A red-shifted photochromic sulfonylurea for the remote control of pancreatic beta cell function

Article abstract of DOI:10.1039/c5cc01224d

Azobenzene photoresponsive elements can be installed on sulfonylureas, yielding optical control over pancreatic beta cell function and insulin release. An obstacle to such photopharmacological approaches remains the use of ultraviolet-blue illumination. Herein, we synthesize and test a novel yellow light-activated sulfonylurea based on a heterocyclic azobenzene bearing a push-pull system.

Full text of DOI:10.1039/c5cc01224d

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A red-shifted photochromic sulfonylurea for the
remote control of pancreatic beta cell function†
Cite this: Chem. Commun., 2015,
1, 6018
5
a
a
b
b
a
J. Broichhagen, J. A. Frank, N. R. Johnston, R. K. Mitchell, K. S˘ mid,
c
c
b
a
b
Received 9th February 2015,
Accepted 21st February 2015
P. Marchetti, M. Bugliani, G. A. Rutter, D. Trauner* and D. J. Hodson*
DOI: 10.1039/c5cc01224d
www.rsc.org/chemcomm
Azobenzene photoresponsive elements can be installed on sulfonyl-
Spurred on by recent studies of ortho- or para-substituted
9
–11
ureas, yielding optical control over pancreatic beta cell function and azobenzenes,
we therefore devised a novel approach for
insulin release. An obstacle to such photopharmacological approaches the synthesis of wavelength-tuned photopharmaceuticals with
remains the use of ultraviolet-blue illumination. Herein, we synthesize red-shifted photochromism. An AzoSulfonylurea based on
and test a novel yellow light-activated sulfonylurea based on a hetero- glimepiride was achieved by installing a heterocyclic aromatic
cyclic azobenzene bearing a push–pull system.
unit, rather than sterically bulky electron-donating halogen or
amine moieties (Scheme 1).
Type 2 diabetes (T2D) is a modern pandemic currently affecting
Starting with the deacetylation of acetazolamide (1) in
B6% of the global population. This disease is characterized by refluxing HCl, heterocycle 2 was obtained that could be further
diminished insulin secretion from pancreatic beta cells, which diazotized with in situ generated HNO . Trapping the resulting
2
together with peripheral resistance to the secreted hormone, diazonium salt with N,N-diethylaniline generated sulfonamide
1
leads to defective glucose homeostasis. The resulting elevated azobenzene 3. Finally, reaction with cyclohexyl isocyanate
glucose concentration drives a variety of complications including yielded JB558 via acylation of the sulfonamide, giving unprece-
heart disease, cancer, retinal degeneration, and nerve and vascular dented access to a sulfonylurea containing a heterocyclic
2
problems.
azobenzene. While yields were reduced compared to the previously
6
While current medical treatments work well, they are associated described JB253 (37% versus 97%), this was most likely due to the
with complications largely due to off-target or persistent actions.
3
Moreover, they are unable to recreate pulsatile insulin release,
4
a more effective signal for glucoregulation. Thus, T2D is ideally
suited to photopharmacology, which harnesses the precision of light
5
to spatiotemporally deliver drug activity. We have recently shown
that a sulfonylurea possessing an azobenzene photoresponsive
element (a.k.a. AzoSulfonylurea) can be used to optically control
beta cell function and insulin release via its effects on ATP-sensitive
potassium (KATP) channels and Exchange Protein directly Activated
6
by cAMP 2A (Epac2A) signaling.
However, a significant barrier to the use of such ‘azo-drugs’
for T2D treatment is their ultraviolet-blue absorption spectra,
increasing phototoxicity and limiting tissue penetration due
7
to photon scattering. By contrast, visible/near infrared wave-
8
lengths demonstrate better penetrance in the body.
a
Department of Chemistry and Center for Integrated Protein Science, LMU Munich,
Munich, Germany. E-mail: dirk.trauner@lmu.de
Scheme 1 (a) Structures of glimepiride (Glim) and the original blue light-
responsive AzoSulfonylurea JB253 for comparison. (b) The logic of a red-shifted
AzoSulfonylurea. Following illumination with green–yellow light (1), the Azo-
Sulfonylurea binds Epac2A, closing KATP channels (2) and opening voltage-
b
c
Section of Cell Biology and Functional Genomics, Department of Medicine,
Imperial College London, London W12 0NN, UK. E-mail: d.hodson@imperial.ac.uk
Department of Clinical and Experimental Medicine, Islet Cell Laboratory,
University of Pisa, Pisa, Italy
2+
2+
v
dependent Ca channels (Ca ) (3). This allows optical control of Ca influx (4)
and insulin secretion (5). (c) Synthesis of the AzoSulfonylurea JB558 that can be
switched from the trans- to the cis-isomer using green/yellow light.
