The replacement of His(4) in angiotensin IV by conformationally constrained residues provides highly potent and selective analogues

Article abstract of DOI:10.1021/jm900651p

The histidine residue in angiotensin IV was replaced by various conformationally constrained amino acids. The substitution of the His ⁴-Pro⁵ dipeptide sequence by the constrained Trp analogue Aia-Gly, in combination with β²

Full text of DOI:10.1021/jm900651p

5612 J. Med. Chem. 2009, 52, 5612–5618
DOI: 10.1021/jm900651p
The Replacement of His(4) in Angiotensin IV by Conformationally Constrained Residues Provides
Highly Potent and Selective Analogues
†
Aneta Lukaszuk, Heidi Demaegdt, Debby Feytens, Patrick Vanderheyden, Georges Vauquelin, and Dirk Tourwe*
‡
†
‡
‡
,†
ꢀ
†Department of Organic Chemistry, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium, and ‡Department of Molecular and
Biochemical Pharmacology, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium
Received May 18, 2009
The histidine residue in angiotensin IV was replaced by various conformationally constrained amino
acids. The substitution of the His4-Pro5 dipeptide sequence by the constrained Trp analogue Aia-Gly,
in combination with β2hVal substitution at the N-terminus, provided a new stable analogue H-(R)-
β2hVal-Tyr-Ile-Aia-Gly-Phe-OH (AL-40) that is a potent ligand for the Ang IV receptor IRAP and
selective versus AP-N and the AT1 receptor.
Introduction
To precisely study the mechanism by which Ang IV exerts
its biological effects, there is great need to improve the
metabolic stability of Ang IV and to prepare analogues that
are highly selective for the AT4 receptor.
The renin-angiotensin system (RAS) mediates important
physiological functions including body water and electrolyte
homeostasis, blood pressure, sexual behavior, and the regula-
tion of gland hormones. These functions are mediated by
angiotensin II (Ang II), which interacts with the AT1 receptor
subtype. The hexapeptide angiotensin IV (Ang IV, H-Val-
Tyr-Ile-His-Pro-Phe-OH), derived from Ang II by cleavage of
the two N-terminal amino acids, binds to the AT1 and AT2
receptors with low affinity. A specific AT4 receptor1-4 has
been identified as the cystinyl aminopeptidase (EC3.4.11.3,
CAPa), also denoted as insulin regulated aminopeptidase
(IRAP).5,6 Among a wide range of physiological actions,
Ang IV is responsible for cognitive enhancement, improving
memory acquisition,7,8 and inhibition of pharmacologically
induced seizures9 as well as for vascular and renal actions.10
Ang IV exerts its effects by binding to AT4 receptors, which
are widely distributed across tissues. Three hypotheses for
explaining the effects of the AT4 receptor ligands were
proposed: (i) Ang IV acts as a potent IRAP inhibitor, which
prolongs the action of endogenous promnestic peptides,11,12
(ii) Ang IV might modulate glucose uptake by modulating
trafficking of GLUT4,5,13 or (iii) IRAP might also act as a
receptor and trigger intracellular processes.4,5,14,15 Moreover,
it has been suggested that aminopeptidase N (AP-N) is also a
potential target of Ang IV.16
We previously reported a study in which each amino acid
in Ang IV, except His, was replaced by its β2-homo- or
β3-homoanalogue.17 β-Homoamino acids diverge from
R-amino acids by having an extra methylene group (homo)
either between the carboxyl group and the R-carbon (β3-
homoamino acid) or between the amine group and the
R-carbon (β2-homoamino acid).18 Incorporation of such
β-homoamino acids has been used to create peptidomimetics
that not only retain biological activity19 but that are also
resistant to proteolysis.20-30 Our study resulted in the devel-
opment of H-β2hVal-Tyr-Ile-His-Pro-β3hPhe-OH 1 (AL-11),
a potent, selective, and stable AT4 ligand, in which β2hVal
provides selectivity for IRAP versus AP-N, and stability
against its aminopeptidase activity and in which β3hPhe is
responsible for selectivity toward the AT1 receptor.17
Because histidine was the only residue in the Ang IV
sequence for which the β2- and β3-homoamino acid analogues
are difficult to obtain,31 this amino acid was not modified in
our previous study.
