Cyclopamine, a Potent Antagonist of the Hedgehog Signaling Pathway

       The connection between the natural product cyclopamine and research into the basic developmental biology associated with the hedgehog pathway is a very interesting story (indeed!) that has been eloquently relayed in detail by Giannis, a leading researcher in the field. These investigations have recently culminated in the disclosure of small-molecule inhibitors of the hedgehog signaling pathway that show great promise as clinically relevant therapeutic agents for the treatment of certain types of cancer. In this post, we’ll look briefly at the role of the hedgehog pathway in tumorigenesis and examine two medicinal chemistry efforts to develop potent and orally active antagonists of the hedgehog pathway.

Overview of the Hedgehog Signaling Pathway

          The term ‘hedgehog’ is derived from the discovery of a gene in the fruit fly that caused larvae to grow a coat of spines on their undersides when mutated. These larvae apparently resembled the spiny mammals for which the hedgehog gene was eventually named. The hedgehog gene encodes for three varieties of hedgehog proteins (Sonic or SHH is the best studied), all of which are ligands for the membrane-bound receptor Patched (PTCH). Binding of a doubly lipid-modified SHH ligand to PTCH induces sequestration and degradation of the receptor, which leads to release of the 7-transmembrane domain protein, Smoothened (SMO), from intracellular compartments. In the absence of a hedgehog ligand, the role of PTCH is to block the function of SMO. With PTCH internalized, SMO can now enter the primary cilia where it stimulates the translocation and activation of transcription factors (Gli1-3) that control expression of Sonic hedgehog target genes and are implicated in the development of some cancers. Loss-of-function mutations in PTCH and activating mutations in SMO have been identified in patients with basal cell carcinoma.
          The precise molecular mechanism by which the 12-transmembrane-spanning receptor Patched suppresses the activity of Smoothened is not well characterized. Scott and Corcoran noted that PTCH is related structurally to a cholesterol transporter and provided evidence to suggest that this receptor modulates SMO activity by acting like a sterol pump that regulates the distribution of some unidentified endogenous oxysterol SMO ligand. They show that specific oxysterols are required for SHH pathway signal transduction and that a subset of these can maximally activate SHH target gene transcription through SMO. However, the identity of the putative endogenous SMO ligand remains unknown. Moreover, Beachy and co-workers have shown with competition and binding studies that oxysterols indirectly activate SMO and that the regulatory event probably occurs upstream of SMO. Delineation of the precise molecular mechanism that connects SMO to Gli’s remains an exciting and active area of biochemical research.

          Since aberrant activation of the hedgehog pathway is associated with malignancy, inhibition of this signaling network is a potential pharmacological intervention against cancer. Cyclopamine is a naturally occurring antagonist of the hedgehog pathway that has been shown to directly bind to SMO and inhibit its activity by a conformational change. However, the development of cyclopamine as an anticancer therapy is hampered by its thermodynamically favorable conversion to veratramine under acidic conditions. This transformation involves ring opening of the spirotetrahydrofuran followed by aromatization of the D-ring. Veratramine is inactive as a SMO antagonist and is known to induce side-effects stemming from serotonergic and other off-target activities. In order to overcome this obstacle and to generally improve cyclopamine’s drug properties, researchers at Infinity Pharmaceuticals homologated the steroidal D-ring by one methylene unit. This modification, in conjunction with A-ring derivatization to incorporate a sulfonamide or a fused pyrazole, delivered a chemically stable and orally active SMO antagonist (IPI-926) that completely regressed tumor formation in a medulloblastoma allograft model after daily oral administration (40 mg/kg). The partial chemical synthesis of IPI-926 and related analogues from cyclopamine, along with the semisynthesis of cyclopamine by the laboratory of Giannis, will be highlighted in subsequent posts.

          Additional chemical classes of SMO inhibitors have also appeared in the literature. These synthetic non-steroidal SMO ligands are exemplified by Genentech’s GDC-0449 (vismodegib) and most are comprised of a biaryl framework substituted with a benzamide. Published clinical results indicate that vismodegib exhibits antitumor activity in patients afflicted with basal-cell carcinoma. However, this class of compounds generally exhibits low aqueous solubility and is plagued by high plasma protein binding that can be attributed to high lipophilicity. Solubility/lipophilicity issues were overcome by medicinal chemists at Pfizer who recently disclosed the structure of PF-4449913, an orally bioavailable inhibitor of SMO that has been advanced to human clinical studies. Collectively, these favorable results encourage the pursuit of SMO antagonists as a means to discover and invent novel anticancer therapies.