Development of Photoswitchable Tethered Ligands that Target the μ‐Opioid Receptor

Converting known ligands into photoswitchable derivatives offers the opportunity to modulate compound structure with light and hence, biological activity. In doing so, these probes provide unique control when evaluating G‐protein‐coupled receptor (GPCR) mechanism and function. Further conversion of such compounds into covalent probes, known as photoswitchable tethered ligands (PTLs), offers additional advantages. These include localization of the PTLs to the receptor binding pocket. Covalent localization increases local ligand concentration, improves site selectivity and may improve the biological differences between the respective isomers. This work describes chemical, photophysical and biochemical characterizations of a variety of PTLs designed to target the μ‐opioid receptor (μOR). These PTLs were modeled on fentanyl, with the lead disulfide‐containing agonist found to covalently interact with a cysteine‐enriched mutant of this medically‐relevant receptor.


Introduction
Throughout the past decade, extensive research has been conducted in expanding the repertoire of photoswitchable ligands that interact with a range of biological targets. [1]The attractiveness of photoswitchable probes stems from the ability to use light to reversibly change their chemical structure and/or properties.Importantly, this change may also alter the inherent biological activity of these compounds, resulting in photocontrol of ligand activity. [1]Such spatial, temporal and non-invasive control may be beneficial in enhancing kinetic and dynamic investigations. [2]This includes overcoming limitations of nonuniform start times in kinetic studies, as well as exploring receptor-ligand interactions and associated conformational changes in dynamic studies. [2]These investigations are particularly important for understanding the mechanisms and interactions of medically-relevant receptors, including G-proteincoupled receptors (GPCRs). [3]pproximately 34 % of clinically approved drugs exert their mechanism of action by modulating GPCR signaling. [4]Despite the successful application into clinics of drugs that target this class of receptors, there is still a knowledge gap in the research surrounding GPCR mechanisms and interactions. [5]This knowl-edge gap is particularly evident for the μ-opioid receptor (μOR).The μOR is notably targeted for pain relief, with drugs such as fentanyl and morphine available on the medical market.However, despite its importance, this GPCR plays a leading role in the current opioid epidemic. [6]In order to better understand the μOR, a wide range of valuable research has been accomplished over decades. [7]In the field of photopharmacology, fentanyl was developed by Trauner et al. [8] and later by our group [9] into photoswitchable ligands that enabled photocontrol of a μOR ionotropic and metabotropic response, respectively (Figure 1).
For ionotropic photocontrol, the trans-isomer (blue light irradiation) of photofentanyl 2 (PF2) was found to trigger μORmediated potassium influx through G-protein-coupled inwardrectifying potassium (GIRK) channels, while its respective cisisomer (360 nm irradiation) retracted this μOR activation. [8]For metabotropic photocontrol, a photoswitchable ligand that is named here as fentanyl azopyrazole 1 (FAPz 1) displayed Figure 1.Structures of previously reported photoswitch-containing ligands that target the μOR (left and right), modeled on the fentanyl pharmacophore (middle).Previous work by Trauner et al. [8] attached an azobenzene photoswitch to the core structure of fentanyl, named photofentanyl 2 (PF2), while previous work by our group (König et al. [9] ) attached an azopyrazole photoswitch unit (FAPz 1).When compared to fentanyl, FAPz 1 contains a methylene insertion (+ CH 2 ), which was reported to improve the photophysical properties of FAPz 1. [9] significant receptor efficacy differences between its respective trans-(528 nm irradiation) and cis-isomers (365 nm irradiation) in a G-protein activation assay (IP-One®). [9]In order to further exploit the possibilities of FAPz 1 as a biochemical tool, this photoresponsive ligand was modified in this work into a range of tethered fentanyl azopyrazole (tFAPz) derivatives.These compounds were designed to contain a reactive group that could covalently interact with the μOR (Figure 2).More specifically, these fentanyl derivatives were designed in accordance with a class of covalent photoswitchable molecules known as photoswitchable tethered ligands (PTLs), which offer several advantages that are discussed in this paper.7d] To date, there have been no PTLs that target μOR, and as a result, the work herein describes findings in the pursuit of these compounds.

