chemical tools that have been used in developmental biology research, focusing on methods that enable spatiotemporal control of gene function and the visualization of embryonic patterning. Limitations of current approaches and future challenges are also discussed.

    View details for DOI 10.1039/b703023c

    View details for Web of Science ID 000257018800002

    View details for PubMedID 18568156

  • Gene regulation technologies in zebrafish MOLECULAR BIOSYSTEMS Esengil, H., Chen, J. K. 2008; 4 (4): 300-308

    Abstract

    Achieving the potential of zebrafish models in biomedical research is contingent on the development of reverse-genetic resources. This review describes current technologies for genetic perturbations in zebrafish, including an ecdysone receptor-based system that permits conditional transgene expression. Such methodologies promise to enable new zebrafish models for interrogating human physiology and disease.

    View details for DOI 10.1039/b718447f

    View details for Web of Science ID 000254162200004

    View details for PubMedID 18354783

  • Light-controlled gene silencing in zebrafish embryos NATURE CHEMICAL BIOLOGY Shestopalov, I. A., Sinha, S., Chen, J. K. 2007; 3 (10): 650-651

    Abstract

    Functional genomic studies in zebrafish frequently use synthetic oligonucleotides called morpholinos that block RNA splicing or translation. However, the constitutive activity of these reagents limits their experimental utility. We report here the synthesis of a photoactivatable morpholino targeting the no tail (ntl) gene. This caged reagent permits spatiotemporal gene regulation in vivo and the photochemical generation of functionally mosaic organisms.

    View details for DOI 10.1038/nchembio.2007.30

    View details for Web of Science ID 000249642700013

    View details for PubMedID 17717538

  • Small-molecule regulation of zebrafish gene expression NATURE CHEMICAL BIOLOGY Esengil, H., Chang, V., Mich, J. K., Chen, J. K. 2007; 3 (3): 154-155

    Abstract

    The zebrafish has emerged as a versatile model organism for biomedical research, yet its potential has been limited by a lack of conditional reverse-genetic tools. Here we report a chemically inducible gene expression technology that has orthogonality to vertebrate signaling processes, high induction levels, and rapid kinetics. Coupled with tissue-specific promoters, this system provides multidimensional control of gene expression and will enable new models of human disorders and diseases.

    View details for DOI 10.1038/nchembio858

    View details for Web of Science ID 000244336600010

    View details for PubMedID 17237798

  • Smoothened signal transduction is promoted by G protein-coupled receptor kinase 2 MOLECULAR AND CELLULAR BIOLOGY Meloni, A. R., Fralish, G. B., Kelly, P., Salahpour, A., Chen, J. K., Wechsler-Reya, R. J., Lefkowitz, R. J., Caron, M. G. 2006; 26 (20): 7550-7560

    Abstract

    Deregulation of the Sonic hedgehog pathway has been implicated in an increasing number of human cancers. In this pathway, the seven-transmembrane (7TM) signaling protein Smoothened regulates cellular proliferation and differentiation through activation of the transcription factor Gli. The activity of mammalian Smoothened is controlled by three different hedgehog proteins, Indian, Desert, and Sonic hedgehog, through their interaction with the Smoothened inhibitor Patched. However, the mechanisms of signal transduction from Smoothened are poorly understood. We show that a kinase which regulates signaling by many "conventional" 7TM G-protein-coupled receptors, G protein-coupled receptor kinase 2 (GRK2), participates in Smoothened signaling. Expression of GRK2, but not catalytically inactive GRK2, synergizes with active Smoothened to mediate Gli-dependent transcription. Moreover, knockdown of endogenous GRK2 by short hairpin RNA (shRNA) significantly reduces signaling in response to the Smoothened agonist SAG and also inhibits signaling induced by an oncogenic Smoothened mutant, Smo M2. We find that GRK2 promotes the association between active Smoothened and beta-arrestin 2. Indeed, Gli-dependent signaling, mediated by coexpression of Smoothened and GRK2, is diminished by beta-arrestin 2 knockdown with shRNA. Together, these data suggest that GRK2 plays a positive role in Smoothened signaling, at least in part, through the promotion of an association between beta-arrestin 2 and Smoothened.

    View details for DOI 10.1128/MCB.00546-06

    View details for Web of Science ID 000241252300018

    View details for PubMedID 16908539

  • Purmorphamine activates the Hedgehog pathway by targeting Smoothened NATURE CHEMICAL BIOLOGY Sinha, S., Chen, J. K. 2006; 2 (1): 29-30

    Abstract

    Hedgehog (Hh) signaling is an important regulator of embryonic patterning, tissue regeneration, stem cell renewal and cancer growth. A purine derivative named purmorphamine was previously found to activate the Hh pathway and affect osteoblast differentiation through an unknown mechanism. We demonstrate here that purmorphamine directly targets Smoothened, a critical component of the Hh signaling pathway.

