Metabolically stable apelin-analogues, incorporating cyclohexylalanine and homoarginine, as potent apelin receptor activators

Article abstract of DOI:10.1039/d1md00120e

High blood pressure and consequential cardiovascular diseases are among the top causes of death worldwide. The apelinergic (APJ) system has emerged as a promising target for the treatment of cardiovascular issues, especially prevention of ischemia reperfusion (IR) injury after a heart attack or stroke. However, rapid degradation of the endogenous apelin peptides in vivo limits their use as therapeutic agents. Here, we study the effects of simple homologue substitutions, i.e. incorporation of non-canonical amino acids l-cyclohexylalanine (l-Cha) and l-homoarginine (l-hArg), on the proteolytic stability of pyr-1-apelin-13 and apelin-17 analogues. The modified 13-mers display up to 40 times longer plasma half-life than native apelin-13 and in preliminary in vivo assay show moderate blood pressure-lowering effects. The corresponding apelin-17 analogues show pronounced blood pressure-lowering effects and up to a 340-fold increase in plasma half-life compared to the native apelin-17 isoforms, suggesting their potential use in the design of metabolically stable apelin analogues to prevent IR injury. This journal is

Full text of DOI:10.1039/d1md00120e

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RESEARCH ARTICLE
Metabolically stable apelin-analogues,
incorporating cyclohexylalanine and
homoarginine, as potent apelin receptor
activators†
Cite this: RSC Med. Chem., 2021, 12,
1402
Kleinberg X. Fernandez, a Conrad Fischer, ‡a Jennie Vu,b Mahmoud Gheblawi,bc
Wang Wang,bd Samantha Gottschalk, a Xavier Iturrioz,§efg
a
Catherine Llorens-Cortés,efg Gavin Y. Ouditbc and John C. Vederas
*
High blood pressure and consequential cardiovascular diseases are among the top causes of death
worldwide. The apelinergic (APJ) system has emerged as a promising target for the treatment of
cardiovascular issues, especially prevention of ischemia reperfusion (IR) injury after a heart attack or stroke.
However, rapid degradation of the endogenous apelin peptides in vivo limits their use as therapeutic
agents. Here, we study the effects of simple homologue substitutions, i.e. incorporation of non-canonical
amino acids L-cyclohexylalanine (L-Cha) and L-homoarginine (L-hArg), on the proteolytic stability of pyr-1-
apelin-13 and apelin-17 analogues. The modified 13-mers display up to 40 times longer plasma half-life
than native apelin-13 and in preliminary in vivo assay show moderate blood pressure-lowering effects. The
corresponding apelin-17 analogues show pronounced blood pressure-lowering effects and up to a 340-
fold increase in plasma half-life compared to the native apelin-17 isoforms, suggesting their potential use
in the design of metabolically stable apelin analogues to prevent IR injury.
Received 6th April 2021,
Accepted 21st June 2021
DOI: 10.1039/d1md00120e
rsc.li/medchem
elabela.3–6 Apelin peptides have been shown physiologically
as influential regulators of various metabolic functions,
Introduction
Cardiovascular diseases are one of the leading causes of
mortality in the world, accounting for 31% of deaths,
globally.1,2 However, the human body is equipped with the
apelinergic system, an endogenous cardioprotective hormone
system which depends on the G-protein coupled apelin
receptor (APJR) and the natural substrates, apelin and
including cardiovascular output,7 fluid homeostasis,8 and
carbohydrate and fat metabolism.9 In addition, various types
of cancers, such as gastroesophageal, glioblastoma, prostate,
colon, and oral squamous cell carcinoma, show elevated
expression of apelin. Apelin peptides in cancer diseases play a
role in tumor neo-angiogenesis through induction of cell
proliferation and migration, and the high apelin expression
suggests its use as a biomarker in cancerous tissues.10
Recently, researchers have also found that the apelin system
is involved in age-associated sarcopenia, where apelin levels
were negatively correlated with age in rodents and humans.11
This endogenous hormone is initially expressed as a 77-
amino acid long prepropeptide, which is further processed
into active apelin-55, apelin-36, apelin-17 and apelin-13/pyr-1-
apelin-13 isoforms.12,13 However, in circulation, the biological
half-life of the shorter isoforms is usually quite limited (<5
min) due to the activity of various proteases (Scheme 1).14
Previously, we have studied the rapid degradation of apelin
by angiotensin-converting enzyme 2 (ACE2) at the C-terminal
phenylalanine residue,15 neprilysin (neutral endopeptidase
24.11, NEP) at the “RPRL” region,12,16 and plasma kallikrein
(KLKB1).17 The latter protease cleaves apelin-17 between
Arg14/Arg15 (Scheme 1). Various strategies have been tested
a Department of Chemistry, University of Alberta, 11227 Saskatchewan Drive NW,
Edmonton, Alberta, T6G 2G2, Canada. E-mail: john.vederas@ualberta.ca
b Department of Physiology, University of Alberta, 8440-112 Street NW, Edmonton,
Alberta T6G 2B7, Canada
c Mazankowski Alberta Heart Institute, University of Alberta, 8440-112 St. NW,
Edmonton, Alberta, T6G 2B7, Canada
d Department of Medicine, University of Alberta, 8440-112 Street NW, Edmonton,
Alberta T6G 2B7, Canada
e Laboratory of Central Neuropeptides in the Regulation of Body Fluid Homeostasis
and Cardiovascular Functions, INSERM, 1050, Paris, F-75005, France
f Center for Interdisciplinary Research in Biology (CIRB), College de France, Paris,
F-75005, France
g CNRS, UMR 7241, Paris, F-75005, France
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1md00120e
‡ Present address: Department of Physical Sciences, Barry University, 11300 NE
2nd Ave, Miami Shores FL 33161, USA.
