Specificity and Speed: Tethered Photopharmacology - Biochemistry

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Specificity and Speed: Tethered Photopharmacology Philipp Leippe, Julia Koehler Leman, and Dirk Trauner Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00687 • Publication Date (Web): 06 Sep 2017 Downloaded from http://pubs.acs.org on September 7, 2017

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Specificity and Speed: Tethered Photopharmacology

Philipp Leippe,a Julia Koehler Leman,b,c and Dirk Traunera,d*

a

Department of Chemistry and Center for Integrated Protein Science Munich, Ludwig-Maximilians-Universität

München, Butenandtstr. 5-13, 81377 Munich, Germany b c

Center for Computational Biology, Flatiron Institute, Simons Foundation, 162 Fifth Avenue, New York, NY 10010

Department of Biology and Center for Genomics and Systems Biology, New York University, NY 10003

d

Department of Chemistry, New York University, Silver Center, 100 Washington Square East, Room 712, USA

Abstract Genetics and pharmacology are often seen as two distinct approaches to interrogate, elucidate, and manipulate biological systems. The former is renowned for its precision whereas the latter for its fast kinetics, reversibility, and practicality. Here, we show that both can be joined as “Tethered Pharmacology”, wherein a genetically programmed bioconjugation site provides selectivity and a tethered pharmacophore provides function. The speed of onset, and especially cessation, of pharmacological activity can be greatly enhanced by incorporating photoswitches and using light as the trigger (“Tethered Photopharmacology”). Genetically encoded, tethered photopharmacology is a variant of Optogenetics and could even play a role in medicine wherever gene therapy is viable. However, gene therapy may not be necessary if sufficiently selective tethering strategies can be developed that operate on wild-type receptors.

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The identification of receptors in complex biological networks and the elucidation of their functional roles remains one of the most important goals in physiology. Alongside imaging techniques, genetics and pharmacology have proven the most useful methods in this regard. Both have distinct advantages and disadvantages and have historically played different roles.

Before the advent of molecular cloning, receptors were mostly characterized and classified with small molecules that functioned as agonists or inhibitors. Classical examples include the opioid receptors (µ, κ, δ) or the ionotropic glutamate receptors (AMPA, kainate, and NMDA), each of which have reasonably selective ligands to control their function. The deciphering of the action potential would not have been possible without blockers that could distinguish between potassium and sodium channels (TEA and TTX, respectively). However, despite these undeniable successes, pharmacology often lacked resolution, since its effects are concentration dependent and affinities to closely related receptor subtypes can be very similar. This has made the identification of receptor subtypes and the elucidation of their roles in complex biological systems with pharmacology alone very challenging. Starting in the late 1980s, molecular cloning could be used to clarify many uncertainties surrounding the targets of pharmacology.1 Using the high resolution of genetics and the power of whole genome sequencing, a wealth of new receptors and receptor subtypes was discovered. Molecular cloning and genome sequencing also uncovered numerous orphan receptors, for which endogenous or exogenous ligands have not yet been identified or are available. In terms of function, genetic methods can affect receptors as knock-outs (e.g. using CRISPR/Cas9)2 or knock-downs (e.g. with RNAi).3 In addition, genetics provides spatial selectivity through promotors that regulate the expression of a protein of interest in a particular type of cell. Despite the undeniable power of genetics, pharmacology (“chemical genetics”)4 still retains some advantages. The most obvious one is its practicality and relevance to human medicine. The other important advantage lies in the kinetics of pharmacology since the effect of a small molecule can become apparent within seconds. This is much faster than genetic methods for perturbation, such as inducible Cre/lox recombination or RNAi. Useful as these techniques are, their onset is limited by the time it takes to express a target gene or neutralize its mRNA, respectively. This can take hours to days, depending on the complexity of the biological system. In addition, genetic manipulation is usually irreversible, which is not the case with 2 ACS Paragon Plus Environment