    View details for DOI 10.1038/nchembio.753

    View details for Web of Science ID 000233971600010

    View details for PubMedID 16408088

  • Activity-dependent internalization of smoothened mediated by beta-arrestin 2 and GRK2 SCIENCE Chen, W., Ren, X. R., Nelson, C. D., Barak, L. S., Chen, J. K., Beachy, P. A., de Sauvage, F., Lefkowitz, R. J. 2004; 306 (5705): 2257-2260

    Abstract

    Binding of Sonic Hedgehog (Shh) to Patched (Ptc) relieves the latter's tonic inhibition of Smoothened (Smo), a receptor that spans the cell membrane seven times. This initiates signaling which, by unknown mechanisms, regulates vertebrate developmental processes. We find that two molecules interact with mammalian Smo in an activation-dependent manner: G protein-coupled receptor kinase 2 (GRK2) leads to phosphorylation of Smo, and beta-arrestin 2 fused to green fluorescent protein interacts with Smo. These two processes promote endocytosis of Smo in clathrin-coated pits. Ptc inhibits association of beta-arrestin 2 with Smo, and this inhibition is relieved in cells treated with Shh. A Smo agonist stimulated and a Smo antagonist (cyclopamine) inhibited both phosphorylation of Smo by GRK2 and interaction of beta-arrestin 2 with Smo. beta-Arrestin 2 and GRK2 are thus potential mediators of signaling by activated Smo.

    View details for DOI 10.1126/science.1104135

    View details for Web of Science ID 000225950000047

    View details for PubMedID 15618519

  • Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened GENES & DEVELOPMENT Chen, J. K., Taipale, J., Cooper, M. K., Beachy, P. A. 2002; 16 (21): 2743-2748

    Abstract

    The steroidal alkaloid cyclopamine has both teratogenic and antitumor activities arising from its ability to specifically block cellular responses to vertebrate Hedgehog signaling. We show here, using photoaffinity and fluorescent derivatives, that this inhibitory effect is mediated by direct binding of cyclopamine to the heptahelical bundle of Smoothened (Smo). Cyclopamine also can reverse the retention of partially misfolded Smo in the endoplasmic reticulum, presumably through binding-mediated effects on protein conformation. These observations reveal the mechanism of cyclopamine's teratogenic and antitumor activities and further suggest a role for small molecules in the physiological regulation of Smo.

    View details for DOI 10.1101/gad.1025302

    View details for Web of Science ID 000179027900002

    View details for PubMedID 12414725

  • Small molecule modulation of Smoothened activity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chen, J. K., Taipale, J., Young, K. E., Maiti, T., Beachy, P. A. 2002; 99 (22): 14071-14076

    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.

    View details for DOI 10.1073/pnas.182542899

    View details for Web of Science ID 000178967400021

    View details for PubMedID 12391318

  • Medulloblastoma growth inhibition by Hedgehog pathway blockade SCIENCE Berman, D. M., Karhadkar, S. S., Hallahan, A. R., Pritchard, J. I., Eberhart, C. G., Watkins, D. N., Chen, J. K., Cooper, M. K., Taipale, J., Olson, J. M., Beachy, P. A. 2002; 297 (5586): 1559-1561

    Abstract

    Constitutive Hedgehog (Hh) pathway activity is associated with initiation of neoplasia, but its role in the continued growth of established tumors is unclear. Here, we investigate the therapeutic efficacy of the Hh pathway antagonist cyclopamine in preclinical models of medulloblastoma, the most common malignant brain tumor in children. Cyclopamine treatment of murine medulloblastoma cells blocked proliferation in vitro and induced changes in gene expression consistent with initiation of neuronal differentiation and loss of neuronal stem cell-like character. This compound also caused regression of murine tumor allografts in vivo and induced rapid death of cells from freshly resected human medulloblastomas, but not from other brain tumors, thus establishing a specific role for Hh pathway activity in medulloblastoma growth.

    View details for Web of Science ID 000177697300059

    View details for PubMedID 12202832

  • Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine NATURE Taipale, J., Chen, J. K., Cooper, M. K., Wang, B. L., Mann, R. K., Milenkovic, L., Scott, M. P., Beachy, P. A. 2000; 406 (6799): 1005-1009

    Abstract

    Basal cell carcinoma, medulloblastoma, rhabdomyosarcoma and other human tumours are associated with mutations that activate the proto-oncogene Smoothened (SMO) or that inactivate the tumour suppressor Patched (PTCH). Smoothened and Patched mediate the cellular response to the Hedgehog (Hh) secreted protein signal, and oncogenic mutations affecting these proteins cause excess activity of the Hh response pathway. Here we show that the plant-derived teratogen cyclopamine, which inhibits the Hh response, is a potential 'mechanism-based' therapeutic agent for treatment of these tumours. We show that cyclopamine or synthetic derivatives with improved potency block activation of the Hh response pathway and abnormal cell growth associated with both types of oncogenic mutation. Our results also indicate that cyclopamine may act by influencing the balance between active and inactive forms of Smoothened.