§ Present address: Université Paris Saclay, CEA, INRAE, Medicines and
Technologies for Health Department, SIMoS, Gif-sur-Yvette, 9119, France.
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Scheme 1 Native apelin-13 and apelin-17 with different protease cleavage sites (KLKB1: plasma kallikrein, NEP: neprilysin, ACE2: angiotensin-
converting enzyme 2).
to enhance proteolytic stability of apelin-related peptides,
including insertion of non-canonical amino acids,18 D-amino
acids,19,20 and N- and C- methylation.12 Two apelin analogues
incorporating non-canonical amino acids have been reported
to be resistant to cleavage by ACE2, while still remaining
active against ischemia reperfusion injury both in vivo and
ex vivo.21 In addition, internal lactamization has been used
as a modification technique which results in conformational
restriction and improved stability through prevention of
peptidase action.22,23 To improve the pharmaceutical
properties – plasma stability, cardiac inotropy, hypotensive
apelin agonists.32 Combined research efforts led to peptidic
and non-peptidic apelin agonists, which have shown
increased metabolic stability and improved receptor binding
abilities.
A recently published X-ray crystal structure of an inactive
modified APJR in complex with an inactive peptidic derivative
(AMG3054) suggests binding interactions of the receptor with
substrates at a minimum of two sites.18 Two approaches,
amino acid substitution and macrolactamization, were used
in combination to rigidify the apelin analogue and enable
better fit into the APJ-derived protein (Scheme 1). Among
these modifications, L-cyclohexylalanine (L-Cha) and
L-homoarginine (L-hArg) were introduced in positions 9 and
10 from the C-terminus within receptor binding site 2
(termed ‘RPRL’ according to the native amino acid
sequence).18 Despite an apparent good fit, the impact upon
agonistic vs. antagonistic interaction of AMG3054 on the
native receptor was not disclosed in this publication. Our
current study examines the possibility of metabolic
stabilization and receptor activation of such homologue-
substituted apelin peptides by dissecting the individual and
combined binding contribution of these substitutions. Since
these substitutions are within the RPRL binding motif, even
minute spatial changes in this region will likely affect
receptor binding and overall conformation change that
defines the extent of downstream G-protein signaling.
Starting from first generation analogues 1 and 6 (Scheme 2),
already shown to possess higher proteolytic resistance,15 we
synthesized nine derivatives, 3–5 (apelin-13 analogues) and
8–13 (apelin-17 analogues), incorporating methylation and
L-Cha and/or L-hArg in positions 9/10. Using enzyme and
human plasma assays, we determined the metabolic stability
effects, and binding affinity
–
of apelin peptides,
acylation24,25 using fatty acids, PEGylation17,26 and more
recently the addition of
a fluorocarbon chain to the
N-terminus of apelin peptides20 have been used. Respectively
modified apelin analogues have been shown effective in
circulatory
diseases
including
alloimmune-mediated
vasculopathy and abdominal aortic aneurysm27,28 and in
hyponatremia.29 In addition to peptide-based apelin
analogues, non-peptide APJR agonists have also been
synthesized. One of the first reported peptidomimetic small
molecules was E339-3D6, which showed great selectivity
against other GPCRs and reasonable affinity for the APJR.30
Nevertheless, in combination with lower receptor affinity to
native apelin peptides and the molecule having
a
comparatively high molecular weight, E339-3D6 was not
classed as drug-like. However, further studies have identified
a promising small molecule designated CMF-019.31 This
molecule possesses many desirable characteristics, such as
low molecular weight (455 Da), high nanomolar APJR affinity,
and increased cardiac contractility and vasodilation; allowing
for exploration of novel syntheses of new small molecule
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Scheme 2 Apelin derivatives studied in this paper: ACE2-resistant analogues (1, 6), NEP-stabilized compounds (2, 7), and current title apelin
analogues (3–5, 8–13) implementing an ACE2-resistant C-terminus (L-p-bromophenylalanine, aminoisobutyric acid, L-norleucine). Pyroglutamic
acid abbreviated as pGlu.
of these compounds and further characterized their receptor
activation properties through means of Ca2+-mobilization
capacity. In vivo (mouse) blood pressure tests showed
significant and prolonged blood pressure-lowering effects of
the apelin-17 series (compounds 8–13), suggesting that they
can serve as candidates to assist the design of metabolically
stable and physiologically active apelin analogues as
therapeutic agents.
reactions in urea cycles40,41 or through L-arginine:glycine
amidinotransferase (AGAT) catalysis,42 contributing to its role
as a biomarker.
Apelin analogue sets 3–5 and 8–13 were synthesized on
trityl-resin using Fmoc-chemistry. First, Fmoc-p-BrPhe-OH
was loaded onto 2-chlorotrityl chloride resin and extended by
solid phase peptide synthesis until the octapeptide stage,
from the C-terminus, was obtained. Incorporation of the
appropriate Arg/Leu modifications were completed after this
stage, and the 13-mer and 17-mer analogues were extended
to the tridecapeptide and heptadecapeptide stage,
respectively. Since the peptides are also intended to address
stability toward NEP cleavage, we compared their metabolic
and physiological features to the most potent NEP-stabilized
analogues from a previous series (2 and 7).12 All peptides
were analyzed using LC-MS and MALDI-TOF and purified
using an analytical HPLC.
