International Journal of Molecular Sciences Article Reversible Photocontrol of Dopaminergic Transmission in Wild-Type Animals Carlo Matera 1,2,3 , Pablo Calvé 4,† , Verònica Casadó-Anguera 5,† , Rosalba Sortino 1,2,† , Alexandre M. J. Gomila 1,2 , Estefanía Moreno 5 , Thomas Gener 4 , Cristina Delgado-Sallent 4 , Pau Nebot 4 , Davide Costazza 1 , Sara Conde-Berriozabal 6 , Mercè Masana 6 , Jordi Hernando 7 , Vicent Casadó 5 , M. Victoria Puig 4 and Pau Gorostiza 1,2,8, * 1 Institute for Bioengineering of Catalonia (IBEC), the Barcelona Institute for Science and Technology, 08028 Barcelona, Spain 2 Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), 28029 Madrid, Spain 3 Department of Pharmaceutical Sciences, University of Milan, 20133 Milan, Italy 4 Hospital del Mar Medical Research Institute (IMIM), Barcelona Biomedical Research Park, 08003 Barcelona, Spain 5 Department of Biochemistry and Molecular Biomedicine, Faculty of Biology, Institute of Biomedicine, University of Barcelona, 08028 Barcelona, Spain 6 Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Institute of Neuroscience, University of Barcelona, IDIBAPS, CIBERNED, 08036 Barcelona, Spain 7 Department of Chemistry, Autonomous University of Barcelona (UAB), 08193 Cerdanyola del Vallès, Spain 8 Catalan Institution for Research and Advanced Studies (ICREA), 08010 Barcelona, Spain * Correspondence: pau@icrea.cat † These authors contributed equally to this work. Citation: Matera, C.; Calvé, P.; Casadó-Anguera, V.; Sortino, R.; Abstract: Understanding the dopaminergic system is a priority in neurobiology and neuropharma- Gomila, A.M.J.; Moreno, E.; Gener, T.; cology. Dopamine receptors are involved in the modulation of fundamental physiological functions, Delgado-Sallent, C.; Nebot, P.; and dysregulation of dopaminergic transmission is associated with major neurological disorders. Costazza, D.; et al. Reversible However, the available tools to dissect the endogenous dopaminergic circuits have limited specificity, Photocontrol of Dopaminergic Transmission in Wild-Type Animals. reversibility, resolution, or require genetic manipulation. Here, we introduce azodopa, a novel Int. J. Mol. Sci. 2022, 23, 10114. photoswitchable ligand that enables reversible spatiotemporal control of dopaminergic transmission. https://doi.org/10.3390/ We demonstrate that azodopa activates D1 -like receptors in vitro in a light-dependent manner. More- ijms231710114 over, it enables reversibly photocontrolling zebrafish motility on a timescale of seconds and allows separating the retinal component of dopaminergic neurotransmission. Azodopa increases the overall Academic Editor: Giuseppe Di Giovanni neural activity in the cortex of anesthetized mice and displays illumination-dependent activity in individual cells. Azodopa is the first photoswitchable dopamine agonist with demonstrated efficacy Received: 3 August 2022 in wild-type animals and opens the way to remotely controlling dopaminergic neurotransmission for Accepted: 31 August 2022 fundamental and therapeutic purposes. Published: 4 September 2022 Publisher’s Note: MDPI stays neutral Keywords: azobenzene; behavior; brainwave; dopamine; GPCR; in vivo electrophysiology; optogenetics; with regard to jurisdictional claims in optopharmacology; photochromism; photopharmacology; photoswitch; zebrafish published maps and institutional affil- iations. 1. Introduction Dopamine receptors (DARs) are members of the class A G protein-coupled recep- Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. tor (GPCR) family and are prominent in the vertebrate central nervous system (CNS). This article is an open access article Their primary endogenous ligand is the catecholaminergic neurotransmitter dopamine, distributed under the terms and a metabolite of the amino acid tyrosine. Among the many neuromodulators used by the conditions of the Creative Commons mammalian brain to regulate circuit function and plasticity, dopamine (Figure 1a) stands Attribution (CC BY) license (https:// out as one of the most behaviorally powerful [1,2]. Dopaminergic neurons are critically creativecommons.org/licenses/by/ involved in diverse vital CNS functions, including voluntary movement, feeding, reward, 4.0/). motivation, sleep, attention, memory, and cognition. The extracellular