Photoswitchable Antagonists for a Precise Spatiotemporal Control of β2-Adrenoceptors

Article abstract of DOI:10.1021/acs.jmedchem.0c00831

β2-Adrenoceptors (β2-AR) are prototypical G-protein-coupled receptors and important pharmacological targets with relevant roles in physiological processes and diseases. Herein, we introduce Photoazolol-1-3, a series of photoswitchable azobenzene β2-AR ant

Full text of DOI:10.1021/acs.jmedchem.0c00831

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Article
Photoswitchable antagonists for a precise
2
spatiotemporal control of #-adrenoceptors
Anna Duran Corbera, Juanlo Catena, Marta Otero Viñas, Amadeu Llebaria, and xavier Rovira
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00831 • Publication Date (Web): 20 Jul 2020
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Photoswitchable antagonists for a precise spatiotemporal
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control of β -adrenoceptors
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Anna Duran-Corbera†, Juanlo Catena†§, Marta Otero-Viñas‡, Amadeu Llebaria†* and Xavier
Rovira‡ξ*
†
MCS, Laboratory of Medicinal Chemistry, Institute for Advanced Chemistry of Catalonia
(IQAC-CSIC), 08034, Barcelona, Spain.
§
SiMChem, Service of Synthesis of High Added Value Molecules, Institute for Advanced
Chemistry of Catalonia (IQAC-CSIC), 08034, Barcelona, Spain.
‡
Molecular Photopharmacology Research Group, The Tissue Repair and Regeneration
Laboratory (TR2Lab), Faculty of Sciences and Technology, University of Vic – Central
University of Catalonia, 08500, Vic, Spain.
KEYWORDS: β -adrenoceptor, GPCR, light-regulated drugs, receptor photopharmacology.
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ABSTRACT
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β -adrenoceptors (β -AR) are prototypical G protein-coupled receptors and important
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pharmacological targets with relevant roles in physiology and disease. Herein, we introduce
Photoazolol-1-3, a series of photoswitchable azobenzene β -AR antagonists that can be
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reversibly controlled with light. These new photochromic ligands are designed following the
azologization strategy, with a p-acetamido azobenzene substituting the hydrophobic moiety
present in many β -AR antagonists. Using a FRET biosensor-based assay, a variety of
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photopharmacological properties are identified. Two of the light-regulated molecules show
potent β -AR antagonism and enable a reversible and dynamic control of cellular receptor
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activity with light. Their photopharmacological properties are opposite, with Photoazolol-1 being
more active in the dark whereas Photoazolol-2 demonstrates higher antagonism upon
illumination. In addition, we provide a molecular rationale for the interaction of the different
photoisomers with the receptor. Overall, we present innovative tools and a proof of concept for
the precise control of β -AR by means of light.
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INTRODUCTION
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Photopharmacology is an emerging field of research based on the use of light-regulated drugs.1
In the past years, a number of photo-controllable molecules have been reported, showing their
ability to modulate the activation state of several G protein coupled receptors (GPCRs).2
Interestingly, unprecedented research involving GPCR photopharmacology with diffusible drugs
has demonstrated the performance of this chemical approach to dynamically manage
physiological conditions in rodents, including the abolition of the physical and emotional
symptoms of persistent pain.3,4 These and other recent studies highlight the enormous potential
of photopharmacology for the study of GPCR roles in physiological processes and the
development of future precise drugs.5
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Mainly two different chemical strategies have been used in photopharmacology to design
diffusible molecules that allow a spatiotemporal localization of the drug action. The first, named
compound caging, is based on the transformation of biologically active molecules into inactive
compounds through the attachment of a photolabile protecting group to their chemical structure,
which abolishes its binding within the receptor. The protecting groups are irreversibly cleaved
upon illumination, thus causing a local release of the drug that subsequently acts following
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conventional pharmacology patterns. Therefore, the activity of caged compounds is not
reversible, which limits the temporal regulation of the bioactive molecule once released and may
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cause possible side effects in adjacent tissues. The second approach is based on the
introduction of a photochromic moiety within a drug structure to reversibly adjust its activity with
light.7,8 From the four most typical classes of photochromic moieties (azobenzenes,
dihydropyrans, diarylethenes, and fulgides) azo-based compounds have been widely applied in
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the context of GPCR photopharmacology to date. The reversible conversion of the compound
between two states by light illumination produces changes in polarity, geometry, and end-to-end
distance. These light-regulated changes in the molecule can be designed to alter its functional
properties as a ligand (e.g. agonist/antagonist character) or the accessibility to its binding site
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within the receptor (affinity). In principle, with photoisomerizable molecules the selectivity of the
drug action can be finely tuned by a rapid thermal relaxation to the most stable state or using a