Results and discussion
SPPS synthesis
Starting from the ACE2-stable series (compounds 1 and 6),15
we attempted to achieve higher metabolic stabilization
through relatively conservative homologue substitution.
Inspired by the literature peptide AMG3054,18 it seemed
interesting to determine whether L-hArg and/or L-Cha
substitution would lead to increased proteolytic stability and
improved receptor activation. This approach has the
advantage of affordable and commercially available amino
acids that can be readily incorporated in good yield through
solid phase peptide synthesis (SPPS). Additionally, there is
literature precedent that the use of L-homoarginine and
L-cyclohexylalanine may have intrinsic advantages with
respect to cardiovascular activity of the resulting analogues.
L-Cha has been shown to increase regulatory function in body
fluid and blood pressure homeostasis, and vasodilation
activity when it was substituted for Phe in an atrial
natriuretic peptide.33–35 On the other hand, L-hArg is an
independent protective biomarker,36,37 where lower L-hArg
plasma levels were correlated with renal failure38 and
reduced nitric oxide bioavailability.39 It should also be noted
that L-hArg can be formed metabolically through the
replacement of ornithine for lysine in liver enzyme-catalyzed
In vitro NEP stability
First, the in vitro NEP stability of the Arg/Leu analogues was
compared to the ACE2-resistant first generation analogues 1
and 6 and their NEP-stabilized pendants 2 and 7 (Fig. S1 and
S2,† Table 1). Analogues were incubated with recombinant
human NEP (rhNEP, Sino biological) at 37 °C for up to 24 h,
and the extent of degradation was analyzed via LC-MS due to
the overlapping elution patterns of peptide fragments. The
ACE2-resistant (A2) analogues 1 and 6 showed comparable
NEP degradation rates to native apelin isoforms, as
previously reported.12 When examining the effects of Arg/Leu
modifications, all new analogues (3–5, 8–10) showed
improved proteolytic stability to NEP compared to the A2
analogues (Fig. S1 and S2,† Table 1). Overall, Arg/Leu-
substituted apelin-17 analogues 8–13 showed increased
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Table 1 Metabolic features of reference apelins and homologue-substituted apelins. Experiments were done in triplicates. Errors represent standard
error of the mean (S.E.M.)
Analogue
t1/2 human NEP (h)
t1/2 human plasma (h)
t1/2 mice plasma (h)
Plasma protein binding efficacy (%)
1
2
3
4
5
6
7
8
Apelin13A2
NMeLeu13A2
hArg13A2
Cha13A2
hArgCha13A2
Apelin17A2
NMeLeu17A2
hArg17A2
Cha17A2
hArgCha17A2
NMeCha17A2
hArgNMeCha17A2
hArgNMeLeu17A2
10.31 0.01
40.27 0.01
19.01 0.01
22.07 0.01
21.91 0.02
10.95 0.03
>48
40.59 0.01
47.03 0.01
46.39 0.01
0.81 0.20
3.54 0.12
1.31 0.42
1.77 0.34
2.58 0.19
0.90 0.17
8.29 0.21
3.06 0.04
4.34 0.03
6.28 0.02
2.90 0.03
3.41 0.05
5.63 0.08
57 10
55 10
0.99 0.11
9
10
11
12
13
0.52 0.12
0.44 0.07
0.36 0.25
0.38 0.21
65
68
69
9
8
8
stability compared to pyr-1-apelin-13 analogues 3–5.
Substitutions with L-Cha and L-hArg, for both the pyr-1-
apelin-13 and apelin-17 analogues, showed higher stability in
the isolated NEP assay, but did not show as much resistance
as the NEP-stabilized N-Me-Leu analogues 2 and 7.
bulkiness, had lower ability in stabilizing the peptide against
proteases, compared to modifications on the backbone alone.
The most stable apelin-17 analogue 10 (hArgCha17A2) still
displayed 25% decreased plasma half-life compared to the
NEP-stabilized analogue 7 (Table 1).
In vitro analogue stability – human plasma
In vitro analogue stability – mice plasma
We then examined the in vitro plasma stability of these
analogues (Fig. 1, Table 1). Triplicate experiments were
performed in human blood plasma and the extent of
degradation was analyzed via LC-MS. Arg/Leu substitution
had relatively similar impacts on the different apelin
isoforms. All pyr-1-apelin-13 (Fig. S3†) and apelin-17
analogues (Fig. 1) showed enhanced stability compared to
In addition, the in vitro murine plasma stability of four target
analogues was tested and compared to the NEP-stabilized
analogue 7 (Fig. S4,† Table 1). Triplicate experiments were
performed in murine blood plasma and analyzed via LC-MS
to determine the extent of degradation. Generally, proteolytic
stability is significantly lower due to the higher metabolic
rate and renal clearance in rodents compared to humans.43
Compared to the NEP-stabilized NMeLeu17A2 analogue 7
(∼60 min), all the apelin-17 analogues (10–13) showed lower
rates of proteolytic stability in murine plasma (≤40 min),
with analogue 10 displaying the highest stability of the
apelin-17 isoforms. Nevertheless, the enhanced in vitro
plasma stabilities of these Arg/Leu-modified analogues to
both NEP and non-specific plasma proteolysis, compared to
the ACE2-resistant isoforms, were encouraging for
exploration of the physiological activities of each analogue in
future mouse model studies.
the ACE2-resistant analogues
1 and 6, with apelin-17
analogues 8–13 showing higher stability compared to the pyr-
1-apelin-13 isoforms 3–5. This suggests that NEP proteolysis
appears to be more significant in the degradation of pyr-1-
apelin-13 analogues than the apelin-17 counterparts.