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pharmacology. As such, genetics is good at addressing “what?” and “where?”, but not so precise with respect too “when?”. In this perspective, we argue that the benefits of genetics and pharmacology can be combined by covalently tethering a drug to a receptor. The attachment site of the tether can be genetically programmed by introducing a tag that facilitates a subsequent bioconjugation (Table 1). This tag can be a natural amino acid with distinct reactivity, such as a cysteine, an unnatural amino acid that undergoes bioorthogonal “click chemistry”, or a self-labeling protein domain. These modifications are introduced through site-directed mutagenesis, Amber suppression, or fusion of two genes to create a chimeric protein. The corresponding bioconjugations require different labeling reagents that undergo the reaction at different speeds and with different levels of selectivity (see below). Tethered pharmacology provides acuteness in spatial terms and can have a relatively fast onset. Its termination, however, is slow and defined by receptor desensitization and recycling. These limitations can be overcome with tethered photopharmacology. In this variant of optogenetics, a synthetic photoswitch is introduced that changes the accessibility or efficacy of the tethered ligand in a light-dependent fashion. The photoswitch, most often an azobenzene molecule, changes its configuration upon irradiation, e.g. from an elongated trans form to a bent cis-form. This change can be reversed thermally or through irradiation with light of a different wavelength. The switching can be made very fast in both directions, allowing for the activation and deactivation of receptors within milliseconds.

Several covalent tethering strategies have been described: these are distinguished by the bioconjugation chemistry and the resulting distance between attachment site and the site of pharmacological activity. Fig 1 and Table 1 provide an overview of available bioconjugation methods and schematic architectures of some of the constructs obtained.


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Table 1: Bioconjugation techniques used in tethered photopharmacology. The techniques shown in entry 1-5 operate on genetically engineered receptors, whereas PAL/TCP (entry 6) targets native, unmodified receptors. POI = protein of interest. Red: Photoswitch. Green: leaving group that is not incorporated in the adduct after bioconjugation. a)Compared to glycine.


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Figure 1: Tethered Photopharmacology A) Use of a PTL to control metabotropic a glutamate receptor with light. B) photo-BOLT for the optical control of the MEK1-kinase. C) PORTL applied to metabotropic glutamate receptors. D) DART and its potential extension to photoswitchable ligands (uPORTL). The earliest and so far most popular approach is the photoswitchable tethered ligand (PTL), which was introduced in the late 1970s5 and generalized in 2004.6 It relies on cysteinemaleimide chemistry to form the covalent bond.6 The photoswitch resides mostly in the tether, holding the ligand “at arm’s length” in one form and allowing it to reach the binding site in the other form. A PTL that operates on a metabotropic glutamate receptor, mGluR2, is outlined in Fig. 1A.7 The corresponding PTL is shown in Table 1, entry 1, in both configurations of the photoswitch. Following bioconjugation via Michael addition, the ligand, a glutamate derivative, cannot reach the binding site in the trans form of the photoswitch, whereas it can effectively bind to the extracellular Venus flytrap-like domain in the cis form. This activates the receptor and leads to downstream signaling. Related PTLs have been developed for a range different receptors, including potassium channels (Kv, TREK, HyLighter),6,8,9 ionotropic glutamate receptors (kainate, NMDA),10,11 metabotropic glutamate receptors7, pentameric ligand-gated ion channels (nAChR)12 and trimeric ligand-gated ion channels (P2X)13. These have found several applications in the in vivo optical control of biological systems, contributing to the development of Optogenetics.14–16 The advantage of the PTL strategy is that the engineered cysteines represent a minimal structural change in the receptor protein. This ensures that its expression, trafficking, and 5 ACS Paragon Plus Environment

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function are as similar as possible to the native receptor. In addition, the genetic manipulation needed to change a single amino acid is straightforward. One potential disadvantage is that many reactive cysteines are present on the surface of cells, limiting the selectivity of bioconjugation. Although this issue can be overcome by affinity labeling (vide infra), It still represents a potential pitfall. Moreover, cysteine bioconjugation is limited to extracellular applications, since maleimides are incompatible with the high concentrations of glutathione present inside cells. Finally, maleimides are susceptible to slow hydrolysis at physiological pH, which seriously hampers their applicability in larger tissues and organs. As shown below, this limitation can be overcome by a more robust bioconjugation chemistry. A second method to covalently attach a ligand to a protein was introduced in 2015 by Chin and was termed bioorthogonal ligand tethering (BOLT, Fig. 1B).17 It makes use of engineered unnatural amino acids, introduced through Amber codon suppression technology. These contain a strained double or triple bond and undergo bioconjugation with tethered ligands through click chemistry. A photoswitchable version, termed photo-BOLT, was also introduced, wherein an azobenzene photoswitch was incorporated into the linker: a photoswitchable inhibitor of the kinase MEK1 was bolted to the protein using Fox’ bioorthogonal tetrazine Hetero-Diels-Alderchemistry (Fig. 1B: Table 1, entry 2).18 Again, the advantage of this method is that a relatively small change - the unnatural amino acid - is introduced to the protein of interest. However, the chemistry of bioconjugation, at least the tetrazine method, introduces a relatively large moiety in the near vicinity of the site of pharmacological action and places geometrical constrains on the photoswitchable tethered ligand making the rational design of these systems more difficult. In addition, nonsense suppression is technically challenging, especially in eukaryotic cells, as it requires the introduction of an engineered tRNA/tRNA-synthetase pair, addition of a cellpermeable unnatural amino acid, and mutation of the gene of interest. Although the modification of eukaryotic transmembrane proteins with Amber suppression technology has been achieved,19 photo-BOLT has not yet been applied to receptors or ion channels. However, as the technology becomes increasingly popular, it is likely, that these applications will emerge. To make tethered photopharmacology more general, practical, and suitable for in vivo studies, we recently introduced an extension of the PTL concept: the photoswitchable orthogonal remotely-tethered ligand (PORTL).20 In this case, the tether is attached far away from the ligand binding site to a self-labeling protein tag, such as a SNAP-,21 CLIP-,22 or HALO-tag,23 which is genetically fused to the receptor of interest. These tags are convenient to use and do not


