From the Division of Research, Dyax Corp., Cambridge,
Massachusetts 02139 and ¶ Human Genome Sciences, Inc.,
Rockville, Maryland 20850
Received for publication, December 18, 2002, and in revised form, February 21, 2003
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ABSTRACT |
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Angiotensin-converting enzyme 2 (ACE2), a
recently identified human homolog of ACE, is a novel
metallocarboxypeptidase with specificity, tissue distribution, and
function distinct from those of ACE. ACE2 may play a unique role in the
renin-angiotensin system and mediate cardiovascular and renal function.
Here we report the discovery of ACE2 peptide inhibitors through
selection of constrained peptide libraries displayed on phage.
Six constrained peptide libraries were constructed and selected against
FLAG-tagged ACE2 target. ACE2 peptide binders were identified and
classified into five groups, based on their effects on ACE2 activity.
Peptides from the first three classes exhibited none, weak, or moderate inhibition on ACE2. Peptides from the fourth class exhibited strong inhibition, with equilibrium inhibition constants
(Ki values) from 0.38 to 1.7 µM.
Peptides from the fifth class exhibited very strong inhibition, with
Ki values <0.14 µM. The most potent
inhibitor, DX600, had a Ki of 2.8 nM.
Steady-state enzyme kinetic analysis showed that these potent ACE2
inhibitors exhibited a mixed competitive and non-competitive type of
inhibition. They were not hydrolyzed by ACE2. Furthermore, they did not
inhibit ACE activity, and thus were specific to ACE2. Finally, they
also inhibited ACE2 activity toward its natural substrate angiotensin I, suggesting that they would be functional in vivo. As
novel ACE2-specific peptide inhibitors, they should be useful in
elucidation of ACE2 in vivo function, thus contributing to
our better understanding of the biology of cardiovascular regulation.
Our results also demonstrate that library selection by phage display
technology can be a rapid and efficient way to discover potent and
specific protease inhibitors.
One major control mechanism for blood pressure homeostasis is the
renin-angiotensin system, in which angiotensin-converting enzyme
(ACE)1 is a vital player.
ACE, a zinc metallopeptidase, promotes blood pressure elevation at
least in part by cleaving the inactive angiotensin I (Ang I) to the
vasoconstrictor Ang II (1) and inactivating the vasodilator bradykinin
by cleavage (2). Its role in regulating blood pressure and renal
function is underscored by the effective clinical use of ACE inhibitors
in the treatment of hypertension and other cardiovascular diseases.
ACE2 is a recently identified human homolog of ACE (3, 4). It contains
a single zinc-binding catalytic domain, which is 42% identical to the
human ACE active domain. Genomic structure comparison suggests that
ACE2 and ACE genes arose by duplication of a common ancestor (3).
Although both ACE2 and ACE are zinc metallopeptidases and
angiotensin-converting enzymes with a membrane-associated and a
secreted form, many differences exist between these two enzymes (for
reviews, see Refs. 5 and 6). First, they are different in enzymatic
activity; ACE2 is a carboxypeptidase, removing the C-terminal residue
from the decapeptide Ang I to form angiotensin-(1-9) (Ang-(1-9)) (3,
4), whereas ACE is a dipeptidase, cleaving the C-terminal dipeptide
from Ang I to form the octapeptide Ang II. Second, ACE2 and ACE have
different substrate specificities; ACE2 cleaves Ang I, Ang
II, apelin-13, apelin-36, dynorphin A-(1-13), and des-Arg bradykinin
(3, 7); ACE cleaves Ang I, Ang-(1-9), bradykinin, and many other
bioactive peptides such as substance P, neurotensin, and enkephalin
(8). Another difference between these two enzymes is the inhibitor
specificity; ACE2 cannot be inhibited by ACE inhibitors (3, 4).
Finally, a difference in tissue expression has been observed; ACE2 is
primarily expressed in the heart, kidney and testis, whereas ACE is
more ubiquitously expressed in tissues including heart, lung, kidney,
colon, small intestine, ovary, testis, prostate, liver, skeletal
muscle, pancreas, and thyroid (3, 4).
