Fluorescent stilbazolium dyes as probes of the norepinephrine transporter: structural insights into substrate binding

Org Biomol Chem. 2012 Nov 21;10(43):8710-9. doi: 10.1039/c2ob26633d.

Abstract

We report the synthesis, binding kinetics, optical spectroscopy and predicted binding modes of a series of sterically demanding, fluorescent norepinephrine transporter (NET) ligands. A series of bulky stilbazolium dyes, including six newly synthesized compounds, were evaluated to determine the effect of extending the molecular probes' 'heads' or 'tails'. Taking advantage of the dyes' characteristic 'turn-on' emission, the kinetic binding parameters, k(on) and k(off) were determined revealing that extension of the molecules' tails is well tolerated while expansion of the head is not. Additionally, a 'headfirst' orientation appears to be preferred over a 'tail-first' binding pose. Further details of the possible binding modes were obtained from the emission spectra of the bound probes. A small range of interplanar twist angles, approximately 35° to 60°, is predicted to produce the observed emission. Docking experiments and molecular modelling support the kinetic and spectroscopic data providing structural insights into substrate binding.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Binding Sites
  • Cells, Cultured
  • Fluorescent Dyes / chemical synthesis
  • Fluorescent Dyes / chemistry*
  • HEK293 Cells
  • Humans
  • Kinetics
  • Ligands
  • Microscopy, Confocal
  • Models, Molecular
  • Molecular Probes / chemical synthesis
  • Molecular Probes / chemistry*
  • Molecular Structure
  • Norepinephrine Plasma Membrane Transport Proteins / chemistry*
  • Pyridinium Compounds / chemical synthesis
  • Pyridinium Compounds / chemistry*
  • Quantum Theory

Substances

  • 4-(4-dimethylaminostyryl)-1-methylpyridinium
  • Fluorescent Dyes
  • Ligands
  • Molecular Probes
  • Norepinephrine Plasma Membrane Transport Proteins
  • Pyridinium Compounds