However, compared to the inherently NEP-stabilized N-Me-
Leu analogues (2 and 7), all methylated and homologue-
substituted pyr-1-apelin-13 and apelin-17 analogues (3–5,
8–13) showed lower levels of plasma stability. This suggests
that the modifications of the side-chains, regardless of the
Fig. 1 In vitro human plasma degradation trends for apelin-17 analogues. (A) Comparison of (6) ACE2-resistant, (7) NMeLeu17A2, and (8–10) novel
Arg/Leu substituted analogues. (B) Comparison of (6) ACE2-resistant, (7) NMeLeu17A2, and (11–13) NMeArg/Leu substituted analogues.
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APJR receptor radioligand binding experiments
heart rate (HR) were continuously monitored in the aorta
cannulated via the right carotid artery. The pyr-1-apelin-13
analogues 3–5 displayed marginal cardiovascular effects (Fig.
S6†). However, all apelin-17 analogues 8–13 showed more
pronounced cardio-physiological effects (Fig. 3). In fact,
analogues 10 and 12 showed quick and stable increase in
heart rate and prolonged blood pressure-lowering effects,
respectively, which are even more profound than the most
potent compound from our previous studies, analogue 7 (ref.
12) (Table 2). These results suggests that, in addition to
enhanced binding to the APJR, L-hArg/L-Cha substitution is
also cardio-physiologically well tolerated and beneficial for
the further development of cardiovascular-active apelin
analogues.
Next, the newly synthesized pyr-1-apelin-13 (3–5) and apelin-
17 (8–13) analogues were compared with the ACE2-resistant
(1, 6) and NEP-stabilized (2, 7) to determine their ability to
compete with
[
125I]-pyr-1-apelin-13 (0.2 nM) binding on
membrane preparations from CHO cells stably expressing the
wild-type rat apelin receptor-EGFP. As shown in Table 2, all
pyr-1-apelin-13 (3–5) analogues showed improved competitive
binding, in comparison with the radiolabeled ligand from
the rat apelin receptor EGFP. These analogues showed lower
pKi values compared to the ACE2-resistant ( pKi = 8.70 0.06
nM) and the NEP-stabilized analogues ( pKi = 10.00
0.17
nM). In addition, the apelin-17 (8–13) analogues showed
comparable pKi values at the nanomolar range compared to
the ACE2-resistant ( pKi = 9.72 0.01 nM) and NEP-stabilized
( pKi = 9.35 0.09 nM) isoforms. In conclusion, these data
show that all the pyr-1-apelin-13 and apelin-17 analogues
were orthosteric ligands as they were able to interact with the
rat APJR at the same binding site as the natural ligand.
Plasma protein-binding assay
Lastly, target analogues, 8, 11, and 13, containing amino acid
modifications at the neprilysin proteolytic cleavage site,
generally display a slightly higher plasma protein binding (65
8 to 69
modified ACE2-resistant analogue 6 (57
8%) than the native (apelin-17),44 the non-
Receptor activation-Ca2+-mobilization assay
10%) and the
NMeLeu17A2 analogue 7 (55 10%) (Table 2). Considering
the improved half-life (t1/2) of the apelin-17 analogues (8–13)
in human plasma compared to the ACE2-resistant isoform 6,
it appears that subtle homologue substitution increases
lipophilicity of apelin and proteolytic stability through
plasma protein binding.45 These results correlate well with
the observed improvement in in vitro plasma stability of
these analogues.
In order to study the extent to which newly derived analogues
trigger downstream APJR activation, a fluorescence coupled
calcium release assay was used with a recombinant human
APJ-Ga16 receptor cell line. All new analogues (3–5, 8–13)
showed comparable pEC50 values (Table 2), suggesting a
similarly strong APJR-activation potential comparable to the
ACE2-resistant (1, 6)15 and NEP-stabilized (2, 7)12 analogues
from previous studies. These results were supported by the
binding affinity values of these compounds (Table 2). The
binding curves (Fig. 2 and S5†) also indicate the agonistic
behaviour to the APJR.
Conclusion
In the current study, apelin analogues modified at the NEP
cleavage site (Arg9-Leu10 in apelin-17) were studied for their
metabolic stability and cardiovascular features. Comparisons
of the in vitro NEP and human- and mice- plasma stability
between the novel analogues (3–5, 8–13) and previous ACE2-
resistant (1, 6)15 and NEP-stabilized (2, 7)12 isoforms revealed
that all newly synthesized analogues showed elevated
Physiological test – blood pressure assay
The newly synthesized analogues, 3–5 and 8–13, were tested
for blood pressure-lowering abilities in anesthetized mice.
Apelin peptide analogues were delivered systematically via
the right internal jugular vein, while blood pressure (BP) and
Table 2 Receptor binding and physiological response data of reference apelins and homologue-substituted apelins. Experiments were done in
triplicates. Errors represent standard error of the mean (S.E.M.)