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require additional cofactors or enzymes to promote the bioconjugation. They are comparable in size to small fluorescent proteins (20-34 kDa) and are tolerated by many receptors either as an N- or a C-terminal fusion. As circular permutated versions of the tags emerge, which can be inserted into loops,24 the versatility of the PORTL approach will increase.

The introduction of such large bioconjugation motifs has consequences for the design of the tethered photoswitches. Since the attachment is remote, they require a long, flexible linker between the reactive moiety and the ligand. These linkers can be several nanometers long, which means that the isomerization of a single azobenzene photoswitch has a negligible effect on their hydrodynamic radius. Instead, the photoswitch is moved into the pharmacophore to change the efficacy or affinity of the ligand upon isomerization. As such, PORTLs can be thought of as diffusible photoswitchable ligands ‘on a long leash’. This leash, i.e. the linker, should not stick to the surface of the protein to ensure that the ligand can effectively reach its binding site. In addition, the linker should be water soluble, highly flexible, and synthetically manageable. Polyethylene glycols chains (PEG chains) fulfill these requirements. They are commercially available in different lengths, which allows for the synthesis of small libraries of PORTLs with increasing linker lengths. The PORTL strategy foregoes the need to carefully screen for cysteine mutations or unnatural amino acids as suitable sites of attachment, as is necessary in the PTL and photo-BOLT approach. The attachment site (a cysteine or aspartate residue) is fixed in the self-labeling protein tags. The PORTL concept was first demonstrated by our group in 2015 using the metabotropic receptor 2 (mGluR2) as a target (Fig. 1C). The tethered agonist, a glutamate-derivative (Table 1, entry 3) turned out to be active in the cis-configuration of the photoswitch. This feature would have been difficult to design computationally and was established experimentally. It has the advantage that the photoswitch is silent after bioconjugation and only activates the receptor upon irradiation. Of course, tethered antagonists, tethered blockers, and tethered positive and negative allosteric modulators that are active in the trans-configuration of the switch (i.e. in the dark), could be equally useful. Fig. 2 shows a model of the SNAP-mGluR2 receptor chimera with a PORTL attached in its dimeric state. One of the tethered ligands is oriented towards the ligand binding site in the Venus flytrap ligand binding domain, whereas the other one is stretched out. The model provides an idea of the relative dimensions of a SNAP tag, the extracellular domain, the seven-


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helix transmembrane domain of the receptor, and the tethered photoswitch. According to this model, it seems unlikely that a ligand tethered to one protomer could reach over to bind to the Venus flytrap domain of the other one, a notion which was recently confirmed experimentally.25

Figure 2: Molecular model of a dimeric SNAP-mGluR2 receptor construct labeled with the PORTL BGAG12. For calibration, two spheres with a 5 nm radius are shown. TMD = 7-helix transmembrane domain (from pdb 4OR2), CRD = cysteine rich domain (pdb 5KZQ), LBD = Venus flytrap ligand binding domain (from pdb 5CNI). The SNAP tag (pdb 3KZY) is shown in orange and the glutamate head group of the PORTL, which features 12 ethylene glycol repeats, in red.


