Development of a high throughput screening assay for inhibitors of hedgehog-heparin interactions

Laura R Daye, Willietta Gibson, Kevin P Williams

Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham, NC , USA

Abstract: The activity of hedgehog ligand is mediated in part by interactions with heparin and heparan sulfate proteoglycans. In order to identify inhibitors that block the interaction of hedgehog with heparin, we have developed a simple and robust high throughput assay based on fluorescence polarization. This assay utilizing fluorescein-labeled heparin binding to sonic hedgehog N-terminal domain (ShhN) protein was developed and optimized in a 384-well format. ShhN bound to fluorescein-labeled heparin with high affinity (K_D = 99 nM) and the interaction was shown to be stable over time and tolerant to dimethyl sulfoxide (DMSO). A panel of unlabeled heparins of varying molecular weights was tested in the assay, with lower IC₅₀ values correlating with heparins of increasing size. The assay was automated into two simple steps, and validation with whole DMSO plates yielded a Z’ factor of 0.56, indicating a robust assay for high throughput screening. We predict that this assay will be suitable for identifying chemical probes of hedgehog ligand-heparin interactions. Further, compounds that disrupt the ShhN interaction with heparin and/or heparan sulfate proteoglycans may be considered as potential therapeutics for those cancers driven by Hh ligand overexpression.

Keywords: hedgehog, heparin, fluorescence polarization, high throughput screening