Analogue
pKi binding affinity (nM) pEC50 Ca2+-mobilization (nM) ΔMABP (mmHg) Duration of hypotensive effect (min)
1
2
3
4
5
6
7
8
9
Apelin13A2
NMeLeu13A2
hArg13A2
8.70 0.06
10.00 0.17
9.17 0.07
9.24 0.10
9.43 0.01
9.72 0.01
9.35 0.09
9.08 0.13
8.80 0.13
8.96 0.15
8.70 0.11
9.19 0.08
8.85 0.11
8.37 0.14
8.17 0.12
8.66 0.17
8.62 0.13
8.43 0.12
8.37 0.14
8.56 0.18
8.17 0.12
8.42 0.10
8.36 0.11
7.79 0.42
7.14 0.20
6.56 0.17
23.88 7.60
9.52 5.60
26.44 12.60
0.23 5.30
15.33 7.70
10.24 2.60
31.98 5.90
40.45 13.30
19.72 6.00
44.90 12.80
28.83 8.00
47.57 4.60
34.69 4.90
60a
26
60a
15
Cha13A2
hArgCha13A2
Apelin17A2
NMeLeu17A2
hArg17A2
57
33
60a
60a
60a
60a
60a
60a
60a
Cha17A2
10 hArgCha17A2
11 NMeCha17A2
12 hArgNMeCha17A2
13 hArgNMeLeu17A2
a
Hypotensive effect maintained for the duration of the experiment.
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Fig. 2 Concentration response (fluorescence) curves of novel analogues 3–5 and 8–13 in comparison to the ACE2-resistant (1, 6) and NEP-
stabilized (2, 7) analogues.
metabolic stability. Blood pressure assays indicate stable and
prolonged blood pressure-lowering effects of the apelin-17
analogue series, especially the 17-mer analogues
incorporating both homologue-substitutions (analogues 8
and 12). In addition, the synthesized apelin derivatives
showed comparable or improved receptor binding and
activation based on the results of the Ca2+-mobilization
assays and APJR radioligand binding experiments. Hence,
these results indicate that relatively conservative homologue-
substitution in positions 9/10 contributes to a moderate
metabolic peptide stabilization, yielding cardiovascular-active
apelin analogues as promising targets for further drug
development for cardiovascular diseases.
distilled over calcium hydride. ACS grade solvents (>99.0%
purity) were used for flash column chromatography with no
further purification. All reactions were monitored by thin
layer chromatography (TLC) using aluminum plates
containing a UV fluorescent indicator (normal SiO2, Merck 60
F
254). Analytical, semi-preparative and preparative scale high
performance liquid chromatography (HPLC) was performed
on a Gilson instrument equipped with 322 pump heads, a
model 171 diode array detector, a FC 203B fraction collector,
and a Reodyne 7725i injector fitted with a 1000 μL sample
loop. Deionized water was filtered through a Milli-Q reagent
water filtration system. HPLC grade acetonitrile and
deionized water were filtered through a Millipore filtration
system under vacuum prior to use. Peptides were purified to
≥95% purity assessed by analytical reinjection.
Characterization. A Perspective Biosystems Voyager Elite
MALDI-TOF MS, with a matrix consisting of 4-hydroxy-α-
cyanocinnamic acid (HCCA), Kratos AEIMS-50, or Bruker 9.4
T Apex-Qe FTICR (high resolution, HRMS, MS/MS, Fig. S7–
S15†) were used to record mass spectra (MS). LC-MS was
performed on an Agilent Technologies 6130 LCMS
instrument.
Experimental section
General information
Solvents, reagents, and purification. Commercially-
available biological and chemical reagents were purchased
from: Caledon, Chem-Impex International Inc., Fisher
Scientific Ltd., R&D Systems Sigma-Aldrich Canada, and VWR
International, and used without further purification, unless
otherwise stated. All solvents were of American Chemical
Society (ACS) grade and used without further purification.
Flame-dried glassware, used for anhydrous reactions, were
subjected to a positive pressure of argon (Ar). Prior to use,
anhydrous reaction solvents were distilled: dichloromethane
was distilled over calcium hydride, and methanol was
1. General apelin analog SPPS elongation method.
A
suspension of resin in DMF (5 mL) was bubbled under argon
gas to swell for 20 min. The N-terminal Fmoc group was
taken off by bubbling 20% piperidine in DMF (3 × 5 mL),
then extensively washing with DMF (3 × 5 mL) after each
individual deprotection. The extent of Fmoc-deprotection was
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Fig. 3 In vivo heart rate (HR), mean arterial blood pressure (MABP), systolic blood pressure (SBP) and diastolic blood pressure (DBP) analyses,
following intravenous injection of synthesized novel analogues (8–13) compared to the ACE2-resistant (6) and NEP-stabilized (7) isoforms in
anesthetized mice (n = 3). Values represent mean S.E.M.
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monitored using TLC, where the dibenzofulvene-piperidine
adduct appearing as a dark purple spot under UV. The
coupling protocol consisted of DIPEA (2.2 equiv) being
added to a solution of Fmoc-protected amino acid (1.1 equiv
compared to resin loading), PyBOP (1.0 equiv), and HOBt
(1.1 equiv) in DMF (5 mL) and stirred for 10 min. The resin
was then washed with DMF (3 × 5 mL), and the extent of
coupling was assessed by cleaving a small sample of resin
with a solution of 95 : 2.5 : 2.5 TFA/TIPS/H2O for 1–2 h, and
MALDI-TOF analysis. To end-cap any unreacted amines, a
solution of 20% acetic anhydride in DMF (5 mL) was added
to the resin and bubbled for 10 min under Ar gas.
