Identifying structural determinants of potency for analogs of apelin-13: Integration of C-terminal truncation with structure-activity

Article abstract of DOI:10.1016/j.bmc.2014.04.001

Apelin peptides function as endogenous ligands of the APJ receptor and have been implicated in a number of important biological processes. While several apelinergic peptides have been reported, apelin-13 (Glu-Arg-Pro-Arg-Leu-Ser-His- Lys-Gly-Pro-Met-Pro-Phe) remains the most commonly studied and reported ligand of APJ. This study examines the effect of C-terminal peptide truncations and comprehensive structure-activity relationship (SAR) for a series of analogs based on apelin-13 in an attempt to develop more potent and stable analogs. C-terminal truncation studies identified apelin-13 (N-acetyl 2-11) amide (9) as a potent agonist (EC₅₀ = 4.4 nM). Comprehensive SAR studies also determined that Arg-2, Leu-5, Lys-8, Met-11, were key positions for determining agonist potency, whereas the hydrophobic volume of Lys-8 was a specific determinate of activity. Plasma stability studies on the truncated 10-mer peptide 28 (EC₅₀ = 33 nM) indicated the primary sites of cleavage occurred between Nle-3 and Leu-4 and also between Ala-5 and Ala-6. These new ligands represent the shortest known apelin peptides with good functional potency.

Full text of DOI:10.1016/j.bmc.2014.04.001

Bioorganic & Medicinal Chemistry 22 (2014) 2992–2997
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identifying structural determinants of potency for analogs
of apelin-13: Integration of C-terminal truncation
with structure–activity
⇑
Yanyan Zhang, Rangan Maitra, Danni L. Harris, Suraj Dhungana, Rodney Snyder, Scott P. Runyon
Organic and Medicinal Chemistry, RTI International, 3040 Cornwallis Rd, PO Box 12194, Research Triangle Park, NC 27709, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Apelin peptides function as endogenous ligands of the APJ receptor and have been implicated in a number of
important biological processes. While several apelinergic peptides have been reported, apelin-13
(Glu-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe) remains the most commonly studied and
reported ligand of APJ. This study examines the effect of C-terminal peptide truncations and comprehensive
structure–activity relationship (SAR) for a series of analogs based on apelin-13 in an attempt to develop
more potent and stable analogs. C-terminal truncation studies identified apelin-13 (N-acetyl 2–11) amide
(9) as a potent agonist (EC50 = 4.4 nM). Comprehensive SAR studies also determined that Arg-2, Leu-5,
Lys-8, Met-11, were key positions for determining agonist potency, whereas the hydrophobic volume of
Lys-8 was a specific determinate of activity. Plasma stability studies on the truncated 10-mer peptide 28
(EC50 = 33 nM) indicated the primary sites of cleavage occurred between Nle-3 and Leu-4 and also between
Ala-5 and Ala-6. These new ligands represent the shortest known apelin peptides with good functional
potency.
Received 13 January 2014
Revised 24 March 2014
Accepted 1 April 2014
Available online 13 April 2014
Keywords:
Apelin
Agonist
Peptide
APJ
Structure–activity
Function
Truncation
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
peptides. While some work has been done in this direction with
recently reported PEGylated apelin peptides, many questions
Apelin peptides have been identified as endogenous ligands for
the G-protein coupled receptor (GPCR) APJ.1 Apelin peptides are
synthesized as a 77-amino acid prepropeptide (preproapelin)
which is conserved across human, mouse, rat and bovine species,
especially at the C-terminus.2 Preproapelin is proteolytically
cleaved to various smaller peptides that act as agonists at the APJ
receptor.1–3 In addition to apelin-36 found in bovine colostrum,
pyroglutamated apelin-13 (Pyr-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-
Pro-Met-Pro-Phe) remains the most commonly studied and
reported ligand of APJ in several species.4 APJ is an emerging drug
target to treat cardiovascular failure, obesity and diabetes, excito-
toxic brain injury, liver fibrosis, angiopathies, pancreatitis, and as
remain unanswered including defining the minimum polypeptide
capable of agonizing APJ.8 In turn, these studies can lead the way
to the design of stable peptidomimetics to study APJ. Thus, there
is great interest in gaining a better understanding of the impor-
tance of amino acid side-chains as pharmacophore elements and
their potential role in stabilizing bioactive conformations of APJ
ligands.
