purmorphamine and cyclopamine

Since its discovery by C. Nüsslein-Volhard and E. F. Wieschaus, hedgehog (hh) signaling has come a long way. Today it is regarded as a key regulator in embryogenesis where it governs processes like cell proliferation, differentiation, and tissue patterning. Furthermore, in adults it is involved in the maintenance of stem cells, and in tissue repair and regeneration. But hh signaling has a second-much darker-face: it plays an important role in several types of human cancers where it promotes growth and enables proliferation of tumor stem cells. The etiology of medulloblastoma and basal cell carcinoma is tightly linked to aberrant hh activity, but also cancers of the prostate, the pancreas, the colon, the breasts, rhabdomyosarcoma, and leukemia, are dependent on irregular hh activity. Recent clinical studies have shown that hh signaling can be the basis of an important new class of therapeutic agents with far-reaching implications in oncology. Thus, modulation of hh signaling by means of small molecules has emerged as a valuable tool in combating these hh-dependent cancers. Cyclopamine, a unique natural product with a fascinating history, was the first identified inhibitor of hh signaling and its story is closely linked to the progress in the whole field. In this review we will trace the story of cyclopamine, give an overview on the biological modes of hh signaling both in untransformed and malignant cells, and finally present potent modulators of the hh pathway-many of them already in clinical studies. For more than 30 years now the knowledge on hh signaling has grown steadily-an end to this development is far from being conceivable.

Topics: Antineoplastic Agents; Hedgehog Proteins; Humans; Neoplasms; Signal Transduction; Veratrum Alkaloids

Smoothened (SMO) is a GPCR that mediates hedgehog signaling. Hedgehog binds the transmembrane protein Patched, which in turn regulates SMO activation. Overactive SMO signaling is oncogenic and is therefore a clinically established drug target. Here we establish a nanoluciferase bioluminescence resonance energy transfer (NanoBRET)-based ligand binding assay for SMO providing a sensitive and high throughput-compatible addition to the toolbox of GPCR pharmacologists. In the NanoBRET-based binding assay, SMO is N terminally tagged with nanoluciferase (Nluc) and binding of BODIPY-cyclopamine is assessed by quantifying resonance energy transfer between receptor and ligand. The assay allowed kinetic analysis of ligand-receptor binding in living HEK293 cells, competition binding experiments using commercially available SMO ligands (SANT-1, cyclopamine-KAAD, SAG1.3 and purmorphamine), and pharmacological dissection of two BODIPY-cyclopamine binding sites. This high throughput-compatible assay is superior to commonly used SMO ligand binding assays in the separation of specific from non-specific ligand binding and, provides a suitable complement to chemical biology strategies for the discovery of novel SMO-targeting drugs. SIGNIFICANCE STATEMENT: We established a NanoBRET-based binding assay for SMO with superior sensitivity compared to fluorescence-based assays. This assay allows distinction of two separate binding sites for BODIPY-cyclopamine on the SMO transmembrane core in live cells in real time. The assay is a valuable complement for drug discovery efforts and will support a better understanding of Class F GPCR pharmacology.

Topics: Binding Sites; Biological Assay; Bioluminescence Resonance Energy Transfer Techniques; Boron Compounds; Cinnamates; Drug Discovery; Gene Knockout Techniques; Hedgehog Proteins; HEK293 Cells; Humans; Ligands; Luciferases; Morpholines; Nanostructures; Purines; Signal Transduction; Smoothened Receptor; Veratrum Alkaloids

