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 (GABAARs) 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.

In the quest to understand brain circuits, controlling neuronal activity with light has become an essential tool to manipulate the balance between excitation and inhibition. [1] However,o ptogenetic tools to inhibit neurons (halorhodopsin pumps, anion-conducting bacterial channelrhodopsins and chlorideconducting ChR2 mutants) [2][3][4] have very limitedc onductance and dynamic responses to depolarization. Caged g-aminobutyric acid(GABA) compounds have been used to inhibits pines and to control seizures, but uncaging is irreversible and their neurotoxicity [5] has been avoided only recently by means of allosteric ligands. [6] Ap owerful alternative is using reversible chemicalp hotoswitches [7][8][9][10] to harness endogenousanion-conductingr eceptor-channels like GABA and glyciner eceptors (GABA A R, GlyR), which mediate inhibitoryn eurotransmission in the mammalian central nervous system. [11] Although some GABA A Rp hotoswitches have been reported based on azobenzene, [12][13][14] this photochromic group displays several shortcomings:i tp rovides incomplete photoconversion due to as ubstantialo verlap of the absorption maxima of cis and trans isomers, and can alter the pharmacophorea ctivity.I ndeed, in all azobenzene derivatives of benzodiazepines (allostericp otentiators of GABA A R) this characteristicp roperty is abolished, as found in the 7-aminos ite of nitrazepam, which is reportedly tolerant of other substitutions. [15] In addition, GABA A Rp hotoswitches described so far are agonistsora ntagonists that interfere with endogenous neurotransmission,n ot pure modulators. [13,16,17] Here we introduce Fulgazepam (compound 4), ad erivative of diazepam based on ad ifferent photochromic group (fulgimide), which shows both quantitativer eversible switching and GABA A Rp otentiation. It is inactive in its open isomera nd potentiates the receptor in its closed isomer without concomitant agonisto ra ntagonist activity,t hus overcoming the above mentioned hurdles and displaying an ideal photopharmacological profile. Most importantly,F ulgazepam works both in vitro and in vivo, which indicates that the compound is devoid of toxicity and has favourable molecularp roperties enabling wide applications.
In contrastt oa zobenzenes, dithienylethenes, fulgides and their fulgimide-nameda mide derivatives generally feature high photostationary states (PSS) with both photoisomersb eing thermally stable. [7,18] As dithienylethenes often lack of switching efficiency and stabilityi np olar solvents due to at wisted intramolecular electron charget ransfer, [20][21][22] we chose fulgi(mi)des as photochromic scaffold in this study.B oths ubtypes can be interconverted between their flexible, less-coloured ring-open and their rigid, more coloured ring-closed isomer upon light-induced conrotatory 6p-electrocyclic rearrangement( Figure 1, Scheme 1). [18,23] Althoughs witching from the open to the closed form is usuallytriggered using UV light, this might be avoided by the isolation and separate application of both isomers. In addition, this ensures the application of quantitative amounts of either the open or the closed form. Thereby,abiological effect can clearly be assigned to one or the other conformation.S ynthetic investigations revealed the beneficial effects of an isopropyl group in the alpha bridge position of the fulgide, as the E-Z isomerization of the open isomer is suppressed due to steric hindrance and consequently only two distinct isomersa re observed ( Figure 1A). [19] One advantage of fulgimides over fulgides is their improved switching in aqueous solutions and high stability. Furthermore, the two-step transformation of fulgides towards fulgimides via nucleophilic ring-opening of the anhydride by ap rimary amine and subsequent recyclization allows the smooth introduction of amino-functionalized biomolecules. [18,19] However,f ew biological applications of fulgi(mi)des are reported. [24][25][26] The transformation of ak nown ligand into ap hotoresponsive molecule can be designed by either extendingt he pharmacophore with ap hotoswitch or via incorporation of the photochromic scaffold as part of the drug's chemical structure. Once introduced, ideally one isomeric statei sbiologically active whereas the other loses its required interactions. In the presented work, both approaches were pursued. On the one hand, af uran-fulgide photochromic scaffold was merged with an amino-benzodiazepine under fulgimide formation ( Figure 1B,l eft panel). A difference in activity can be expected from the different flexi-bility of the isomeric states. On the other hand, af unctionalized diazepine was synthesized aiming for ap hotochromic benzodiazepine core ( Figure 1B,r ight panel). In this case, the difference in activity was expected to be given by the different conjugation of the pharmacophore'sa romatic system upon switching. Unfortunately,t he latter modified pharmacophore (compound 9,F igure 2B)w as inactive in patch-clamp studies (data not shown) and the synthesis towards the photoswitch was not further pursued.
