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ScienceDirect The in vivo chemistry of photoswitched tethered ligands Johannes Broichhagen and Dirk Trauner Nature’s photoreceptors are typically composed of a chromophore that is covalently bound to a receptor protein at the top of a signaling cascade. The protein can function as a G-protein coupled receptor (GPCR), an ion channel, or as an enzyme. This logic can be mimicked with synthetic photoswitches, such as azobenzenes, that are linked to naturally ‘blind’ transmembrane proteins using in vivochemistry. The resulting semisynthetic receptors can be employed to optically control cellular functions, especially in neurons, and influence the behavior of animals with the exquisite temporal and spatial precision of light. Addresses Department of Chemistry, Ludwig-Maximilian-University Munich, and Munich Center for Integrated Protein Science, Butenandtstrasse 5-13, Munich 81377, Germany Corresponding author: Trauner, Dirk ([email protected], [email protected])

Current Opinion in Chemical Biology 2014, 21:121–127 This review comes from a themed issue on In vivo chemistry Edited by Joseph M Fox and Marc S Robillard For a complete overview see the Issue and the Editorial Available online 8th August 2014 http://dx.doi.org/10.1016/j.cbpa.2014.07.008 1367-5931/# 2014 Published by Elsevier Ltd.

Introduction The optical control of biological functions with genetically encoded photoreceptors (optogenetics) has recently become a major area of scientific activity [1]. Originally introduced as a means to control the excitability of neurons, optogenetics has been since expanded to cover other cellular functions, such as motility, proliferation and gene expression. It relies on an ever-growing repertoire of light-sensitive ion channels, GPCRs and enzymes, the successful design of which reflects the fascinating modularity of living systems. In Nature, light-sensitivity can be traced to a relatively small subset of chromophores, which include retinal, flavins (such as flavine mononucleotide) and tetrapyrroles (such as phycocyanobilin). They usually undergo covalent attachment to the apo-protein, either via Schiff-base formation with lysine residues or reaction with cysteine residues. Chemists have been able to mimic this logic and in doing so expand the toolbox of optogenetics. www.sciencedirect.com

A small but growing subdiscipline of optogenetics uses artificial photoswitches that are prepared by chemical synthesis and are attached to genetically encoded bioconjugation motifs in native receptors (optochemical genetics) [2,3,4]. This can be done in several ways [5] amongst which the photoswitched tethered ligand approach (PTL approach) has emerged as the most successful one for applications in vivo [6]. For the purposes of this review, we define in vivo studies as those that take place in whole, living organisms, but we also extend this definition to complex tissues, such as brain slices, and more or less isolated cells (e.g. Xenopus oocytes or dissociated mammalian neurons).

The PTL approach Photochromic tethered ligands (PTLs) consist of three functional and structural parts (Figure 1a): firstly, a functional handle for specific bioconjugation that should work in vivo; secondly, a molecular photoswitch; and thirdly, a ligand that can influence the biological activity of the target protein. These are held together with linkers of varying lengths. The chemical composition of these linkers is often defined by the synthetic pathway chosen, which should be as simple and flexible as possible. Upon isomerization with light, the photoswitch changes its configuration, and the ligand can bind — or bind better — to the active site (Figure 1b). Depending on the pharmacology of the ligand and the attachment site, the PTL can activate the receptor and function as a tethered agonist or displace a natural agonist, thereby operating as a photoswitched tethered antagonist. It is also conceivable that the ligand binds to an allosteric site diminishing or potentiating the activity of a natural agonist. The affinity of the ligand before tethering can be low since, after covalent attachment, the effective local concentration is very high (usually in the millimolar range). Indeed, it is advisable to use low affinity ligands, which allows for withdrawal from the binding site upon photoisomerization. It should be noted that Figure 1 is a schematic representation and that the photoswitch and linkers may well be part of the pharmacophore. The ligand head group could also bind to the ligand-binding site in both states of the tethered photoswitch but with different efficacy. In principle, several ways to attach a PTL to a given protein can be imagined. They range from classical electrophilic protein labeling with cysteine-reactive groups, such as maleimides or haloacetamides, to the use of chemically more stable electrophiles that only react with genetically encoded protein domains, such as SNAP-tags Current Opinion in Chemical Biology 2014, 21:121–127