The blood brain barrier (BBB) is a diffusion barrier that maintains the brain environment. Wip1 is a nuclear phosphatase induced by many factors and involved in various stresses, tumorigenesis, organismal aging, and neurogenesis. Wip1's role in BBB integrity has not been thoroughly investigated. The purpose of the present study was to investigate the effect and mechanism of Wip1 on lipopolysaccharide (LPS)-induced BBB dysfunction and inflammation in an in vitro BBB model. The in vitro BBB model was established by co-culturing human brain-microvascular endothelial cells and human astrocytes and then exposing them to 1μg/ml LPS for 6, 12, 18, 24, and 48h. Wip1 expression was significantly elevated by LPS treatment. Knockdown of Wip1 aggravated the increased permeability and decreased transepithelial electrical resistance, protein expression of ZO-1, and occludin induced by LPS. Wip1 silencing augmented the elevated inflammatory cytokines TNF-α, IL-1β, IL-12, and IL-6 of the BBB induced by LPS, whereas overexpression of Wip1 showed a contrary effect. Sonic hedgehog signaling (SHH) was activated by Wip1 overexpression and inhibited by Wip1 silencing. Additionally, activating or inhibiting the SHH pathway by purmorphamine or cyclopamine, respectively, abolished the Wip1-induced changes in transepithelial electrical resistance and permeability and inflammatory responses in the LPS-injured BBB model. Our results demonstrate that Wip1 may protect the BBB against LPS-induced integrity disruption and inflammatory response through the SHH signaling pathway.

Topics: Astrocytes; Blood-Brain Barrier; Cells, Cultured; Coculture Techniques; Encephalitis; Endothelial Cells; Endothelium, Vascular; Gene Knockdown Techniques; Hedgehog Proteins; Humans; In Vitro Techniques; Lipopolysaccharides; Morpholines; Permeability; Protein Phosphatase 2C; Purines; Recombinant Proteins; RNA Interference; RNA, Small Interfering; Signal Transduction; Veratrum Alkaloids

It has been well known that Hedgehog (Hh) signaling plays an important role in bone development, however, its function in cementogenesis has not yet been reported. This study was intended to elucidate the role of Hh signaling in cementoblast differentiation.. Expression changes of various Hh signaling components and levels of skeletogenic markers (alkaline phosphatase, osteocalcin, osteopontin) and osteogenic transcription factors (RUNX2, Osterix) by Hh signaling modulators during OCCM-30 cementoblast differentiation were determined by quantitative real-time reverse transcriptase polymerase chain reaction. To investigate effects of Hh signaling modulators on the mineralization of cementoblast, alkaline phosphatase and alizarin red S staining were used. Then, the interaction between Hh and BMP signaling during cementoblast differentiation was evaluated using co-treatment of BMP7 and Hh signaling modulators.. We observed the consistent expression of Hh signaling molecules in the OCCM-30, which were up-regulated during cementoblast differentiation. We also found that the treatment of cells with Purmo, an Hh activator, enhanced cementoblast differentiation by increasing the mRNA expression of skeletogenic markers and osteogenic transcription factors, as well as increasing alkaline phosphate activity and mineralization capability. On the contrary, an Hh antagonist, like Cyclo, effectively inhibited cementoblast differentiation. Furthermore, BMP7 promoted cementoblast differentiation through crosstalk with the Hh signaling.. These results suggest that Hh signaling is involved in cementoblast differentiation, and Hh signaling molecules may therefore represent new therapeutic targets in periodontal treatment and regeneration.

Topics: Alkaline Phosphatase; Animals; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cell Differentiation; Cell Line; Cell Survival; Core Binding Factor Alpha 1 Subunit; Dental Cementum; Hedgehog Proteins; Humans; Mice; Mice, Transgenic; Morpholines; Osteocalcin; Osteogenesis; Osteopontin; Purines; RNA, Messenger; Signal Transduction; Sp7 Transcription Factor; Transcription Factors; Up-Regulation; Veratrum Alkaloids

