Discovery and characterization of selective human sphingomyelin synthase 2 inhibitors

Eur J Med Chem. 2017 Aug 18;136:283-293. doi: 10.1016/j.ejmech.2017.04.067. Epub 2017 Apr 25.

Abstract

Sphingomyelin synthase (SMS) is a membrane enzyme that catalyzes the synthesis of sphingomyelin, is required for the maintenance of plasma membrane microdomain fluidity, and has two isoforms: SMS1 and SMS2. Although these isoforms exhibit the same SMS activity, they are different enzymes with distinguishable subcellular localizations. It was reported that SMS2 KO mice displayed lower inflammatory responses and anti-atherosclerotic effects, suggesting that inhibition of SMS2 would be a potential therapeutic approach for controlling inflammatory responses and atherosclerosis. This study aimed to discover a novel small-molecule compound that selectively inhibits SMS2 enzymatic activity. We developed a human SMS2 enzyme assay with a high-throughput mass spectrometry-based screening system. We characterized the enzymatic properties of SMS2 and established a high-throughput screening-compatible assay condition. To identify human SMS2 inhibitors, we conducted compound screening using the enzyme assay. We identified a 2-quinolone derivative as a SMS2 selective inhibitor with an IC50 of 950 nM and >100-fold selectivity for SMS2 over SMS1. The 2-quinolone exhibited efficacy in a cell-based engagement assay. We demonstrated that a more potent derivative directly bound to SMS2-expressing membrane fractions in an affinity selection mass spectrometry assay. Mutational analyses revealed that the interaction of the inhibitor with SMS2 required the presence of the amino acids S227 and H229, which are located in the catalytic domain of SMS2. In conclusion, we discovered novel SMS2-selective inhibitors. 2-Quinolone SMS2 inhibitors are considered applicable for leading optimization studies. Further investigations using these SMS2 inhibitors would provide validation tools for SMS2-relevant pathways in vitro and in vivo.

Keywords: 2-Quinolone derivative; Catalytic domain; Mass spectrometry; Sphingolipid; Sphingomyelin synthase 2 (SMS2).

MeSH terms

  • Animals
  • Dose-Response Relationship, Drug
  • Drug Discovery*
  • Enzyme Inhibitors / chemical synthesis
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / pharmacology*
  • HEK293 Cells
  • High-Throughput Screening Assays
  • Humans
  • Mass Spectrometry
  • Membrane Proteins / antagonists & inhibitors*
  • Membrane Proteins / deficiency
  • Membrane Proteins / metabolism
  • Mice
  • Mice, Knockout
  • Molecular Structure
  • Nerve Tissue Proteins / antagonists & inhibitors*
  • Nerve Tissue Proteins / deficiency
  • Nerve Tissue Proteins / metabolism
  • Small Molecule Libraries / chemical synthesis
  • Small Molecule Libraries / chemistry
  • Small Molecule Libraries / pharmacology*
  • Structure-Activity Relationship
  • Transferases (Other Substituted Phosphate Groups) / antagonists & inhibitors*
  • Transferases (Other Substituted Phosphate Groups) / deficiency
  • Transferases (Other Substituted Phosphate Groups) / metabolism

Substances

  • Enzyme Inhibitors
  • Membrane Proteins
  • Nerve Tissue Proteins
  • Small Molecule Libraries
  • SGMS1 protein, human
  • Transferases (Other Substituted Phosphate Groups)