One in vitro function of ACE2 is the catalysis of Ang I to
Ang-(1-9) (3, 4). In vivo detection of Ang-(1-9) in rat
and human plasma has been described, and the levels are twice that of
Ang II (9-11). Although Ang-(1-9), itself catabolized by
ACE, is considered a competitive inhibitor of ACE (3, 12),
it has been demonstrated to have weak pressor effects in anesthetized rats and dogs, and weak vasoconstricting activity in isolated rat aorta
(9). A recent study2 with
Ang-(1-9) also indicates that Ang-(1-9) is a pressor agent that
potentiates Ang II-mediated vasoconstriction in isolated rat aortic
rings and pressor effects in the awake rat. ACE2 also cleaves Ang II to
produce Ang-(1-7). Ang-(1-7) is proposed to be a vasodilator in
animal studies (13, 14). However, its significance in humans is still
controversial (6). Unlike ACE, ACE2 does not cleave bradykinin.
However, ACE2 cleaves and inactivates des-Arg bradykinin, a local
vasodilator functioning through binding to the B1 receptor expressed
when inflammation or tissue damage occurs (15). In contrast,
bradykinin, cleaved and inactivated by ACE, functions as a systemic
vasodilator through binding to the B2 receptor (15). Based on the
potential in vivo functions of Ang-(1-9) and des-Arg
bradykinin, it is tempting to speculate that ACE2 plays a role in the
regulation of vasomotor tone and blood pressure at least in part
through cleavage of Ang I and des-Arg bradykinin. However, a recent
knock-out mice study demonstrates that disruption of ACE2 in mice does
not alter blood pressure and renal function but leads to increased
levels of Ang II, up-regulation of hypoxia-induced genes, and decreased
cardiac contractility that can be rescued by a second mutation causing
ACE deficiency (16). Thus, ACE2 appears to be essential for regulating
heart function in vivo. However, its role in blood pressure
regulation remains unclear. Animal studies with specific ACE2
inhibitors should provide more information to our understanding of the
physiological roles of ACE2 in cardiovascular regulation.
Here we described the discovery of novel ACE2 peptide inhibitors
through selection of constrained peptides from libraries displayed on
filamentous phage. We discovered very potent ACE2 peptide inhibitors
with Ki values as low as 2.8 nM. These peptides were stable inhibitors, not hydrolyzed by ACE2, and were specific for ACE2. As novel ACE2-specific peptide inhibitors, they
should be useful in elucidation of ACE2 in vivo function.
Materials--
Biotinylated anti-FLAG M2 monoclonal antibody,
FLAG peptide, ACE, angiotensin I, NAD, resazurin, diaphorase, and
captopril were purchased from Sigma. Horseradish peroxidase-conjugated
anti-M13 antibody was purchased from Amersham Biosciences.
Tetramethylbenzidine peroxidase substrate solution was purchased
from Kirkegaard & Perry Laboratories (Gaithersburg, MD).
Ser-Mag streptavidin magnetic beads were purchased from Seradyn
(Ramsey, MN). Fluorogenic ACE2 substrate M-2195 was purchased from
Bachem (King of Prussia, PA). Leucine dehydrogenase was purchased from
Calbiochem. Teprotide (pyro-Glu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro), also
known as bradykinin potentiating factor SQ20881, was purchased from ICN
Pharmaceuticals (Costa Mesa, CA).
Expression and Purification of ACE2--
The cDNA encoding
the extracellular domain of ACE2 was cloned into a baculovirus transfer
vector pA2. A recombinant baculovirus was generated by transfecting
Sf9 cells with the ACE2 expression vector. ACE2 protein (~85
kDa) was purified from conditioned media of Sf9 cells that had
been infected with the recombinant baculovirus. FLAG-tagged ACE2 was
purified by affinity purification from the supernatant of 293 cells
that had been transiently transfected with a mammalian expression
vector expressing the FLAG-ACE2 protein.