Adult stem cells are considered promising candidates for cellular therapies due to their capacity to differentiate and self-renew. Differentiation leads to changes in the metabolism, structure, and gene expression patterns of cells. Hedgehog is one of the pathways that is involved in the enhancement of osteogenesis and chondrogenesis in adult stem cells, but its mechanisms are poorly understood. In this study, we treated adipose tissue-derived stem cells (ADSC) with two well-characterized drugs, purmorphamine (Hedgehog pathway activator) and cyclopamine (Hedgehog pathway inhibitor), and identified mRNAs associated with polysomes in each treatment group to determine the post transcriptional genetic networks governed by the Hedgehog pathway. Activation of the Hedgehog pathway by purmorphamine results in significant upregulation of mRNAs associated with cellular communication and signal transduction. Furthermore, our experiments show that cyclopamine acts late downregulating GLI1 expression in ADSCs but promotes the upregulation of mRNAs associated with energy pathways and metabolism at early times. Through in silico analysis, we identified some miRNAs, such as miR-355, that could regulate these mRNAs association with polysomes and thereby modulate the Hedgehog pathway. Our results suggest that activation of the Hedgehog pathway by purmorphamine also results in a negative regulation of mRNAs in the protein translation machinery.

Topics: Adipose Tissue; Cell Nucleus; Energy Metabolism; Gene Regulatory Networks; Hedgehog Proteins; High-Throughput Nucleotide Sequencing; Humans; Morpholines; Polyribosomes; Purines; Sequence Analysis, RNA; Stem Cells; Veratrum Alkaloids

The sonic hedgehog (Shh) signalling pathway has an important role in the maintenance of various stem cells and organogenesis during development. However, the effect of Shh in skin-derived precursors (SKPs), which have the capacity for multipotency and self-renewal, is not yet clear. The present study investigated the effects of the Shh signalling pathway on the proliferation and self-renewal of murine SKPs (mSKPs).. The Shh signalling pathway was activated by treatment with purmorphamine (Shh agonist) or recombinant Shh in mSKPs. Cyclopamine (Shh antagonist) or GANT-61 (Gli inhibitor) was used to inhibit the pathway. Western blot, qPCR, and immunofluorescence were used to analyse the expression of genes related to self-renewal, stemness, epithelial-mesenchymal transition (EMT) and the Shh signalling pathway. In addition, cell proliferation and apoptosis were examined.. Inhibiting the Shh signalling pathway reduced mSKP proliferation and sphere formation, but increased apoptosis. Activating this signalling pathway produced opposite results. The Shh signalling pathway also controlled the EMT phenotype in mSKPs. Moreover, purmorphamine recovered the self-renewal and proliferation of aged mSKPs.. Our results suggest that the Shh signalling pathway has an important role in the proliferation, self-renewal and apoptosis of mSKPs. These findings also provide a better understanding of the cellular mechanisms underlying SKP self-renewal and apoptosis that allow more efficient expansion of SKPs.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Morpholines; Neoplastic Stem Cells; Purines; Signal Transduction; Skin; Veratrum Alkaloids