Results and Discussion
PTLs have been successfully applied in targeting GPCRs, ion channels and enzymes. [10]These tool compounds are composed of a pharmacophore that is attached to a photoswitch unit, which in turn, is attached to a reactive unit. [11]The two segments that link these 3 components may vary in length.Longer segments result in a greater separation between these components, which may be beneficial in maintaining pharmacophore affinity.A greater separation between these components may also allow for more dramatic displacement of the pharmaco-phore from the binding pocket upon photoisomerization; however, too much flexibility could oppose this effect.Once the PTL is drawn by affinity to its respective binding site, the reactive unit is able to form a covalent bond via a cross-linking reaction, localizing the ligand to its biological target. [12]In doing so, PTLs overcome several limitations of freely diffusible photoswitchable ligands. [12]Covalent localization to the target receptor may improve site-selectivity, minimize off-target interactions, and is resistant towards sample washing or dilution.One of the most established extracellular bioconjugation techniques using PTLs has been the cysteine-maleimide system. [13]In this system, a sulfhydryl group of a cysteine residue near the receptor binding pocket reacts with an electrophilic maleimide moiety installed on the PTL, forming a covalent bond.Since reduced and solventexposed cysteine residues have a low natural abundance in proteins, mutation of amino acids near the binding site to a cysteine residue has commonly been required to allow for bioconjugation. [12]10e] For example, the azide group of FAPz 1 could be directly used in a bioorthogonal approach to covalently attach this fentanyl-based probe to the binding pocket of μOR, [15] however, this may require more complex genetic engineering of μOR.Furthermore, due to the low natural abundance and good reactivity of 'free' sulfhydryl groups, cysteine residues are considered to be one of the most convenient targets for selective bioconjugation. [16]verall, PTLs offer significant advantages as biochemical tools.As a result, it became of interest to expand FAPz 1 that already possesses desirable biochemical and photophysical properties into PTLs. [9]Since FAPz 1 already contains the fentanyl pharmacophore and an azopyrazole photoswitch unit, a reactive group was attached to the photoswitch unit via various linker lengths, resulting in an array of tFAPz derivatives (Figure 2).Various lengths of this linking segment were explored in order to achieve covalent interaction in or near the μOR binding pocket.The longest segment designed in this series was the PEG-4 linker in tFAPz 2 g.This derivative was hypothesized to form covalent interactions more distal to the ligand-receptor binding pocket in accordance with the design approach of photoswitchable orthogonal remotely tethered ligands (PORTLs). [11]Following both PTL and PORTL strategies, a maleimide series was developed (tFAPz 2 a-g) using different synthesis techniques for maleimide attachment to FAPz 1, including the well-known 'click' and 'Staudinger' reaction methods (Figure 2). [17]Even though maleimides have been effectively used in bioconjugation, hydrolysis and other drawbacks provided incentive to synthesize additional PTLs with other reactive groups. [16]The disulfide reactive group is highly selective for cysteine residues via disulfide exchange with free thiol groups.Despite susceptibility to reduction, disulfides have been proven valuable in bioconjugation for targeting GPCRs, [18] and as a result, a disulfidecontaining PTL was designed (tFAPz 1).
Similarly, isothiocyanate tFAPz 3 was developed due to its promising ability to engage in covalent interactions.Isothiocya- nates have been shown to react with both sulfhydryl and amine nucleophiles, depending on pH, with a resistance towards water and alcohol-mediated hydrolysis. [18]It should be mentioned that in previous literature, an isothiocyanate group was attached to the terminus of the phenethyl moiety of fentanyl to form a covalent ligand (FIT or further modifications to SUPERFIT). [19]espite possessing covalent properties, these ligands resulted in selectivity for δOR instead of μOR.Further modifications were explored in other literature work in attempts to obtain a fentanyl-based ligand that covalently targets μOR. [20]1a,b, 21] As a result, the desire to expand the repertoire of fentanylbased covalent probes provided further incentive for the development of the proposed tFAPz ligands.
The synthesis of tFAPz either required the development of individual synthetic routes (tFAPz 1 and tFAPz 2 a, Figure 3) or could be easily accessed from FAPz 1 via a one-pot synthesis reaction (tFAPz 2 b-g and 3, Figure 4, 5 and 6).To obtain tFAPz 1 (Figure 3A), arylazopyrazole 4 that was synthesized according to literature procedures [9] was activated using methanesulfonyl chloride to form 5 in 97 % yield.Nucleophilic substitution with potassium thioacetate resulted in a thioester, which was directly transformed to pyridyl disulfide 6 using Aldrithiol TM -2 in an overall yield of 67 %.Boc-deprotection with trifluoroacetic acid then afforded amine 7 in high yield.Subsequent reductive amination with 1-phenethyl-4-piperidone, afforded intermediate 8 in 88 % yield, which was then acylated to obtain intermediate 9 in 95 % yield.Treatment with cysteamine, following previous literature, [22] afforded tFAPz 1 in 95 % yield.