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The principle of optochemical genetics. (a) Photochromic tethered ligands (PTLs) consist of a bioconjugation handle, a photoswitch and a ligand. (b) After introduction of a reactive cysteine residue into a protein via site-directed mutagenesis and bioconjugation, the PTL can act as an agonist (top) or as an antagonist (bottom). (c) Targets that have succumbed to the PTL approach: ionotropic and metabotropic glutamate receptors (iGluRs, mGluRs), nicotinic acetylcholine receptors (nAChR), GABAA receptors, purinergic receptors (P2X2) and voltage-gated K+ channels (Kv).

or CLIP-tags [8]. The introduction of unnatural amino acids via expansion of the genetic code and their labeling with in vivo-click chemistry is another possible way to introduce PTLs. Powerful as these techniques may be, however, they are in their infancy as far as mammalian neurons and applications in human therapy are concerned. For the in vivo application of PTLs, thiol-maleimide chemistry has proven to be practical and it has a number of advantages compared with other methods for bioconjugation: it only requires the introduction of an accessible cysteine that is slow to oxidize following surface-expression of the protein. This leaves the receptor structure close to its native state, whereas a fusion protein might alter the function and expression of the target receptor. Current Opinion in Chemical Biology 2014, 21:121–127

The drawback of cysteine labeling is the inherent instability of the requisite maleimides and similar electrophiles in the aqueous, nucleophilic environments of organisms and tissues, which could result in unselective labeling and destruction of the PTL. For instance, it is unlikely that maleimides are stable in the presence of serum albumin, which is known to bear a highly reactive cysteine designed to inactivate electrophiles [7]. In addition to this, thiol-maleimide chemistry it is confined to extracellular bioconjugation. The reason for this is the high concentration of intracellular glutathione, which would rapidly inactivate the maleimide. Nevertheless, maleimide chemistry has been a success in vivo, at least as far as applications in cell cultures, zebrafish and rodent eyes are concerned. The PTLs www.sciencedirect.com

Photoswitched tethered ligands Broichhagen and Trauner 123

are typically administered from DMSO stock solutions, which are diluted to micromolar concentrations before application. Due to their small molecular weight, they are quickly distributed and find their extracellular targets within seconds or minutes. They first bind non-covalently with their ligand head group and then undergo fast covalent attachment, provided the photoswitch is in the right configuration [9]. This affinity labeling affords selectivity for the intended cysteines. In addition to this, reactive cysteines elsewhere on the cell surface are rare, which may be another reason why nonselective labeling has never been encountered as a problem. Azobenzenes have emerged as the chemical photoswitches of choice — and for good reasons. Their photostationary states and thermal relaxation kinetics can be readily tuned and they display large changes in geometry and dipole moment upon switching. They can be synthesized with a variety of efficient methods and are usually accessible on scale. In addition, they have the advantage of fast photoswitching (in the picosecond range) preventing intersystem-crossing and singlet oxygen formation, which would be incompatible with in vivo applications. As such, azobenzenes have proven to be highly photostable, going through numerous cycles of photoswitching without noticeable decay (e.g. hundreds of times in electrophysiological preparations).