Appropriate neural patterning of human induced pluripotent stem cells (hiPSCs) is critical to generate specific neural cells/tissues and even mini-brains that are physiologically relevant to model neurological diseases. However, the capacity of signaling factors that regulate 3-D neural tissue patterning in vitro and differential responses of the resulting neural populations to various biomolecules have not yet been fully understood.. By tuning neural patterning of hiPSCs with small molecules targeting sonic hedgehog (SHH) signaling, this study generated different 3-D neuronal cultures that were mainly comprised of either cortical glutamatergic neurons or motor neurons.. Abundant glutamatergic neurons were observed following the treatment with an antagonist of SHH signaling, cyclopamine, while Islet-1 and HB9-expressing motor neurons were enriched by an SHH agonist, purmorphamine. In neurons derived with different neural patterning factors, whole-cell patch clamp recordings showed similar voltage-gated Na(+)/K(+) currents, depolarization-evoked action potentials and spontaneous excitatory post-synaptic currents. Moreover, these different neuronal populations exhibited differential responses to three classes of biomolecules, including (1) matrix metalloproteinase inhibitors that affect extracellular matrix remodeling; (2) N-methyl-d-aspartate that induces general neurotoxicity; and (3) amyloid β (1-42) oligomers that cause neuronal subtype-specific neurotoxicity.. This study should advance our understanding of hiPSC self-organization and neural tissue development and provide a transformative approach to establish 3-D models for neurological disease modeling and drug discovery.. Appropriate neural patterning of human induced pluripotent stem cells (hiPSCs) is critical to generate specific neural cells, tissues and even mini-brains that are physiologically relevant to model neurological diseases. However, the capability of sonic hedgehog-related small molecules to tune different neuronal subtypes in 3-D differentiation from hiPSCs and the differential cellular responses of region-specific neuronal subtypes to various biomolecules have not been fully investigated. By tuning neural patterning of hiPSCs with small molecules targeting sonic hedgehog signaling, this study provides knowledge on the differential susceptibility of region-specific neuronal subtypes derived from hiPSCs to different biomolecules in extracellular matrix remodeling and neurotoxicity. The findings are significant for understanding 3-D neural patterning of hiPSCs for the applications in brain organoid formation, neurological disease modeling, and drug discovery.

Topics: Amyloid beta-Peptides; Cell Culture Techniques; Cells, Cultured; Ectoderm; Electrophysiological Phenomena; Embryoid Bodies; Hedgehog Proteins; Humans; Induced Pluripotent Stem Cells; Male; Matrix Metalloproteinase Inhibitors; Morpholines; N-Methylaspartate; Neural Stem Cells; Neurons; Neurotoxins; Octamer Transcription Factor-3; Purines; Small Molecule Libraries; Synapses; Veratrum Alkaloids

We have examined a number of reagents for their ability to modulate activity of the Hh signaling pathway during embryonic development of Xenopus. In particular we have focused on regulation of events occurring during tailbud stages and later. Two inducible protein reagents based on the Gli1 and Gli3 transcription factors were generated and the activity of these proteins was compared to the Hh signaling pathway inhibitor, cyclopamine, and the activators, Smoothened agonist (SAG) and purmorphamine (PMA). Effectiveness of reagents was assayed using both molecular biological techniques and biological readouts. We found that the small molecule modulators of the Hh pathway were highly specific and effective and produced results generally superior to the more conventional protein reagents for examination of later stage developmental processes.

Topics: Animals; Cyclohexylamines; Embryo Culture Techniques; Embryonic Development; Gene Expression Regulation, Developmental; Hedgehog Proteins; Morpholines; Purines; Signal Transduction; Thiophenes; Veratrum Alkaloids; Xenopus

A complex set of extrinsic and intrinsic signals acts in specific temporal and spatial orders to enable neural differentiation during development. These processes have been extensively studied in animal models, but human neural development remains much less understood. This lack of detailed information about human early neurogenesis is a hindrance for the differentiation of pluripotent stem cell lines into specific neuronal phenotypes. Therefore, it is important to strengthen the interspecies comparative approaches. We describe a novel model system in which in vitro differentiation of human and mouse embryonic stem (ES) cells are temporally aligned to each other and compared with mouse telencephalic neurogenesis in vivo. In this comparative model system, we tested the in vitro role of Hedgehog (Hh) signaling for ES cell-derived telencephalic differentiation. In vivo, Hh signaling mediates dorsal/ventral patterning during early stages of telencephalic development. We monitored the effect of pharmacological modulators of the Hh signaling pathway, purmorphamine-an agonist and cyclopamine-an antagonist of the Smoothened receptor (Smo), on the expression of region-specific transcription factors and signaling molecules relevant for telencephalic development in vivo. Purmorphamine strongly upregulated the expression of telencephalic ventral markers Nkx2.1, Nkx6.2, Lhx6, and Lhx8 in mouse and human cells, thus reflecting the in vivo process of the medial ganglionic eminence patterning and specification. Cyclopamine upregulated the expression of telencephalic dorsal markers, but at lower levels in human compared with mouse cells. Modulation of Smo in vitro differentially affected, in mouse and human cells, the expression of molecules of the Hh pathway, especially the Gli1 and Gli3 effectors, Sonic Hh ligand and Ptch receptors. These results provide evidence for the different default differentiation of mouse and human ES cells and prove the utility of the comparative system for optimizing the directed differentiation of human pluripotent stem cells.