In order to improve synthesis yields, Cu(II)-assisted click chemistry was employed to generate additional maleimidecontaining PTLs (Figure 5).This was achieved using a similar method to that previously reported. [25]Using this approach, the azide of FAPz 1 was reacted with alkyne derivatives that contained a maleimide group to obtain tFAPz 2 f and 2 g.The alkyne used to obtain tFAPz 2 f was synthesized via the reaction of activated 2-maleimidoacetic acid with N-propargylamine (see Supporting Information), while the alkyne used to obtain tFAPz 2 g was commercially obtained.In this assisted cycloaddition reaction, sodium ascorbate was used as an initiating reagent and Cu(II) was chelated with TBTA, which yielded tFAPz 2 f and 2 g in higher yields of 47 % and 46 %, respectively.To obtain isothiocyanate tFAPz 3 (Figure 6), previously synthesized FAPz 1 was directly subjected to a one-pot reaction that has been previously reported. [26]This involved generating an iminophosphorane from the azide of FAPz 1, followed by a condensation reaction with carbon disulfide to afford tFAPz 3 in 42 % yield.
Once synthesized, it was important to determine whether the desirable photophysical properties of FAPz 1 were maintained despite ligand extension into PTLs.Since chemical modifications were conducted at distal sites to the photoswitch unit to obtain the tFAPz derivatives, similar photophysical properties to FAPz 1 were anticipated and obtained (Figure 7 and Supporting Information). [9]Similar to FAPz 1, irradiation wavelengths of 365 nm could be used to obtain the cis-isomers of the tFAPz ligands, while a desirable red-shifted wavelength (528 nm) could be used to obtain the respective trans-isomers.In addition, the tFAPz derivatives displayed resistance towards cycle fatigue for at least 5 isomerization cycles, exhibited comparable photostationary states (PSS) to FAPz 1 and possessed cis-isomer thermal half-lives of at least 3 days in both DMSO and buffer solution.In particular, the long thermal half-lives allow for good photocontrol, as the abundance of either the cis-or trans-isomer can be spatially and temporally modulated by irradiation with either 365 nm or 528 nm, respectively.
Since the PTLs displayed desirable photophysical properties, it was next important to determine whether tFAPz 1, 2 a-g and 3 displayed a potency towards wild-type μOR (μOR wt ).These PTLs were subjected to a ligand-mediated μOR activation screen at   10 μM ligand concentration, using a G-protein activation assay (IP-One®). [27]This assay measures agonist-stimulated accumulation of IP in HEK293T cells, which were transiently co-transfected with the receptor and the hybrid G-protein Gα qi5HA . [27]Each PTL was irradiated prior to biological analysis with the appropriate wavelength in order to obtain either the respective trans-or cisisomer, and were compared to the full agonist reference DAMGO.In this screen, tFAPz 2 a-g and 3 displayed weak receptor activation (SI Table 2).After an IP accumulation period of 3 h, both cis-and trans-isomers of tFAPz 2 b, 2 d and 2 f displayed less than 31 % μOR wt maximum receptor response, while both respective isomers of tFAPz 2 a, 2 c, 2 e, 2 g and 3 displayed no significant μOR activation.These poor activation profiles, espe-cially when compared to fentanyl (100 % response), may be due to suboptimal modifications to the pharmacophore or off-target interactions of the reactive tethering groups; however, compound degradation under the described conditions cannot be entirely excluded.In contrast to these derivatives, the disulfidecontaining tFAPz 1 displayed full agonist activity (E max = 100 %) similar to the efficacy stimulated by fentanyl.As a result, tFAPz 1 was identified as a lead compound in this work.
The use of disulfide-containing compounds to covalently bind to GPCRs have been proven successful in generating stable and functional GPCR-ligand complexes. [18]Once such ligands diffuse via intrinsic affinity into the receptor binding pocket, a chemoselective disulfide exchange proceeds between the disulfide unit of the ligand and a nearby sulfhydryl group of a free cysteine residue. [28]This highly selective reaction reduces the risk of off-target interactions with other nucleophilic amino acids and ultimately, would result in covalent binding to the target site.7d,29] Using this μOR mutant, ligand-mediated activation and binding affinity of tFAPz 1 to μOR M1 were evaluated (Figure 8, Table 1).In order to determine ligand-mediated activation, fulldose response curves of both trans-and cis-isomers of tFAPz 1 were generated using the IP-One® assay.In this study, both isomers of tFAPz 1 behaved as full agonists with potencies of 480 nM (trans-isomer) and 810 nM (cis-isomer).Despite the reduction in potency when compared to fentanyl, both transand cis-isomers were still able to activate the receptor in a nM range, with approximately 2-fold higher activity found for trans-tFAPz 1 than cis-tFAPz 1. Importantly, the previously published efficacy ligand FAPz 1 possessed a lower potency than tFAPz 1 in the same activation assay with wild-type μOR (trans-FAPz 1: EC 50 = 4,700 nM; cis-FAPz 1: EC 50 = 2,300 nM; compare with fentanyl: EC 50 = 2.6 nM). [9]These results suggest that modification of FAPz 1 to tFAPz 1 by replacing the azide moiety with a disulfide unit may be better tolerated for receptor activation.Radioligand binding studies revealed competitive binding of tFAPz 1 to μOR M1 , with K i values of 42 nM and 80 nM obtained for its trans-and cis-isomers, respectively (Table 1).Similar to activation studies, trans-tFAPz 1 displayed approximately 2-fold higher affinity for μOR M1 than cis-tFAPz 1.Furthermore, the former isomer displayed only a 3.8-fold attenuation in binding affinity when compared to fentanyl, which shows that the ligand specifically recognizes μOR M1 .