concentration of Int. J. Mol. Sci. 2022, 23, 10114. https://doi.org/10.3390/ijms231710114 https://www.mdpi.com/journal/ijms mammalian brain to regulate circuit function and plasticity, dopamine (Figure 1a) stands out as one of the most behaviorally powerful [1,2]. Dopaminergic neurons are critically involved in diverse vital CNS functions, including voluntary movement, feeding, reward, motivation, sleep, attention, memory, and cognition. The extracellular concentration of Int. J. Mol. Sci. 2022, 23, 10114 2 of 18 dopamine oscillates following day/night cycles and plays important physiological roles in the regulation of olfaction, retinal function [3], and circadian rhythms [4]. Disentangling these diverse components of DAR signaling and dopaminergic transmission is an unmet dopamine oscillates following day/night cycles and plays important physiological roles in need both of basictheresearch regulation and medicine, of olfaction, because retinal their[3], function abnormal function and circadian leads[4]. rhythms to Disentangling com- plex medical conditions such as Parkinson’s and Huntington’s diseases, schizophrenia, these diverse components of DAR signaling and dopaminergic transmission is an unmet attention deficitneed hyperactivity both of basic disorder, researchTourette’s and medicine,syndrome, because drug abuse, and their abnormal addiction function leads to com- [1]. To date, fiveplex subtypes medical of conditions DARs havesuch been ascloned: D1, Dand Parkinson’s 2, DHuntington’s 3, D4, and D5. diseases, Based onschizophrenia, their coupling to eitherattention deficit hyperactivity Gαs,olf proteins disorder, or Gαi/o proteins, Tourette’s which, syndrome,stimulate respectively, drug abuse, orand addiction [1]. inhibit the production of To the date, five subtypes second messenger of DARs cAMP, have DARsbeenare cloned: D1 , Das classified 2, D D31,-like D4 , and D5 . Based on receptors (D1, D5) or D2-like receptors (D2, D3, D4). However, both classes are known to signalstimulate their coupling to either G αs,olf proteins or G αi/o proteins, which, respectively, or inhibit the production of the second messenger cAMP, DARs are classified as D -like through multiple pathways. Targeting these receptors using specific agonists and antag- 1 receptors (D1 , D5 ) or D2 -like receptors (D2 , D3 , D4 ). However, both classes are known onists allows totomodulate dopaminergic signal through transmission multiple pathways. and dopamine-dependent Targeting these receptors using specific func- agonists tions. Indeed, hundreds of compounds interfering with the dopaminergic and antagonists allows to modulate dopaminergic transmission and dopamine-dependent system have been developed,functions. and manyIndeed,of themhundreds are clinically used to treat of compounds variouswith interfering disorders. They also system the dopaminergic constitute pharmacological tools to study the role of dopamine in synaptic have been developed, and many of them are clinically used to treat various and neuraldisorders. They as circuits [5,6] as well alsotheconstitute mechanisms pharmacological underlyingtools to study the role debilitating dopamine-related of dopamine condi-in synaptic and neural circuits [5,6] as well as the mechanisms underlying tions [7]. However, conventional ligands cannot differentiate among specific neuronal dopamine-related debilitating sub-populations in heterogeneous brain regions where multiple neuronal subtypes exist,neuronal conditions [7]. However, conventional ligands cannot differentiate among specific sub-populations in heterogeneous brain regions where multiple neuronal subtypes exist, thus potentially activating DARs that mediate distinct or even opposing physiological thus potentially activating DARs that mediate distinct or even opposing physiological functions [2]. For this reason, functions [2]. Forthethislack of the reason, circuit lack selectivity is a confounding of circuit selectivity is a confoundingelement elementin in basic basic research and likelyand research cause of cause likely the poor safety of the pooras wellasaswell safety efficacy of many as efficacy dopaminergic of many dopaminergic drugs. drugs. Hence, methods to activate DARs noninvasively with high Hence, methods to activate DARs noninvasively