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The PORTLs are covalently attached via reaction of the protein tags with benzyl guanines, benzyl cytosines, or haloalkanes, respectively (Table 1, entry 3-5). These reagents are chemically stable under physiological conditions, making it possible to apply them in larger tissues, collect the supernatant and use them again for labeling, even after days or even weeks of storage at room temperature.20 The bioconjugation reactions have the selectivity of an enzymatic reaction, go to near completion, have high reaction rates,26 require a relatively low concentration of the PORTL, and have been shown to work well in vivo.27 The tagged proteins can be labelled with fluorophores to monitor their expression and lifetime. A wealth of receptor proteins have been cloned and many are now commercially available, such as SNAP and HALO tagged GPCRs and RTKs.28 They could be readily repurposed, making it likely that many new applications of PORTL will emerge in the future. As far as their reactivity is concerned, the different tags are orthogonal to each other, making it possible to multiplex the optical control over several receptors or use a combination of tethered agonists, antagonists, and modulators. Recently, this orthogonality was demonstrated with the simultaneous dual optical control over two subtypes of metabotropic glutamate receptors. CLIPmGluR2 and SNAP-mGluR7 were expressed in the same cell and labeled with two PORTLs with orthogonal reactivity (a benzyl cytosine and a benzyl guanine) and activation/deactivation wavelengths. Using different wavelengths of light, the receptors could then be activated and deactivated independently from each other.25 Notably, PORTL could also work with unmodified receptors. The tag doesn’t necessarily need to be attached to the receptor itself but can be linked to a protein in its vicinity, e.g. a scaffolding or auxiliary protein. Very recently, Tadross elaborated on this idea by introducing drugs acutely restricted by tethering (DART, Fig. 1D). Here, a non-photoswitchable ligand, such as a AMPA receptor antagonist or an antagonist of a metabotropic acetylcholine receptor, was tethered through a very long PEG linker to a HALO-tag attached to a membrane anchor. By expressing this anchored HALO-tag only in a defined subset of cells, he could show how AMPA receptors contribute to Parkinson’s disease.29 The DART concept could easily be expanded to include photoswitchable ligands. The resulting unfused PORTL (uPORTL) is shown schematically in Fig. 1D. Even more ambitiously, the tag could reside on a different cell as the receptor, e.g. across a synaptic cleft. It will be interesting to find out whether the effective concentration of ligands on very long PEG-linkers will be sufficient to activate receptors at such a distance (2040 nm).30


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Although the combination of genetics and pharmacology is powerful, its application in human medicine is limited since gene therapy, as promising as it is, is still in its infancy. Apart from technical and regulatory issues with viral gene delivery, the immunogenicity of modified or introduced genes needs to be scrutinized in each case. Furthermore, genetically modified receptors are usually overexpressed against a background of native receptors that have not been incapacitated. Although this has not emerged as a major limitation, it could lead, at least in principle, to non-physiological responses that provide a misleading picture of the native system. Therefore, approaches to tethered photopharmacology that do not require genetic modification are of great interest. One possible strategy relies on photoswitchable affinity labels (PALs). Here, the ligand binds non-covalently first and subsequently reacts with native nucleophiles nearby through a tethered weak electrophile. Due the increased effective concentration, this reaction happens at an enhanced rate. Indeed, affinity labeling was already observed in 2007 with PTLs for ionotropic glutamate receptors, where cis-configured maleimides underwent bioconjugation faster than those with the photoswitch in the trans-configuration.31 The PAL-concept was initially proposed for tethered blockers of potassium channels32 and validated experimentally with photoswitchable inhibitors of human carbonic anhydrase.33 It was subsequently applied as Targeted Covalent Photoswitches (TCPs) to kainate receptors by Gorostiza.34 Entry 6 in Table 1 shows the structure of the corresponding molecule, an NHS ester of a photoswitchable maleimideazobenzene-glutamate. Following non-covalent binding, the NHS-ester reacts with specific lysines in the vicinity of the binding site. The PAL concept could be further extended to photoswitchable photoaffinity labels (PPALs) using benzophenones, diazirines etc. as photoactive reactive groups. In this case, the orthogonality of photoswitching and the photochemistry used for bioconjugation needs to be carefully considered. The attachment could also rely on affinity labels wherein the non-covalent recognition element is part of a leaving group that is displaced on the course of covalent bond formation. This approach has been used to label AMPA receptors with fluorophores and could be adapted to attach PORTLs.35,36

Another strategy for the optical control of native receptors could rely on tethering through strong and specific non-covalent interactions, such as the ones that bind an antibody to its receptor. Indeed, this could be achieved by linking photoswitchable ligands to antibodies to form