One of the primary obstacles for studying apelin-13 in vivo is the
rapid degradation of apelin peptides to smaller fragments by
circulating peptidases. One of the major sites of cleavage is between
Pro-12 and Phe-13 through the action of ACE2 and possibly other
enzymes.4 Modification of this site to limit cleavage has been
accomplished by Murza et al.9 A series of unnatural amino acids
were substituted for Pro-12 that led to analogs with good potency
in functional assays and reasonable plasma stability. The study also
revealed the critical importance of Arg-2, Pro-3 and Arg-4, and the
lesser role of Pro-12 as determinants of activity and stability. Fur-
ther, this study led to an improved understanding of the properties
of the C-terminal pocket on apelin-13. This indicated hydrophobic
non-aromatic interactions between the residues were possible,
and confirmed the interaction of the C-terminal Phe-13 with
electron-rich residues in the binding pocket of the APJ receptor
through functional and binding assays of the synthesized peptides.9
a
prophylactic against HIV infection.4,5 Apelin-13 is rapidly
degraded by cleavage between Pro-12 and Phe-13 residues
in vitro by angiotensin converting enzyme related carboxypepti-
dase (ACE II) with high catalytic efficiency.6 Accordingly, in vivo
studies of APJ have been difficult as apelin peptides are extremely
labile in humans.7 In order to better evaluate the multiple
in vivo roles of apelin, new pharmacological tools are required that
possess improved drug-like properties compared to endogenous
⇑
Corresponding author. Tel.: +1 919 316 3842; fax: +1 919 541 6499.
E-mail address: srynyon@rti.org (S.P. Runyon).
http://dx.doi.org/10.1016/j.bmc.2014.04.001
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

Y. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 2992–2997
2993
Considering the success of this effort, it was hypothesized that trun-
cated analogs that eliminated the cleavage sites could provide
improved templates for analog design.
Deletion of Phe-13 (2) caused ꢀ50-fold decrease in activity
(EC50 = 19.8 nM) while deletion of Pro-12 (3) did not cause any fur-
ther reduction in agonist potency (EC50 = 19.2 nM), illustrating an
important role of Phe-13 for agonist potency. These data agree
with previous alanine scanning mutagenesis data demonstrating
that [Glu-1, Ala-13] apelin-13 has pEC50 of 7.08 0.19 using a sim-
ilar calcium mobilization assay.13 Further truncation of apelin-13
by removal of Met-11 (4) decreased activity by >2500-fold com-
pared to apelin-13. Truncations of Pro-10 (5), Gly-9 (6), Lys-8 (7),
and His-7 (8) did not provide analogs with activity as either ago-
nists or antagonists. This demonstrated that peptides truncated
from the C-terminal beyond Met-11 had significantly reduced
activity and suggested that either the overall architecture of the
peptide was being disrupted due to a lack of residues to stabilize
a particular ensemble of secondary structures or that the amino
acid side chains or backbone atoms of C-terminal residues were
participating in important receptor interactions.
C-terminal truncation studies provided 3 (apelin-13, 1–11) as
the shortest active fragment of the parent peptide. Our studies
indicated that pyroglutamate or glutamine in position 1 on ape-
lin-13 did not play a significant role in determining either agonist
potency or efficacy (Table 1). Therefore, Apelin-13 (N-acetyl 2–
11-amide) 9 was tested in which the N-terminal glutamine was
removed from 3 and capped with acetic acid (Table 2). Interest-
ingly, peptide 9 demonstrated a ꢀ5-fold enhancement in agonist
potency over 3, with an EC50 of 4.4 nM. Since previous studies also
indicated that the RPRL motif in apelin-13 was critical for agonist
potency, it was concluded that the 10-mer (9) was the smallest
contiguous fragment that could be obtained from truncating
apelin-13. Confirmation of this hypothesis by mutating Arg-2 to
Ala-2 resulted in peptide 13 that had significantly reduced potency
(EC50 = 1383 nM). Once it was determined that 9 was the shortest
fragment that retained agonist potency, contributions of each side-
chain to secondary structure stabilization and receptor potency
were evaluated.