Thorough washes of the resin with DMG (3 × 5 mL) was
done and the resin was subjected to either Fmoc-
deprotection to continue elongation of the peptide, or
rinsed with CH2Cl2 (3 × 5 mL) and dried thoroughly and
stored at −20 °C under Ar gas.
2. General method for apelin resin cleavage. Resin-bound
apelin analogue (0.05 mmol) was suspended in 95 : 2.5 : 2.5
TFA/TIPS/H2O with shaking under Ar atmosphere for 2–3 h.
The cleaved resin was filtered through glass wool, rinsed
thoroughly with TFA, and the solution was concentrated in
vacuo. Cold diethyl ether (2 × 5 mL) was added to triturate
the crude peptide. The diethyl ether was decanted into a 15
mL Falcon tube and briefly centrifuged to pellet any residual
peptide. The diethyl ether pellet and triturated crude residue
were pooled together and dissolved in 0.1% aqueous TFA.
3. Apelin analogue synthesis. The synthesis and
characterization of the ACE2 resistant (1 and 6) and NEP
resistant (2 and 7) analogues have been previously
described,5 synthetic compounds were prepared according to
literature protocols (ESI†). Through Fmoc-solid phase peptide
synthesis (SPPS), three sets of apelin analogues were
synthesized for pyr-1-apelin-13 (3–5), apelin-17 (8–10) and
apelin-17 NMe (11–13), including L-hAr and/or L-Cha
modifications. To ensure that all Arg/Leu modified apelin
analogues were resistant to ACE2 enzymatic cleavage,
p-bromo-phenylalanine (BrF), 2-aminoisobutyric acid (Aib),
and norleucine (Nle), were previously incorporated in
exchange for the natural C-terminal Phe, Pro and Met
residue, respectively.5 The syntheses of these analogues were
carried out using a 2-chlorotritylchloride resin (loading of 0.8
mmol g−1). Prelude X automated peptide synthesizer (Gyros
protein technologies) was used to synthesize up to Ser, and
subsequent amino acids (1.1 equiv compared to resin
loading, 0.672 mmol) were attached in the following order by
hand: apelin-13: R3, R2, Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH,
and L-pyroglutamic acid (L-pGlu) and apelin-17: R3, R2, Fmoc-
Apelin analogue 3 (hArg13A2). A sample of resin-bound
peptide (0.05 mmol) was cleaved as previously described. The
resulting precipitate was purified using a C18 RP-HPLC
analytical column, eluting at 13.2 min (Fig. S16†). After
lyophilization, the desired peptide was isolated as a white
solid (10 mg, 12%). Monoisotopic MW calculated for C72H115
-
BrN22O16 811.4017, found high resolution (FTICR-ESI-MS)
811.4019 (M + 2H)2+.
Apelin analogue 4 (Cha13A2). A sample of resin-bound
peptide (0.05 mmol) was cleaved as previously described. The
resulting precipitate was purified using a C18 RP-HPLC
analytical column, eluting at 13.3 min (Fig. S16†). After
lyophilization, the desired peptide was isolated as a white
solid (11 mg, 13%). Monoisotopic MW calculated for C70H113
-
BrN22O16 798.3939, found high resolution (FTICR-ESI-MS)
798.3916 (M + 2H)2+.
Apelin analogue 5 (hArgCha13A2). A sample of resin-
bound peptide (0.05 mmol) was cleaved as previously
described. The resulting precipitate was purified using a C18
RP-HPLC analytical column, eluting at 13.1 min (Fig. S16†).
After lyophilization, the desired peptide was isolated as a
white solid (13 mg, 15%). Monoisotopic MW calculated for
C73H117BrN22O16 818.4095, found high resolution (FTICR-ESI-
MS) 818.4074 (M + 2H)2+.
Apelin analogue 8 (hArg17A2). A sample of resin-bound
peptide (0.05 mmol) was cleaved as previously described. The
resulting precipitate was purified using a C18 RP-HPLC
analytical column, eluting at 13.2 min (Fig. S17†). After
lyophilization, the desired peptide was isolated as a white
solid (12 mg, 14%). Monoisotopic MW calculated for C99H164
-
BrN34O20 742.7343, found high resolution (FTICR-ESI-MS)
742.7329 (M + 3H)3+.
Apelin analogue 9 (Cha17A2). A sample of resin-bound
peptide (0.05 mmol) was cleaved as previously described. The
resulting precipitate was purified using a C18 RP-HPLC
analytical column, eluting at 13.1 min (Fig. S17†). After
lyophilization, the desired peptide was isolated as a white
solid (15 mg, 18%). Monoisotopic MW calculated for C97H162
-
BrN34O20 734.0624, found high resolution (FTICR-ESI-MS)
734.0605 (M + 3H)3+.
Apelin analogue 10 (hArgCha17A2). A sample of resin-
bound peptide (0.05 mmol) was cleaved as previously
described. The resulting precipitate was purified using a C18
RP-HPLC analytical column, eluting at 13.8 min (Fig. S17†).
After lyophilization, the desired peptide was isolated as a
white solid (12 mg, 14%). Monoisotopic MW calculated for
C
100H166BrN34O20 747.4062, found high resolution (FTICR-
ESI-MS) 747.4050 (M + 3H)3+
.
