cur-61414 and cyclopamine

Sonic hedgehog (Shh) is the most widely characterized of the three vertebrate Hedgehog homologs, and is essential for proper embryonic development. Shh binds to its receptor, Patched (Ptch1), resulting in the de-repression of Smoothened (Smo). This leads to the activation of Gli2, which regulates the transcription of target genes that include Gli1 and Ptch1. Several synthetic and naturally occurring small-molecule modulators of Smo have been discovered. Shh-signaling antagonists that bind to Smo include cyclopamine, SANT1, and Cur-61414. Shh signaling agonists that bind to Smo include the synthetic small molecules purmorphamine and SAG. Small molecules that inhibit Shh signaling downstream of Smo, GANT58 and GANT61 have also been reported. Robotnikinin inhibits the Shh pathway by directly targeting Shh. Although progress has been made in understanding and modulating Shh signaling, fundamental aspects of Shh signal transduction remain obscure, including the mechanism(s) whereby Ptch1 regulates Smo activity. Small-molecule modulators of Shh signaling provide a means to regulate the activity of a pathway implicated in medulloblastoma, basal cell carcinoma (BCC), pancreatic cancer, prostate cancer and developmental disorders. Several Shh inhibitors have not succeeded in the clinic for unknown reasons, but clinical trials in BCC and pancreatic cancer with the promising Smo antagonists GDC-0449 and IPI-926 are currently underway.

Topics: Anilides; Animals; Dioxoles; Hedgehog Proteins; Humans; Models, Biological; Molecular Structure; Piperazines; Pyrazoles; Pyridines; Receptors, G-Protein-Coupled; Signal Transduction; Smoothened Receptor; Thiophenes; Veratrum Alkaloids

In addition to the potential stem cells offer for regenerative medicine, they also rapidly are becoming a center of focus in oncology. There are several developmental pathways that are involved in the deregulated signaling in stem cells resulting in tumorigenesis. For example, aberrant activation of the Hedgehog (Hh) pathway has been associated with numerous malignancies including basal cell carcinoma, medulloblastoma, prostate, pancreatic and breast cancers. In vivo evidence suggests the antagonism of excessive Hh signaling may provide a route to unique mechanism-based anti-cancer therapies. This review summarizes recent developments in targeting cell-surface proteins and intracellular targets from the Hh pathway with small molecules. Hh signaling is triggered by lipid-modified Hh proteins that exert their activity via a series of transmembrane receptors (Patched, Ptc and Smoothened, Smo). Smoothened (Smo) is a 7-TM protein reported to be the most druggable target in the Hh signaling cascade. We further review several published programs geared towards identification and profiling of synthetic antagonists of Smo. Challenges and perspectives of this approach are also discussed.

Topics: Animals; Dioxoles; Drosophila Proteins; Hedgehog Proteins; Humans; Neoplasms; Patched Receptors; Piperazines; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Signal Transduction; Smoothened Receptor; Veratrum Alkaloids

Desmethylveramiline (1), an aza steroid analogue of veramiline was designed as a surrogate for cyclopamine, a reference antagonist of the Sonic Hedgehog (Shh) pathway. Desmethyveramiline (1) was prepared in seven steps from commercially available Fernholtz acid using the hydroformylation of a terminal olefine as the key step for the construction of the piperidine appendage. In two assays (i) the inhibition of the Shh-induced Gli-dependent luciferase activity in Shh-light2 cells, (ii) the inhibition of the SAG-induced differentiation of the mesenchymal C3H10T1/2 cells, desmethylveramiline (1) is an inhibitor in the μM range comparable to cyclopamine.

Topics: Animals; Cell Line; Cholesterol; Enzyme Inhibitors; Hedgehog Proteins; Mice; Models, Molecular; Molecular Structure; NIH 3T3 Cells; Piperidines; Signal Transduction; Veratrum Alkaloids

Hedgehog signaling regulates Gli transcription factors. Aberrant hedgehog signaling can be oncogenic and drugs that block hedgehog are being tested as anticancer agents. We considered whether hedgehog/Gli signaling may be involved in human bladder transitional cell carcinoma proliferative or invasive behavior.. We stratified the human bladder transitional cell carcinoma lines RT4 (ATCC), 253JP, 253BV, UMUC6 and UMUC3 for relative growth rate by cell counting and for in vitro invasiveness by Matrigel invasion assay. Cells were tested for growth inhibition by the hedgehog blocking drug cyclopamine or the inactive mimic tomatidine. Cell RNA was characterized for hedgehog signaling component expression, including ligands, receptors and signaling mediators, by quantitative reverse transcriptase-polymerase chain reaction. Gli2 expression or activity was modified by Gli2 expression lentiviruses or the Gli inhibitor GANT61. We measured effects on proliferation and invasiveness.. Cell growth rates and invasiveness were stratified into an equivalent order (RT4 <243JP <253BV

Topics: Blotting, Western; Carcinoma, Transitional Cell; Cell Line, Tumor; Dioxoles; Gene Expression Profiling; Hedgehog Proteins; Humans; Kruppel-Like Transcription Factors; Linear Models; Neoplasm Invasiveness; Nuclear Proteins; Piperazines; Pyridines; Pyrimidines; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Tomatine; Urinary Bladder Neoplasms; Veratrum Alkaloids; Zinc Finger Protein Gli2

The seven-transmembrane receptor Smoothened (Smo) transduces the signal initiated by Hedgehog (Hh) morphogen binding to the receptor Patched (Ptc). We have reinvestigated the pharmacological properties of reference molecules acting on the Hh pathway using various Hh responses and a novel functional assay based on the coexpression of Smo with the alpha subunit of the G15 protein in HEK293 cells. The measurement of inositol phosphate (IP) accumulation shows that Smo has constitutive activity, a response blocked by Ptc which indicates a functional Hh receptor complex. Interestingly, the antagonists cyclopamine, Cur61414, and SANT-1 display inverse agonist properties and the agonist SAG has no effect at the Smo-induced IP response, but converts Ptc-mediated inactive forms of Smo into active ones. An oncogenic Smo mutant does not mediate an increase in IP response, presumably reflecting its inability to reach the cell membrane. These studies identify novel properties of molecules displaying potential interest in the treatment of various cancers and brain diseases, and demonstrate that Smo is capable of signaling through G15.

Topics: Animals; Cell Cycle Proteins; Cell Line; Cell Membrane; Dioxoles; DNA-Binding Proteins; Genes, Tumor Suppressor; Hedgehog Proteins; Humans; Mice; Neoplasms; Piperazines; Pyrazoles; Receptors, G-Protein-Coupled; RNA-Binding Proteins; Signal Transduction; Smoothened Receptor; Stem Cells; Trans-Activators; Tumor Suppressor Proteins; Veratrum Alkaloids