Library Construction--
The disulfide-constrained loop peptide
libraries, TN6/6, TN7/4, TN8/9, TN9/4, TN10/9, and TN12/1, were
constructed in MANP, a derivative of M13mp18. This vector has the LacZ
complementation system removed, a bla
(AmpR) gene, and a modified junction between
signal sequence and coding region of gene III. Two unique restriction
sites, NcoI and PstI, were introduced into this
modified junction for easy directional cloning. The variegated DNA
(vgDNA) flanked by constant sequences was synthesized by MorphoSys
(Munich, Germany) using TRIM technology. In TRIM, one can add preformed
trinucleotides allowing complete control of what relative abundance of
each amino acid type is allowed at each variegated position. The vgDNA
was PCR-amplified using a top strand primer containing an
NcoI site and a bottom-strand primer containing a
PstI site, cleaved with NcoI and PstI,
and ligated into similarly cleaved MANP vector. The peptides encoded by
vgDNA and the flanking sequences of each library are shown in Table
I. The TN6/6, TN7/4, TN8/9, TN9/4,
TN10/9, and TN12/1 libraries encode peptide loops of 6, 7, 8, 9, 10, and 12 amino acids (counting the cysteines), respectively.
Two linear libraries Ph.D.-7 and Ph.D.-12 were obtained from New
England Biolabs (Beverly, MA). Ph.D.-7 has 7 variable residues (XXXXXXXGGGSAET), whereas Ph.D.-12 has 12 variable residues
(XXXGGGSAET).
Selection from Libraries--
Peptides from the six
constrained loop libraries and two linear libraries were selected using
FLAG-ACE2 as the target, which can be immobilized to
streptavidin-coated magnetic beads via a biotinylated anti-FLAG
antibody. To remove binders to streptavidin beads, anti-FLAG antibody,
and FLAG peptide, the libraries were depleted 5 times by binding to
FLAG peptide/biotinylated anti-FLAG antibody-immobilized beads before
selection against the target. The depleted libraries were incubated
with 6 µg of FLAG-ACE2 in 300 µl of phosphate-buffered saline (PBS)
for 1 h and then incubated with biotinylated anti-FLAG
antibody-immobilized beads for 1 h. The beads were washed 7 times
with PBS, 0.1% Tween 20 (PBST) to remove unbound phage. The bound
phage were then eluted with FLAG peptide (100 µg/ml) in Tris buffer
(10 mM Tris-Cl, 150 mM NaCl, pH 7.5) for 30 min. Eluted phage were amplified and underwent two more similar rounds
of selection and amplification. In round 1, the six constrained peptide
libraries, TN6/6, TN7/4, TN8/9, TN9/4, TN10/9, and TN12/1, were
selected separately. To accelerate the selection procedures, in the
subsequent rounds of selection, these six libraries were combined into
two pools: pool A composed of TN6/6, TN7/4, and TN8/9, and pool B
composed of TN9/4, TN10/9, and TN12/1. The two linear peptide
libraries, Ph.D.-7 and Ph.D.-12, were combined as Ph.D.-7/12, from the
beginning of selection.
Screening for ACE2-binding by Phage ELISA--
Phage enriched
from the third round of selection were screened by phage ELISA for ACE2
binding. Immulon 2 96-well plates were coated with streptavidin for
1 h at 37 °C and subsequently coated with biotinylated
anti-FLAG antibody for 1 h at room temperature. Half of the plates
were further coated with FLAG-ACE2 as the target plates, and the other
half were coated with FLAG peptide as the background plates. The amount
of each protein or peptide coated was 100 ng per well. The coated
plates were then incubated for 1 h with 1:2 diluted overnight
phage cultures that were made by inoculating phage from individual
plaques into bacteria cells. After washing 7 times with PBST, the
plates were incubated with horseradish peroxidase-conjugated anti-M13
antibody for 1 h, washed 5 times, developed with
tetramethylbenzidine solutions, and read at 630 nm with an ELISA plate reader.
DNA Sequencing and Peptide Synthesis--
DNA sequences encoding
displayed peptides of positive phage binders were amplified by PCR and
sequenced by automatic sequencing. Based on the motif sequences
identified by sequence cluster analysis, representative peptides from
each motif were synthesized. The crude peptides were ordered from
Sigma. The peptides were then cleaved from resin with trifluoroacetic
acid, purified using reverse phase-high pressure liquid chromatography,
oxidized, and lyophilized. The purity of each oxidized peptide was
greater than 90%. The peptides were dissolved in dimethyl sulfoxide
(Me2SO) at a stock concentration of 25 mM, aliquoted, and stored at ACE2 Enzyme Assays Using Synthetic Substrate--
The enzymatic
activity of ACE2 was assayed using a fluorogenic substrate, M-2195,
7-methoxycoumarin-Tyr-Val-Ala-Asp-Ala-Pro-Lys(2,4-dinitrophenyl)- OH.
