Neutral ceramidase-active site inhibitor chemotypes and binding modes

Abstract

Ceramides impact a diverse array of biological functions and have been implicated in disease pathogenesis. The enzyme neutral ceramidase (nCDase) is a zinc-containing hydrolase and mediates the metabolism of ceramide to sphingosine (Sph), both in cells and in the intestinal lumen. nCDase inhibitors based on substrate mimetics, for example C6-urea ceramide, have limited potency, aqueous solubility, and micelle-free fraction. To identify non-ceramide mimetic nCDase inhibitors, hit compounds from an HTS campaign were evaluated in biochemical, cell based and in silico modeling approaches. A majority of small molecule nCDase inhibitors contained pharmacophores capable of zinc interaction but retained specificity for nCDase over zinc-containing acid and alkaline ceramidases, as well as matrix metalloprotease-3 and histone deacetylase-1. nCDase inhibitors were refined by SAR, were shown to be substrate competitive and were active in cellular assays. nCDase inhibitor compounds were modeled by in silico DOCK screening and by molecular simulation. Modeling data supports zinc interaction and a similar compound binding pose with ceramide. nCDase inhibitors were identified with notably improved activity and solubility in comparison with the reference lipid-mimetic C6-urea ceramide.

Keywords: Colorectal cancer; Neutral ceramidase; Neutral ceramidase inhibitor; nCDase.

Figure 1:

nCDase inhibitory compounds (SB-17, SB-22, SB-26) show little or no activity for activity against alkaline and acid ceramidase microsomes at concentrations of 5uM or 50uM. Hydrolysis of C16 ceramide substrate to sphingosine was measured by LC-MS/MS, as described in Materials and Methods, and expressed as pmol/g of protein/min.

Figure 2.

A. IC50 curve for isothiazole SB-37 by RBM assay. B. Lineweaver Burk plot of SB-37 indicating a substrate competition mode of action. Enzyme was incubated with nCDase inhibitor SB-37 (0.2, 0.5, and 5μM) and substrate added at the final concentration of 0.75, 1.25, 2.5, 5, 10, 20, and 40μM in Triton X100 buffer. C. 293 cells were transiently transfected with wild type (WT) and inactive mutant human nCDase CMV expression constructs and ceramidase activity measured by RBM assay, with and without SB-26 (30uM).

Figure 3.

A. Predicted consensus pose for 14 out of 17 isothiazole nCDase inhibitors. B. Modeled pose for ceramide from Airola et al. (1). Proposed Zn²⁺ interactions in dashed cyan and hydrogen bonding in dashed magenta. Docked ligands in orange, ceramide in green, protein residues in gray.

Figure 4.

Comparison of docked poses for ligand SB-26 (#26; orange) with SB-34, SB-37, and SB-17 (#34, #37, #17; magenta).

Figure 5.

(Left) Ligand RMSD plots from three independent 100 ns MD simulations (replicates) for SB-26 docked with nCDase. Solid colored lines represent moving boxcar averages (window size = 100 frames) for each trajectory. (Right) Frame overlay plot (1000 evenly spaced frames) for SB-26 (orange) and zinc (cyan) from one MD simulation. Protein residues hidden for clarity.

Figure 6.

A. Proposed binding geometries for compound SB-26 (left, orange), SB-17 (middle, magenta), and SB-37 (right, magenta) with nCDase in comparison to the previously published modeled pose for ceramide (green) from Airola et al. (1). B. Protein-ligand van der Waals energies, on a per-residue basis (termed footprints) for all four species (SB-26, SB-17, SB-37, ceramide).