with high spatiotemporalspatiotemporal resolu- resolution are tion are requiredrequired both for research both and therapeutic for research and therapeutic purposes. purposes. Figure 1. Design,Figure structure, and photochromism 1. Design, of azodopa. (a) structure, and photochromism Chemical(a)structure of azodopa. Chemicalof dopamine structure of dopamine and representative (semi)rigidified derivatives: rotigotine (nonselective agonist), A68930 (D and representative (semi)rigidified derivatives: rotigotine (nonselective agonist), A68930 (D11 agonist), and dinapsoline (D1 agonist). (b) 2D and 3D chemical structure of the photochromic dopamine ligand Int. J. Mol. Sci. 2022, 23, 10114 3 of 18 azodopa (trans and cis isomers). Essential pharmacophoric features for D1 receptor binding are highlighted: blue spheres represent cationic site points and H-bond donors, i.e., the protonated amino function that can form a salt bridge and a hydrogen bond with Asp103 and Ser107, respectively; red spheres represent H-bond acceptors, i.e., the hydroxyl groups of the catechol ring that can interact with Ser198, Ser199, and Ser202; yellow spheres represent hydrophobic elements, i.e., the aromatic ring that can form π–π interactions with Phe288 and Phe289. The mutual position and orientation of such pharmacophoric features in the receptor-bound conformation should affect binding affinity and efficacy of the ligands. (c) Photochromic behavior of azodopa (50 µM) studied with steady-state spectroscopy in aqueous (PBS, pH 7.4) and organic (DMSO) solutions. As lifetimes of cis-hydroxyazobenzenes are very short in polar protic solvents, no changes in the absorption spectrum of aqueous solutions of azodopa could be observed after illumination with 365 nm light (3 min). (d) Photochromic behavior of azodopa (30 µM) investigated by transient absorption spectroscopy in water (only representative traces are shown for the sake of clarity; see Figure S7 for the full experiment). Transient absorption time traces were measured at different wavelengths upon excitation of trans-azodopa with a 5 ns pulsed laser at λ = 355 nm (3 mJ/pulse energy) and 25 ◦ C. Thermal relaxation half-life of the cis isomer (200 µs) was estimated by applying an exponential one-phase decay model (GraphPad Prism 6). Inset: Transient absorption spectrum of trans-azodopa upon pulsed irradiation at λ = 355 nm recorded at t = 0 µs. X-values represent wavelength (nm), Y-values represent ∆A (arbitrary units, AU). Pursuing a traditional pharmacological approach would hardly pay off in such a physi- ological scenario, because a drug generally affects its target in multiple CNS regions at once and its effect is slowly reversible. Light is an unparalleled input signal to noninvasively ma- nipulate biological systems in precisely designated patterns, and photopharmacology [8–10], which relies on molecular photoswitches to regulate bioactive compounds, has already been successful in GPCRs [11–16], ion channels [17,18], and enzymes [19,20]. The possibility of using light as an external stimulus to manipulate specific populations of dopaminergic neurons has generated enormous interest in neurobiology since the invention of opto- genetics [21,22], in particular to elucidate the basis of complex behavioral and cognitive processes [23,24]. However, the achievements of optogenetics rely on the overexpression of exogenous proteins that lack critical aspects of endogenous GPCR signaling, including their native ligand binding sites, downstream molecular interactions, and other elements that can affect receptor dynamics. Thus, current optogenetic tools may provide partial, or sometimes inaccurate, insights into biological processes. In addition, the application of genetic manipulation techniques to human subjects is still importantly hampered by safety (possible immune responses against the gene transfer), regulatory, and economic issues. Complementary approaches to address some of these limitations have been pro- posed. Trauner, Isacoff, and collaborators developed light-gated DARs through a combined chemical–genetic method in which a weakly photoswitchable ligand was tethered via a maleimide–thiol conjugation to a genetically engineered cysteine residue at the target receptor in order to improve photocontrol [25]. They also used a genetically targeted membrane anchor to tether a dopaminergic ligand via SNAP-tag labeling [15]. Unlike the exogenous optogenetics tools described above, light-gated receptors bear a single-point mutation and provide a nearly physiological study system, but they still require gene delivery and overexpression. Genetics-free methods have also been described. For instance, Etchenique, Yuste, and collaborators developed a caged dopamine compound based on ruthenium–bipyridine chemistry and used it to activate dendritic spines with two-photon excitation [26]. More recently, Gmeiner et al. described two caged DAR antagonists that can serve as valuable tools for light-controlled blocking of D2 /D3 receptors [27]. However, notwithstanding its virtues, uncaging is an irreversible chemical process, while a photophar- macological modulation based on reversible, byproduct-free molecular photoswitches has important advantages in vivo. Noteworthy in this regard is the work published by König and collaborators, who developed a set of photochromic small molecules by incorpora- tion of dithienylethenes and fulgides into known dopamine receptor ligands [28]. Two of those compounds, named 29 and 52, showed an interesting isomer-dependent (open vs. Int. J. Mol. Sci. 2022, 23, 10114 4 of 18 closed form) efficacy at activating D2 receptors, although neither in situ photoswitching nor in vivo validation of their effects were reported. We present here the design, synthesis, and photopharmacological characterization of the first chemical tool that enables the reversible photocontrol of native dopamine receptors in wild-type animals. Notably, it can be used to separate the retinal component of dopaminergic neurotransmission in zebrafish and manipulate brain waves in mice. 2. Results 2.1. Design Strategy and Synthesis Aryl azo compounds, especially azobenzenes, have emerged as the photoswitches of choice in photopharmacology because of their physical and chemical properties, which make them especially suitable for biological applications [29,30]. An elegant strategy for the incorporation of an azobenzene into a bioactive ligand [11] relies on the isosteric replacement of the two-atom linker between the two aromatic rings with a diazene unit (–N=N–) [11,20,31–33], which entails minimal perturbation of pharmacophore and drug- like properties, thus accounting for the success of this so-called azologization approach [31]. However, to the best of our knowledge, this strategy is not applicable to any of the known dopamine agonists, since the scaffold of an isomerizable aryl azo compound is not directly conceivable in their structure. In the quest for a freely diffusible drug-like dopaminergic photoswitch, we noticed that most agonists (especially to D1 receptors, see Figure 1a) are rigid or semi-rigid, con- formationally restrained structures in which essential pharmacophoric features are held in their mutual position [34]. Indeed, two main routes can be identified in the early devel- opment of dopamine agonists, namely, the rigidification of the dopamine molecule and the dissection of apomorphine, one of the first potent dopamine agonists to be found [35]. Dopamine rigidification led to the discovery of potent agonists, whereas the apomorphine de-rigidification generally reduced its efficacy [36,37]. It stands then to reason that govern- ing the geometry of such structures would enable the control of their biological effects. This could be achieved by “building” upon a semi-rigid and photoisomerizable molecular frame the structural elements required for DAR activation and using light as external control signal. This approach can be likened to a “photoswitch decoration”, in which pharma- cophore groups are introduced into the structure of a molecular photoswitch to design a light-controlled bioactive compound. Common features in the dopamine agonist pharma- cophore model are the following: (I) a cationic site point (an amino group) that forms a salt bridge with an aspartic acid residue in the receptor’s third transmembrane helix (TM3); (II) one or two hydrogen bond acceptor/donor sites (e.g., hydroxy groups), that interact with serine residues in TM5; and (III) an aromatic ring system that takes part in π–π inter- actions with hydrophobic