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antibody-photoswitch conjugates (APCs). With the advent of antibody-drug-conjugates (ADCs) the requisite bioconjugation chemistry has been developed to a very high degree of sophistication.37 Like PORTLs, APCs would require long, flexible tethers since antibodies are very large molecules and the site of attachment is far removed from binding site of the photoswitchable ligand. To alleviate these spatial concerns and increase the effective concentration, the photoswitches could also be tethered to smaller probes that provide strong and selective binding: nanobodies, monobodies, affibodies, anticalins, designed ankyrin repeat proteins (DARPins), or any other chemically modifiable protein that can take over the role of a full-size antibody.38 Aptamers, i.e. affinity probes based on polynucleotides, tethered to a photoswitchable ligand are another option.39 The challenge here lies in identifying strong binders that do not unduly affect the function of their target receptor. Finally, one could link a photoswitchable ligand to a second small molecule that binds to an allosteric site with high affinity.

In summary, we have shown that tethered photopharmacology is a useful approach to controlling biological systems with light. In the genetically programmed version, it is a subdiscipline of optogenetics. Indeed, our paper on SPARK, the synthetic photoswitchable azobenzene regulated potassium channel, was published very early in the in the history of this field.6 Although this Perspective has focused on transmembrane receptors, many intracellular proteins could be targeted with tethered photopharmacology, especially with PORTL and photoBOLT. The further development of these techniques will rely on progress in bioconjugation chemistry and the availability of photoswitchable ligands with useful photophysical, pharmacokinetic, and pharmacodynamic properties. The linkers between these ligands and the reactive moieties for covalent attachment also need to be thoroughly investigated. An interesting question is how remotely a ligand can be tethered to a binding site with a particular type of linker. To increase the effective concentration, the linkers could be branched (dendritic) to mount several photoswitches onto a single site of attachment. While this would make the molecules quite large and could hamper their biodistribution, the success of therapeutic antibodies provides us with optimism that this might not be a limitation. Indeed, biologically active molecules linked to photoswitches, such as antibody photoswitch conjugates, are also likely to play a useful role in physiology in future years, perhaps even in human medicine.


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Acknowledgements We are grateful to the SPP1926 (P.L. and D.T.), the SFB1032 (P.L. and D.T.) and the Center for Integrated Protein Science Munich (CIPSM) (P.L. and D.T). We are also thankful for stimulating discussions to Dr. Johannes Broichhagen and to Dr. Bryan Matsuura for proofreading.

Corresponding Author *Dirk Trauner, [email protected]


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Biochemistry

For Table of Contents Only


Entry

Name

Bioconjugation Tag (Size)

LabelingBiochemistry Reagent

Reaction Type HOOC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

NH2 COOH

1

PTL

HN

photoswitch trans

engineered cysteine

O

POI-SH (47 Da)a

addition

H N

N O

O

hν1

H N

N

O

N

O

N

O

maleimide

hν2 or kBT

O

O

N

N H

N

photoswitch cis NH2

HOOC 2

photo-BOLT

COOH

strained alkene or alkyne O

POI

N

POI

N N

(248 Da)a

(182 Da)a

3

PORTL

F

N H N

N

O

N

SNAP-tag, reactive Cys POI-SNAP-SH (20 kDa)

N

N N N

H N

N

N H

cycloadditon -elimination

F F

O N

HN

N H

tetrazine

I

O O

O

O

O

O

O

NH2

O

N

4

PORTL

N

CLIP-tag, reactive Cys

N N N

H N

N

HN

R

O

substitution

O

H N N H

O

O

O

O substitution

HOOC

O

NH2

POI-CLIP-SH (20 kDa)

O

N

benzyl guanine

O

O

NH2

benzyl cytosine

COOH

5

6

PORTL DART

PAL/TCP

HALO-tag, reactive Asp

O

Cl

POI-HALO-COOH (34 kDa)

O N

POI-NH2

R

POI

NH

etc.

substitution

O

chloroalkane

native nucleophile POI-SH

H N

O

N

O

O O

O

N

ACS Paragon N-hydroxy succinimidePlus ester

N N

H N O

Environment

condensation N

N

O N H NH2 HOOC

COOH

Page 18 of 21

Page 19 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Biochemistry

ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Page 20 of 21

5 nm SNAP tag

BGAG12 LBD

CRD

TMD


Page 21 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Biochemistry

bioconjugation tag

receptor of interest

photoswitch remotely tethered ligand


Specificity and Speed: Tethered Photopharmacology - Biochemistry

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