Topics: Animals; Antigens, Differentiation; Body Patterning; Cells, Cultured; Embryonic Stem Cells; Gene Expression; Gene Expression Profiling; Gene Expression Regulation, Developmental; Hedgehog Proteins; Humans; Mice; Morpholines; Neurogenesis; Purines; Receptors, G-Protein-Coupled; Signal Transduction; Smoothened Receptor; Telencephalon; Transcription Factors; Veratrum Alkaloids

Sonic hedgehog (Shh)/Gli pathway plays an important regulatory role on the neuroepithelial cells (NEc) proliferation in the dorsal regions of the developing vertebrate Central Nervous System. The aim of this paper was to analyze the effect of the Shh/Gli signaling pathway activation on the proliferation dynamics and/or the spatial organization of the NEc proliferation activity during early stages of the developing chick optic tectum (OT). In ovo pharmacological gain and loss of hedgehog function approaches were complemented with in vivo electroporation experiments in order to create ectopic sources of either Shh or Gli activator (GliA) proteins in the OT. NEc proliferating activity was analyzed at ED 4/4.5 by recording the spatial co-ordinates of the entire population of mitotic NEc (mNEc) located along OT dorsal-ventral sections. Several space signals (numerical sequences) were derived from the mNEc spatial co-ordinate records and analyzed by different standardized non-linear methods of signal analysis.. In ovo pharmacologic treatment with cyclopamine resulted in dramatic failure in the OT expansion while the agonist purmorphamine produced the opposite result, a huge expansion of the OT vesicle. Besides, GliA and Shh misexpressions interfere with the formation of the intertectal fissure located along the dorsal midline. This morphogenetic alteration is accompanied by an increase in the mNEc density. There is a gradient in the response of NEcs to Shh and GliA: the increase in mNEc density is maximal near the dorsal regions and decrease towards the OT-tegmental boundary. Biomathematical analyses of the signals derived from the mNEc records show that both Shh and GliA electroporations change the proliferation dynamics and the spatial organization of the mNEc as revealed by the changes in the scaling index estimated by these methods.. The present results show that the Shh/Gli signaling pathway plays a critical role in the OT expansion and modelling. This effect is probably mediated by a differential mitogenic effect that increases the NEc proliferation and modulates the spatial organization of the NEc proliferation activity.

Topics: Animals; Cell Proliferation; Chick Embryo; Electroporation; Gene Expression Regulation, Developmental; Hedgehog Proteins; Morpholines; Neuroepithelial Cells; Neurogenesis; Oncogene Proteins; Purines; Signal Transduction; Superior Colliculi; Teratogens; Trans-Activators; Veratrum Alkaloids; Zinc Finger Protein GLI1