different light wavelength to switch on and off its activity. In addition, the concentration of the
active isomer can be adjusted by modulating the light power or the wavelength used.7,10 Today,
the GPCR photopharmacological toolbox includes a variety of pharmacological and chemical
strategies, including photoswitchable tethered ligands,11 ligands acting as agonists or
antagonists depending on the applied light,9,12 photoswitchable dualsteric ligands,13 light-
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regulated ligands based on dithienylethenes and fulgides, ligands that can be activated using
two-photon excitation with near-infrared light,15 and ligands more active in the dark16,17 and
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others in the less stable photoisomeric state upon illumination,18 among others. Overall, the
development of photoswitches to regulate GPCRs with light is an emerging field and may open
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new avenues for the treatment of many diseases with unmet needs in innovative ways that are
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not possible with classical pharmacology.1,5
GPCRs are major pharmacological targets accounting for around one third of the
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marketed drugs. Among this family, β-adrenoceptors are prototypical GPCRs and molecules
directed towards these receptors, either agonists or antagonists, are first-line treatments for
important diseases, such as several heart dysfunctions, asthma, anxiety, urinary incontinence,
migraine and glaucoma, among others.20,21 Therefore, developing light-regulated strategies to
activate or inactivate β-adrenoceptors in a controlled manner can be of great research and
therapeutic interest. To date, several studies have been directed to control the activity of β2-
adrenoceptors (β -AR) with light. Indeed, caged derivatives based on adrenergic receptor
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agonists were developed. Also, a caged version of the antagonist Timolol has been recently
synthesized crosslinked on a polymer surface to build contact lenses, which may have
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applications for the treatment of intraocular pressure in patients with glaucoma. Using a very
different approach, a research presented a chimeric β -AR construct that could be controlled by
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light (opto-β AR). This construct was composed of the light-sensitive region of rhodopsin and
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the G -coupling region of β -AR. The authors demonstrated that this biotechnological tool
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could be applicable for the light-control of cyclic adenosine monophosphate (cAMP) levels in
cell cultures, native tissues and even to modulate behavior in freely moving mice.
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Despite the interesting prospects of caged compounds and light-regulated chimeric
constructs, major drawbacks, such as the irreversible nature of caged compounds or the need
to genetically modify the organism, hamper their precise use in physiological environments. In
this context, using a new approach based on photoisomerizable molecules targeting β -AR
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could provide a valid alternative with therapeutic and research potential. The present work
provides the first proof of concept for reversible β -AR photopharmacology. We present two
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photoswitchable compounds that antagonize β -AR agonist activity in a light-dependent manner.
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Biological in vitro testing highlights the successful development of two compounds with
promising pharmacological properties that can be reversibly controlled in opposite ways, either
increasing or reducing their β -AR activity upon illumination.
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RESULTS AND DISCUSSION
Design, Synthesis and Photochemical Characterization of Photoswitchable β2-
adrenoceptor antagonists.
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The chemical design of photoswitchable compounds targeting β -AR was based on the 3-
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aryloxypropan-2-olamine molecular scaffold commonly found in β-adrenoceptor antagonists
(Figure 1). Noticeably, these molecules are constituted by an aromatic ring connected to an
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ethanolamine backbone through an oxymethylene bridge. It is well described that the
ethanolamine moiety plays an essential role on the ligand-protein interaction, considering that it
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forms H-bond interactions with key residues on the binding pocket. Additionally, compounds
incorporating the oxymethylene bridge on their structure have been signaled as β-adrenoceptor
antagonists. Therefore, the relevance of the oxyaminoalcohol substructure in ligand function
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highlights it as the so-called molecular fingerprint of β-adrenoceptor antagonists. On the other
hand, the hydrophobic moiety admits certain variations as it can integrate multiple aromatic
fused rings or heteroaromatic substructures (e.g. carbazole and indole). Consequently, we
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identified the hydrophobic core as suitable for the azologization strategy. We proposed three
different molecules, where the naphthalene of propranolol and the tricyclic carbazole moiety of
carazolol are substituted for an azobenzene moiety. The designed azobenzenes are structural
isomers, where the only difference relies on the aromatic substitution pattern of the
oxyaminoalcohol with respect to the N=N double bond (Figure 1).