Substitution of Met-11 with hydrophobic residues like Phe (10,
EC50 = 4.7 nM) or Leu (12 EC50 = 1.9 nM) provided peptides with
potent agonist activity. However, a reduction in steric volume
through substitution with Val provided 11 with an EC50 of
691 nM and Emax ꢀ63%, indicating that hydrophobic volume was
playing a role in both receptor function and potency at position
11 consistent with past studies.9 The calculated volume of Met,
Leu, Phe, and Val residues are: 92.9 Å3, 92.5 Å3, 114 Å3, and
87.3 Å3 respectively suggesting a steric volume threshold must
be exceeded at this site to effectively interact with the APJ recep-
tor. Substitutions at Gly-9 were not conducted at this time and
could form the basis of future interrogation of the effect of hydro-
phobic volume at this position in conjugation with other changes.
Identification of peptide 12 (EC50 = 1.9 nM) suggested that sub-
stitution of Leu in position 11 for Met would provide a more chem-
ically tractable and metabolically stable residue to further pursue
SAR and in vitro studies (Table 2). Previous studies indicated a
limited role for the side-chains of Ser-6 and His-7 in [Glu-1]-
apelin-13.13 Substitution of Ser-6 and His-7 with Ala in the shorter
10-mer (12) resulted in peptide 14 having an EC50 of 1.1 nM and a
max efficacy of 104% confirming the limited role of Ser-6 and His-7
for APJ activation or secondary structural stabilization.
One of the primary goals for probe development throughout
this study was to limit the total number of amino acids and flexible
side-chains so that key receptor interactions could be identified
and a more comprehensive pharmacophore defined. In support of
this strategy, Ala was substituted for Pro-3 (15) causing a 16-fold
reduction in potency compared to 12, supporting the probable role
of Pro-3 in orienting the secondary structure of the RPRL motif.
Replacement of Arg-4 (16), Leu-5 (17) Lys-8 (18), and Pro-10 (19)
with Ala resulted in a 5-fold, 492-fold, 335-fold, and 124-fold
A comprehensive pharmacophore for apelin and its proteolytic
fragments has not yet emerged although there have been recent
advances. Conformational preferences of apelin-17 have been
studied using NMR at various temperatures and these studies indi-
cate that the fragment Arg-6 to Leu-9 (RPRL sequence in apelin-13)
contains a highly structured backbone and has an increased pro-
pensity to form b-turns.10 It is well recognized that peptide turn
motifs often correlate with biological activity for a diverse set of
GPCR receptors. Macaluso and co-workers have examined the role
of turn motifs in APJ receptor antagonists by analyzing apelin
(aqueous solution) molecular dynamics trajectories. In doing so,
they described a possible role for these motifs in the RPRL region
of apelin-13.11 This effort produced di-RPRL two-ring antagonists
with a high turn character in each of the cyclized-CRPRLC-rings
with intervening spacers resulting in a purported antagonist with
moderate binding affinity.12
The translation of features found in large peptides to small
molecule peptidomimetics remains a significant challenge. This is
because identification of the bioactive secondary structure or shape
of the backbone and the requisite three-dimensional display of key
amino acid side-chains is often uncertain from both experimental
and computational perspectives. One of the goals of this study
was the identification of analogs that could provide additional
SAR information for apelin-13 analogs and suggest sites that were
either altering the secondary structure of the modified peptide, or
were disrupting key ligand-receptor interactions. Such a paradigm
would be of value for ultimately transitioning from peptidergic
ligands towards small molecule analogs for the APJ receptor.
In order to improve the probability of successfully transitioning
from a peptide core to a small molecule template, use of C- and or
N-terminal truncations can assist with identification of the small-
est bioactive core. Unfortunately, this only provides information
for activity of key peptide structural features that are present in
a small fragment of contiguous residues. Rather, key determinants
of activation might also be present in non-contiguous strings of
early (N-terminal) and late (C-terminal) chain residues requiring
a thorough experimental dissection of the larger peptide fragment.
In this study we focused on identifying the smallest active frag-
ment of apelin-13 that avoids the ACE2 cleavage site with the goal
of understanding SAR on the truncated analogs. This was initially
accomplished by producing a number of truncated apelin peptides
and assessment of their potency using a functional assay for APJ.
