Probing seco-steroid inhibition of the hedgehog signaling pathway.

Calcitriol, vitamin D3 (VD3), and structurally related VD3 analogues are inhibitors of Hh signaling in multiple contexts and are promising anti-cancer agents in Hh-dependent forms of cancer; however, the cellular mechanisms through which these compounds regulate Hh signal transmission are not clearly defined. Previous studies in this area have implicated both Smoothened, a key mediator of Hh signaling, and the vitamin D receptor (VDR) as potential mediators of Hh inhibition for this class of seco-steroids. We have performed a series of in vitro studies to more fully probe the cellular mechanisms that govern seco-steroid-mediated inhibition of Hh signaling. Our results support a role for both the Hh and VDR pathways in this process, as well as the possibility that other, as yet unidentified proteins, are also central to seco-steroid-mediated inhibition of Hh signaling.

Repurposing the Clinically Efficacious Antifungal Agent Itraconazole as an Anticancer Chemotherapeutic.

Itraconazole (ITZ) is an FDA-approved member of the triazole class of antifungal agents. Two recent drug repurposing screens identified ITZ as a promising anticancer chemotherapeutic that inhibits both the angiogenesis and hedgehog (Hh) signaling pathways. We have synthesized and evaluated first- and second-generation ITZ analogues for their anti-Hh and antiangiogenic activities to probe more fully the structural requirements for these anticancer properties. Our overall results suggest that the triazole functionality is required for ITZ-mediated inhibition of angiogenesis but that it is not essential for inhibition of Hh signaling. The synthesis and evaluation of stereochemically defined des-triazole ITZ analogues also provides key information as to the optimal configuration around the dioxolane ring of the ITZ scaffold. Finally, the results from our studies suggest that two distinct cellular mechanisms of action govern the anticancer properties of the ITZ scaffold.

Vitamin D3 analogues that contain modified A- and seco-B-rings as hedgehog pathway inhibitors.

The hedgehog (Hh) signaling pathway is a developmental signaling pathway that has been implicated as a target for anti-cancer drug development in a variety of human malignancies. Several natural and synthetic vitamin D-based seco-steroids have been identified as potent inhibitors of Hh signaling with chemotherapeutic potential. These include the previously characterized analogue 4, which contains the northern CD-ring/side chain region of vitamin D3 (VD3) linked to an aromatic A-ring mimic through an ester bond. To further explore structure-activity relationships for this class of VD3-based Hh pathway inhibitors, we have designed, synthesized and evaluated several series of compounds that modify the length, composition, and stereochemical orientation of the ester linker. These studies have identified compounds 54 and 55, which contain an amine linker and an aromatic A-ring incorporating a para-phenol, as new lead compounds with enhanced potency against the Hh pathway (IC50 values = 0.40 and 0.32 µM, respectively).

Structure-activity relationships for vitamin D3-based aromatic a-ring analogues as hedgehog pathway inhibitors.

A structure-activity relationship study for a series of vitamin D3-based (VD3) analogues that incorporate aromatic A-ring mimics with varying functionality has provided key insight into scaffold features that result in potent, selective Hedgehog (Hh) pathway inhibition. Three analogue subclasses containing (1) a single substitution at the ortho or para position of the aromatic A-ring, (2) a heteroaryl or biaryl moiety, or (3) multiple substituents on the aromatic A-ring were prepared and evaluated. Aromatic A-ring mimics incorporating either single or multiple hydrophilic moieties on a six-membered ring inhibited the Hh pathway in both Hh-dependent mouse embryonic fibroblasts and cultured cancer cells (IC50 values 0.74-10 µM). Preliminary studies were conducted to probe the cellular mechanisms through which VD3 and 5, the most active analogue, inhibit Hh signaling. These studies suggested that the anti-Hh activity of VD3 is primarily attributed to the vitamin D receptor, whereas 5 affects Hh inhibition through a separate mechanism.