Another successful approach for enhancing the receptor
selectivity and stability of biologically active peptides is
the incorporation of conformationally constrained amino
acids.32,33 The conformations of the side chains in a peptide
are highly important for biological activity and the introduc-
tion of constrained side chains is at the basis of the design in χ-
space, which resulted in many potent peptidomimetics.34,35
The conformations of the amino acid side chains are described
by the torsional angles χ1, χ2,...35 For aromatic residues such
as Phe, Tyr, Trp, or His, three low energy conformations exist:
gauche (-) (χ1=-60°), trans (χ1=180°), and gauche (þ) (χ1=
þ60°). These low energy conformations can be constrained by
structural modifications that limit the rotation around the
CR-Cβ (and Cβ-Cγ) bond.35,36
*To whom correspondence should be addressed. Phone: (þ32) 2 629
32 95. Fax: (þ32) 2 629 33 04. E-mail: datourwe@vub.ac.be.
a Abbreviations: Aba, 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-
one; Aia, 4-amino-1,2,4,5-tetrahydro-indolo[2,3-c]-azepin-3-one; AP-N,
aminopeptidase N; BSA, bovine serum albumin; CAP, cystinyl amino-
peptidase; CHO cells, Chinese hamster ovary cells; DIOZ, 4-isopropyl-
5,5-diphenyloxazolidin-2-one; DIPEA, diisopropylethylamine; DMEM,
Dulbecco’s modified essential medium; EDTA, ethylenediaminetetraace-
tic acid; GLUT4, insulin-regulated glucose transporter; HEK293 cells,
human embryonic kidney cells; IRAP, insulin regulated aminopeptidase;
L-Leu-pNA, L-leucine-p-nitroanilide; PBS, phosphate-buffered saline;
Spi, 4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-carboxylic acid;
TBTU, O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium tetrafluoro-
borate; TES, triethylsilane; TFA, trifluoroacetic acid; TFMSA, trifluoro-
methanesulfonic acid; Tic, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic
acid; Tris, tris(hydroxymethyl)aminomethane.
We now describe the results of the replacement of His4 in
Ang IV by conformationally constrained aromatic residues.
In this study, we selected two types of constraints (Figure 1).
r
pubs.acs.org/jmc
Published on Web 08/27/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18 5613
Figure 1. Conformationally constrained aromatic residues used as His replacements in Ang IV.
Table 1. Inhibition of Enzyme Activity by Ang IV Analogues in Membranes of Transfected HEK 293 Cells
HEK293þIRAP
pKi ( SD
HEK293þAP-N
code
Angiotensin IV: H-Val-Tyr-Ile-His-Pro-Phe-OH
pKi ( SD
Ki (AP-N)/Ki (IRAP)
2
3
4
5
6
H-Val-Tyr-Ile-Spi-Pro-Phe-OH
H-Val-Tyr-Ile-Tic-Pro-Phe-OH
H-Val-Tyr-Ile-Aia-Gly-Phe-OH
6.29 ( 0.09
6.66 ( 0.01
6.74 ( 0.03
7.30 ( 0.04
7.90 ( 0.06
8.07 ( 0.05
6.89 ( 0.03
7.26 ( 0.06
7.25 ( 0.14
7.28 ( 0.03
7.56 ( 0.21
7.00 ( 0.15
5.52 ( 0.26
5.61 ( 0.09
5.07 ( 0.12
5.69 ( 0.04
5.53 ( 0.19
6.10 ( 0.61
5.32 ( 0.49
5.40 ( 0.37
6.08 ( 0.02
4.97 ( 0.23
5.23 ( 0.04
5.35 ( 0.15
5
11
50
50
H-Val-Tyr-Ile-Aba-Gly-Phe-OH
H-(R)-β2hVal-Tyr-Ile-Aba-Gly-Phe-OH
H-(R)-β2hVal-Tyr-Ile-Aia-Gly-Phe-OH
H-(R)-β2hNle-Tyr-Ile-Aba-Gly-Phe-OH
H-(R)-β2hNle-Tyr-Ile-Aia-Gly-Phe-OH
H-Val-Tyr-Ile-His-Pro-Phe-OH49
H-Nle-Tyr-Ile-His-Pro-Phe-OH
250
100
33
7 (AL-40)
8
9
100
14
Ang IV
[Nle1]Ang IV
1 (AL-11)
10
200
200
