Small molecule inhibitors of Smoothened ciliary localization and ciliogenesis

Abstract

Vertebrate Hedgehog (Hh) signals involved in development and some forms of cancer, such as basal cell carcinoma, are transduced by the primary cilium, a microtubular projection found on many cells. A critical step in vertebrate Hh signal transduction is the regulated movement of Smoothened (Smo), a seven-transmembrane protein, to the primary cilium. To identify small molecules that interfere with either the ciliary localization of Smo or ciliogenesis, we undertook a high-throughput, microscopy-based screen for compounds that alter the ciliary localization of YFP-tagged Smo. This screen identified 10 compounds that inhibit Hh pathway activity. Nine of these Smo antagonists (SA1–9) bind Smo, and one (SA10) does not. We also identified two compounds that inhibit ciliary biogenesis, which block microtubule polymerization or alter centrosome composition. Differential labeling of cell surface and intracellular Smo pools indicates that SA1–7 and 10 specifically inhibit trafficking of intracellular Smo to cilia. In contrast, SA8 and 9 recruit endogenous Smo to the cilium in some cell types. Despite these different mechanisms of action, all of the SAs inhibit activation of the Hh pathway by an oncogenic form of Smo, and abrogate the proliferation of basal cell carcinoma-like cancer cells. The SA compounds may provide alternative means of inhibiting pathogenic Hh signaling, and our study reveals that different pools of Smo move into cilia through distinct mechanisms.

Repurposing the Antihelmintic Mebendazole as a Hedgehog Inhibitor

“…The inhibitory effect of microtubule inhibitors on Hh signaling has been demonstrated by unbiased screens that have identified such agents indirectly, as inhibitors of GLI activity, or by direct inhibition of cilia formation and function (33,56,57). Thus far, the anti-microtubule agents identified in these screens are either highly toxic, or at early stages of characterization.…”

The roles of evolutionarily conserved functional modules in cilia-related trafficking

“…Heralding such a possibility, a recent study identified small molecules that selectively affect smoothened (a Hedgehog signalling protein) cilia-related trafficking, and these could thus be employed as anti-tumour compounds 131 .…”

Förster resonance energy transfer-based cholesterolysis assay identifies a novel hedgehog inhibitor

“…Hedgehog’s oncogenic activity has galvanized small molecule discovery efforts, resulting in the recent approval of vismodegib (Erivedge) for the treatment of advanced basal cell carcinoma [5]. Vismodegib, like several other Hh inhibitors under clinical study, interferes with transduction of the Hh signal by antagonizing a cell surface receptor called Smoothened [6, 7]. While a major milestone, alternative approaches to combat aberrant Hh activity are worth pursuing for combination therapy and to contend with clinical resistance to vismodegib [8-10].…”