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Figure 1. Design of photoswitchable azobenzene β -AR antagonists Photoazolols (PZLs). Left
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panel, prototypical β-adrenoceptor antagonists. Right panel, designed photoisomerizable
molecules following the azologization strategy.
Importantly, the vast majority of ligands targeting β -AR are chiral. In fact, different
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pharmacological behavior has been distinguished for the enantiomers, with better β -AR
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pharmacological properties always observed for the (S)-eutomers. Therefore, and according
to the structure of potent ligands we aimed to synthesize the (S)-enantiomers of the designed
azobenzenes. Finally, all azobenzene compounds were designed with a p-acetamido
substituent, which was introduced with the objective to obtain photochromic ligands with
appropriate photochemical properties.28
Scheme 1. Synthesis of Photoazolol-1–3.a
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a
Reagents and conditions: (a) Oxone, H O/DCM 1:1, r.t, 2 h; (b) p-Acetamidoaniline, AcOH,
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r.t, 48 h, 25-41%; (c) BBr , DCM, 0 ºC to r.t, 24 h, 95%; (d) (I) NaNO , aq HCl, 0 ºC, 5 min; (II)
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Phenol, aq NaOH, 0 ºC, 30 min 63%; (e) (R)-Epichlorohydrin, K CO , butanone, reflux, 48 h,
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9%-quantitative; (f) i-PrNH , 12 h, r.t, 29-56%;(g) 2-Aminophenol, AcOH, r.t, 48 h, 16-21% (h)
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2S)-Glycidyl tosylate, K CO , DMF, r.t, 12 h; (i) i-PrNH , 90 ºC, 48 h, 33%.
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The synthetic routes developed to produce photo-controlled β -AR antagonists are
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depicted in Scheme 1. All three routes share an analogous intermediate, which is the phenolic
azobenzene (5, 6 and 10). Direct diazotization of the p-acetamidoaniline (4) followed by reaction
with phenol yielded phenylazophenolic intermediate 6. Nevertheless, synthesis of the meta- and
ortho-acetamido intermediates 5 and 10 was conducted via the typical Mills reaction involving
condensation of appropriate anilines and aromatic nitroso compounds. This alternative
procedure was explored after the attempted diazotization of the respective aminophenols proved
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unsuccessful. Thus, ortho-phenolic azobenzene 10 was obtained by oxidation of the p-
acetamidoaniline (4) to the corresponding nitroso compound (9), followed by Mills condensation
with 2-aminophenol. On the other hand, it is worth noting that in order to produce m-phenolic
azobenzene 5 a methoxy protected intermediate 3 had to be synthesized. The condensation of
the nitrosoacetamide with 3-aminophenol proceeded with very low yields. In consequence, 3-
methoxyaniline (1) was oxidized to the nitroso derivative (2) and reacted with the p-
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acetamidoaniline (4) to yield azobenzene 3. Subsequent O-demethylation in 3 using BBr led to
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the desired azobenzene intermediate 5 in good yield.
Once the key intermediates were synthesized, the following steps required the use of
enantioselective reactions to afford the (S)-enantiomers of PZL-1–3. In order to obtain the
proposed light-regulated β -AR antagonists, we initially followed a commonly reported route for
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the synthesis of β -AR antagonists. The phenolic azobenzenes were alkylated by direct
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reaction with (R)-epichlorohydrin. This reaction, using either acetone or butanone as a solvent,
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proceeds with inversion of configuration to yield the (S)-oxiranes. The epoxides were finally
opened by nucleophilic attack of isopropylamine. This route provided azobenzenes PZL-2 and
-3 with good enantiomeric purity. In contrast, the ortho-substituted product was found to be
partially racemized. The exact reasons for this behavior in the o-diazenylphenol 10 remain
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unknown. In order to overcome this difficulty, a different enantioselective route reported for the
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production of (2S)-propranolol was explored. This new approach consists in a one-pot reaction
of phenol 10 using (2S)-glycidyl tosylate and isopropylamine as main reagents. Regioselective
displacement of the tosylate moiety by phenol under mild basic conditions afforded the
epoxyether intermediate (11) that was not isolated. Epoxide opening was thereafter effected by
refluxing the reaction mixture with isopropylamine to give PZL-1 with good enantiomeric purity.