Cleavage of this substrate at the C-terminal Lys residue by ACE2
removes the 2,4-dinitrophenyl moiety that quenches the fluorescence of
the 7-methoxycoumarin moiety, thus resulting in increased fluorescence. For the initial screen of inhibitors from ACE2 binders, each peptide was incubated with 20 nM ACE2 for 10 min at room
temperature at a concentration of either 100 µM or 2 mM in 100 mM Tris-HCl, pH 7.4, 0.1% Tween. The
amount of Me2SO was kept the same in each sample. Following
incubation, the substrate M-2195 was added to achieve a final
concentration of 50 µM, and the plates were read immediately on a SpectraMAX Gemini fluorescence spectrophotometer at an
excitation wavelength of 328 nm and an emission wavelength of 392 nm.
Fluorescence was monitored at 36-s intervals for 15 min. For
IC50 determination, increasing concentrations of peptide inhibitors (0-100 µM) were incubated with 20 nM ACE2 prior to the addition of the substrate M-2195 (50 µM). For Ki determination, 7 nM ACE2 was incubated with the peptide inhibitors ranging
from 0.1- to 5-fold, the IC50 value of individual
inhibitor. The concentrations of added substrate M-2195 ranged from 14 to 50 µM. The inhibition assay of ACE was carried out
essentially in the same way as that of ACE2 with the same substrate
M-2195.
ACE2 Enzyme Assays Using Natural Substrate Ang I--
ACE2
activity toward its natural substrate was measured by an assay based on
a spectrofluorometric enzyme-coupled system. ACE2 hydrolyzes Ang I
(NH2-DRVYIHPFHL-COOH) to produce Ang-(1-9) (NH2-DRVYIHPFH-COOH) and leucine. The released leucine can
then be monitored by the activity of leucine dehydrogenase with
concomitant conversion of NAD+ to NADH. The production of
NADH is coupled to the diaphorase-catalyzed reduction of resazurine to
resorufin, which can be monitored on a fluorescence reader.
In the inhibition assay, ACE2 was incubated with the
peptide inhibitor or ACE inhibitor for 10 min at room temperature in reaction buffer consisting of 100 mM Tris, pH 8, 0.01%
Tween, 4 mM NAD, 25 µM resazurin, 0.1 unit/ml
leucine dehydrogenase, and 0.1 unit/ml diaphorase. The amount of
Me2SO was kept the same in each sample. The substrate Ang I
was subsequently added, and following an additional 30 min of
incubation, the plate was read on a SpectraMAX Gemini fluorescence
spectrophotometer at an excitation wavelength of 565 nm and an emission
wavelength of 585 nm. Fluorescence was monitored at 1-min intervals for
2 h. The IC50 and Ki values were
determined similarly as described for the assay with the synthetic substrate.
Measurement of Binding Affinities by BIAcore--
The binding
affinities of the selected peptides for ACE2 were measured using a
BIAcore 3000. ACE2 (250 nM) in 50 mM acetate, pH 4.0, was coupled to the dextran surface of a CM5 sensor chip by the
standard
N-hydroxysuccinimide/1-ethyl-3-(dimethylaminopropyl)-carbodiimide coupling procedure to a ligand density of 5425 response units. A flow
cell containing blocked dextran was used as a control. Experiments were
performed in 100 mM Tris, pH 8.0, plus 0.01% Tween 20. Serially diluted peptide solutions (500, 250, 125, 62.5, and 31.3 nM) were injected at 20 µl/min for 2 min using the
kinject function. Following a 3-min dissociation, the surface was
regenerated with a quick inject of 1 M NaCl for 25 s
at 50 µl/min. Sensorgrams were fit by global analysis using the
BIAevaluation software 3.1 for a Langmuir 1:1 interaction. The
equilibrium dissociation constants (Kd) were
calculated from kinetic rate constants (Kd = koff/kon).
