Abstract 2997: Dynarrestin, a novel dynein inhibitor that does not block ciliogenesis

Introduction: Aberrant hedgehog (Hh) pathway activation contributes to the pathogenesis of multiple cancers. Currently available Hh pathway inhibitors target Smoothened (Smo) which can acquire mutations causing drug resistance. Therefore, a major challenge is to identify compounds that inhibit Hh signaling downstream of Smoothened. Methods: We conducted a high-throughput screening (HTS) campaign of 337,000 small molecules using a Hh-dependent differentiation assay of C3H10T1/2 cells into osteoclasts. Primary active screening hits were validated by a cell-based assay cascade including motor neuron differentiation from mouse embryonic stem cells, Gli reporter activation in ShhLight2 cells and proliferation of heterozygous Patched (PTCH+/-) medulloblastoma (MB) cells. Smo binding was monitored in membrane-based Cyclopamine competition assays. To identify the underlying target identification of promising hits, affinity chromatography was performed followed by label-free quantitative (LFQ) tandem mass spectrometry. Effects on cytoplasmic dynein were examined in Smo trafficking and in vitro microtubules motility assays as well as by live cell imaging of labeled endosome movement. Results: We identified dynarrestin, a novel aminothiazole that potently blocks Hh signaling and the proliferation of Hh-dependent PTCH+/- MB tumor cells (IC50∼70nM). Dynarrestin does not bind to Smo and is able to suppress the Hh pathway in the presence of the Smo agonist SAG and following the knockdown of the Hh pathway suppressor SUFU indicating that dynarrestin inhibits Hh signaling downstream of both Smo and SUFU. Chemical LFQ proteomics identified cytoplasmic dynein as the target of dynarrestin. Dyneins convert energy from ATP hydrolysis into motor activity and are essential for many cellular processes, including Hh signaling. Unlike other dynein inhibitory molecules, e.g. Ciliobrevins, dynarrestin inhibits Smoothened trafficking but not ciliogenesis. We further demonstrate that dynarrestin reversibly interferes with endosome movement, proper mitotic spindle orientation, and dynein-based microtubule translocation in vitro without blocking ATP hydrolysis. Conclusions: Dynarrestin provides a novel dynein inhibitor with distinct specificity that uniquely facilitates probing dynein function. Given its multiple contributions to Hh signaling, mitosis and other cellular events, dynein remains a highly attractive target for medicinal chemistry programs aimed at exploiting and modulating its complex, poorly understood chemical cycle to interfere with different aspects of activity and function. We believe that unraveling dynein-dependent cellular processes by dynarrestin has great potential for development into novel anti-cancer drugs controlling Hh signaling downstream of Smoothened. Citation Format: Matthias Baumann, Susanne Hoing, Ting-Yu Yeh, Nancy Martinez, Peter Habenberger, Lea Kremer, Hannes CA. Drexler, Philipp Kuchler, Peter Reinhardt, Axel Choidas, Mia-Lisa Zischinsky, Gunther Zischinsky, Stephanie A. Ketcham, Lydia Wagner, Peter Nussbaumer, Slava Ziegler, Bert Klebl, Trina Schroer, Hans R. Scholer, Herbert Waldmann, Jared L. Sterneckert. Dynarrestin, a novel dynein inhibitor that does not block ciliogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2997.