To characterize Fulgazepami nv itro, patch clamp experiments were performed on cells transiently expressing a 1 b 2 g 2 subunits of the GABA A receptor.T his receptor possesses the canonical benzodiazepine allosteric site and its EC 50 forG ABA is about 8 mm. [14] The effects of the fulgimide-based benzodiazepine derivatives 3 and 4 on the receptor's functionw ere studied upon co-application of 0.5 mm GABA, that is, ac oncentration below the EC 50 (close to EC 3 )t hat allows to observea llosteric potentiationofG ABA A R-mediated currents. [34] Application of compound 4a (open isomer)( 10 mm)c aused no significant effect on GABA A -mediated currents, while application of 4b (closed isomer), generated by pre-illumination with UV light (365 nm), induced an increase of GABA A -mediated current amplitudes ( Figure 4A). Thus, the isomers of compound 4 interactd ifferently with GABA A Rs, being inactive in the open form and potentiatingi nt he closed form. Analysis of as eries of dose-responsec urves established that the EC 50 for 4b was 13 mm (n = 6; Figure 4B). Figure 4C demonstrates that UV illumination switches the conformation of 10 mm com-pound 4 (from 4a to 4b)a nd increases the amplitude of GABA-induced currents by 228 AE 41 %( Figure 4D; n = 11). Compound 3a in its open state co-applied withG ABA (0.5 mm)i nduced astrong potentiationofGABA A R-mediated currents (Figure S3A). This potentiation was not sensitive to illumination by UV light and subsequent isomerization to the closed isomer 3b (Figure S3 B) and the kinetics of compound 3b's development (slow wash-in and slow wash-out) was similar to the one of 4b.A pplication of 10 mm of 3a increased the current amplitude by 292 AE 65 %, while 50 mm of 3a increasedt he current amplitude by 544 AE 107 %( n = 11). The EC 50 of 3a was 12 mm, similar to the one of compound 4b (Figure S3 C, n = 11). The degree of GABA A Rp otentiation induced by 3a markedly varied for different cells (cf. Aa nd Bi nF igure S3). As imilar behavior was observed for 4b.W es uggest that this effect reflectst he variability in the EC 50 of GABA in different cells, as it has been shown that allosteric potentiation decreasesa thigh GABA concentrations. [35] The outstanding photopharmacological profile of Fulgazepam (4)a sG ABA A Rp otentiator prompted further assays in vivo. Studies in zebrafish larvae show that the compound alters their behavior depending on isomerization and that this effect can be maintained over time in the absence of illumination. As both compound states 4a and 4b are stable in the dark, larvae behaviors could be studied using pre-illuminated  (Figure 3). Pre-illuminated compound 4b alteredi nadose dependent manner the behavior of undisturbed larvae.I np articular,100 mm 4b evoked an increase in swimmingd istance ( Figure 5B,t op). The effect of subsequenti llumination was also studied. For all concentrations of 4a,U Vl ight (hence, photoconversion to 4b)s ignificantly increased larvae motility,a nd this effect was potentiated during the following dark period and reduced to vehicle levels upon illumination with visible light ( Figure 5A and Figure 5B, bottom). Therefore, these changes in larvae motilitya re triggered by conformational changes of compound 4 rather than by natural photoresponsive behaviors. As ignificant increase in larvae activity,a bove vehicle levels, is observed when 4b isomerisation is potentiated (duringa nd after UV illumination) and lowers to naturala ctivity when 4a is recovered with green illumination independently of the initial Fulgazepam state that is administered to larvae.