Once the smallest peptide with reasonable potency was identified,
a series of peptides with substitutions at Arg-2, Arg-4, Lys-8, and
combinations thereof were produced and functionally evaluated.
2. Results and discussion
2.1. Structure–activity relationships
Systematic truncation studies of apelin-13 have been limited
with only two truncated peptides previously produced and studied
by Murza et al.9 It was further reported that cleavage of apelin-13 at
either position 5 or 8 resulted in inactive fragments of apelin-13.13
Both N-acetyl-apelin-13 (8–13) and N-acetyl-apelin-13 (5–13)
displayed pEC50 values of <4.5 M.13 Thus, we focused on the impor-
tance of amino acids 6–13 on apelin-13 because evidence already
suggested that the RPRL motif on apelin-13 prefers to form b-turns
and is critical for activity.10,12 As a result, seven analogs were
synthesized and tested by progressively deleting C-terminal resi-
dues using a functional calcium mobilization assay for APJ.13,14
The activities of these analogs are summarized in Table 1.

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Y. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 2992–2997
Table 1
Activity of truncated apelin peptides
Peptide
Peptide sequence
Activities
a
b
1
2
3
4
5
6
7
8
9
10
11
12
13
EC50 (nM)
Emax (%)
Pyr-apelin-13 1
Apelin-13
Pyr
Q
Q
Q
Q
Q
Q
Q
Q
R
R
R
R
R
R
R
R
R
P
P
P
P
P
P
P
P
P
R
R
R
R
R
R
R
R
R
L
L
L
L
L
L
L
L
L
S
S
S
S
S
S
S
S
Sc
H
H
H
H
H
H
H
Hc
K
K
K
K
K
K
Kc
G
G
G
G
G
Gc
P
P
P
M
M
M
Mc
P
F
F
0.42 0.03
0.87 + 0.4
100
100
100
100
P
Apelin-13 (1–12) 2
Apelin-13 (1–11) 3
Apelin-13 (1–10) 4
Apelin-13 (1–9) 5
Apelin-13 (1–8) 6
Apelin-13 (1–6) 7
Apelin-13 (1–5) 8
Pc
20
19
4
4
P
Pc
2350 1100
>3000
>3000
>3000
>3000
a
EC50 was calculated by running various concentrations of each peptide against APJ in a functional calcium mobilization assay, fitting resulting data using non-linear
regression (GraphPad, Prism software).
b
Emax was expressed as a percentile of maximum response from peptide analog over pyr-apelin-13 control in calcium mobilization assay.
C-terminal amide.
c
Table 2
Activity data for apelin peptides
Peptide
Position
Peptide sequence
Activities
EC50 (nM)
a
1
2
3
4
5
6
7
8
9
10
11
Emax (%)
9
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
R
R
R
R
A
R
R
R
R
R
R
P
P
P
P
P
P
A
P
P
P
P
R
R
R
R
R
R
R
A
R
R
R
L
L
L
L
L
L
L
L
A
L
L
S
S
S
S
H
H
H
H
H
A
A
A
A
A
A
K
K
K
K
K
K
K
K
K
A
K
G
G
G
G
G
G
G
G
G
G
G
P
P
P
P
P
P
P
P
P
P
A
Mb
Fb
Vb
Lb
Lb
Lb
Lb
Lb
Lb
Lb
Lb
4.4 1.0
4.7 2.2
691 268
1.9 0.4
1383 18
1.1 0.3
98
106
63
100
86
104
103
103
104
67
10
11
12
13
14
15
16
17
18
19
S
A
A
A
A
A
A
31
6
8.9 0.1
936 170
637 214
237 29
93
Arginine 2 modifications
20
21
Ac
Ac
K
P
P
R
R
L
L
A
A
A
A
K
K
G
G
P
P
Lb
Lb
494 48
>3000
101
n/a
K(TFA)
Arginine 4 modifications
22
23
Ac
Ac
R
R
P
P
Nle
K
L
L
A
A
A
A
K
K
G
G
P
P
Lb
Lb
3.2 1.4
1.7 0.7
100
105
Leucine 5 modifications
24 Ac
R
R
P
P
K
K
F
L
A
A
A
A
K
G
G
P
P
Lb
Lb
44 30
93
97
Lysine 8 modifications
25
Ac
Nle
2.2 0.9
Combinations
26
27
28
29
Ac
Ac
Ac
Ac
R
R
R
R
P
P
P
P
Nle
Nle
Nle
Aoc
Ho Phe
Aoc
L
A
A
A
A
A
A
A
A
K
K
Nle
A
G
G
G
G
P
P
P
P
Lb
Lb
Lb
Lb
73 101
539 129
33 15
100
93
103
40
L
>3000
a
Emax was expressed as a percentile of maximum response from peptide analog over pyr-apelin-13 control in calcium mobilization assay.