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Following the synthesis of the desired compounds, the photochemical properties of PZL-
1–3 were evaluated. To be able to effectively control β -AR with light, it is an essential requisite
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to find specific light parameters to interconvert trans and cis azobenzenes in both directions with
high isomeric conversion ratios and in a relatively fast manner with respect to the receptor
activation kinetics. Results from the photochemical characterization of the obtained
azobenzenes are summarized in Table 1. General tendencies can be observed for the three
azobenzenes. Suitable isomerization from the thermostable trans to the cis configuration occurs
when applying near ultraviolet light (365/380 nm). Compounds can also be back-isomerized to
their thermally stable isomer using green-yellow light (525/550 nm) (Figure 2 and S1–S5).
Table 1. Photochemical properties of Photoazolols-1-3a
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PSS380b PSS550b
% cis) (% trans)
Compound λtransa (nm) λcisa (nm) t (min)
a
½
(
PZL-1
PZL-2
PZL-3
356
350
360
428
430
440
72.6
152.1
169.4
86.2
87.7
94.3
71.4
77.5
86.2
0
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a
b
Determined at 50 µM in aqueous buffer + 0.5 % DMSO, 25 ºC. Photostationary state (PSS)
1
ratios were determined at 12 ºC by H-NMR after illumination (380/550 nm) of a 100 µM sample
in D O.
2
Importantly, conversion ratios from trans to cis isomers are higher than 86%. Nevertheless,
back-isomerization occurs with lower efficiency, especially for PZL-1 and -2, with conversions
ranging from 71 to 77 %. This can be explained through the shape of the trans isomers UV-Vis
spectra. This family of compounds presents a strong π-π* band near 360 nm under dark
conditions and a shoulder n-π* band around 440 nm (Figure 2). The n-π* transition is forbidden
by symmetry in trans azobenzenes, which frequently leads to a spectrum with a very weak n-π*
absorption band. Therefore, the presence of this slightly prominent n-π* band in the trans isomer
UV-Vis spectrum suggests that this transition is not completely forbidden for these compounds.
This results in a non-negligible absorption of the trans isomers at these higher wavelengths and
can be considered a particular feature on the UV-Vis spectra of the described azobenzenes. In
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any case, this absorption hinders a more efficient light-triggered transition from cis towards the
more thermodynamically stable trans isomer.
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Moreover, thermal relaxation in aqueous media is relatively slow, considering that all
measured half-life times at 25 ºC are longer than 1h (Table 1) and G protein activation happens
on the second timescale.31 Finally, photoisomerization of all compounds was found to be
reversible and molecules photostable over the application multiple light cycles (Figure 2, S1 and
S2).
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Figure 2. Photochemical evaluation of PZL-1. (A) 2D and 3D chemical structures of the
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photoisomers of PZL-1. (B) UV-Vis absorption spectra of PZL-1 under different light conditions.
1
(
C) Photostationary state (PSS) quantification by H-NMR. Samples were continuously
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illuminated using 380 nm and 550 nm light sources. Chemical shift variations on the methyl of
the acetamide group were followed. (D) Multiple cis/trans isomerization cycles (380/550 nm)
show the stability of the compound over 45 minutes of light application. (E) Half-lifetime
estimation of cis-PZL-1 at 25 ºC; absorbance was measured at 364 nm.
Photopharmacological characterization of compounds.
β -AR are primarily coupled to the G protein, which upon activation causes an increase
2
s
of the intracellular concentration of cAMP through the regulation of adenylate cyclase.32
Consequently, the biological activity of all compounds was evaluated through their ability to
block the increase of intracellular cAMP induced by the addition of an agonist. An assay suitable
to be employed under different illumination conditions was developed using a genetically
encoded Epac CFP-YPF FRET sensor that allowed to dynamically monitor intracellular
33
concentrations of cAMP. Briefly, a HEK 293 cell line, which endogenously expresses β -AR,
2
was transfected with the cAMP sensor and a single clone was selected to establish a stable cell
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line. These cells, which homogeneously express the target receptor and the cAMP sensor, were
used to evaluate ligand light-dependent activities (Figure 3A).