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/c5cc01224d
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presence of a less reactive sulfonamide intermediate, as predicted by trans-JB558, presumably due to the momentary stationary state
the lower pK value for 3 (7.36, Fig. S1, ESI†) and JB558 (2.35, see ESI†). favoring some continued cis-isomerization (Fig. S3, ESI†).
JB558 possessed a red-shifted absorption spectrum (lmax We next assessed the photoswitching properties of JB558 in
26 nm) in DMSO (Fig. 1a), and could be repeatedly photocon- native beta cells where sulfonylurea-mediated KATP channel–
a
=
5
2
+
verted to its cis-state with green–yellow light (l = 520 nm) (Fig. 1b). Epac2A signaling is intimately linked to voltage-dependent Ca
Thermal back relaxation occurred rapidly in the dark and switching channel (VDCC) activity and insulin exocytosis.
1
3–16
As expected,
2
+
kinetics were within the millisecond range (tcis = 64.9 Æ 1.5 ms; JB558 was able to evoke large increases in intracellular Ca
t
trans = 410.8 Æ 12.6 ms), without obvious decomposition (Fig. 1b). concentrations in B60% of beta cells following exposure to yellow
JB558 was stable in the presence of Escherichia coli azoreductase, an (l = 561 Æ 5 nm) (Fig. 3a and b), but not violet (l = 405 Æ 5 nm)
enzyme expected to limit oral bioavailability through diazene light (Fig. 3c). These effects were potentiated using a high concen-
cleavage in the intestine (Fig. S2, ESI†).
tration of glimepiride (Fig. 3d), and abrogated using diazoxide
To determine the binding affinity of JB558 to the KATP channel (Fig. 3e) to force open the K channel pore. Repeated switching of
ATP
2+
subunit SUR1, as well as Epac2A, [3H]-glibenclamide displacement cytosolic Ca concentrations could be achieved in the same islet
and FRET assays were performed. While trans-JB558 bound SUR1 following a brief period of dark exposure to induce trans-JB558
with B10 000-fold less affinity than glimepiride (IC50(trans-JB558) accumulation (Fig. 3f).
=
37.3 mM; IC50(Glim) = 1.8 nM) (Fig. 2a), it was able to strongly
and light-dependently activate an Epac2-camps biosensor containing
1
2
the sulfonylurea binding domain (Fig. 2b–d).
+
Electrophysiological recordings of K currents in HEK293T-
SUR1-Kir6.2 cells revealed partial KATP channel blockade by
Fig. 1 (a) UV-Vis spectra of JB558 in DMSO following illumination with
l = 520 nm (green) or under dark-adapted conditions (black). (b) Robust
photoswitching between cis- and trans-JB558 induced with l = 520 nm and
dark, respectively.
2+
Fig. 3 (a) JB558 increases intracellular Ca concentrations in 59% of
beta cells residing within rodent islets of Langerhans following illumination
with l = 561 nm (scale bar = 75 mm) (n = 8 islets). (b) Photoswitching is
rapid following exposure to l = 561 nm. (c) As for (b), but showing the
absence of photoswitching with l = 405 nm. (d) A high concentration of
2+
glimepiride (Glim) augments JB558-stimulated Ca rises. (e) Diazoxide
2
+
Fig. 2 (a) trans-JB558 and glimepiride (Glim) displace [3H]-glibenclamide
from SUR1 (n = 3 repeats). (b) Glimepiride decreases FRET (shown here as
an increase in R/Rmin) in HEK293T cells expressing full length Epac2-camps
(Dz) reverses cis-JB558-induced Ca fluxes. (f) Reversible manipulation
2+
of Ca transients can be achieved in the same islet following thermal back
relaxation of JB558 in the dark (5 min between stimulation 1 and 2). Traces
represent n = 6–10 recordings from 3 animals. Islets were maintained in
5 mM D-glucose throughout.
(n = 32 cells). (c) As for (b) but cis-JB558 (l = 561 nm) (n = 41 cells). (d) As
for (c) but trans-JB558 (dark) (n = 37 cells). Values represent mean Æ s.e.m.
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Similar to the results observed in rodent tissue, cis-JB588
Communication
2
+
was able to confer light-sensitivity on Ca -spiking activity in
human pancreatic islets (Fig. 4a–c), and this effect could be
reversed following 5 min relaxation in the dark (Fig. 4d).