residues in TM6 [38–40]. On these grounds, we devised a photo- switchable dopamine agonist by decorating an azobenzene molecule with two hydroxyl substituents (II) on one phenyl ring, and an amino group (I) connected through a short linker to the other phenyl ring (III) (Figure 1b). This molecule, that we named azodopa, carries the main DAR binding determinants and enables to change their relative position upon photoisomerization (Figure 1b). Azodopa was synthesized in two steps via an azo- coupling reaction between commercially available 2-(2-(dimethylamino)ethyl)aniline and 1,2-dihydroxybenzene (see supplementary Scheme S1 and Supplementary Materials, SM, for detailed synthetic procedures and physicochemical characterization). 2.2. Photochemical Characterization An essential condition for azodopa to function as a photoswitchable dopamine ligand is that it photoisomerizes between two different configurations (trans and cis). We first characterized it by steady-state UV–Vis absorption spectroscopy (Figure 1c), which revealed an absorption maximum at 370 nm in aqueous solution (PBS, pH 7.4) and at 380 nm in DMSO. It is known that the thermal cis-to-trans isomerization of 4-hydroxyazobenzenes follows a very fast kinetics in polar protic solvents via solvent-assisted proton transfer Int. J. Mol. Sci. 2022, 23, 10114 5 of 18 tautomerization, whereas it proceeds more slowly in aprotic and nonpolar solvents [41]. In agreement, no changes in the absorption spectrum of azodopa could be detected with a conventional UV–Vis spectrophotometer upon illumination at 365 nm in aqueous buffer because of the short lifetime of its cis isomer in this medium, likely because of the presence of a non-negligible concentration of azonium cation at physiological pH and/or the forma- tion of hydrogen-bonded complexes with the solvents that accelerate the thermal cis-to-trans isomerization via tautomerization, and only a small change could be recorded in a polar but aprotic solvent such as DMSO, where the tautomerization is still possible, although less pronounced (Figure 1c). No measurements were performed in nonpolar solvents (e.g., toluene) because of the very poor solubility of our compound in this kind of media. We next determined the optimal isomerization wavelength and lifetime of cis-azodopa by means of transient-absorption spectroscopy with ns-time resolution. Upon pulsed excitation of azodopa in aqueous media with UV and violet light, an instantaneous and remarkable decrease of the absorption signal was detected, which can be ascribed to the depletion of the trans ground electronic state due to the photoinduced formation of the corresponding cis isomer (Figure 1d) [41,42]. The initial absorption value was quickly recovered because of the fast thermal cis-to-trans back-isomerization which restores the initial concentration of the trans isomer with an estimated half-life of about 200 µs (Figure 1d). Thus, since conversion to the trans form occurs almost immediately after turning the light off, we per- formed all biological experiments under continuous illumination. Overall, our absorption spectroscopy studies showed that azodopa undergoes trans-to-cis photoisomerization upon illumination with UV and violet light and it spontaneously reverts to its full trans isomer in a fraction of a millisecond once the light is switched off. Other mechanisms, such as excited-state intramolecular proton transfer, could also play a role. In any case, azodopa should allow fast regulation of DAR activity using a single illumination source to induce trans-to-cis isomerization. 2.3. In Vitro Pharmacology We next tested the effects of azodopa on D1 -like receptors, for which freely diffusible photoswitchable agonists have not been reported. First, we evaluated the binding affinity of azodopa in mammalian D1 -like and D2 -like receptors using competitive radioligand binding experiments performed either in the dark or under illumination at 365 nm (see SM for details) [43,44]. Azodopa displayed a good capacity to bind to D1 -like and D2 -like recep- tors, with higher affinity in the trans configuration in both cases. In particular, for D1 -like receptors, we calculated an almost fourfold decrease in affinity at 365 nm (Kd dark = 600 nM, Kd 365 nm = 2200 nM) (supplementary