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We firstly evaluated the activity of the developed molecules under different light
conditions. Dose-response curves were performed for PZLs 1-3 supplemented with 3 nM
cimaterol in dark conditions and under continuous illumination with violet light (Figure 3B, 3D
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and S6). Cimaterol was selected as a β -AR agonist for the assays considering that it is a potent
2
ligand and has proved to be stable in aqueous solution, even after 2h of continuous illumination
34
with near ultraviolet light (Figure S7). Remarkably, our results show that both PZL-1 and PZL-2
are potent β -AR antagonists with nanomolar activity (Table 2). On the other hand, their
2
structural isomer PZL-3 showed negligible antagonism (Figure S6). Strikingly, the light-
dependent properties of the two active azobenzenes presented an opposite behavior (Figure
3A). Whereas the most thermodynamically stable configuration of PZL-1 was found to be more
potent (trans-on compound; Table 2), the cis-PZL-2 demonstrated a significantly higher
inhibition compared to its trans form (cis-on compound; Table 2). Indeed, PZL-2 was found to
be approximately 3.6 times more active upon illumination than in the dark. The light-induced
shift in activity was noticeably higher for PZL-1, with 17-fold shift of the inhibitory potency
measured for the compound in dark conditions.
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Finally, we aimed to corroborate that the measured antagonism for the two active
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azobenzenes was directly linked to β -AR. To do so, we performed analogous experiments on
2
cells treated with forskolin, which induces an increase on cAMP concentration by activating
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adenylate cyclase instead of β -AR. In these experiments, no significant decrease of cAMP
2
levels was observed for PZL-1 or PZL-2 (Figure S8), which demonstrates that the described
antagonism is specific to the activation of β -AR by cimaterol.
2
Figure 3. Light-dependent β -AR inhibition of PZL-1 and PZL-2. (A) Representation of the two
2
distinct photopharmacological behaviors observed for PZL-1 (trans-on) and PZL-2 (cis-on).
Dose-response curves of PZL-1 (B) and PZL-2 (D) with a constant concentration of the agonist
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cimaterol (3 nM) in the dark and under constant violet light (380 nm). Dose-response curves of
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cimaterol in the presence of PZL-1 (C) and PZL-2 (E) in the dark and under constant violet light
(380 nm). Data are shown as the mean ± SEM of four independent experiments in duplicate.
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To better characterize the light-dependent pharmacology of PZL-1 and PZL-2, dose-
response curves of the agonist were measured in the presence of different concentrations of
antagonist, both in dark and light conditions (Figure 3C, 3E, S9 and S10). The results confirm a
competitive antagonism for both compounds and a light-triggered modulation of their potency.
In particular, the addition of 10 nM PZL-1 displaces 37-fold the dose-response curve of cimaterol
to the right when cells are kept in the dark, whereas the EC variation is not significantly different
50
to the control without PZL-1 when the cells are illuminated (Table S1), thus validating the trans-
on activity of this compound. On the other hand, the addition of 1 µM PZL-2 shifts 20-fold the
dose-response curve of cimaterol when cells are illuminated with violet light, whereas the curve
displacement is significantly lower when cells are kept in the dark (Table S2). This further
confirms the cis-on activity of PZL-2. For both compounds, a progressive increase on the
concentration of the assayed compound produces equipotent displacements of the agonist
curve and an increase of the agonist EC (Figure S9 and S10). Additionally, these experiments
50
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show no saturation of the inhibitory effect and no significant decrease of the maximal efficacy or
basal activity, which is again consistent with a competitive antagonist pharmacological activity.
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Table 2. Pharmacological Data of photoswitchable β -AR antagonists and propranolol.
2
0
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9
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DARK
IC (nM)
LIGHT
Cmpd
PZL-1
SEM
0.44
35.73
-
IC (nM)
SEM
10.21
23.20
-
Shifta
17.41
0.28
-
SEM
4.41
0.05
-
50
50
1.72*
593.88****
>2000
0.55
28.94
160.64
>2000
0.48
PZL-2
PZL-3
Propranolol
0.13
0.11
0.97
0.21
a
Relation between the measured IC50 in light and dark conditions respectively. Statistical
differences from light IC50 values are denoted for adjusted p values as follows: *p<0.05 and
***p<0.0001.
*
The development of compounds with opposing light-dependent pharmacology is of great
interest since it provides a toolbox of compounds that enable the control of β -AR upon
2
illumination in different manners. Thus, PZL-1, blocking β -AR activity in the dark and allowing
2
the receptor to be activated upon illumination, might be useful for certain research applications
that require spatiotemporal activation of specific receptors while the rest remain inactive. In
contrast, cis-on molecules like PZL-2, with increased inhibitory potency upon illumination, would
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allow a strict inactivation of a subset of receptors during a specific time while the rest remain
under physiological conditions, which opens the door to the development of precise medical
applications without side effects in other tissues and organs.
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Dynamic and reversible photocontrol of β -AR.