Since tFAPz 1 displayed significant affinity and activation profiles towards μOR M1 , it was crucial to determine whether this lead PTL was able to covalently bind to μOR M1 .Specific binding of [ 3 H]diprenorphine was determined, according to previous procedures, [28,30] for membranes pre-treated with trans-or cis-tFAPz 1. Corresponding findings were compared to a control homogenate that was incubated with the reversible ligand naloxone or fentanyl.For membranes that were treated with tFAPz 1, the determined specific binding indicates the amount of blocked receptor binding sites by covalently bound ligand after washing and exposure to excess radioligand.In comparison Table 1.Ligand-mediated activation and radioligand binding with μOR M1 .
The superiority of the trans-isomer over the cis-isomer was also reflected by the kinetics of covalent binding.While cis-tFAPz 1 displayed covalent binding to the receptor with a half-life (t 1/2 ) of 23.8 min, a t 1/2 of 12.5 min was obtained for trans-tFAPz 1.This result indicates 2-fold faster kinetics for the latter isomer (Figure 9, SI Table 3).These findings were further validated by the observation that trans-tFAPz 1 decreased specific binding by 51 % after 15 min, while cis-tFAPz 1 only decreased specific binding by 27 % in the same amount of time.Since differences in covalent binding properties were observed between the isomers of tFAPz 1, it may be inferred that the differing geometry of each isomer was maintained upon diffusion into the receptor binding pocket and during the process of ligand-receptor complexing.Overall, these findings establish tFAPz 1 as an attractive probe that can localize and covalently bind to μOR M1 .

Conclusions
The work described herein focuses on the development of PTLs, modeled on the potent μOR agonist fentanyl.The newly synthesized ligands displayed ideal photophysical properties that were similar to that of the previously reported FAPz 1, including long thermal half-lives (of at least 3 days) and cycle performance resistance (for at least 5 cycles) in both buffer and DMSO systems. [9]Furthermore, respective cis-isomers could be accessed with 365 nm irradiation and respective trans-isomers could be accessed using a desirable red-shifted wavelength of 528 nm.
These findings not only establish tFAPz 1 as a photoswitchable agonist that can form covalent ligand-receptor complexes in this system but demonstrate that the structural differences between isomers were maintained upon covalent interaction with μOR M1 .Future work may consist of minor structural modifications to tFAPz 1 in order to further enhance biological differences between isomers.10e] Due to the high binding affinity of trans-tFAPz 1 (K i = 42 nM) and cis-tFAPz 1 (K i = 80 nM), structural modifications to slightly reduce affinity may be beneficial. As a result, the development and confirmation of tFAPz 1 as a covalent and photoswitchable μOR probe provides a foundation to further expand and improve the repertoire of tFAPz ligands.Furthermore, this covalent probe may be beneficial in future biochemical investigations surrounding μOR and fentanyl-based structure relations, with the added advantage of utilizing the photoswitchable properties of tFAPz 1 when desired.

Figure 4 .
Figure 4. Synthesis of tFAPz 2 b-2 e via an adapted Staudinger reaction.Yields of isolated products are shown.Please refer to Supporting Information for detailed synthesis methods.

Figure 5 .
Figure 5. Synthesis of tFAPz 2 f-2 g via Cu(II)-mediated click reaction.Yields of isolated products are shown.Please refer to Supporting Information for detailed synthesis methods.
[a] IP-One accumulation assay (Cisbio).[b]The isomer shown in brackets has a greater potency than its respective isomer.[c] Maximum receptor activation in % � S.E.M. relative to the full effect of DAMGO.[d] Number of individual experiments, each performed in duplicate.[e] Data is derived from experiments conducted with the μOR wild-type receptor.[f] Binding data to μOR M1 was determined by competition binding with [ 3 H]diprenorphine.[g] Number of individual experiments, each performed in triplicate.