Selection of ACE2 Peptide Binders--
Six constrained loop
peptide libraries, TN6/6, TN7/4, TN8/9, TN9/4, TN10/9, and TN12/1, and
two linear peptide libraries Ph.D.-7 and Ph.D.-12, were used for
selection against FLAG-ACE2 target. After incubation with libraries in
solution, the target was immobilized to streptavidin-coated magnetic
beads via biotinylated anti-FLAG antibody. The bound phage were eluted
with FLAG peptide. After three rounds of selection, the fraction of
input, which was calculated as the total amount of output phage divided
by the total amount of input phage, increased from 10
To identify positive phage binders, the eluted phage from the third
round of selection were screened by ELISA. The target plates were
sequentially coated with streptavidin, anti-FLAG antibody, and
FLAG-ACE2, and the background plates were sequentially coated with
streptavidin, anti-FLAG antibody, and FLAG peptide. Phage ELISA of
selected isolates (n = 1916) from constrained peptide libraries showed that ~32% of the isolates were ACE2 binders, with
target to background signal ratios
The ELISA positive isolates (n = 613) from constrained
libraries were sequenced. The amino acid sequences of the encoded
peptides were analyzed for shared motifs and, as shown in Fig.
1, 10 major motifs were found. Some
clusters were found in multiple libraries, some were found exclusively
in one library. For example, the
ALFCV(D/E)F and
RXXXRDSRC motifs were found in both
TN6/6 and TN10/9 libraries (Fig. 1, A and D); the
(F/Y)C(F/L/I)(D/E)F motif, similar to the
ALFCV(D/E)F motif, was found in TN8/9 and TN10/9
libraries (Fig. 1B); the
DXCXTWXXPC motif was found in TN7/4 and TN8/9 libraries (Fig. 1I); and
the CF(D/E)W(E/D) motif was identified in the TN7/4, TN8/9, and TN12/1 libraries (Fig. 1F). Whereas the
(D/E)C(E/D)WXX(F/W) and
CXPXRXXPWXXC
motifs were found only in the TN12/1 library (Fig. 1, C and
J), the
CXTXDCV motif in
the TN6/6 library (Fig. 1E) and the
(Y/W)EXCH(W/Y)XP and
KECKFGYXXCLXW
motifs were found in the TN8/9 library (Fig. 1, G and
H). Based on these consensus motifs and the number of
isolates occurring per sequence, 23 peptides representing these 10 motifs were synthesized.
Screening of Peptide Binders for ACE2
Inhibitors--
The 23 peptides synthesized as ACE2 peptide binders
were further screened for ACE2 inhibitors by assays using fluorogenic substrate M-2195. For initial screening of inhibitors, ACE2 (20 nM) was incubated with each peptide at 100 µM
prior to the addition of 50 µM substrate. Based on their
effects on ACE2 enzyme activity, the peptides were classified into 5 groups with none (
Some peptides from the same motif showed either no or weak inhibition.
Peptides from the ALFCV(D/E)F,
(F/Y)C(F/L/I)(D/E)F, and
(D/E)C(E/D)WXX(F/W) motifs fell
into these categories (Table II and Fig. 1). Peptides from each of the CF(D/E)W(E/D),
CXTXDCV, and
KECKFGYXXCLXW motifs showed none, weak, and moderate inhibition, respectively. Whereas the strong peptide inhibitors were from the
RXXXRDSRC, (Y/W)EXCH(W/Y)XP, and
DXCXTWXXPC motifs, the very strong peptide inhibitors were exclusively from the
CXPXRXXPWXXC
motif identified from the TN12/1 library.
Among the very strong inhibitors (DX512, DX513, DX599,
DX600, DX601, and DX602), DX600 was the most potent inhibitor with a
Ki of 2.8 nM (Table II and Fig.
2). Kinetic analyses of DX600 by Dixon
plots (Fig. 2) indicated a mixed inhibition pattern consisting of
competitive and non-competitive components, with the maximum velocity
(Vmax) reduced, and the apparent Michaelis constant (Km) increased. The other very strong and
strong inhibitors also showed a similar inhibition pattern (data not shown).
