most effective. The effects of BETP and BMTP were likely to be
GLP-1R mediated, as they were almost absent in CHO cells
without GLP-1R overexpression (Figure S3a) and, like
GLP-1(7–36)NH -induced responses, could be reduced by in-
2
hibiting G-protein signaling intermediates including Epac2
(
ESI09) and phospholipase C (U73122) (Figure S3b–e). Surpris-
2
+
ingly, BETP was the only PAM that enhanced the Ca re-
sponse to GLP-1(7–36)NH when pre-incubated, as previously
2
[2a]
described. This may reflect depletion of accessible intracellu-
2
+
lar Ca stores at a rate dependent on the intrinsic activity of
the PAM (Figure S4).
PAMs increase receptor affinity for extracellular ligand, cou-
[12]
pling to intracellular effectors, or both. We employed a time-
resolved Fçrster resonance energy transfer (TR-FRET) approach
to measure ligand-receptor binding in real-time, whereby FITC-
conjugated orthosteric ligand binding to SNAP-GLP-1R self-
[13]
labeled with Lumi4-Tb leads to an increase in FRET. For fur-
ther details of this technique, see Section 6.6 in the Supporting
Information. We determined that GLP-1R PAMs substantially
slow down dissociation of GLP-1(7–36)NH -FITC, increasing res-
2
[
14]
Figure 2. Prearranged PAMs potently enhance GLP-1R signaling. a) Allosteric
enhancement of GLP-1(9–36)NH (“9–36”) cAMP responses (30 min incuba-
tion; n=4) (4-parameter logistic fit shown). b) As for (a), but with exen-
din(9–39) (ex9-39) (n=5). c) Allosteric enhancement of 10 mm GLP-1(9–
idence time (Figure 3a), and also decreasing the rate of asso-
ciation (Figure 3b). The overall effect was to increase binding
2
affinity of GLP-1(7–36)NH -FITC approximately fivefold (Fig-
2
ure 3c). trans-SETP and trans-SMTP increased the residence
time and binding affinity more than their cis-counterparts.
The GLP-1R undergoes extensive internalization after agonist
stimulation, and ongoing cAMP generation by internalized re-
3
2
6)NH -induced b-arrestin2 (barr2) recruitment (30 min incubation; n=5).
d) cAMP responses to indicated PAM or prearranged PAM concentration in
the absence of orthosteric ligand (30 min incubation; n=3). e) Cytosolic
2
+
Ca responses in Calcium 6 dye-loaded cells, expressed relative to baseline
fluorescent signal (60 min incubation; n=7). f) Area under curve (AUC) de-
termined from (e). *P<0.05, **P<0.01, ***P<0.001; one- or two-way
randomized block ANOVA followed by either Sidak’s or Tukey’s post-hoc
test. Values represent the mean + or Æ SEM. Except where indicated, PAMs
or prearranged PAMs were applied at 10 mm.
[16]
ceptors may play a role in GLP-1R signaling. We hypothe-
sized that the PAMs described here, which unlike peptide li-
gands are membrane-permeating, might access and modulate
the behavior of pre-internalized ligand–receptor complexes
(
Figure 3d). After complete GLP-1R internalization with 100 nm
The GLP-1R signals primarily through the generation of
GLP-1(7–36)NH -FITC (Figure 3e) and washout of remaining ex-
2
[
11]
cyclic adenosine monophosphate (cAMP). As expected, BETP
potently augmented the cAMP responses to the GLP-1 break-
tracellular ligand, a steady reduction in FRET was detected, in-
dicative of ligand–receptor dissociation within endosomes.
However, when BETP, BMTP, and prearranged PAMs were ap-
plied to the cell immediately post-washout, marked reductions
in dissociation from endosomal receptors were observed (Fig-
ure 3 f,g). This effect was most marked with trans-SMTP.
down product and weak agonist GLP-1(9–36)NH (Figure 2a),
2
but not fully active GLP-1(7–36)NH (Figure S1 in the Support-
2
ing Information). BETP also conferred agonist activity on the
GLP-1R orthosteric antagonist exendin(9–39) (Figure 2b). Al-
though cis-SETP allosterically enhanced cAMP responses, it
was outperformed by trans-SETP, which displayed a threefold
increase in potency as a GLP-1R PAM (as indicated by EC50
values in Table S1). Similar results were seen with BMTP and
SMTP, suggesting that the replacement of ethyl sulfoxide with
its methyl counterpart is well tolerated. Potentiation of GLP-
In the present study, we describe a photopharmacology-in-
spired strategy for the rational design of potent GLP-1R PAMs
[3]
based upon the allosteric photoswitch PhotoETP. By restrict-
ing degrees of freedom using stilbenes, it could be shown that
compounds prearranged as their trans-isomer were more
[6]
potent than other PAMs, including BETP. Such findings likely
reflect the requirement for bound PAMs to undergo fine con-
formational changes at the allosteric site for full activation in
1
(9–36)NH -induced b-arrestin2 recruitment to the GLP-1R was
2
also greatest with trans-isoforms (Figure 2c).
[6b]
PAMs stabilize active receptor conformations and can
behave as agonists, even in the absence of the orthosteric
ligand. When BETP, BMTP, and related prearranged PAMs were
applied in pure agonist mode, cAMP responses were greatest
with trans-diastereomers (Figure 2d). There was, however, no
detectable increase in b-arrestin2 recruitment with any PAM
response to orthosteric binding.
Prearrangement may cir-
cumvent this by stabilizing molecule dynamics, allowing PAMs
to adopt an allosteric ’ON’ state when unbound. Moreover, re-
placement of the ethyl sulfoxide moiety with a methyl sulfox-
ide maintained full activity, whilst promoting cheaper and
faster access to PAMs and prearranged PAMs from commercial-
ly available chemicals in high yields. Although the differences
in signaling described here for the prearranged PAMs may
seem small, it should be noted that GLP-1R PAMs allow less
(
Figure S2). We also investigated the ability of these com-
2
+
pounds to liberate Ca from internal stores. In the absence of
2
+
GLP-1, BMTP induced strong cytosolic Ca rises, more so than
BETP (Figure 2e,f). SETP and SMTP trans-isomers were again
[2b]
active GLP-1 metabolites to signal at the orthosteric site,
ChemistryOpen 2017, 00, 0 – 0
3
ꢀ 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ
Jones, Ben J.