50
H-(R)-β2hVal-Tyr-Ile-His-Pro-β3hPhe-OH17
H-(R)-β2hVal-Tyr-Ile-His-Pro-Phe-OH17
Figure 2. Inhibition of the enzymatic activity in membrane homogenates of HEK293 cells transfected with human IRAP (9) or AP-N (1)
(corresponding to 1.5ꢀ105 cells/incubation) with compounds 5, 6, 7, and 9. Cells were incubated at 37 °C with 1.5 mM L-Leu-pNA in the
absence (control activity) or presence of increasing concentrations of compound. The rate of L-Leu-pNA cleavage (corresponding to enzyme
activity and expressed as a percentage of control) was calculated by linear regression analysis of the absorption (at 405 nm) vs time curves with
measurements made every 5 min (between 10 and 50 min). The pKi values of all peptide-enzyme combinations are given in Table 1.
1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid (Tic)
and 4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine-6-car-
boxylic acid (Spi), analogues of Phe and His, respectively,37,38
restrict the side chain conformation to a χ1 of -60° or þ60°.39
The 2-azepinone-type residues 4-amino-1,2,4,5-tetrahydro-
2-benzazepin-3-one (Aba) and 4-amino-1,2,4,5-tetrahydro-
indolo[2,3-c]-azepin-3-one (Aia), which are analogues of Phe
and Trp, respectively, restrict the side chain conformation to a
χ1 of þ60° or 180°.36,40,41 Because the latter constraint is
obtained by linking the aromatic ring in the side chain to the

5614 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18
Lukaszuk et al.
Figure 3. Interaction of the compounds with the AT1 receptor (left) and dose-response curve for 6 (right). CHO cells stably transfected with
the AT1 receptor (CHO-AT1) were incubated for 40 min at 37 °C with 10-5 M of compound and 1.5 nM [3H]Valsartan. The full dose
competition to determine the pKi was performed for 6. Data refer to specific binding of [3H]Valsartan (expressed as percent of control binding,
i.e., binding in medium only), calculated by subtracting nonspecific binding in the presence of 1 μM Candesartan from total binding.
nitrogen of the succeeding amino acid through a methylene
bridge,36 the Pro5 residue of Ang IV was replaced by Gly.
Those constraints have been successfully applied in peptides
and peptide mimetics.35,36,38,42-45 Following our previous
finding that metabolic stability is linked to modification
of the Val1 residue, the incorporation of these conforma-
tional constraints at position four of Ang IV is combined
with the use of (R)-β2hVal and (R)-β2hNle residues at posi-
tion one.
being more potent than our best analogue so far, peptide 1.
These two compounds exhibited at least a hundred-fold
IRAP selectivity versus AP-N. In contrast to native Ang
IV in which the replacement of Val1 by Nle results in an
increase in IRAP versus AP-N selectivity (Table 1), this is not
the case for the β2hNle-containing analogues 8 and 9.
Binding to the AT1 Receptor. The affinity of the Ang IV
analogues for the AT1 receptor was evaluated on intact
CHO-AT1 cells by measuring the ability of 10-5 M of
compound to inhibit the specific binding of the selective
AT1 receptor antagonist Valsartan (1.5 nM). Except for 6,
none of the analogues inhibited Valsartan binding and hence
showed no affinity for the AT1 receptor (Figure 3).