Binding Affinity of ACE2 Inhibitors--
The binding
affinity of the very strong peptide inhibitors was measured by BIAcore
as described under "Experimental Procedures." Sensorgrams were
analyzed using the simultaneous association and dissociation 1:1
Langmuir fitting model. For all measurements, the
Stability of ACE2 Inhibitors--
To test the stability of ACE2
peptide inhibitors, DX600 and DX512 peptides were individually
incubated with ACE2 (20 nM) for 10 min or 20 h at room
temperature prior to the addition of M-2195. The results showed that
incubation of ACE2 with DX600 or DX512 for up to 20 h did not
affect the inhibitory activities of these peptides (Fig.
4). Analysis by liquid
chromatography/mass spectrometry showed that ACE2 did not hydrolyze
DX600 peptide after 20 h of incubation with ACE2 at room
temperature (data not shown). Taken together, these results indicated
that DX600 and other peptides were stable ACE2 inhibitors not
hydrolyzed by ACE2.
Specificity of ACE2 Inhibitors--
To determine whether the
identified peptide inhibitors were specific to ACE2, we analyzed the
effect of DX600 on the enzyme activity of the other ACE. In this assay,
increasing concentrations of DX600 peptide were incubated with ACE2 (20 nM) or ACE (7.5 nM) prior to the addition of
the substrate M-2195. DX600, which greatly inhibited ACE2 activity with
a Ki of 2.8 nM, did not inhibit ACE
activity at concentrations up to 100 µM (Fig. 5A). Similarly, other ACE2
inhibitors including DX512 and DX513 did not inhibit ACE (data not
shown). These results indicated that DX600 and other peptide inhibitors
were specific ACE2 inhibitors.
Likewise, the effect of an ACE inhibitor on ACE2 activity was also
tested. As shown in Fig. 5B, teprotide, the ACE peptide inhibitor, inhibits the activity of ACE at 7 nM with an
IC50 of about 38 nM but did not inhibit ACE2
activity at concentrations up to 100 µM. Captopril,
another ACE inhibitor, also showed no inhibition on ACE2 activity (data
not shown), which was consistent with reports from others (3).
Inhibition on ACE2 Activity toward Its Natural
Substrate Ang I--
The inhibitory activities of identified peptides
were determined based on ACE2 assays using synthetic fluorogenic
substrate. Such inhibition on ACE2 activity toward the synthetic
substrate may not necessarily correspond to the inhibition on ACE2
toward its natural substrate. Thus, to determine whether the identified peptide inhibitors also inhibit ACE2 toward its natural substrate Ang
I, we developed an ACE2 assay using Ang I as the substrate based on a
spectrofluorometric enzyme-coupled system, which is superior to high
pressure liquid chromatography-based assays that are discontinuous and
time consuming. This assay is based on the cleavage of the C-terminal
leucine from Ang I substrate after ACE2 catalysis. The leucine
formation is then monitored by the activity of leucine dehydrogenase
with concomitant conversion of NAD+ to NADH. The production
of NADH is coupled to the diaphorase-catalyzed reduction of resazurine
to resorufin, which can be monitored on a fluorescence reader. Before
its use in ACE2 inhibition studies, experiments were undertaken to
verify that the observed inhibition was not due to the other two
enzymes (leucine dehydrogenase and diaphorase) in the assay (data not shown).
The most potent inhibitor DX600 was tested by this assay. Kinetic
analysis of DX600 showed a Ki of 2.8 nM
and a mixed inhibition pattern consisting of competitive and
non-competitive components as demonstrated by Dixon plot (Fig.
6), which were similar to the results
from ACE2 assays using the synthetic substrate. These results indicated
that DX600 inhibited ACE2 activity toward its natural substrate with
the same potency as inhibition on ACE2 toward the synthetic substrate.
Another peptide inhibitor, DX512, was also tested by this assay. As for
DX600, the Ki and inhibition pattern for DX512 were
similar to those when using the synthetic substrate (data not
shown).
Because ACE2 and ACE share the same natural substrate Ang I, we wanted
to know if ACE inhibitors may also inhibit ACE2 activity in this assay.
Two ACE inhibitors, teprotide and captopril, were tested for inhibition
on ACE2 activity toward Ang I. As shown in Fig.