C-terminal amide.
b
reduction in activity respectively. This demonstrated that the most
critical residues for potency were Arg-2, Leu-5, Lys-8, Pro-10, and
Leu-11. Importantly, modification of Lys-8 (18) with Ala reduced
efficacy to 67% of apelin-13 suggesting a potential role for Lys-8
in driving agonist function for this class of ligands.
Having developed a generic template of critical resides on our
truncated 10-mer (12) we began to further investigate the
more specific roles for the side-chains of Arg-2, Arg-4, Leu-5, and
Lys-8. Modification of Arg-2 with Lys (20, EC50 = 494 nM) and
Lys-TFA (21, EC50 >3000 nM) which modify basicity, significantly
reduced potency (Table 2). This indicated that the positive charge
of Arg-2 was an important structural feature for either receptor
potency or conformational stabilization. Substitution of Arg-4
however was much more forgiving than Arg-2. Replacement of
Arg-4 with Ala (16) only reduced agonist potency by 5-fold
suggesting that the positive charge of Arg-4 was not a critical fea-
ture for either receptor interaction or conformational stabilization.
Substitution of Arg-4 with Lys (23, EC50 = 1.7 nM) or nor-leu (22,
EC50 = 3.2 nM) induced minimal alteration in agonist potency or
efficacy suggesting the charge or polarity of Arg-4 was not likely
a critical pharmacophore feature.
There is significant evidence supporting the critical role Leu-5
plays in apelin potency.15 This also appeared to be true for the
10-mer analog (17) where Leu-5 to Ala lowered activity by 492-
fold. Replacement of Leu-5 with Phe (24, EC50 = 44 nM) also
reduced agonist potency by 23-fold compared to 12 (Table 2).
When amino-octanoic acid (27) was substituted for Leu-5, agonist
potency was reduced by 168-fold compared to 22. In addition, Leu-
5-homo-Phe (26) caused a 22-fold reduction in agonist potency
compared to 22. This suggested a rather limited tolerance to steric
bulk at position 5.
One of the remaining positions to be fully evaluated was posi-
tion 8. Our goal was to determine if the positive charge of lysine
was involved in either a critical receptor interaction or played a

Y. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 2992–2997
2995
role in stabilizing secondary structure. Modification of Lys-8 to Nle
(25, EC50 = 2.2 nM) afforded a peptide having similar activity as 22
suggesting that Lys-8 is not involved in forming critical
interactions.
Utilizing the information gained from our study of individual
residues, we began to combine some of the most favorable substi-
tutions into single peptides. Peptide 28 removed two positive
charges by substituting Nle for Arg-4 and Nle for Lys-8 in a trun-
cated 10 mer. This resulted in a compound having full efficacy
and moderate potency (28, EC50 = 33 nM, 103% Emax). When the
hydrophobic volume of position 8 was reduced through substitu-
tion of Ala, potency and efficacy was lost (29, EC50 = >3000 nM).
Considering the reasonable potency of 28 and the lack of a defined
ACE2 cleavage site we began to investigate the utility of this as an
in vivo probe.
2.2. Plasma stability
Having developed peptides that removed the ACE2 cleavage site
and had reasonable potency, investigations were conducted to
identify additional cleavage sites using a plasma stability study.
Peptide 28 was selected for evaluation because it was one of the
smallest apelin peptides to date without the known ACE2 site that
had favorable potency and efficacy. In addition, it had replaced
Arg-4 and Lys-8 with hydrophobic residues that could potentially
improve stability. Peptide 28 was incubated in rat serum and ali-
quots were analyzed for percent parent remaining at 2, 5, 15, 30,
and 60 min. From the rapid disappearance of parent peptide it
was clear that proteolytic cleavage was still occurring but at sites
other than the ACE2 site leading to a short t1/2 ꢁ 2 min (Fig. 1).
