Identifying Small Molecule Inhibitors Of Fascin 1 Using Fragment-Based Drug Discovery

The actin-bundling protein fascin 1 is markedly overexpressed in a range of invasive tumors and is believed to play a critical role in cancer cell metastasis. Targeting fascin is however very challenging owing to its mechanism of protein-protein interaction and a lack of knowledge regarding the crucial actin-binding sites. By combining a fragment-based approach, biophysical assay screening and X-ray crystallography, we have been able to identify and optimize novel fascin 1 inhibitors that show nanomolar affinity in biochemical binding and bundling assays. We now show the development of functional cell assays in which the lead fascin inhibitor compounds have been tested. For the first time we disclose the structures of the most advanced compounds and show their effects in 2D and 3D cell culture. Fascin 1 binds and cross-links filamentous actin (F-actin) into parallel bundles that are used in the formation of dynamic cellular protrusions (such as lamellipodia and filopodia) used during cell migration, and in the formation of invadopodia used by tumor cell lines to degrade the tumor extracellular matrix (ECM). Whereas fascin 1 expression is low or absent in normal epithelia, its expression is dramatically increased in a variety of invasive tumor types including colon, lung, ovarian and pancreas, with increased expression being an independent prognostic indicator of poor clinical outcome in the most aggressive and metastatic tumor types. Knockdown of fascin expression has been shown to reduce tumor cell invasion both in vitro and in vivo , thereby highlighting fascin as an important drug discovery target. Small molecule inhibitors of fascin 1 were identified by screening our fragment library (~1000 compounds) using surface plasmon resonance (SPR). X-ray co-crystallography with these hit compounds showed four distinct ligand binding sites within the fascin protein. Optimized site 2 binders induce a substantial conformational change, with the deeply enclosed pocket between fascin domains 1 and 2 opening a channel that accesses the surface of the protein. A virtual screen identified the compound BDP-00010834, which binds in this pocket with an SPR K d of 29.7µM and also inhibits (IC 50 =50µM) the functional activity of fascin measured in an F-actin bundling assay. Structure-based optimization in combination with X-ray co-crystallography has enabled the generation of compounds with more than 300-fold increase in both binding affinity and functional activity over this screening hit. Our current best-in-series compounds include BDP-00013544 (K d =29nM, IC 50 =185nM). These lead compounds have now been tested in a number of cell based invasion assays including both 2D and 3D cultures. Citation Format: Daniel Croft, Stuart Francis, Justin Bower, Andrea Gohlke, Gillian Goodwin, Christopher Gray, Jennifer Konczal, Sophie Macconnachie, Patricia McConnell, Laura McDonald, Heather McKinnon, Mokdad Mezna, Charles Parry, Nikki Paul, Angelo Pugliese, Alexander Schuettelkopf, Laura Machesky, Martin Drysdale. Identifying small molecule inhibitors of Fascin 1 using fragment-based drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-228. doi:10.1158/1538-7445.AM2017-LB-228