Receptors and cells targeted with PTLs Over the last decades, a variety of PTLs have been developed and some have been demonstrated to work in live animals (Figure 2). The very first application of PTLs dates back to the late 1970s, when azobenzenes were used by Erlanger and Lester to control nicotinic acetylcholine receptors [10]. To this end, a benzylic bromide called QBr was synthesized and tethered to a cysteine obtained via reduction of an extracellular disulfide bond. While this system was successful in controlling the transmembrane potential of electroplaques and rat myoballs, it could not be applied in mammalian neurons since the modern techniques of heterologous expression were not yet available [11,12]. Thus, it took until 2004 when Trauner and Kramer introduced the synthetic photoisomerizable azobenzene-regulated K+ channel, SPARK, that PTLs made an impact on neurobiology [13]. SPARK consists of the PTL MAQ attached to a Shaker-type potassium channel. It could be used to silence neuronal firing in rat hippocampal neurons upon irradiation with UV-A light. The approach was subsequently expanded to other types of voltage-gated potassium channels, demonstrating one of the major advantages of the PTL approach: the possibility to optically control native receptors, minimally altered via cysteine mutation, in a subtype selective way (Figure 3). A similar approach was taken to unravel the role of TREK1-potassium channels (KCNQ2) in www.sciencedirect.com

GABAB-receptor signaling, which, like most potassium channels, lack selective blockers [14]. Beginning in 2006, the concept was expanded to glutamate receptors. Attachment of the photoswitchable agonist L-MAG, which comes in three different versions (LMAG0/1/2, Figure 2), to the kainate receptor GluK2 yielded the light-gated ionotropic glutamate receptor LiGluR [15]. Irradiation of LiGluR with 380 nm elicited neuronal firing, which could be turned off by irradiation with 500 nm light. Several variants of this molecular device have been developed. For instance, the attachment site could be slightly moved to convert the PTL from a tethered agonist to a tethered antagonist [6]. Conversely, attachment of L-MAG to a channel chimera that is selective for potassium ions resulted in inhibition instead of activation upon irradiation with UV-light (HyLighter) [16]. L-MAG was also applied to study gating in ionotropic glutamate receptor homomers and heteromers. Ultrafast switching provided a real-time measure of gating and revealed that partially occupied receptors can activate without desensitizing [17]. A small chemical alteration of L-MAG, viz. the replacement of an amide moiety by an amine, yielded L-MAG460, which elicited action potentials upon irradiation with visible light (Figure 2) [18]. This is advantageous when phototoxicity is a concern, e.g. in the restoration of vision. Very recently, L-MAG2p and LMAGA2p were introduced, which could be switched to the active cis-form via two-photon excitation. The latter contains a naphthalene ‘antenna’ with a large two-photon cross-section (Figure 2) [19]. Attachment of L-MAG(A)2p to the engineered kainate receptor used in LiGluR allows for efficient two-photon activation of neurons and astrocytes with subcellular resolution using near-infrared light (820 nm). Metabotropic glutamate receptors have also succumbed to the PTL approach [20]. This required a change in the stereochemistry of the tethered glutamate, converting LMAG to D-MAG (Figure 2). Attachment of D-MAG0 to metabotropic glutamate receptors of subtypes 2, 3 and 6 resulted in G-protein coupled receptors that could be activated with UV-A light (LimGluR). LimGluR inhibits neuronal firing in dissociated neurons and brain slices as a consequence of its effects on G-protein coupled inward rectifying potassium channels and the intracellular concentration of cAMP. A tethered antagonist was also developed. Attachment of D-MAG1 to the same cysteine resulted in robust photoantagonism. Pentameric ligand-gated ion channels can be controlled with PTLs as well [21]. Two different molecules, MAACh and MAHoCh were developed for nicotinic acetylcholine receptors (Figure 2). Both are based on choline and incorporate azobenzenes but differ in their Current Opinion in Chemical Biology 2014, 21:121–127

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Current Opinion in Chemical Biology 2014, 21:121–127

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Current Opinion in Chemical Biology

PTLs that have been used in optochemical genetics.