2.3. Metabolic cleavage sites
Figure 2. Cleavage of peptide 28 in rat plasma.
Since 28 was rapidly cleaved in serum and devoid of the known
ACE2 cleavage site on apelin-13, we investigated which positions
were metabolically labile so that future analogs could be rationally
designed. Serum treated samples were passed through a reverse
phase capillary C18 column using nano ESI-MS system monitored
by an Orbitrap mass spectrometer. The time-course study clearly
showed three major peaks at 10 min, 25 min and 38 min. The
peaks at 10 min and 25 min were new peaks coming from serum
treatment that could be metabolites of 28 (Fig. 2). Mass spectra
analysis of these three peaks showed [M+H]+ of 427.2656,
611.3879 and 1061.6824 for peaks at ꢀ10 min, ꢀ25 min and
ꢀ38 min, respectively (Fig. 2). With the aid of peptide ladder
predictions, these three peaks were mapped to be the cleaved
N-terminal products of Ac-RPnle (Fragment 2), Ac-RPnleLA
(Fragment 1) and parental 28 (Ac-RPnleLAAnleGPL-amide) as
indicated in Figure 2. This prediction was later on confirmed by
tandem MS/MS fragmentation. While it was not totally surprising
that a primary cleavage occured between two adjacent alanines,
future structural modification studies might be used to address
these labile sites.
3. Conclusion
Truncation of apelin-13 from both the C- and N-terminus
resulted in a series of 10-mer peptides that displayed potent ago-
nist activity comparable to parental apelin-13. Presence of an
ACE2 cleavage site between Pro-12 and Phe-13 on apelin-13 leads
to rapid cleavage of this peptide with a very short half-life. The
analogs presented herein provide improved templates to design
better in vivo probes of the APJ receptor.9 In addition to removing
an important metabolic liability, this study confirmed the impor-
tance of Met-11, Arg-2, and Leu-5 as determinants of functional
potency at the APJ receptor. Our results provide experimental
SAR data involving side-chain substitutions on the shortest apelin
peptide reported to date. These findings also provide insights into
the spatial preferences for ligand-receptor interactions and will aid
in the future design and discovery of synthetic ligands for APJ
receptor.
8.0×108
Peptide 28
6.0×108
4.0×108
2.0×108
0
4. Experimental procedures
4.1. Materials
Compounds 2–5 were purchased from AnaSpec (Fremont, CA).
0
2
5
15
All resins, Fmoc-amino acids,
were purchased from AAPPTec (Louisville, KY) and Chem-Impex
international Inc. (Wood Dale, IL). All organic solvents were
D-amino acids, coupling reagents
Time (min)
Figure 1. Plasma stability of peptide 28.

2996
Y. Zhang et al. / Bioorg. Med. Chem. 22 (2014) 2992–2997
purchased from VWR (Radnor, PA). CHO cells expressing APJ
receptor/G q16 were utilized as described in our previous publica-
tion. Cell culture medium was procured from Sigma (St. Louis, MO),
96-well plates were from Corning Corporation (Corning, NY).
Fluo-4 AM dye was from Invitrogen (Eugene, OR).
with Eksigent nanoLC-Ultra (Eksigent, Dublin, CA). Peptides were
separated on a HALO C18 (2.7
m, 75 mm  102 mm) Pico Frit col-
a
l
umn (New Objective, Woburn, MA) using water with 0.1% formic
acid as mobile phase A (MPA) and acetonitrile with 0.1% formic acid
as mobile phase B (MPB). Initial nano-LC condition of consisted of
10% MPB, which was linearly increased to 60% MPB over the course
of 55 min at a flow rate of 450 nl/min. All MS data were acquired in
positive ion nano-ESI data dependent mode with iteration of 1
FTMS and 3 IT-MS/MS cycles at MS resolution of 7500 and a total
LC–MS run time of 75 min. The source temperature was set at
200 °C and the capillary voltage of 3.5 kV was used for the sample
analysis. Orbitrap Velos was calibrated using Thermo LTQ-Velos
calibration solution prior to sample analysis. LC–MS/MS data were
processed using Xcalibur (Thermo Scientific, San Jose, CA).