Results
Synthesis. The conformationally constrained amino acids
Tic and Spi were prepared from Phe and His, respectively, by
a Pictet-Spengler condensation with formaldehyde accord-
ing to literature procedures.37,38 The Aba-Gly and Aia-Gly
dipeptide analogues were prepared as described in our pre-
vious publications.36,41 The β2-homoamino acids (R)-β2hVal
and (R)-β2hNle were synthesized by an asymmetric amino-
methylation reaction using the DIOZ chiral auxiliary.46,47
All amino acid analogues were Boc-protected and incorpo-
rated into the Ang IV sequences by solid phase peptide
synthesis using a Merrifield resin. The peptides were purified
by semipreparative HPLC to a purity >98%.
Enzyme Activity. Determination of the aminopeptidase
catalytic activity was performed in membrane homogenates
of HEK293 cells transiently transfected with human IRAP
or AP-N and was based on the cleavage of the substrate
L-leucine-p-nitroanilide (L-Leu-pNA) into L-leucine and
p-nitroaniline in the presence of increasing concentrations
of the Ang IV analogues.48 The Ang IV analogues produced
a concentration-dependent inhibition of the catalytic acti-
vity. The results of the inhibition of enzyme activity by the
Ang IV analogues are presented in Table 1, and selected
curves are shown in Figure 2.
For the analogues containing native Val at position 1, a
comparison with the data for native Ang IV reveals that the
substitution of His4 by Tic in 3 or Spi in 2 results in a drop in
inhibitory potency both for IRAP and AP-N. The 2-benza-
zepinone constraint on the contrary leads to a compound
with comparable potency as Ang IV in case of the Aba-Gly
substitution (5) and to a slightly less potent compound in the
case of the Aia-Gly substitution (4). The best results were
obtained for the analogues in which β2-homoamino acids
were combined with the 2-benzazepinone constraint. The
(R)-β2hVal-containing analogues 6 and 7 displayed very
similar pKi values for inhibition of IRAP, the latter one
A full dose-inhibition study was conducted for 6 and a
pKi=6.85 ( 0.11 was determined.
Stability Experiments. Stability experiments were per-
formed in membrane homogenates of CHO-K1 cells con-
taining endogenous IRAP. Preincubations of the mem-
branes with different concentrations of compound were
carried out for 40 min at 37 °C in the presence or absence
of metal chelators. Then a competition binding assay was
performed by adding [3H]Ang IV (3 nM) for 30 min at 37 °C.
Nonspecific binding was measured with Ang IV (10 μM). In
the presence of the chelators, ethylenediaminetetraacetic
acid (EDTA) and 1,10-phenantroline, which have the ability
to block IRAP, AP-N, and other metalloprotease activity,
no metabolic breakdown was taking place.50 In the absence
of these chelators, peptides susceptible to metalloprotease
degradation will be rapidly metabolized.
Omission of metal chelators during the preincubation step
resulted in a rightward shift of the 4 and 5 inhibition curves
(Figure 4), suggesting that these compounds were quickly
metabolized by IRAP and/or other metalloproteases present
in the cell membranes.
In contrast all analogues containing a β2-homoamino acid
at the N-terminus (6-9) were stable under these conditions.
Discussion
In this study, we examined the potential of the replace-
ment of His4 for obtaining more stable and more IRAP-
selective Ang IV analogues. The introduction of conforma-
tionally constrained amino acid analogues is a powerful
method to confer receptor selectivity and metabolic stabi-
lity to the modified peptides.31,32 Therefore we have
replaced His4 in Ang IV by several constrained analogues
of His, Phe, and Trp.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18 5615
Figure 4. Stability experiments performed in membrane homogenates of CHO-K1 cells. Membranes were preincubated for 40 min at 37 °C
with increasing concentrations of compounds in the presence (9) or absence (2) of chelators. Then cells were incubated for 30 min with 3 nM
[3H]AngIV. Data refer to specific binding of [3H]AngIV (expressed as percent of control binding), calculated by subtracting nonspecific binding
in the presence of 10 μM unlabeled Ang IV from total binding.