    View details for Web of Science ID 000089020200048

    View details for PubMedID 10984056

  • The identification of myriocin-binding proteins (vol 6, pg 221, 1999) CHEMISTRY & BIOLOGY Chen, J. K., Lane, W. S., Schreiber, S. L. 1999; 6 (6): R186-R186
  • COMBINATORIAL SYNTHESIS AND MULTIDIMENSIONAL NMR-SPECTROSCOPY - AN APPROACH TO UNDERSTANDING PROTEIN-LIGAND INTERACTIONS ANGEWANDTE CHEMIE-INTERNATIONAL EDITION IN ENGLISH Chen, J. K., Schreiber, S. L. 1995; 34 (9): 953-969
  • 2 BINDING ORIENTATIONS FOR PEPTIDES TO THE SRC SH3 DOMAIN - DEVELOPMENT OF A GENERAL-MODEL FOR SH3-LIGAND INTERACTIONS SCIENCE Feng, S. B., Chen, J. K., Yu, H. T., Simon, J. A., Schreiber, S. L. 1994; 266 (5188): 1241-1247

    Abstract

    Solution structures of two Src homology 3 (SH3) domain-ligand complexes have been determined by nuclear magnetic resonance. Each complex consists of the SH3 domain and a nine-residue proline-rich peptide selected from a large library of ligands prepared by combinatorial synthesis. The bound ligands adopt a left-handed polyproline type II (PPII) helix, although the amino to carboxyl directionalities of their helices are opposite. The peptide orientation is determined by a salt bridge formed by the terminal arginine residues of the ligands and the conserved aspartate-99 of the SH3 domain. Residues at positions 3, 4, 6, and 7 of both peptides also intercalate into the ligand-binding site; however, the respective proline and nonproline residues show exchanged binding positions in the two complexes. These structural results led to a model for the interactions of SH3 domains with proline-rich peptides that can be used to predict critical residues in complexes of unknown structure. The model was used to identify correctly both the binding orientation and the contact and noncontact residues of a peptide derived from the nucleotide exchange factor Sos in association with the amino-terminal SH3 domain of the adaptor protein Grb2.

    View details for Web of Science ID A1994PT13200054

    View details for PubMedID 7526465

  • SH3 DOMAIN-MEDIATED DIMERIZATION OF AN N-TERMINAL FRAGMENT OF THE PHOSPHATIDYLINOSITOL 3-KINASE P85 SUBUNIT BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Chen, J. K., Schreiber, S. L. 1994; 4 (14): 1755-1760
  • STRUCTURAL BASIS FOR THE BINDING OF PROLINE-RICH PEPTIDES TO SH3 DOMAINS CELL Yu, H. T., Chen, J. K., Feng, S. B., Dalgarno, D. C., Brauer, A. W., Schreiber, S. L. 1994; 76 (5): 933-945

    Abstract

    A common RXL motif was found in proline-rich ligands that were selected from a biased combinatorial peptide library on the basis of their ability to bind specifically to the SH3 domains from phosphatidylinositol 3-kinase (PI3K) or c-Src. The solution structure of the PI3K SH3 domain complexed to one of these ligands, RKLPPRPSK (RLP1), was determined. Structure-based mutations were introduced into the PI3K SH3 domain and the RLP1 ligand, and the influence of these mutations on binding was evaluated. We conclude that SH3 domains recognize proline-rich motifs possessing the left-handed type II polyproline (PPII) helix conformation. Two proline residues directly contact the receptor. Other prolines in the ligands appear to function as a molecular scaffold, promoting the formation of the PPII helix. Three nonproline residues consisting of combinations of arginine and leucine interact extensively with the SH3 domain and appear to confer ligand specificity.

    View details for Web of Science ID A1994NA89000018

    View details for PubMedID 7510218

  • BIASED COMBINATORIAL LIBRARIES - NOVEL LIGANDS FOR THE SH3 DOMAIN OF PHOSPHATIDYLINOSITOL 3-KINASE JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Chen, J. K., Lane, W. S., Brauer, A. W., Tanaka, A., Schreiber, S. L. 1993; 115 (26): 12591-12592

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