Glutamate Transporter Inhibitors with Photo‐Controlled Activity

Abstract

Glutamate is an important signaling molecule in the nervous system and its extracellular levels are regulated by amino acid transporters. Studies on the role of glutamate transport have benefitted from the development of small molecule inhibitors. Most inhibitors, however, cannot be remotely controlled with respect to the time and place of their action, which limits their application in biological studies. Herein, the development and evaluation of inhibitors of the prokaryotic transporter Glt Tk with photo-controlled activity, enabling the remote, reversible, and spatiotemporally resolved regulation of transport is reported. Based on a known inhibitor, seven inhibitors, bearing a photoswitchable azobenzene moiety, are designed and synthesized. The most promising photo-controlled inhibitor, shows in its non-irradiated form, an IC 50 of 2.5 ± 0.4 μm for transport by Glt Tk . Photoswitching results in a reversible drop of potency to an IC 50 of 9.1 ± 1.5 μm. This 3.6-fold difference in activity is used to demonstrate that the transporter function can be switched on and off reversibly through irradiation. As a result, this inhibitor could be a powerful tool in studying the role of glutamate transport by precisely controlling the time, and the specific tissue or groups of cells, in which the inhibitor is active.

Structural ensemble of a glutamate transporter homologue in lipid nanodisc environment

“…Fully loaded state. At the concentration of 300 mM Na + used in the experiments presented here, the apparent K d for L-aspartate is 120 nM 32 , and therefore addition of 50 µM L-aspartate to 5.6 µM nanodiscs is expected to lead to substrate saturation. The cryo-EM structure of Glt Tk in the holo (Asp) state is symmetrical with all three transport domains in an intermediate-outward position ( Figs.…”

“…Cryo-EM structures of Glt Tk in nanodiscs. Purified Glt Tk was reconstituted into nanodiscs using the MSP2N2 scaffold protein 31 and a mixture of E.coli polar lipids and egg PC (3:1 (w/w)), a lipid composition that supports robust transport activity of the protein in proteoliposomes 11,32 . Glt Tk -nanodiscs were concentrated to 4.5-9.0 µM, and supplemented with 300 mM Na + .…”

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.…”

“…[1][2][3][4][5] Its scope has been demonstrated with av ast array of biological targets ranging from enzymes [6][7][8][9] to elements of the cytoskeleton, [10] voltage-and ligand-gated ion channels, [11][12][13][14][15][16] Gp rotein-coupled receptors (GPCRs), [17][18][19][20] and transporters. [21][22][23] Amongst other photoswitchable molecules,azobenzenes have emerged as the photoswitch of choice due to av ariety of reasons,i ncluding fast photoswitching, good photostationary states,l ack of phototoxicity,alarge change in dipole moment and steric bulk upon switching, compatibility with physiological conditions,f atigue-resistance,t unability,a nd the relative ease of their chemical synthesis. [1] Several research groups have developed anumber of ingenious ways to red-shift the photoswitching of azobenzenes and modulate their thermal bistability and conformational preferences.…”

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.…”