The substitution of His by its analogue Spi (2) or by the Phe
analogue Tic (3), both of which adopt a gauche (þ) conforma-
tion within a peptide sequence,39 led to a substantially
decreased ability to inhibit the hydrolysis of Leu-p-NA by
IRAP.39 This indicates that a gauche (þ) (χ1=þ60°) orienta-
tion of the His side chain results in a less efficient interaction
with IRAP. The replacement of the His-Pro dipeptide frag-
ment by Aba-Gly on the contrary, which restricts the side
chain conformation to a χ1 of þ60° or 180°,36,40,41 is leading to
a compound (5) with a potency comparable to that of Ang IV.
Because the gauche (þ) orientationwas excludedbythe results
of 2 and 3, it can be concluded that the trans orientation of the
aromatic side chain at position 4 of Ang IV is preferred and
that the imidazole ring is not a requirement. In fact, an indole
ring is also accepted at this position, the Aia-Gly substitution
(4) leading to a compound that is only slightly less potent.
Importantly, these substitutions maintain the selectivity for
IRAP versus AP-N. As can be expected, however, the analo-
gues that were not modified at the N-terminus were not stable
against the aminopeptidase activity of IRAP. Our strategy to
introduce (R)-β2hVal was also successful in this study. Ana-
logues 6 and 7 do not only have an increased affinity for
IRAP, but also a good selectivity versus AP-N, and are
metabolically stabilized. Binding studies reported by Sardi-
nia51 indicated that [Nle1]Ang IV had about a 1000-fold
higher affinity than native AngIV. This was, however, not
confirmed in the studies by Lew,11 where a factor of only 10
was observed. Our results do not indicate an increased
potency in the enzyme assay but an increased IRAP versus
AP-N selectivity for [Nle1]Ang IV (Table 1). The change of
β2hVal in analogues 6 and 7 to β2hNle in 8 and 9 did not result
in analogues having a better profile.
These results indicate that neither the Pro5 ring nor the His4
imidazole ring are required for binding to IRAP. Previous
studies have demonstrated that the entire His-Pro-Phe se-
quence in Ang IV can be replaced by o-aminomethylphenyl
acetic acid.52 However, potency, selectivity, and stability were
less than for the analogues reported here.
Furthermore, the affinity of the reported compounds for
the AT1 receptor was studied by measuring the ability of
10-5 M of compound to compete with the binding of the
nonpeptide AT1 receptor antagonist [3H]Valsartan (1.5 nM)
to recombinant CHO cells stably expressing the AT1 receptor.
None of the peptides bound to the AT1 receptor at this
concentration, except 6. This compound shows a binding
affinity (pKi = 6.85 ( 0.11) comparable to that of Ang IV
(pKi=6.39 ( 0.01) (Figure 3). Our previous studies17 showed
that the C-terminal part of Ang IV is crucial for AT1 receptor
binding, the introduction of a β3hPhe residue being respon-
sible for the loss of AT1 affinity. In the present study, the
C-terminus was not modified. Therefore the conformational
constraints introduced at position 4 are also responsible for

5616 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18
Lukaszuk et al.
IRAP vs AT1-selectivity for all analogues except 6. Remark-
ably, 6 with a sequence H-(R)-β2hVal-Tyr-Ile-Aba-Gly-Phe-
OH, was the only analogue showing weak AT1 affinity, in
contrast to the related Val1-containing analogue 5, and to 7,
which contains an indole ring instead of the benzene ring in 6.
