The Hedgehog (Hh) pathway was first discovered in a Drosophila genetic screen because of its role in patterning the body of the animal (Nusslein-Volhard et al., Nature (1980) 287:795-801). Shortly afterward, mammalian homologues of the Hedgehog gene were cloned and characterized in chick and mouse (Echelard et al., Cell (1993) 75:1417-1430; Riddle et al., Cell (1993) 75:1401-1416; Roelink et al., Cell (1994) 76:761-775). There are three Hh vertebrate homologues, named Sonic Hedgehog (Shh), Desert Hedgehog (Dhh) and Indian Hedgehog (Ihh), with Shh being implicated in limb development and neural tube development (Bitgood et al., Curr. Biol. (1996) 6:298-304; Chiang et al., Nature (1996) 383:407-413; St. Jacques et al., Genes Dev. (1999) 13:2072-2086; Zhang et al., Cell (2001) 106:781-792). Using both the Drosophila and the mouse models to define how the Hh pathway transduced its signal, it was revealed that this is a multifactorial and unconventional pathway (Hooper et al., Nat. Rev. Mol. Cell. Biol. (2005) 6:306-317; Huangfu et al., Dev. (2006) 133:3-14). In vertebrates, signal transduction in Hh pathway begins by the Shh ligand binding to Patched (Ptc1) a 12 transmembrane receptor. Ptc1 is coupled to and represses a signaling polypeptide Smoothened (Smo), a 7 transmembrane, G-protein coupled receptor. In the presence of Shh, the Ptc1 inhibition on Smo is released, and Smo transduces the Shh signal, activating downstream pathway components. However, in the absence of Shh, Ptc1 represses Smo signaling, and no signal transduction takes place.
The final downstream effector of the Hh pathway is the transcription factor Gli. There are 3 Gli proteins in vertebrates, Gli1, Gli2 and Gli3, and the Gli polypeptides take their name from their discovery as genes amplified in glioblastoma (Kinzler et al., Science (1987) 236:70-73; Bai et al, Dev. Cell (2004) 6:103-115; Motoyama et al, Dev. Biol. (2003) 259:150-161). The three Gli proteins share high homology in the zinc finger domain, but have limited homology outside of this region (Matise and Joyner Oncogene (1999) 18: 7852-7859). Gli 1 is a transcriptional activator, while Gli2 and Gli3 are bifunctional and can function as a transcriptional activator or, when proteolytically processed, a transcriptional repressor (Dai et al., J. Biol. Chem. (1999) 12:8143-8152). In general, Gli1 expression is restricted to proliferating cells adjacent to tissues expressing Shh. Gli2 and Gli3 are broadly expressed in proliferating cells exposed to lower concentrations of Shh (Hui et al., Dev. Biol. (1994) 162:402-413).
In the cytoplasm, Gli is complexed with the protein Suppressor of Fused and may be tethered to the microtubule cytoskeleton (Methot and Basler, Dev. (2000) 127:4001-4010; Chen et al., Mol. Cell. Biol. (2005) 25:7042-7053; Preat, Genetics (1992) 132:725-736). Upon transduction of a Shh signal, Gli is released from the complex and migrates to the nucleus. There Gli binds to specific sites in the genome and induces gene expression. It is interesting to note that some of the transcripts produced by the Gli transcription factors are components of the Hh pathway itself, such as Gli1, Ptc1 and Hedgehog interacting protein (Hip).
Disruption of the Hh pathway causes developmental abnormalities in embryogenesis and cancer in the adult. For example, in development, Shh mutations in a mouse model have a dramatic embryonic phenotype with lack of anterior and posterior limb polarity, lack of lung mesoderm and most ventral CNS motorneurons (Chiang et al., Nature (1996) 383:407-413). Mouse models of Ptc1 mutations can be embryonic lethal when homozygous, and display severe developmental defects similar to that of the Shh mutants when heterozygous (Goodrich et al., Science (1997) 277:1109-1113). For the Gli genes, a mouse that is homozygous for a Gli1 mutant with deleted zinc-finger domains develops normally (Matise et al., Development (1998) 125:2759). In contrast, Gli2 mutants without zinc-finger domains show developmental defects in the ventral CNS, lung, vertebrae and bones (Matise et al, supra; Motoyama et al, Nat. Genet. (1998) 20:54-57). Double mutants that are Gli1−/− and Gli2−/+ have a milder phenotype than Gli2−/− and have relatively normal limb development, but die at birth or shortly after. (Park et al., Development (2000) 127:1593-1605). Mice containing a Gli2+/−; Gli3−/− mutant have skeletal abnormalities that are more severe than either mutant alone (Mo et al., Development (1997) 124:113-123).
As mentioned previously, Gli was named for its discovery in brain cancer (glioblastoma). In Basal Cell Nevus Syndrome or Gorlin's syndrome, loss of function of Ptc1 leads to a predisposition to pediatric medulloblastoma and basal cell carcinoma (BCC) of the skin, the most common pediatric brain tumor and the most common type of skin cancer in the Caucasian population (Goodrich and Scott, Neuron (1998) 21:1243-1257). Gli1 overexpression is frequently found in BCC patients with Gorlin's syndrome and in non Gorlin's patients where the BCC has arisen spontaneously (Fan et al, Nat. Med. (1997) 3:788-792). Disruption of the Shh-Gli pathway is has also been described in a number of adult cancers which are discussed in more detail below.
The primary cilium is a hair-like appendage extending from the surface of a cell. This specialized structure, with a unique microtubular cytoskeleton (axoneme) and a surrounding membrane, is assembled and maintained by the intraflagellar transport machinery. Recent work has shown that primary cilia concentrate receptors and signal transduction components that have vital roles in development. In particular, evidence in the developing neural tube and limb bud has shown that genes encoding the IFT motors and the IFT particle subunits are required for Shh signaling. It has been found that the Shh signaling components, Patched (Ptc), Smo, Suppressor of fused and Gli transcription factors, concentrate in primary cilia.
Compounds which specifically affect the Hh pathway are few. Cyclopamine is a steroidal alkaloid derived from plants that antagonizes Smo and is currently in phase I clinical trials (Curis, Cambridge Mass.). Three other compounds have been described to act downstream of Smo in the Hh pathway, but their targets and mechanism of action are currently unclear (Lauth et al., Proc. Natl. Acad. Sci. USA (2007) 104:8455-8460; Lee et al., Chembiochem (2007)8:1916-1919).
Thus the Hh pathway is important for normal development of the embryo and carcinogenesis in the adult, indicating there is a great need for compounds and methods of using such compounds of the present disclosure for the alleviation and study of cancers and developmental disorders of the Hh pathway.