2
Probably the most interesting and biologically unexplored capacity of photoisomerizable
drugs acting on GPCRs is their ability to modulate the receptor activity over time using light as
an externally operated regulatory control element of biological activity. For this to happen, it is
necessary that the differential pharmacological effect of cis and trans states can be reverted and
dynamically governed by means of light. In the present work, we addressed whether PZL-1 and
PZL-2 were able to control the β -AR activation state with temporal precision.
2
First, we evaluated the time evolution of the receptor activity after treatment with the two
active azobenzenes in dark and light conditions. To address this question, cells were incubated
for 45 minutes with cimaterol and PZL-1 or PZL-2 in the dark and under violet illumination. After
the incubation period, the activity of β -AR was continuously measured in the dark for 30 min
2
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(
Figure S11). As expected, the experiments where PZL-1 and PZL-2 were kept in dark
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conditions showed a steady evolution of receptor activity over time. This indicates that both trans
isomers have reached an equilibrium after 45 min incubation. However, results after light
application highlighted a remarkably different behavior for the two azobenzenes. At time zero,
PZL-1 shows a significantly higher inhibition of the receptor in dark conditions, consistent with
the reduced activity measured for the cis isomer (Figure 3B and S11A). Continuous tracking of
receptor activity for 30 min demonstrated that cis-PZL-1 was spontaneously isomerizing to its
thermodynamically stable trans isomer, as the antagonism was gradually restored over time. At
the end of measurements, both illuminated and dark experiments showed similar levels of
receptor activity. This suggests that PZL-1 completely back-isomerized in the course of 30
minutes in the cell assay system, which is much faster than the measured relaxation time for
the compound in in a 0.5% DMSO buffer solution (Figure 2E). The increased thermal relaxation
rate of cis-PZL-1 in these experiments evidences a distinct photochemical behavior of the
compound when it is enclosed in a more physiological environment. On the other hand, PZL-2
was more efficient antagonizing the activity of cimaterol under violet illumination, which is
aligned with its described cis-on behavior (Figure 3C and S11B). Interestingly, the antagonism
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observed for cis-PZL-2 at time zero was maintained for the 30 min measured after illumination.
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This suggests that the interaction with the receptor stabilizes PZL-2 in its cis isomer and leads
to a reduced thermal relaxation rate to trans-PZL-2.
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From the previous experiments a question arises concerning the light-triggered
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reversibility of PZL-1 and PZL-2, considering whether light is able to destabilize the ligand-
receptor interaction once the active isomer is bound to β -AR. To address the temporal control
2
of β -AR with light, in a subsequent series of experiments we aimed to assess the capacity of
2
PZL-1 and PZL-2 to dynamically modulate the receptor activation state using cycles of violet
and green light (Figure 4). Interestingly, we found that receptor function could be efficiently
controlled in a reversible manner by the photoswitchable antagonists reported here through the
application of intercalated light cycles at 380 nm and 550 nm. This β -AR reversible control in
2
vitro was observed for at least 3 consecutive cycles. In particular for the trans-on PZL-1, close
to a complete inhibition of the agonist was measured when it was co-added with cimaterol in the
dark (Figure 4). This inhibitory effect was largely diminished when violet light was applied and
could be restored upon the application of green light. The cis-on antagonist PZL-2 showed a
decrease of almost 30% on receptor activation when violet light was applied (Figure 4). This
significant effect was completely reversed upon illumination with green light and this ON/OFF
cycle could be efficiently repeated several times with the same result. The described light-
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dependent effects were not observed when both azobenzenes were assayed in dark conditions,
where the ligand activity remained stable throughout the course of the experiment (Figure S12).
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Figure 4. Real-time optical control of β -AR. (A) Time course quantification of intracellular cAMP
2
challenged with the β -AR agonist cimaterol in the presence of PZL-1 (orange dots) and PZL-2
2
(blue dots). Purple and green boxes correspond to 10 min illumination breaks using 380 nm and
550 nm lights, respectively. (B) Receptor activity values measured for the different light
conditions. Data are shown as the mean ± SEM of three to four independent experiments.
These experiments demonstrate the potential and versatility of the developed
photopharmacological tools to control the activity of β -AR, not only in space but also in time.
2
Indeed, one of the main differences between reversible photoswitches and other drug delivery
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methods is that the activity of reversible ligands can be pulsed with several ON/OFF cycles.