This observation is difficult to explain but indicates that subtle
changes in the peptide main chain, combined with side chain
modifications, can influence the interaction with the AT1
receptor while not having a major effect on IRAP affinity.
performed in DMF/CH2Cl2 (1v:1v) using TBTU (3 equiv) and
DIPEA (6 equiv), and the reaction was monitored using the
Kaiser test. The peptides were cleaved from the resin by treating
it with TFA/TFMSA/TES (20:2:3). The peptides were purified
by RP-HPLC on a SUPELCO DiscoveryBIO wide pore pre-
parative C18 column, and the pure peptides were then lyophi-
lized. Purities and structures were confirmed using analytical
RP-HPLC and ESI-MS.
Peptide 7. The peptide was obtained with a yield of 3.8%.
HPLC: tR=13.02 min (purity > 99%); MS (ESI): (M þ Hþ)
calculated: 810.40; (M þ Hþ) found: 810.40.
The remaining peptides were synthesized and characterized
according to the method described above. Purities, yields,
HPLC, and MS data can be found in the Supporting Informa-
tion.
Conclusion
In this study we used a new approach to develop Ang IV
analogues that are selective for IRAP versus AP-N and versus
the AT1 receptor and that are resistant to aminopeptidase
degradation. The replacement of the His4-Pro5 dipeptide
sequence by residues that have a side chain constrained in
the trans conformation was very effective to provide this
selectivity. In combination with a (R)-β2hVal substitution to
provide metabolic stability, the new ligand H-(R)-β2hVal-
Tyr-Ile-Aia-Gly-Phe-OH, 7, was developed with improved
potency compared to the previously reported 1. This ligand
contains a constrained Trp residue as a replacement for His4.
It will be an improved tool to study the physiological roles of
Ang IV and IRAP. These results further demonstrate the
power of conformational constraintsas a toolto overcomethe
well-known limitations of natural peptides.
Cell Culture, Transient Transfection, and Membrane Prepara-
tion.17 CHO-K1, CHO-AT1, and HEK293 cell lines were cul-
tured in 75 and 500 cm2 culture flasks in Dulbecco’s modified
essential medium (DMEM) supplemented with L-glutamine
(2 mM), 2% (v/v) of a stock solution containing 5000 IU/mL
penicillin, and 5000 μg/mL streptomycin (Invitrogen, Merelbeke,
Belgium), 1% (v/v) of a stock solution containing nonessential
amino acids, 1 mM sodium pyruvate, and 10% (v/v) fetal bovine
serum (Invitrogen, Merelbeke, Belgium). The cells were grown
in 5% CO2 at 37 °C until confluent.
HEK293 cells were transiently transfected with plasmid
DNA, pCIneo containing the gene of human IRAP (kindly
obtained from Prof. M. Tsujimoto, Laboratory of Cellular
Biochemistry, Saitama, Japan) or pTEJ4[38] carrying the com-
plete human AP-N cDNA.54 The transient transfection was
performed as described previously with 8 μL/mL Lipofect-
AMINE (Invitrogen, Merelbeke, Belgium) and 1 μg/mL plasmid
DNA.54 After transfection, the cells were cultured for 2 more
days. IRAP and AP-N transfected HEK293 cells displayed a
10 and 8 times higher enzyme activity than nontransfected cells.
CHO-K1 cell and transfected HEK293 cell membranes were
prepared as described previously.48 In short, the cells were
harvested with 0.2% EDTA (w/v) (in phosphate-buffered saline
(PBS), pH 7.4) and centrifuged for 5 min at 500g at room
temperature. After resuspending in PBS, the number of cells
was counted and they were washed. The cells were then homo-
genized in 50 mM Tris-HCl (at pH 7.4) using a Polytron (10 s at
maximum speed) and Potter homogenizer (30 strokes at 1000 rpm)
and then centrifuged for 30 min (30000 g at 4 °C). The pellet was
resuspended in 50 mM Tris-HCl, centrifuged (30 min 30000g at
4 °C), and the supernatant was removed. The resulting pellets
were stored at - 20 °C until use.