4.2. Peptide synthesis
Ala scanning and truncated peptides were synthesized by stan-
dard Fmoc/HBTU chemistry on solid phase using a FOCUS XC
(AAPPTec, Louisville, KY) peptide synthesizer. Wang-resin or Rink
MBHA resin was used to generate peptides having a C-terminal
acid or amide as indicated. A 5-fold excess of Fmoc-amino acids
and HBTU and a 10-fold excess of DIEA were used in the coupling
reaction for 30 min. Twenty percent piperidine in DMF was used to
remove Fmoc protecting groups during the synthesis. Peptide
cleavage from solid support and side-chain deprotection were
simultaneously achieved using cleavage cocktail containing
TFA/water/TIS/phenol of 92.5:2.5:2.5:2.5 for 3 h. Crude peptides
were precipitated and triturated by cold ether from cleavage
cocktail and purified by HPLC.
4.6. Plasma stability assay
Plasma stability assays measure the conversion or degradation
of compounds in plasma.16 The test peptides were incubated at
1 lM in rat plasma adjusted to pH 7.4, and DMSO concentration
was limited to less than 2.5%. Samples were incubated at 37 °C
and terminated at 0, 2, 5, 15, 30, and 60 min after initiation with
acetonitrile. The samples were clarified by centrifugation and
ready for LC–MS/MS studies. The amount of 28 in each sample
was monitored on Waters Acquity UPLC/AB-Sciex API 4000 Q-Trap
system using a previously developed MRM (multiple reaction
monitoring) method. The peak area representing 28 was quantified
with Analyst software. Metabolite ID searches were performed
using Thermo LTQ-Velos Orbitrap LC–MS/MS. The serum treated
samples were passed through a reverse phase capillary C18 column
on nano ESI-MS system and monitored by the orbitrap mass
spectrometer.
4.3. Peptide purification and analytical analysis
Crude peptides were purified on Agilient preparative reverse
phase HPLC system using a Spirit™ peptide 120 C18 5 lm
25 Â 2.12 column (AAPPTec, Louisville, KY). The pure peak corre-
sponding to the right mass of the peptide was collected and HPLC
solvent was evaporated under nitrogen. The purity of peptides was
analyzed using analytical column Spirit™ peptide 120 °C18 5 lm
25 Â 0.46 column (AAPPTec) with a gradient of 5–45% acetonitrile
over 30 min. Retention time, calculated mass and actual mass
obtained from ESI-MS are reported in Table 1 Supplementary
information.
5. Author contributions
4.4. APJ functional assay
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
CHO cells stably expressing APJ-Gaq16 were generated previ-
ously and maintained with 150 mg/L geneticin and hygromycin
in Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium sup-
plemented with 10% fetal bovine serum (FBS) and 1% penicillin and
streptomycin (p/s).14 The calcium mobilization assay to assess APJ
function was performed as described previously.14 Briefly, 40,000
cells were plated in each well of a black Costar 96-well optical bot-
tom plate and grown for 24 h at 37 °C. Medium was removed and
cells were washed with Fluo-4 buffer (HBSS buffer with 20 mM
Acknowledgment
This work is supported by internal funding from RTI
International.
Supplementary data
HEPES, 1% BSA, 10
lM probenecid, pH 7.4). Then 2 lM Fluo-4 AM
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.04.001.
dye in 225 L Fluo-4 buffer was added to each well and incubated
l
for 1 h at 37 °C. A series of peptide analog dilutions were made at
10Â concentration and placed in a 96-well plate. At the end of
incubation, both compound plate and the plate with cells were
inserted to a Flexstation III plate-reader. Agonist-mediated changes
in fluorescence were monitored at Ex 488 nm and Em 525 nm for
60 s. Data collected on a Flexstation III plate-reader were imported
to Prism software (GraphPad, La Jolla, CA). The maximum change in
fluorescence after agonist addition was used to determine APJ
activity. Nonlinear regression analysis was performed to fit data
and obtain maximum response (Emax), EC50, correlation coefficient
(r2) and other parameters using GraphPad Prism software. The data
were reported from at least three independent experiments per-
formed in duplicate as mean standard error.
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