Synthesis and characterization of a new two photon excitable acid sphingomyelinase FRET probe

Bioorg Med Chem. 2021 Aug 15:44:116303. doi: 10.1016/j.bmc.2021.116303. Epub 2021 Jul 13.

Abstract

Recently, FRET probes for acid sphingomyelinase (ASM) have enabled the observation of enzyme activity in intact cells for the first time. Here we present an ASM FRET probe specifically optimized for 2-photon excitation. To facilitate probe characterization and comparison to the previous probe, we mixed the two intact probes with defined amounts of the probes' ceramide cleavage products and mounted them on lipid beads. Directly excited NBD FRET acceptor fluorescene proved to be a useful means of reference and showed that the new probe is brighter, albeit only moderately, than the previous one. The new probe was then used to detect inhibition by various ASM inhibitors microscopically for the first time. Also in cells, directly excited acceptor fluorescence proved to be a useful parameter in addition to FRET to visualize inhibition of ASM.

Keywords: Antidepressants; Enzyme inhibitors; FRET probes; Förster resonance energy transfer (FRET); Sphingolipids; Two-photon microscopy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Coumarins / chemical synthesis
  • Coumarins / chemistry
  • Coumarins / pharmacology*
  • Dose-Response Relationship, Drug
  • Enzyme Inhibitors / chemical synthesis
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / pharmacology*
  • Fluorescence Resonance Energy Transfer*
  • Humans
  • Molecular Structure
  • Photons*
  • Sphingomyelin Phosphodiesterase / analysis*
  • Sphingomyelin Phosphodiesterase / metabolism
  • Structure-Activity Relationship

Substances

  • Coumarins
  • Enzyme Inhibitors
  • coumarin
  • SMPD1 protein, human
  • Sphingomyelin Phosphodiesterase