Experimental Section
General. L-Leu-pNA was obtained from Sigma-Aldrich
(Bornem, Belgium) and p-nitroaniline from VWR International
(Leuven, Belgium). All other reagents were of the highest grade
commercially available. CHO-K1 cells were kindly obtained
from the Pasteur Institute (Brussels, Belgium). [3H]Valsartan
was kindly supplied by Novartis (Basel, Switzerland), and
ꢀ
3
[ H]Angiotensin IV was obtained from G. Toth, Biological
Research Center (Szeged, Hungary).53
Fmoc-Phe-Wang resin, CH2Cl2, TES, and Fmoc-protected
amino acids were obtained from Fluka (Bornem, Belgium),
TBTU was from SennChemicals (Gentilly, France), and DMF
and DIPEA were from Sigma-Aldrich (Bornem, Belgium).
Analytical RP-HPLC was performed using an Agilent 1100
Series system (Waldbronn, Germany) with a Supelco Discovery
BIO wide pore (Bellefonte, PA) RP C-18 column (25 cm ꢀ
4.6 mm, 5 μm) using UV detection at 215 nm. The mobile phase
(system 1: water/acetonitrile) contained 0.1% TFA. The stan-
dard gradient consisted of a 20 min run from 3 to 97%
acetonitrile at a flow rate of 1 mL/min. Preparative HPLC
was performed on a Gilson apparatus and controlled with the
software package Unipoint. The reversed-phase C18-column
(Discovery BIO wide pore 25 cmꢀ21.2 mm, 10 μm) was used
under the same conditions as the analytical RP-HPLC but with
a flow rate of 20 mL min-1. Mass spectrometry (MS) was
recorded on a VG Quattro II spectrometer using electrospray
(ESP) ionization, and data collection was done with Masslynx
software.
Enzyme Assay.17 Determination of the aminopeptidase cata-
lytic activity was based on the cleavage of the substrate
L-leucine-p-nitroanilide (L-Leu-pNA)48 into L-leucine and
p-nitroaniline. This latter compound displays a characteristic
light absorption maximum at 405 nm. Pellets, prepared as
described above, were thawed and resuspended using a Polytron
homogenizer in enzyme assay buffer containing 50 mM Tris-
HCl (pH 7.4), 140 mM NaCl, 0.1% (w/v) bovine serum albumin
(BSA), and 100 μM phenyl methyl sulfonyl fluoride. The
incubation mixture comprised 50 μL of membrane homogenate,
200 μL of L-Leu-pNA (1.5 mM), and 50 μL of enzyme assay
buffer alone or with test compound. The amount of membrane
homogenate corresponded to 1.5ꢀ105 transfected HEK293 cells
in each well. Assays were carried out at 37 °C in 96-well plates
(Medisch Labo Service, Menen, Belgium), and the formation
of p-nitroaniline was followed by measuring the absorption at
405 nm every 5 min between 10 and 50 min in a Tecan M200 96-
well reader. The enzymatic activities were calculated by linear
regression analysis of the time-wise increase of the absorption.
Stability Experiments.17 The stabilities of the compounds
were compared in the presence of CHO-K1 cell membranes.
Membrane pellets were thawed and resuspended using a Poly-
tron homogenizer in 50 mM Tris-HCl (pH 7.4) enzyme assay
Each peptide was at least 98% pure as assessed by analytical
RP-HPLC. The molecular weights were confirmed by ESI-MS
(Supporting Information).