2+
To link photocontrol of Ca levels with insulin secretion, batches
of rodent islets were incubated with JB558 and exposed to either
dark (no illumination) or light (l = 560 Æ 10 nm). JB558-treated islets
kept under dark conditions were no different to controls (5 mM
glucose-alone) (Fig. 5a), suggesting that the observed stationary state
Fig. 5 (a) JB558-treated islets respond to illumination with l = 560 nm by
increasing insulin secretion (**P o 0.01 and NS, non-significant versus
Con; one-way ANOVA). (b) Incubation with JB558 for 1 h did not adversely
affect cell viability versus dimethyl sulfoxide (DMSO), as assessed by
KATP channel block was insufficient to elicit exocytosis. By contrast,
irradiation dramatically stimulated insulin release (Fig. 5a). Finally,
cytotoxicity assays demonstrated that JB558 did not adversely affect
cell viability, as assessed using the vital stain calcein and the necrosis the ratio of propidium iodide (dead) : calcein (live) fluorescence (positive
indicator propidium iodide (Fig. 5b and c).
control; Triton X-100) (NS, non-significant versus Con; one-way ANOVA).
c) Representative images of islets stained with calcein and propidium
(
The data presented here outline a synthetic route for the
production of AzoSulfonylureas with red-shifted photochromism.
Consistent with its sulfonylurea backbone, JB558 was able to bind
SUR1 and activate Epac2A. Formation of cis-JB558 occurred with
green–yellow light (l = 520–561 nm), and thermal back relaxation in
iodide. In all cases, n = 36 islets per treatment group from 6 animals. Values
represent mean Æ s.e.m.
A more plausible explanation is the inactivation of beta cell
the dark yielded trans-JB558. While photoconversion between Epac2A-signaling, which may lag behind that of JB558 due to
2
+ 1,17
cis- and trans-forms was rapid in solution, it was slower in the persistent mobilization of intracellular Ca .
Such tissue
tissue setting, taking minutes for reisomerization. An effect of Fluo-2 effects may be desirable for the development of photophar-
excitation on the isomer equilibrium cannot be excluded, although maceuticals, since pulsed illumination would reduce photo-
2+
illumination at l = 491 nm per se was unable to evoke Ca rises toxicity, while sustaining compound activity to match long-lasting
4
in either Fluo-2- or Fura2 (l = 340 nm/380 nm)-loaded islets (dozens of minutes) insulin peaks. Indeed, JB558 displayed almost
(Fig. S4, ESI†).
3-fold more potency than its blue-light activated predecessor
JB253, most likely due to slower back-relaxation during the light
6
pulses used in the secretion assays.
+
Neither were we able to detect photoswitching of K currents in
HEK293T cells overexpressing KATP channels, free from orthogonal
wavelengths (Fig. S5, ESI†). This was likely because HEK293T cells
do not express sufficient Epac2A to allow JB558 to properly toggle
13,15,18
KATP activity,
and/or the inability to deliver sufficient illumina-
tion using the non-coherent source on our patch-clamp setup
5
À1
À1
(
e520nm(JB558) = 1.14 Â 10 mol cm ; see ESI†).
Nonetheless, we clearly show that JB558 light-dependently
binds Epac2A, allowing optical control of cell function and insulin
secretion with l = 560 nm in the most physiologically-relevant
testbed, viz. the islets of Langerhans. Thus, JB558 represents a
blueprint for red-shifted AzoSulfonylureas based upon heterocyclic
azobenzenes. Further studies are now warranted to improve
isomerization kinetics in tissue to improve the use of JB558 as a
research tool for rapid KATP channel manipulation. Importantly,
similar synthetic approaches may also be applicable to other
clinically-relevant azobenzene-possessing compounds where steric
19
hindrance may affect molecule motion e.g. neuromodulators,
20,21
22
23
neurotransmitters,
enzymes and antibiotics.
J.B. was supported by a European Foundation for the Study
of Diabetes (EFSD) Albert Renold Young Scientist Fellowship
and a Studienstiftung des deutschen Volkes PhD studentship.
2+
Fig. 4 (a) JB558 increases intracellular Ca concentrations in human
beta cells in response to illumination with 561 nm to induce cis-formation N.R.J. was supported by a Diabetes UK RW and JM Collins
(
5
scale bar = 50 mm). (b) Photoswitching is rapid following exposure to
61 nm and can be potentiated with glimepiride (Glim). (c) cis-JB588
activates 54% of beta cells (n = 4 islets). (d) Reversible manipulation of
Studentship (12/0004601). J.A.F. was supported by a Collaborative
Research Centre Grant (SFB1032). G.A.R. was supported by Well-
come Trust Senior Investigator (WT098424AIA), MRC Programme
2+
Ca
5 min between stimulation 1 and 2). Traces represent n = 3–9 recordings
from a single donor. Islets were maintained in 5 mM D-glucose throughout. Society Wolfson Research Merit Awards. D.T. was supported by an
rises following thermal back relaxation of JB558 in the dark
(MR/J0003042/1), Diabetes UK Project Grant (11/0004210) and Royal
(
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D.J.H. was supported by a Diabetes UK R.D. Lawrence Research
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