Identification of Small-Molecule Inhibitors of Neutral Ceramidase (nCDase) via Target-Based High-Throughput Screening

There is interest in developing inhibitors of human neutral Ceramidase (nCDase) because this Enzyme plays a critical role in colon Cancer. There are currently no potent or clinically effective inhibitors for nCDase reported to date, so we adapted a fluorescence-based Enzyme activity method to a high-throughput screening format. We opted to use an assay whereby nCDase hydrolyzes the substrate RBM 14-16, and the addition of NaIO4 acts as an oxidant that releases umbelliferone, resulting in a fluorescent signal. As designed, test compounds that act as Ceramidase inhibitors will prevent the hydrolysis of RBM 14-16, thereby decreasing fluorescence. This assay uses a 1536-well plate format with excitation in the blue spectrum of LIGHT energy, which could be a liability, so we incorporated a counterscreen that allows for rapid selection against fluorescence artifacts to minimize false-positive hits. The high-throughput screen of >650,000 small molecules found several lead series of hits. Multiple rounds of chemical optimization ensued with improved potency in terms of IC₅₀ and selectivity over counterscreen assays. This study describes the first large-scale high-throughput optical screening assay for nCDase inhibitors that has resulted in leads that are now being pursued in crystal docking studies and in vitro drug metabolism and pharmacokinetics (DMPK).