Photoswitchable Inhibitor of a Glutamate Transporter

Abstract

Excitatory amino acid transporters clear glutamate from the synaptic cleft and play a critical role in glutamatergic neurotransmission. Their differential roles in astrocytes, microglia, and neurons are poorly understood due in part to a lack of pharmacological tools that can be targeted to specific cells and tissues. We now describe a photoswitchable inhibitor, termed ATT, that interacts with the major mammalian forebrain transporters EAAT1-3 in a manner that can be reversibly switched between trans (high-affinity) and cis (low-affinity) configurations using light of different colors. In the dark, ATT competitively inhibited the predominant glial transporter EAAT2 with ∼200-fold selectivity over the neuronal transporter EAAT3. Brief exposure to 350 nm light reduced the steady-state blocker affinity by more than an order of magnitude. Illumination of EAAT2 complexed with ATT induced a corresponding increase in the blocker off-rate monitored in the presence of glutamate. ATT can be used to reversibly manipulate glutamate transporter activity with light and may be useful to gain insights into the dynamic physiological roles of glutamate transporters in the brain, as well as to study the molecular interactions of transporters with ligands.

Glutamate Transporter Inhibitors with Photo‐Controlled Activity

“…Surprisingly, p ‐CF 3 ‐azo‐TBOA , reported earlier, was in our hands unstable and small shifts in the spectra upon five cycles of irradiation were observed (Figure S45, Supporting Information). When determining the PSS upon irradiation in DMSO by 1 H NMR spectroscopy, formation of side products was observed (Figures S43 and S44, Supporting Information), which was not observed before .…”

“…Surprisingly, p ‐CF 3 ‐azo‐TBOA , reported earlier, was in our hands unstable and small shifts in the spectra upon five cycles of irradiation were observed (Figure S45, Supporting Information). When determining the PSS upon irradiation in DMSO by 1 H NMR spectroscopy, formation of side products was observed (Figures S43 and S44, Supporting Information), which was not observed before . However, it must be noted that we used different wavelengths of irradiation (312 nm and 365 nm vs 350 nm) and the shifts in the spectrum are mainly observed for switching in DMSO and not in 50 m m KPi buffer (pH 7.4).…”

“…To overcome this limitation, control over the activity of the inhibitor with an external stimulus would be highly desirable as it would allow to reversibly turn the inhibitor on and off at specific organs, tissues and cells at any chosen time and in a reversible manner. Such a remotely controlled inhibitor would contribute to a better understanding of the role of the glutamate transporters in health and disease, as also exemplified by a recent report by Trauner and Kavanaugh in which one of the molecules also reported here was evaluated on human EAATs …”

“…Due to the difference in electronic properties and structure, all the substituents likely influence both the biological activity of cis and trans isomers and the photochemical properties such as the maximum wavelength of absorption, photo‐stationary states (PSS) and half‐life of the cis isomer. Finally, we also sought to evaluate the p ‐CF 3 substituted compound, which is the closest to the original TFB‐TBOA structure, inspired by a recent report by Trauner and Kavanaugh . In their study, differences in activity between trans and cis isomers were observed on oocytes overexpressing either EAAT1, EAAT2, or EAAT3 by measuring membrane voltage.…”

Sign Inversion in Photopharmacology: Incorporation of Cyclic Azobenzenes in Photoswitchable Potassium Channel Blockers and Openers

“…Freely diffusible azobenzene photoswitches that are active in their trans-form have been developed for av ariety of targets.T hese include GPCRs,s uch as the m-opioid receptor, [17] the M1 muscarinic receptor, [54] the sphingosine phosphate receptor S1PR1, [55] and the metabotropic glutamate receptor mGluR5, [18] and ion channels,s uch as GA-BAA, [56,57] a7n AChR, [14] and ionotropic glutamate receptors. [15,37,58] Dark active photopharmaceuticals have also been used to optically control transporters,such as GAT1, [21] EAAT1-3, [22,23] and F 1 F 0 -ATPase, [59] as well as enzymes. [60] Given the success of CAL, LAB-QA, CLOGO,a nd the glutamate diazocine derivatives LAB-Glu [36] and Glu brAzo1/ 2, [61] which target NMDAr eceptors and kainate receptors, respectively,i ts eems likely that the photopharmacological sign inversion of trans-active azobenzenes through substitution with diazocines is ag enerally applicable concept.…”