Pro-OH,
Fmoc-Arg(Pbf)-OH,
Fmoc-Gln(Trt)-OH,
Fmoc-
Apelin analogue 11 (NMeCha17A2). A sample of resin-
bound peptide (0.05 mmol) was cleaved as previously
described. The resulting precipitate was purified using a C18
RP-HPLC analytical column, eluting at 21.3 min (Fig. S18†).
After lyophilization, the desired peptide was isolated as a
white solid (14 mg, 17%). Monoisotopic MW calculated for
Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Phe-OH, and Fmoc-
Lys(Boc)-H. Where R2 is Fmoc-Arg(Pbf)-OH or Fmoc-
homoArg(Pbf)-OH, and R3 is Fmoc-Leu-OH, Fmoc-Cha-OH,
Fmoc-N-Me-Leu-OH or Fmoc-N-Me-Cha-OH. Completeness of
each coupling step was checked with matrix assisted laser
desorption/ionization (MALDI) coupled with time of flight
mass spectrometry (TOF-MS).
C
100H163BrN34O20 747.4076, found high resolution (FTICR-
ESI-MS) 747.4062 (M + 3H)3+
.
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Apelin analogue 12 (hArgNMeCha 17A2). A sample of
resin-bound peptide (0.05 mmol) was cleaved as previously
described. The resulting precipitate was purified using a C18
RP-HPLC analytical column, eluting at 21.5 min (Fig. S18†).
After lyophilization, the desired peptide was isolated as a
white solid (14 mg, 17%). Monoisotopic MW calculated for
of apelin peptide to internal standard was calculated based
on the area under the curve. The 0 h incubation ratio was
used to compare the apelin peptide/internal standard ratios
for the time experiments.
6. APJR radioligand binding experiments. Membrane
preparations from CHO cells, which stably express the wild-
type rat APJ receptor-EGFP, were prepared as described from
previous literature.47 Crude membrane preparations (1 μg
total membrane mass/assay) were incubated at 20 °C for 3 h
with 0.2 nM [125I]-pyr-1-apelin-13 (monoiodinated on Lys8
with the Bolton–Hunter reagent, PerkinElmer, Wellesley, MA,
USA) in binding buffer (50 mM HEPES, 5 mM MgCl2, pH 7.5,
BSA 1%) alone or in the presence of the different analogues
at various concentrations. The reaction was quenched with
the addition of 4 mL of cold binding buffer, and the product
was filtered on Whatman GF/C filters and washed with 5 mL
of cold binding buffer. Quantification of radioactivity was
C
100H165BrN34O20 564.3025, found high resolution (FTICR-
ESI-MS) 564.3103 (M + 4H)4+
.
Apelin analogue 13 (hArgNMeLeu17A2). A sample of resin-
bound peptide (0.05 mmol) was cleaved as previously
described. The resulting precipitate was purified using a C18
RP-HPLC analytical column, eluting at 21.2 min (Fig. S18†).
After lyophilization, the desired peptide was isolated as a
white solid (16 mg, 19%). Monoisotopic MW calculated for
C98H161BrN34O20 554.3025, found high resolution (FTICR-ESI-
MS) 554.2950 (M + 4H)4+
.
4. In vitro protease experiments, neprilysin degradation
assay. Neprilysin (SinoBiological, Wayne, PA, USA) was
thawed on ice for 10 min. 1 μL of 0.76 mg mL−1 NEP was
diluted with 350 μL Buffer NEP-B to 2 ng μL−1, then the
solution was incubated at 37 °C for 10 min. Six solutions of
each pyr-1-apelin-13 (3–5) and apelin-17 (8–10) analogues
were prepared as 1 mM solution in H2O, then were added to
NEP/Buffer NEP-B solution and incubated at 37 °C. Aliquots
(1 μL) were removed at 0, 2, 10, 20, 48 h and quenched prior
to LC-MS quantification. Assays were performed in triplicate
(n = 3) for each time point.
determined using
a
Wizard 1470 Wallac
γ
counter
(PerkinElmer, Turku, Finland). GraphPad Prism 6 was then
used to analyze the binding experiment data.
7. Ca2+-mobilization assay. A 96-well assay plate of frozen
Chem-5 cells stably expressing the human APJ GPCR (Ready-
to-Assay™ APJ assay, EMDMillipore, Burlington, MA) was
thawed according to the manufacturer's protocol. Cells were
loaded for 1 h with FLIPR Calcium 6-QF fluorescent indicator
dye (Molecular Devices, USA) in assay buffer [Hanks balanced
salt solution (HBSS), 20 mM HEPES, 0.2% DMSO, 2.5 mM
probenecid, pH 7.6], washed three times with assay buffer,
then returned to the incubator for 10 min before the assay
on a fluorometric imaging plate reader (FLIPR; Molecular
Devices, Sunnyvale, CA, USA). Maximum change in
fluorescence over baseline was used to determine agonist
response. Dose–response curve data were fitted to a four-
parameter logistic equation using PRISM (GraphPad, USA)
from which the pEC50 values were calculated.
5. Isolation and quantification of apelin peptides from
plasma. Quantification of peptides in plasma were adapted
from a published protocol.46 20 μL of plasma was pre-
portioned into microcentrifuge tubes and incubated at 37 °C.
