8-Hydroxyquinoline derivatives suppress GLI1-mediated transcription through multiple mechanisms.

Aberrant activation of the Hedgehog (Hh) signaling pathway has been observed in various human malignancies. Glioma-associated oncogene transcription factor 1 (GLI1) is the ultimate effector of the canonical Hh pathway and has also been identified as a common regulator of several tumorigenic pathways prevalent in Hh-independent cancers. The anti-cancer potential of GLI1 antagonism with small molecule inhibitors has demonstrated initial promise; however, the continued development of GLI1 inhibitors is still needed. We previously identified a scaffold containing an 8-hydroxyquinoline as a promising lead GLI1 inhibitor (compound 1). To further develop this scaffold, we performed a systematic structure-activity relationship study to map the structural requirements of GLI1 inhibition by this chemotype. A series of biophysical and cellular experiments identified compound 39 as an enhanced GLI1 inhibitor with improved activity. In addition, our studies on this scaffold suggest a potential role for SRC family kinases in regulating oncogenic GLI1 transcriptional activity.

Structure-based virtual screening identifies an 8-hydroxyquinoline as a small molecule GLI1 inhibitor.

The glioma-associated family of transcription factors (GLI) have emerged as a promising therapeutic target for a variety of human cancers. In particular, GLI1 plays a central role as a transcriptional regulator for multiple oncogenic signaling pathways, including the hedgehog (Hh) signaling pathway. We undertook a computational screening approach to identify small molecules that directly bind GLI1 for potential development as inhibitors of GLI-mediated transcription. Through these studies, we identified compound 1, which is an 8-hydroxyquinoline, as a high-affinity binder of GLI1. Compound 1 inhibits GLI1-mediated transcriptional activity in several Hh-dependent cellular models, including a primary model of murine medulloblastoma. We also performed a series of computational analyses to define more clearly the mechanism(s) through which 1 inhibits GLI1 function after binding. Our results strongly suggest that binding of 1 to GLI1 does not prevent GLI1/DNA binding nor disrupt the GLI1/DNA complex, but rather, it induces specific conformational changes in the overall complex that prevent proper GLI function. These results highlight the potential of this compound for further development as an anti-cancer agent that targets GLI1.

Truncated Itraconazole Analogues Exhibiting Potent Anti-Hedgehog Activity and Improved Drug-like Properties.

We conducted a structure-activity relationship study to explore simplified analogues of the itraconazole (ITZ) scaffold for their ability to inhibit the hedgehog (Hh) signaling pathway. These analogues were based on exploring the effects of chemical modifications to the linker and triazolone/side chain region of ITZ. Analogue 11 was identified as the most potent compound in our first generation, with an IC50 value of 81 nM in a murine Hh-dependent basal cell carcinoma. Metabolic identification studies led us to identify truncated piperazine (26) as the major metabolite in human liver microsomes (HLMs) and an improved Hh pathway inhibitor (IC50 = 22 nM). This work verifies that continued truncation of the ITZ scaffold is a practical method to maintain potent anti-Hh activity while also reducing the molecular weight for the ITZ scaffold and achieving improved pharmacokinetic properties.

Development of posaconazole-based analogues as hedgehog signaling pathway inhibitors.

Inhibition of the hedgehog (Hh) signaling pathway has been validated as a therapeutic strategy to treat basal cell carcinoma and holds potential for several other forms of human cancer. Itraconazole and posaconazole are clinically useful triazole anti-fungals that are being repurposed as anti-cancer agents based on their ability to inhibit the Hh pathway. We have previously demonstrated that removal of the triazole from itraconazole does not affect its ability to inhibit the Hh pathway while abolishing its primary side effect, potent inhibition of Cyp3A4. To develop structure-activity relationships for the related posaconazole scaffold, we synthesized and evaluated a series of des-triazole analogues designed through both ligand- and structure-based methods. These compounds demonstrated improved anti-Hh properties compared to posaconazole and enhanced stability without inhibiting Cyp3A4. In addition, we utilized a series of molecular dynamics and binding energy studies to probe specific interactions between the compounds and their proposed binding site on Smoothened. These studies strongly suggest that the tetrahydrofuran region of the scaffold projects out of the binding site and that π-π interactions between the compound and Smoothened play a key role in stabilizing the bound analogues.