Figure S8 and Table S1). Since the activation of D1 -like receptors promotes cyclic adenosine monophosphate (cAMP) formation and phosphoryla- tion by ERK1/2, we also investigated the ability of azodopa to behave as a D1 -like agonist by studying cAMP accumulation (Figure 2a) and ERK1/2 phosphorylation (Figure 2b) in cells overexpressing human D1 receptors, both in the dark and under continuous illumi- nation at 365 nm (to compare the “full trans” vs. the “cis-enriched” form, respectively). Intracellular cAMP accumulation was measured in HEK-293T cells transiently transfected with D1 receptors in a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. We found that azodopa induced cAMP accumulation in a dose- and light-dependent manner (Figure 2a). In particular, we observed substantial differences between the two forms at 5 µM and 10 µM, with the trans form being significantly more effective at inducing cAMP production than the cis-enriched form at the same concentrations. When cells were co-treated with the D1 -like antagonist SKF83566 (300 nM), the effect of azodopa was largely reduced or even disappeared, indicating that azodopa activates D1 receptors. In cAMP assays performed in non-transfected HEK-293T cells used as negative control, azodopa (10 µM), dopamine (1 µM), and SKF38393 (300 nM) did not produce any effect (Figure S9). ERK1/2 phosphorylation was measured in HEK-293T cells transiently transfected with D1 by Western blot analysis using phospho-ERK1/2 antibody. The application of azodopa (10 µM) promoted ERK1/2 phosphorylation to an extent that was significantly different 2022, 23, x FOR PEER REVIEW 6 of 18 transiently transfected with D1 by Western blot analysis using phospho-ERK1/2 antibody. Int. J. Mol. Sci. 2022, 23, 10114 6 of 18 The application of azodopa (10 μM) promoted ERK1/2 phosphorylation to an extent that was significantly different between the two conditions (dark vs. light). The full trans form displayed an efficacy 3.6-fold greater than the cis-enriched form (Figure 2b), in agreement between the two conditions (dark vs. light). The full trans form displayed an efficacy with the observations in cAMP accumulation assays. Again, co-treatment of the cells with 3.6-fold greater than the cis-enriched form (Figure 2b), in agreement with the observations SKF83566 abolished azodopa in cAMP responses, accumulation showing assays. that Again, the effectsof co-treatment observed the cells were mediatedabolished with SKF83566 by D1. Althoughazodopa we did not thoroughly characterize the pharmacological activity responses, showing that the effects observed were mediated by D profile, 1 . Although we azodopa displayed D1-mediated did not thoroughlyphotoactivity characterize the(blocked by SKF83566) pharmacological activityalso in azodopa profile, zebrafishdisplayed (see later). D1 -mediated photoactivity (blocked by SKF83566) also in zebrafish (see later). Figure 2. In vitro pharmacological Figure 2. In vitro pharmacological characterizationcharacterization of azodopa. (a) ofEffect azodopa.on D(a) Effect on Dadenylyl 1-mediated 1 -mediated adeny- lyl cyclase activation. cAMP accumulation experiments cyclase activation. cAMP accumulation experiments in HEK-293T cells transiently transfected with in HEK-293T cells transiently transfected D1 and treated with with D1 andconcentrations different treated with different concentrations of azodopa, in the dark of azodopa, (black bars) in the dark (black or under bars) or under illumina- tion (purple bars),illumination in the presence (purple bars), (gray in the area) presence or not (white (gray area)area) of aorDnot (white 1-like area) antagonist receptor of a D1 -like receptor (SKF83566). Valuesantagonist (SKF83566). are represented Values arevs. in percentage represented basal levels in percentage of cAMP. Data vs. basal levels ± are mean ofS.E.M. cAMP. Data are mean ± S.E.M. (6 experiments performed (6 experiments performed in quadruplicate). Statisticalindifferences quadruplicate). wereStatistical analyzed differences by two-way were analyzed ANOVA followed by Tukey’s post hoc test (*** p < 0.001 vs. basal; * p < 0.05 vs. basal; ^^^ p < 0.001 by two-way ANOVA followed by Tukey’s post hoc test (*** p < 0.001 vs. basal; * p < 0.05 vs. basal; vs. dark; ^^ p < 0.01ˆˆˆvs. p