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

Abstract

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).

Keywords: HTS; colon cancer; fluorescence; neutral ceramidase; pharmacological inhibitors.

Figure 1.

(A) Scatter plot of data from nCDase Inhibitor Primary HTS. Each dot graphed represents the activity result of a well containing test compound (black dots) or controls (red and green dots). (B) titration result of the pharmacological control compound, C6 urea-ceramide. N = 5 separate experiment, N=4 wells per replicate point, error bars are shown. The average calculated IC₅₀ was 17.23 ± 7.0 μM.

Figure 2.

(A-B) The scatter plots of data from confirmation and conterscreen. Each dot graphed represents the activity result of a well containing test compound (black dots) or controls (red and green for high and low controls, respectively). The red dotted lines represent Hit cutoff percentage. (A) Confirmation screen (B) Couterscreen (C) Venn diagram of compounds found active in the confirmation and counterscreen. Of the 2,497 compounds tested, 125 compounds confirmed activity and 117 appear to be selective / active in confirmation screen and inactive in the counterscreen assay.

Figure 3.

Active compound potency and efficacy. Active compounds from screening (#17, #22, B5, TSA) and an analog of compound 22 (#26) were dose titrated using the RBM-ceramide assay to establish IC50 potency and percent maximal inhibition efficacy. The IC₅₀ is shown in micromolar concentration for each.

Figure 4.

Confirmation of active compound potency and efficacy using orthogonal FRET and NBD-ceramide HPLC assays. (A) FRET assay confirmation of prioritized compound series #17 and #26 with TSA control reference compound identified in pilot HTS screening. (B) NBD-ceramide HPLC dose titration confirmation of compounds #17, #26, B5 and TSA control. HPLC separation of fluorescent substrate, product and test compound based on retention time removes potential compound fluorescent interference. The IC₅₀ is shown in micromolar concentration for each.