Therefore, the use of molecular photoswitches to reversibly control specific receptors may be
used to resemble the dynamic control exerted in physiological regulatory processes. This
particularity can also be extremely useful to uncover the role of a particular protein in a biological
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35
process or to adapt a therapeutic treatment to a right time window and rhythm. Interestingly,
the adaptation of a FRET-biosensor to a photopharmacology assay has proven very useful to
measure the functional activity of photoswitchable compounds in an end point mode, but it has
also permitted to monitor the evolution of the ligand and light effects in the temporal dimension
in a very simple manner. This has provided additional information on the interactions established
between the ligand in both isomeric forms and the receptor. Moreover, this novel approach in
photopharmacology has also enabled an assessment on the dynamic control of receptor activity
through light cycles, which is essential for further research applications with PZL-1 and -2.
Binding modes of active photoisomers.
To gain insight in the molecular interactions leading to the light-dependent effects of
photoswitchable β -AR antagonists we performed a computational study. We examined the
2
binding mode of the different photoisomerizable molecules presented in this work using the
36
crystal structure of the human β -AR in complex with carazolol (PDB code: 2RH1). This
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structure was chosen for the following reasons: first, it corresponds to the human β -AR, the
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same that was used in pharmacological assays; second, carazolol was used in the rational drug
design of the azobenzene-based molecules investigated; and third, this crystal structure
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displays the highest resolution among all available β -AR structures.
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Both, conventional rigid docking and induced fit protocols were used to introduce the
small molecule in the pocket. Due to the planar geometry of the tricyclic carbazole moiety of
carazolol, it was required the introduction of some flexibility in the receptor to allocate larger
structures, such as the cis azobenzene. These procedures were previously validated by re-
introducing carazolol in the empty pocket of the receptor with very similar binding mode in
comparison to the crystal structure (Figure 5A and S13D). The calculations performed with PZL-
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did not retrieve any positive result neither by rigid docking nor by the induced fit protocol, which
is in accordance with the lack or very low activity measured for this compound in
pharmacological assays (Figure S6). For PZL-1 and PZL-2, several poses were obtained for
both cis and trans isomers.
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Journal of Medicinal Chemistry
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Figure 5. Binding mode of the active isomers of PZL-1 and PZL-2 in the crystal structure of
human β -AR in complex with carazolol (PDB code: 2RH1). (A) Rigid docking of carazolol in the
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empty receptor serves as a validation of the procedure. (B) Binding mode of trans-PZL-1 within
the orthosteric binding site of β -AR determined by rigid docking. (C) Binding mode of cis-PZL-
2
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within the orthosteric binding site of β -AR determined by induced fit. Two amino acid positions
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(
Asp1133.32 and Asn3127.39) are highlighted due to their importance in the binding of β -AR
2
antagonists through a network of hydrogen bonds (represented by green lines).
One of the most important molecular determinants for the affinity and activity of β -AR
2
competitive antagonists is the formation of a hydrogen bond network with the amino acids
Asp1133.32 and Asn3127.39 (Figure 5). In our calculations, these interactions are conserved for
all active photoisomers except for trans-PZL-2, which lacks one hydrogen bond with Asn3127.39
(Figure S13). This may suggest that during the dynamics of the receptor the mentioned
hydrogen bond is not as stable as when the molecule is in its cis form, in which this interaction
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is consistently found. This molecular feature may account for the light-triggered activity increase
observed in pharmacological assays. Regarding PZL-1, both trans-PZL-1 and cis-PZL-1
configurations are forming the hydrogen bond network (Figure 5B and S13E). However, in trans-
PZL-1 these interactions are completely aligned with those found in the crystal structure for
carazolol, whereas cis-PZL-1 requires repositioning of several amino acids and the central part
of the ligand. Interestingly, the cis-PZL-1 interaction is found to be very similar to that of the cis-
PZL-2 (Figure 5C), which is around 100-fold less potent than that of trans-PZL-1. These data
suggest that both, cis-PZL-1 and cis-PZL-2, may have very similar binding modes and thereafter
present similar functional activities. Another remarkable difference found for the cis- binding of
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both molecules is the parallel offset aromatic interaction of the azobenzene ring with F2906.52
.