The Smoothened receptor (Smo) mediates hedgehog (Hh) signaling critical for development, cell growth, and migration, as well as stem cell maintenance. Aberrant Hh signaling pathway activation has been implicated in a variety of cancers, and small-molecule antagonists of Smo have entered human clinical trials for the treatment of cancer. Here, we report the biochemical characterization of allosteric interactions of agonists and antagonists for Smo. Binding of two radioligands, [(3)H]3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-{[3-(4-pyridinyl)-phenyl]methyl}-1-benzothiophene-2-carboxamide (SAG-1.3) (agonist) and [(3)H]cyclopamine (antagonist), was characterized using human Smo expressed in human embryonic kidney 293F membranes. We observed full displacement of [(3)H]cyclopamine by all Smo agonist and antagonist ligands examined. N-[(1E)-(3,5-Dimethyl-1-phenyl-1H-pyrazol-4-yl)methylidene]-4-(phenylmethyl)-1-piperazinamine (SANT-1), an antagonist, did not fully inhibit the binding of [(3)H]SAG-1.3. In a functional cell-based beta-lactamase reporter gene assay, SANT-1 and N-[3-(1H-benzimidazol-2-yl)-4-chlorophenyl]-3,4,5-tris(ethyloxy)-benzamide (SANT-2) fully inhibited 3-chloro-4,7-difluoro-N-[trans-4-(methylamino)cyclohexyl]-N-{[3-(4-pyridinyl)phenyl]methyl}-1-benzothiophene-2-carboxamide (SAG-1.5)-induced Hh pathway activation. Detailed "Schild-type" radioligand binding analysis with [(3)H]SAG-1.3 revealed that two structurally distinct Smoothened receptor antagonists, SANT-1 and SANT-2, bound in a manner consistent with that of allosteric modulation. Our mechanism of action characterization of radioligand binding to Smo combined with functional data provides a better understanding of small-molecule interactions with Smo and their influence on the Hh pathway.

Topics: Anilides; Animals; Benzamides; Benzimidazoles; beta-Lactamases; Binding Sites; Binding, Competitive; Cell Line; Cell Membrane; Cyclohexylamines; Genes, Reporter; Humans; Kinetics; Mice; Molecular Structure; Morpholines; NIH 3T3 Cells; Piperazines; Purines; Pyrazoles; Pyridines; Radioligand Assay; Receptors, G-Protein-Coupled; Recombinant Proteins; Smoothened Receptor; Thiophenes; Tomatine; Transfection; Veratrum Alkaloids

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.

Topics: Animals; Cell Membrane; Cell Proliferation; Cells, Cultured; Dose-Response Relationship, Drug; Hedgehog Proteins; Mice; Morpholines; NIH 3T3 Cells; Osteoblasts; Purines; Receptors, G-Protein-Coupled; Signal Transduction; Smoothened Receptor; Stem Cells; Tissue Engineering; Trans-Activators; Veratrum Alkaloids

Topics: Animals; Bone Development; Cell Differentiation; Cell Proliferation; Drosophila Proteins; Embryonic Development; Embryonic Induction; Gene Expression Regulation, Developmental; Hedgehog Proteins; Humans; Morpholines; Nerve Tissue Proteins; Neurons; Purines; Receptors, Cell Surface; Signal Transduction; Trans-Activators; Veratrum Alkaloids

Previously, a small molecule, purmorphamine, was identified that selectively induces osteogenesis in multipotent mesenchymal progenitor cells. In order to gain insights into the mechanism of action of purmorphamine, high-density oligonucleotide microarrays were used to profile gene expression in multipotent mesenchymal progenitor cells treated with either purmorphamine or bone morphogenetic protein-4 (BMP-4). In contrast to BMP-4 treatment, purmorphamine activates the Hedgehog (Hh) signaling pathway, resulting in the up- and downregulation of its downstream target genes, including Gli1 and Patched. Moreover, the known Hh signaling antagonists, cyclopamine and forskolin, completely block the osteogenesis and Glimediated transcription induced by purmorphamine. These results demonstrate that purmorphamine is a small molecule agonist of Hedgehog signaling, and it may ultimately be useful in the treatment of bone-related disease and neurodegenerative disease.

Topics: Alkaline Phosphatase; Animals; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Cluster Analysis; Colforsin; Fibroblasts; Gene Expression Profiling; Gene Expression Regulation, Developmental; Hedgehog Proteins; Mice; Mice, Inbred C3H; Morpholines; Oligonucleotide Array Sequence Analysis; Osteogenesis; Purines; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Trans-Activators; Transcription, Genetic; Veratrum Alkaloids