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Photoswitched tethered ligands Broichhagen and Trauner 125

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PTLs allow for genetic targeting and subtype selectivity. (a) Selective activation of a receptor subtype is difficult to achieve with classical pharmacology. (b) Following site-directed mutagenesis and bioconjugation, a specific receptor subtype can be put under light control, which allows for dissection of its function.

linker lengths. MAACh functions as a photoswitchable agonist when tethered to an a3b4 receptor mutant, whereas attachment of MAHoCh to a4b2 resulted in photo-antagonism. The light-activated nicotinic acetylcholine receptors (LinAChRs) functioned well in Xenopus oocytes but have not yet been implemented in neurons. Another class of ion channels that have been converted to photoreceptors using PTLs are GABAA receptors [22]. Attachment of MPC088, a derivative of the well-known anesthetic propofol, to the a1b2g2 receptor subtype yielded a light-sensitive GABAA receptor (Figure 2). Irradiation with 440 nm light resulted in potentiation of

GABA induced chloride currents, an effect that could be reversed by irradiation with UV-light. In cerebellar slices, this molecular device afforded bidirectional photomodulation of Purkinje cell firing. Finally, PTLs have been applied to purinergic receptors (P2X2) [23]. Attachment of MAQ, which has been previously used for potassium channels, and its close derivative MEA-TMA to engineered cysteines of P2X2 receptors created an optical gate independent of the ATP-binding site (Figure 2). This is an example of a PTL that targets an allosteric site and inhibits the activity of a channel as a photoswitchable blocker as opposed to an

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LiGluR in the retina. The cysteine mutant of the kainate receptor was introduced into the retina of blind mice via transfection with an adeno-associated virus. The activity of the explanted retina was monitored with multielectrode array recordings. (a) Retinal response to 380 nm and 500 nm light in the absence of the PTL L-MAG0. (b) Retinal response after addition of the PTL. www.sciencedirect.com

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126 In vivo chemistry

antagonist. As in the case of LiGluR, HyLighter, LimGluR, the optogated P2X2 receptor could be used to control action potential firing in dissociated neurons.

Applications in live animals As shown above, PTLs have found numerous in dissociated neurons and brain slices, but they also function in live animals. For instance, when introduced into sensory neurons in zebrafish larvae, activation of LiGluR blocks the escape response to touch in a reversible fashion [24]. Perturbation with LiGluR was also instrumental for elucidating the role of the so-called Kolmer-Agduhr neuron, whose activity was shown to provide necessary tone for forward swimming [25]. Finally, LiGluR has found initial applications in mammals. Expression of LiGluR in the retina of blind mice and administration of L-MAG via intraocular injection close to the retina resulted in the restoration of light dependent activity and visually guided behavior (Figure 4) [26]. This was done with the original variant of LiGluR, which requires 360 nm light for activation and is therefore not ideal for vision restoration. However, red-shifted versions using L-MAG460 (vide supra) are available and could be used in the future for this purpose.

Conclusions In recent years, synthetic photoswitches have been increasingly used to optically control biological functions (photopharmacology). Although the majority of these applications concern freely diffusible molecules [21,27– 30], covalently attached photoswitched ligands have also been found to be effective in vivo. They have the added advantage of genetic encoding via engineered cysteine residues, which makes it possible to target them to specific cell types and tissues. Bioconjugation techniques that rely on larger protein domains (SNAP-tags and CLIP-tags) or click chemistry using genetically encoded unnatural amino acids [31] and potentially allow for milder labeling conditions have not yet been implemented. It is likely, however, that they will also contribute to the further development of optochemical genetics.

Acknowledgements JB is grateful to the Studienstiftung des deutschen Volkes for a PhD studentship. We also thank the Center Integrated Proteins Science, Munich, and the European Research Council (ERC Advanced Grant 268795) for financial support.

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1.

Deisseroth K: Optogenetics. Nat Methods 2011, 8:26-29.

2. Fehrentz T, Scho¨nberger M, Trauner D: Optochemical genetics.  Angew Chem Int Ed Engl 2011, 50:12156-12182. An extensive review on the control of biological function with light using synthetic photoswitches. Current Opinion in Chemical Biology 2014, 21:121–127

3.