Peptide Synthesis. The synthesis of all peptides was carried
out by solid-phase peptide synthesis using tert-butoxycarbonyl
(Boc) N-terminal protected amino acids. The peptides were
synthesized on Boc-Phe Merrifield resin (0.57 mmol/g) and side
chain protecting groups were: Tyr(2,6-di-Cl-Bzl), His(Tos). The
removal of the Boc protection was carried out in 50% TFA in
CH2Cl2 (2ꢀ10 min) and 10% TEA in CH2Cl2 (2ꢀ5 min) was
used for neutralization. Coupling of amino acids (3 equiv) was

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18 5617
buffer, and the assays were carried out in polyethylene 24-well
plates (Elscolab, Kruibeke, Belgium). Preincubations were car-
ried out for 40 min at 37 °C in 250 μL containing 150 μL of
membrane homogenate (corresponding with 4ꢀ105 CHO-K1
cells), 50 μL enzyme assay buffer without or with 30 mM EDTA/
600 μM 1,10-phenantrolin, and 50 μL enzyme assay buffer
without or with the different compounds or unlabeled Ang IV
(60 μM for nonspecific binding). Then the binding assay was
initiated by adding 50 μL of enzyme assay buffer containing
[3H]Ang IV (18 nM, without or with 30 mM EDTA/600 μM
1,10-phenanthrolin), and the mixture was further incubated for
30 min at 37 °C. Final chelator concentrations (when present)
were 5 mM EDTA and 100 μM 1,10-phenantrolin, the final
[3H]Ang IV concentration was 3 nM, and the final unlabeled
ligand concentrations are indicated in Figure 3. After incuba-
tion, the mixture was vacuum filtered using an Inotech 24-well
cell harvester through GF/B glass fiber filters (Whatman) pre-
soaked in 1% (w/v) BSA. After drying, the radioactivity re-
tained in the filters was measured (after adding 3 mL of
scintillation liquid (Optiphase Hisafe)) using a β-counter
(Perkin-Elmer). [3H]Ang IV was characterized previously.53
AT1 Receptor Binding.17 Chinese hamster ovary cells stably
expressing the human angiotensin II AT1 receptor (CHO-
AT1)55 were used to test the affinity of the compounds for the
AT1 receptor. Before the experiment, the plated cells were
washed twice with PBS buffer at room temperature (0.5 mL
per well) and then incubated with DMEM medium (400 μL) for
15 min at 37 °C. Next, a competition binding was performed
by incubating the cells with concentrations of compound 10-5
(50 μL) and [3H]Valsartan (final concentration of 1.5 nM, 50 μL)
for 40 min at 37 °C. Nonspecific binding was measured with
Candesartan (final concentration of 1 μM, 50 μL). After in-
cubation, cells were washed 3 times with cold PBS (4 °C). The
cell bound radioactivity in each well was subsequently solubi-
lized with 500 μL of sodium hydroxide (0.2 M) and counted for
3 min in a liquid scintillation counter after addition of 3 mL of
scintillation liquid (Optiphase Hisafe, Perkin-Elmer).
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Data Analysis.17 All experiments were performed at least two
times with duplicate determinations each. The calculation of
IC50 values from competition binding (or enzyme inhibition)
experiments were performed by nonlinear regression analysis
using GraphPad Prism 4.0. The equilibrium dissociation con-
stants (Ki values) of the tested compounds in the binding and
enzyme assays were calculated using the equation Ki=[IC50/(1 þ
[L]/K)] in which [L] is the concentration of free radioligand
(binding) or free substrate concentration (enzyme assay) and K
the equilibrium dissociation constant (KD) of [3H]Ang IV (from
saturation binding experiments) or the Michaelis-Menten con-
stant (Km) for substrate cleavage.56
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D.; Misicka, A.; Karoyan, P.; Vanderheyden, P.; Vauquelin, G.;
Tourwe, D. Beta-homo-amino acid scan of angiotensin IV. J. Med.
Chem. 2008, 51, 2291–2296.
Acknowledgment. We thank the “The Fund for Scientific
Research;Flanders” (FWO Vlaanderen) for financial sup-
port. Heidi Demaegdt is a postdoctoral researcher, Debby
Feytens is a research assitant of the “Fund for Scientific
Research;Flanders” (FWO Vlaanderen), and Patrick Van-
derheyden is holder of a VUB research fellowship. We are
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grateful to G. Toth and E. Szemenyei (Biological Research
Center, Szeged, Hungary) for the tritiation of AngIV.
Supporting Information Available: Characterization of com-
pounds 2-9. This material is available free of charge via the
Internet at http://pubs.acs.org.
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