Optical Control of GABAA Receptors with a Fulgimide-Based Potentiator

Abstract

Optogenetic and photopharmacological tools to manipulate neuronal inhibition have limited efficacy and reversibility. We report the design, synthesis, and biological evaluation of Fulgazepam, a fulgimide derivative of benzodiazepine that behaves as a pure potentiator of ionotropic γ-aminobutyric acid receptors (GABA_A Rs) and displays full and reversible photoswitching in vitro and in vivo. The compound enables high-resolution studies of GABAergic neurotransmission, and phototherapies based on localized, acute, and reversible neuroinhibition.

Keywords: GABAA Receptors; fulgimides; in vivo; photopharmacology.

Figure 1

A) Furan‐fulgimide in its open and closed isomeric state interconvertible by illumination with UV and visible light.18, 19 B) Left: Pharmacophore nitrazepam and its extension towards a photochromic fulgimide. Right: Derivatization towards a photochromic diazepine fulgide hybrid.

Figure 2

A) Synthesis of fulgimide‐nitrazepam 3 a and its iso‐fulgimide derivative 4 a. We performed the reaction of furano‐fulgide 2 25 with amino‐nitrazepam 1 27 upon addition of dicyclohexylcarbodiimide (DCC), diisopropylethylamine (DIPEA), and 1‐hydroxybenzotirazole (HOBt) in methanol, to afford the desired benzodiazepine‐furano‐fulgimide 3 a and its iso‐fulgimide derivative 4 a. B) Synthesis of the highly functionalized furan 7 and its diazepine formation towards compound 9.21, 24, 25, 26

Figure 3

A) Illumination induced ring‐closing (4 b) and ring‐opening (4 a) of iso‐fulgimide 4 (Fulgazepam). B) Photochromic properties of iso‐fulgimide 4 (50 μm) measured in DMSO. Left: Spectral evolution of 4 a (open isomer; grey spectrum) upon illumination with 365 nm and re‐opening of 4 b (closed isomer; purple spectrum) upon illumination with 528 nm. Right: Cycle performance of 4 upon alternate illumination with 365 nm (ring closing) and 528 nm (ring opening) detected at 518 nm (λ _max closed isomer). UV–Vis absorption spectrum and cycle performance of isofulgimide 4 upon illumination with 365 nm and 505 nm. Black arrows indicate the spectral evolution upon illumination. Dotted black arrows label isosbestic points indicating a clear two component switching. After 10 s illumination at λ=365 nm the closed‐PSS was reached and 93 % of the closed‐isomer accumulated. Quantitative reopening was achieved within 120 s illumination at λ=505 nm or 528 nm, respectively. Both compounds show sufficient fatigue resistance for photopharmacological experiments over ten measured cycles upon alternate illumination with 365 nm for closing and 528 nm for opening.

Figure 4

The effect of compounds 4 a and 4 b on GABA_A‐mediated currents. A) Upper panel: representative traces of currents induced by application of GABA 0.5 μM and by mixture of GABA 0.5 μM with 4 a 10 μM; lower panel: representative traces of currents induced by application of GABA 0.5 μM and by mixture of GABA 0.5 μM with 4 b 10 μM. Durations of applications of GABA and compound 4 are indicated by black bars above the traces. B) Cumulative dose‐response curve for compound 4 b (n=6). C) Representative traces demonstrating the effect of 4 a photoswitching on the amplitude of GABA‐induced currents. On the left: current was induced by application of GABA 0.5 μM; on the right: at the same trace current was induced subsequently by GABA 0.5 μM, by mixture of GABA with 4 a 10 μM under visible light and upon illumination with UV light (4 b). Duration of UV illumination is indicated by a violet rectangle. Note the prominent increase of the GABA‐induced current in the presence of 4 during illumination with UV light, which triggers ring‐closing (4 b). D) Cumulative graph representing mean relative amplitude of currents induced by application of GABA 0.5 μM (black column), GABA 0.5 μM+4 a 10 μM (green column) and GABA 0.5 μM+4 b 10 μM (violet column) upon illumination with UV light (n=11).

Figure 5

A) Effect of Fulgazepam in wild type zebrafish larvae. One‐minute trajectories of average swimming distances (n=12 per treatment) are shown for vehicle (1 % DMSO) and three different concentrations of compound 4, starting with pre‐irradiated solutions of 4 a (top) and 4 b (bottom). For the first 20 minutes, larvae were undisturbed in complete darkness (relaxation period, RP), therefore maintaining compound 4 stable states. Following RP larvae were illuminated with three consecutive cycles of visible light (500 nm) and UV (365 nm) with discrete dark between each wavelength. Colored areas show standard error of the mean (S.E.M.). B) Top: Quantification of swimming distances over the last 5 minutes of the RP (darkness) from two independent experiments (n=24 per treatment) for both pre‐illuminated compounds 4 a (green trace) and 4 b (violet trace) and vehicle (1 % DMSO). Bottom: Quantification of total distance swam after light periods (UV and visible light) for compound 4 and vehicle (n=12 per treatment). *p‐value<0.05, ****p‐value<0.0001. Colored areas show standard deviation (S.D.).