Szymanski W, Beierle JM, Kistemaker HA, Velema WA, Feringa BL: Reversible photocontrol of biological systems by the incorporation of molecular photoswitches. Chem Rev 2013, 113:6114-6178.

4. 

Kramer RH, Mourot A, Adesnik H: Optogenetic pharmacology for control of native neuronal signaling proteins. Nat Neurosci 2013, 16:816-823. A recent review on the optical control of native neuronal receptors and the use in exploreing the basic funcions and dysfunctions of the brain. 5.

Browne LE, Nunes JP, Sim JA, Chudasama V, Bragg L, Caddick S, Alan North R: Optical control of trimeric P2X receptors and acid-sensing ion channels. Proc Natl Acad Sci U S A 2014, 111:521-526.

6.

Numano R, Szobota S, Lau AY, Gorostiza P, Volgraf M, Roux B, Trauner D, Isacoff EY: Nanosculpting reversed wavelength sensitivity into a photoswitchable iGluR. Proc Natl Acad Sci U S A 2009, 106:6814-6819.

7.

Kratz F: Albumin as a drug carrier: design of prodrugs, drug conjugates and nanoparticles. J Control Release 2008, 132:171183.

8.

Gautier A, Juillerat A, Heinis C, Correa IR, Kindermann M, Beaufils F, Johnsson K: An engineered protein tag for multiprotein labeling in living cells. Chem Biol 2008, 15:128-136.

9.

Gorostiza P, Volgraf M, Numano R, Szobota S, Trauner D, Isacoff EY: Mechanisms of photoswitch conjugation and light activation of an ionotropic glutamate receptor. Proc Natl Acad Sci U S A 2007, 104:10865-10870.

10. Bartels E, Wassermann NH, Erlanger BF: Photochromic activators of the acetylcholine receptor. Proc Natl Acad Sci U S A 1971, 68:1820-1823. 11. Lester HA, Krouse ME, Nass MM, Wassermann NH, Erlanger BF: A covalently bound photoisomerizable agonist: comparison with reversibly bound agonists at Electrophorus electroplaques. J Gen Physiol 1980, 75:207-232. 12. Chabala LD, Lester HA: Activation of acetylcholine receptor channels by covalently bound agonists in cultured rat myoballs. J Physiol 1986, 379:83-108. 13. Banghart M, Borges K, Isacoff E, Trauner D, Kramer RH: Lightactivated ion channels for remote control of neuronal firing. Nat Neurosci 2004, 7:1381-1386. 14. Sandoz G, Levitz J, Kramer RH, Isacoff EY: Optical control of endogenous proteins with a photoswitchable conditional subunit reveals a role for TREK1 in GABA(B) signaling. Neuron 2012, 74:1005-1014. 15. Volgraf M, Gorostiza P, Numano R, Kramer RH, Isacoff EY, Trauner D: Allosteric control of an ionotropic glutamate receptor with an optical switch. Nat Chem Biol 2006, 2:47-52. 16. Janovjak H, Szobota S, Wyart C, Trauner D, Isacoff EY: A lightgated, potassium-selective glutamate receptor for the optical inhibition of neuronal firing. Nat Neurosci 2010, 13:1027-1032. 17. Reiner A, Isacoff EY: Tethered ligands reveal glutamate receptor desensitization depends on subunit occupancy. Nat Chem Biol 2014, 10:273-280. 18. Kienzler MA, Reiner A, Trautman E, Yoo S, Trauner D, Isacoff EY: A  red-shifted, fast-relaxing azobenzene photoswitch for visible light control of an ionotropic glutamate receptor. J Am Chem Soc 2013, 135:17683-17686. A paper on a red-shifted photochromic tethered ligand that works with visible light (445–500 nm), which is desirable for deep tissue penetration. The resulting light-activated glutamate receptor (LGluR) was employed to trigger ionic currents with green light, while relaxation and deactivation occurred quickly in the dark. 19. Izquierdo-Serra M, Gascon-Moya M, Hirtz JJ, Pittolo S, Poskanzer KE, Ferrer E, Alibes R, Busque F, Yuste R, Hernando J et al.: Two-photon neuronal and astrocytic stimulation with azobenzene-based photoswitches. J Am Chem Soc 2014, 136:8693-8701. 20. Levitz J, Pantoja C, Gaub B, Janovjak H, Reiner A, Hoagland A,  Schoppik D, Kane B, Stawski P, Schier AF et al.: Optical control of www.sciencedirect.com