This particular interaction is found to be T-shaped for carazolol, trans-PZL-1 and the majority of
β2-AR antagonists crystallized so far. Moreover, an additional hydrogen bond is formed between
the two cis molecules and the hydroxyl group of Y3087.35 residue in the upper part of the pocket,
which may compensate the loss of affinity in other regions (Figure 5C and S13E). Altogether,
these data provide a molecular rationale aligned with the measured activities for the novel β2-
AR photoantagonists. Indeed, the nanomolar activity of the trans-PZL-1 would suggest a very
similar interaction pattern with the receptor compared to the co-crystalized carazolol. On the
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Journal of Medicinal Chemistry
other hand, the cis isomers of both active azobenzenes interact with a noticeably different
binding mode which decreases their activity but also provides an original new mode of
interaction with the receptor. The novel binding mode comprises valuable knowledge that will
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facilitate the future design of new cis-on β -AR photoswitches. Further studies will be necessary
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to shed light over these novel interactions and to apply the highlighted concepts to the
development of improved cis-on compounds.
CONCLUSIONS
The first proof of concept for a reversible β -AR photopharmacological approach is
2
presented in this work. Following the azologization strategy, the hydrophobic moiety present in
the majority of the β -AR antagonists was substituted for a p-acetamido azobenzene which led
2
to the development of two β -AR potent antagonists named Photoazolol-1 and -2. Interestingly,
2
PZL-1 and -2, which are structural isomers, showed opposing light-regulated pharmacological
properties. Trans-PZL-1 was found to be the most active isomer whereas for PZL-2, higher
antagonism was observed on its cis form. Of note, we demonstrated that a dynamic control of
the receptor activation with light is possible for both molecules. Additionally, a molecular
rationale is provided to further explore the development of molecules with new
photopharmacological and photochemical properties, such as red-shifted photoswitches that
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can be controlled with deep-penetrating wavelengths. Therefore, we have developed two
chemical probes with complementary and reversible photochromic behavior, which opens the
door to a broad range of future research applications. Further studies will be necessary to
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evaluate the selectivity of these and other photoswitchable β -AR antagonists for possible
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therapeutic uses and in vivo experiments. This confirmation would open the door to the study of
specific roles of β -AR in pathophysiological processes, such as those involved in anxiety.
2
Nonetheless, future research to shed light on the molecular mechanisms of β -AR signaling
2
using fluorescence microscopy in combination with receptor photoswitching are assured.
Overall, these potent tools enable the control of a prototypical class A GPCR enlarging the
photopharmacological toolbox of compounds targeting the GPCR large family of proteins, which
includes a substantial number of therapeutic targets.
EXPERIMENTAL SECTION
Synthetic General Methods. All starting materials were obtained from commercial sources and
used without further purification unless otherwise stated. Anhydrous solvents were obtained
TM
from a solvent purification system (PureSolv-EN ) and kept under a nitrogen atmosphere.
Butanone was dried over activated molecular sieves prior to use. Reactions were monitored by
TLC on silica gel (60F, 0.2 mm, ALUGRAM Sil G/UV254 Macherey-Nagel) and visualized with
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254 nm UV light. Flash column chromatography was carried using silica-gel 60 (Panreac, 40-63
1
2
3
4
µm mesh) or by means of a Biotage Isolera One automated system with Biotage SNAP columns.
Reactions under microwave irradiation were carried out in a CEM Discover Focused™
Microwave reactor. Reactions were performed in 10 mL sealed glass vessels. Analytical HPLC
was performed on a Thermo Ultimate 3000SD (Thermo Scientific Dionex) coupled to a PDA
detector and Mass Spectrometer LTQ XL ESI-ion trap (Thermo Scientific) or on a Waters 2795
Alliance coupled to a DAD detector (Agilent 1100) and an ESI Quattro Micro MS detector
(Waters); HPLC columns used were ZORBAX Eclipse Plus C18 (4.6 x 150 mm; 3.5μm) and
ZORBAX Extend-C18 (2.1 x 50 mm, 3.5 µm) respectively. HPLC purity was determined using
the following binary solvent system: 5% ACN in 0.05% formic acid for 0.5 minutes, from 5 to
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100% ACN in 5 minutes, 100% ACN for 1.5 minutes, from 100 to 5% ACN in 2 minutes and 5%
ACN for 2 minutes. The flow rate was 0.5 mL/min, column temperature was fixed to 35 ºC and
wavelengths from 210-600 nm were registered. The purity of all compounds was determined to
be > 95%. NMR spectroscopy was performed using a Varian-Mercury 400 MHz spectrometer.
Chemical shifts are reported in δ (ppm) relative to an internal standard (non-deuterated solvent
signal). The following abbreviations have been used to designate multiplicities: s, singlet; d,
doublet; t, triplet; m, multiplet; br, broad signal; dd, doublet of doublet; dt, doublet of triplet; ddd,
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