5 μL of apelin peptide (1 mM) was added to each 20 μL
plasma vial and then incubated at 37 °C. The plasma
solution was then quenched with 25 μL 6 M GuHCl, 300 μL
80% ACN/H2O and 5 μL of internal standard (1 mM Dans-
YVG peptide). The plasma sample was then vortexed before
centrifuging at 13 200g for 5 min. The supernatant was
transferred to a 1 mL microcentrifuge tube and evaporated.
Samples were reconstituted with 150 μL 0.1% acetic acid (v/v)
in water and loaded onto a pre-equilibrated C18 spin column,
which had previously been wet with 2 × 150 μL 30%
acetonitrile in 0.1% aqueous TFA and 2 × 150 μL 0.1%
aqueous acetic acid (v/v) respectively, centrifuging at 280g for
2 min between each 150 μL aliquot. Quenched plasma assays
were centrifuged at 280g until the sample was loaded. The
resultant filtrate was reloaded onto the column with 150 μL
of 0.1% aqueous acetic acid (v/v) and centrifuged at 280g for
another 2 min, then the filtrate was discarded. The desired
peptides were washed with 2 × 150 μL 5% MeOH in water
with 1% acetic acid (v/v) and eluted from the column using
100 μL 60% MeOH in water with 10% acetic acid (v/v). The
eluate was injected and analyzed using LC-MS quantification.
To analyze the remaining apelin peptides in plasma,
incubations of pyr-1-apelin-13 (1–5) and apelin-17 (6–13)
analogues were quenched at different time points (1, 3, 6, 24
h), worked up and analyzed as previously described. The ratio
8. Blood pressure assays. All animal studies were conducted
according to the Canadian Council for Animal Care
guidelines and approved by the Animal Care and Use
Committee at the University of Alberta. Male wildtype mice
purchased from Jackson Laboratories (Bar Harbor, ME) were
anesthetized with 1.5% isoflurane/oxygen. The body
temperature was maintained at 36 °C by a heating pad and
monitored. A PV loop catheter (model 1.2F from Scisense,
Transonic) was used to cannulate the aorta via the right
carotid artery, in order to continuously record arterial blood
pressure and heart rate (LabScribe 2.0, Scisense). Peptides
1–13 (1.4 μM kg−1 body weight) or the same volume of saline
was injected via the right jugular vein (n = 3). Results are
reported as heart rate (HR), mean arterial blood pressure
(MABP), systolic blood pressure (SBP), and diastolic blood
pressure (DBP) S.E.M. Blood pressure traces were analyzed
with LabScribe2 (iWorx Systems Inc., Dover, NH), and plotted
with Origin 2018 (OriginLab, Northampton, MA).
9. Plasma protein binding assay. This assay was ran
following a published protocol.44,48 Briefly, 1 mM DMSO
stock solutions of each analogue were diluted to a final
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concentration of 100 μM, with protease inhibitor stabilized
(EDTA and MLN-4760) 55% pooled human plasma. 200 μL of
this solution was dialyzed in a 96-well equilibrium dialysis
apparatus against 200 μL of PBS buffer (pH 7.4) at 37 °C and
120 rpm for 3 h. Thereafter, 25 μL of retentate was diluted
with 25 μL of PBS buffer, and 25 μL of dialysate was diluted
with 25 μL of pooled plasma, and cooled on ice for 10 min.
All samples were quenched with 10% TFA in acetonitrile (200
μL), and 1 mM Dans-YVG peptide (5 μL) was added (internal
standard). Samples were vortexed and centrifuged (11 200g)
for 5 min. The supernatant was transferred to another
microcentrifuge tube and evaporated, yielding a residue
which was then reconstituted in 100 μL of 0.1% aqueous
TFA. C18 spin columns (pre-equilibrated) were used (see
point 5) prior to quantification with LC-MS.
6 A. Pauli, M. L. Norris, E. Valen, G. L. Chew, J. A. Gagnon, S.
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Apelin in the control of body fluid homeostasis and
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12 S. M. K. McKinnie, W. Wang, C. Fischer, T. McDonald, R. K.
Kalin, X. Iturrioz, C. Llorens-Cortès, G. Y. Oudit and J. C.
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Conflicts of interest
The authors do not claim any immediate conflicts of interest.
Acknowledgements
We thank Jing Zheng, Béla Reiz, Dr. Angelina Morales-Izquierdo,
and Dr. Randy Whittal (University of Alberta Mass Spectrometry
Facility) for assistance with mass spectrometry characterization
and analyses of peptides and peptide fragments. We are grateful
to Gareth Lambkin for his assistance with the Ca2+-mobilization
assays. This research was greatly supported by the Canadian
Institutes of Health Research (CIHR Grant 136921), the Natural
Sciences and Engineering Council of Canada (NSERC), Alberta
Innovates Health Solutions (CF, AIHS), and the French National
Institute for Health and Medical Research (INSERM) and the
College de France.
13 S. L. Pitkin, J. J. Maguire, T. I. Bonner and A. P. Davenport,
International Union of Basic and Clinical Pharmacology.
LXXIV. Apelin receptor nomenclature, distribution,
pharmacology, and function, Pharmacol. Rev., 2010, 62,
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14 A. G. Japp, N. L. Cruden, D. A. B. Amer, V. K. Y. Li, E. B.
Goudie, N. R. Johnston, S. Sharma, I. Neilson, D. J. Webb,
I. L. Megson, A. D. Flapan and D. E. Newby, Vascular effects
of apelin in vivo in man, J. Am. Coll. Cardiol., 2008, 52,
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