Probing the functional limits of the norepinephrine transporter with self-reporting, fluorescent stilbazolium dimers

Abstract

A series of stilbazolium dimers were synthesized and investigated as sterically demanding ligands targeting the norepinephrine transporter (NET). The environmentally sensitive fluorescence of the dyes enables their use as self-reporting ligands; binding to and displacement from NET can be monitored by fluorescence microscopy.

Characteristic Fluorescence Response of (6‐Hydroxy‐2‐naphthyl)ethenyl Pyridinium Dyes with Bovine Serum Albumin

“…The ICD measurements were carried out to monitor the absorbance of far ultraviolet range, which reflect the conformation changes of peptide chains of protein induced by the interaction with HNEP. In the ICD spectra, the intensity of the double minimums reflects 58.8% of the α‐helical structure in the BSA, close to a value in the literature (Figure (b)). The binding of the HNEP's to BSA results in a deepening of the trough at 209 nm, which reflects an increase in the helix content of BSA with the conformational changes.…”

Evaluation of [11C]TAZA for amyloid β plaque imaging in postmortem human Alzheimer's disease brain region and whole body distribution in rodent PET/CT

“…Previous reports have revealed micromolar affinities for NET uptake of stilbene and N , N ‐dimethylaminophenyldiazenyl derivatives containing the guanidino group (Hadrich et al ., ) and stilbene dimers (Smith et al ., ). NET existence in BAT has been reported in both rodents (Okuyama et al .…”

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

“…As previously reported for A1, D1, and H1, introduction of the probes results in a rapid increase in emission intensity as a result of binding to NET. 4,5,7,8 In the case of A1 and H1 this initial rapid rise is followed by a more gradual increase due to probe transport and intracellular accumulation in mitochondria. 4,5 Thus, NET binding and uptake can be temporally and spatially resolved by confocal microscopy.…”

“…These results are consistent with our previous report on related stilbazolium dimers. 8 Using the representative poses obtained from AutoDock Vina, we next performed geometry optimizations (MMFF, Spartan 08) on A1 and H1 in the NET binding pocket with the following flexible residues: ASP75, ASP473, PHE72, PHE317, PHE329, and TYR152 (Fig. 11).…”

“…2), may bind to, but are not transported via NET. 8 These probes extended to approximately 35 Å in length, demonstrating that inclusion of a bulky 'tail' appears to be a key feature that limits transport activity while still permitting probe binding to NET. The difference in length between A1 and D1 is approximately 20 Å and it is likely that substrates with dimensions intermediate to A1 and D1 may exhibit both binding to and transport via NET.…”