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metabotropic glutamate receptors. Nat Neurosci 2013, 16:507516. A recent paper on the application of the photochromic tethered ligands (PTL) principle to G-protein coupled receptors of family C, viz. metabotropic glutamate receptors 2, 3 and 6. Light gated metabotropic glutamate receptors (LimGluRs) were shown to work in rodent brain slices and zebrafish. 21. Tochitsky I, Banghart MR, Mourot A, Yao JZ, Gaub B, Kramer RH, Trauner D: Optochemical control of genetically engineered neuronal nicotinic acetylcholine receptors. Nat Chem 2012, 4:105-111. 22. Yue L, Pawlowski M, Dellal SS, Xie A, Feng F, Otis TS, Bruzik KS, Qian H, Pepperberg DR: Robust photoregulation of GABA(A) receptors by allosteric modulation with a propofol analogue. Nat Commun 2012, 3:1095. 23. Lemoine D, Habermacher C, Martz A, Mery PF, Bouquier N, Diverchy F, Taly A, Rassendren F, Specht A, Grutter T: Optical control of an ion channel gate. Proc Natl Acad Sci U S A 2013, 110:20813-20818. 24. Szobota S, Gorostiza P, Del Bene F, Wyart C, Fortin DL, Kolstad KD, Tulyathan O, Volgraf M, Numano R, Aaron HL et al.: Remote control of neuronal activity with a light-gated glutamate receptor. Neuron 2007, 54:535-545.

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25. Wyart C, Del Bene F, Warp E, Scott EK, Trauner D, Baier H, Isacoff EY: Optogenetic dissection of a behavioural module in the vertebrate spinal cord. Nature 2009, 461:407-410. 26. Caporale N, Kolstad KD, Lee T, Tochitsky I, Dalkara D, Trauner D, Kramer R, Dan Y, Isacoff EY, Flannery JG: LiGluR restores visual responses in rodent models of inherited blindness. Mol Ther 2011, 19:1212-1219. 27. Banghart MR, Mourot A, Fortin DL, Yao JZ, Kramer RH, Trauner D: Photochromic blockers of voltage-gated potassium channels. Angew Chem Int Ed Engl 2009, 48:9097-9101. 28. Stein M, Breit A, Fehrentz T, Gudermann T, Trauner D: Optical control of TRPV1 channels. Angew Chem Int Ed Engl 2013, 52:9845-9848. 29. Broichhagen J, Jurastow I, Iwan K, Kummer W, Trauner D: Optical control of acetylcholinesterase with a tacrine switch. Angew Chem Int Ed Engl 2014, 53:7657-7660. 30. Scho¨nberger M, Trauner D: A photochromic agonist for muopioid receptors. Angew Chem Int Ed Engl 2014, 53:3264-3267. 31. Blackman ML, Royzen M, Fox JM: Tetrazine ligation: fast bioconjugation based on inverse-electron-demand DielsAlder reactivity. J Am Chem Soc 2008, 130:13518-13519.

Current Opinion in Chemical Biology 2014, 21:121–127

The in vivo chemistry of photoswitched tethered ligands.

Nature's photoreceptors are typically composed of a chromophore that is covalently bound to a receptor protein at the top of a signaling cascade. The ...
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