In summary,w eh ave achievedt he functionalization of the benzodiazepine nitrazepam via extension by af ulgimide and report an ew photochromic potentiator of GABA A Rs. The synthesized fulgimides 3 and 4 (Fulgazepam) display good photochromic properties andh igh photostationary states. Both fulgimides preserve the GABA A Rp otentiator behavior that is char-acteristic of benzodiazepines, indicatingt hat it is ap harmacologically tolerables ubstitution, in contrastt oa zobenzenes at the same position. Remarkably,b oth fulgimides are photochromic but only Fulgazepam (4)e nables controlling the pharmacological activitywith light. The open conformation of Fulgazepam (4a)d oes not influence the amplitude of GABA A Rcurrents in vitro, while switching to its closed form 4b with UV light strongly potentiates them. The open (4a)a nd closed (4b)c onformation of iso-fulgimide 4 produce different behavioral outcomes in zebrafish larvae.T he ring-open isomer 4a does not alter larvae swimming activities, neither appliedd irectly nor obtained by illumination cycles, and the closed conformation 4b increases larvae motilityi nadose dependent manner both during prolonged dark periodsa nd under UV illumination. Hence, Fulgazepam photoswitching reversibly controls the behavior of larvae, producing high activity swimming upon UV illumination, which persists forc ontinuous dark periods, and reducing activity to control levels with visible light illumination.
GABA A Rs mediate fast inhibition of neural activity and are determinant in cognition, learning, and memory. [36][37][38] Malfunction of these receptors leads to epilepsy,a nxiety, depression and sleep disorders. [37] Clinical treatments with GABA A Rm odulators have limited efficacy and adverse side effects, [39,40] which We have recently developed an azobenzene-nitrazepam based compound (Azo-NZ1) that allows photo-modulation of GABA A receptors. [14] In trans-configuration, this compound selectively interacts with the chloride-permeable channel causing inhibition of GABA-induced currents, while UV-induced transition to the cis-state results in channel unblocking andrestoring the current amplitude. Such unexpected action for ad erivative of benzodiazepine (a GABA A Rp otentiator) was explained by molecular docking calculations and mutagenesis analysisi ndicating that the 2' residue of the channel-forming transmembrane TM2 domain is the target of Azo-NZ1 blocking action.
In contrastt oA zo-NZ1, the GABA A Rm odulator presented here (Fulgazepam)d oes produce potentiation of GABA-induced currents.T his GABA A Rp hotoswitch displays unique characteristics as ad irect result of its photochromic (fulgimide) and pharmacological (diazepam)m oieties:( i) the fulgimide scaffold imparts complete reversible switching of Fulgazepam's conformation;( ii)both Fulgazepam states are stable and can be readily obtained by illumination with light of the appropriate wavelengths;( iii)Fulgazepam is sufficiently soluble in aqueous solution and effectively photocontrols endogenousG A-BA A Rs in vitro-ini ts closed form it is ap urep otentiator of GA-BA A Rs withouta gonist or antagonist activity;( iv) Fulgazepam does not display toxicity in zebrafish and allowsc ontrolling their behavior with light. These outstanding molecular properties enable dissecting the mechanismso fG ABAergic neurotransmission at high spatiotemporal resolution, and pursuing novel phototherapies based on localized, acute,a nd reversible neuroinhibition.

Experimental Section
All animal experiments were conducted according to the EU Directive 2010/63/EU on the protection of animals used for scientific purposes. According to this directive, zebrafish are considered vertebrates and therefore subject to legislation governing animal testing, but embryos and non-independently feeding form larvae are excepted. Figure 5. A) Effect of Fulgazepam in wild type zebrafish larvae. One-minute trajectories of average swimmingd istances (n = 12 per treatment) are shown for vehicle(1% DMSO)a nd threedifferent concentrationso fc ompound 4,s tarting with pre-irradiated solutions of 4a (top) and 4b (bottom). For the first 20 minutes, larvae were undisturbedi nc omplete 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 discreted ark between each wavelength. Colored areas show standard error of the mean (S.E.M.).B )T op:Quantification of swimmingd istances over the last 5minuteso ft he RP (darkness) from two independent experiments (n = 24 per treatment) for both pre-illuminated compounds 4a (green trace) and 4b (violettrace) andvehicle (1 %D MSO). 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.).