7, the IC50 of DX600 was ~8
nM, whereas the IC50 of the ACE peptide
inhibitor teprotide was over 700 µM, and the
IC50 of the small compound inhibitor captopril was over 1 mM. This further indicated that DX600 was a specific ACE2
inhibitor.
Here we report novel ACE2 specific peptide inhibitors discovered
through selection of peptides from libraries displayed on M13
filamentous phage. Six constrained peptide libraries were constructed
and used for selection against FLAG-ACE2 target. In parallel, two
commercially available linear peptide libraries were also used for
selection. Surprisingly, no ACE2-binding phage were identified from the
linear libraries; all ACE2 binders were obtained from the constrained
loop libraries. Sequence analysis of positive phage isolates identified
10 motifs. The 23 representative peptides derived from these motifs
showed a range of inhibitory properties from none to very potent.
Peptides derived from half of the motifs exhibited either no or weak
inhibition, whereas peptides from the other half of the motifs
exhibited moderate, strong, or very strong inhibition, showing that
selection of peptide libraries for binders to enzymes by phage display
technology is a rapid and efficient way to discover enzyme inhibitors.
Interestingly, the most abundantly isolated motifs such as
ALFCV(D/E)F,
(F/Y)C(F/L/I)(D/E)F, and CF(D/E)W(E/D)
were very poor inhibitors. In contrast, the highly inhibitory sequences
such as those from the
CXPXRXXPWXXC motif were each seen as unique sequences. Peptides derived from the
same motif are likely to bind ACE2 at the same site and thus share
similar binding and inhibitory properties. However, due to the slight
sequence variations between consensus or non-consensus residues,
peptides from the same motif could show either no or weak inhibition.
Such examples included peptides derived from the
ALFCV(D/E)F,
(F/Y)C(F/L/I)(D/E)F, and
(D/E)C(E/D)WXX(F/W) motifs.
Similarly, the extent of inhibition among peptides from the same
inhibitory motif could vary significantly. This is well illustrated by
the peptides from the
CXPXRXXPWXXC
motif. Although all of the peptides derived from this motif showed very
strong inhibition, the inhibitory potency varied, with
Ki values ranging from 2.8 to 139 nM.
Thus, in order to find the best inhibitor, it is necessary to
synthesize and test many peptides from an inhibitory motif.
The most potent ACE2 inhibitor was derived from the
CXPXRXXPWXXC
motif. Interestingly, a recent study of ACE2 biological
substrates identifies a consensus sequence of
P-X(1-3)-P-hydrophobic, where hydrolysis
occurs between proline and the hydrophobic amino acid (7). Although our
inhibitory motif
CXPXRXXPWXXC
shows resemblance to the substrate consensus sequence, three key
differences exist. First, there is a conserved basic amino acid
arginine lying between the two prolines in the inhibitory motif but not
in the substrate consensus. Second, the space between two prolines is
different: four residues in the inhibitory motif and one to three
residues in the substrate consensus. Third, the inhibitory sequence is constrained by disulfides whereas the substrate is not. Probably because of these differences, peptides from this inhibitory motif were
stable inhibitors and not hydrolyzed by ACE2, indicating that they were
not better ACE2 substrates than the assay substrate M-2195 but true inhibitors.
As expected, these peptides also inhibited ACE2 enzymatic activity
toward its natural substrate, Ang I. For DX600 and DX512, the
Ki values determined by using Ang I were similar to
those determined by using M-2195. Thus, these peptides were functional
in inhibiting ACE2 toward its natural substrate. However, they were not
inhibitory on ACE, which shares great sequence homology and the same
natural substrate Ang I with ACE2. Likewise, the ACE inhibitors such as
the peptide inhibitor teprotide and the D-benzylsuccinic
acid derivative captopril specifically inhibited ACE but not ACE2.
Thus, although ACE2 and ACE share a homologous catalytic domain, they
are structurally distinct.
Kinetic analyses of the strong and very strong ACE2
inhibitors by Dixon plots showed that these inhibitors exhibited a
mixed competitive and non-competitive type of inhibition, with
Vmax reduced and apparent Km
increased. These data suggest that the inhibitors bind to a site
adjacent to the active site in a manner that interferes with substrate
binding. Interestingly, ACE inhibitors such as captopril also exhibit a
mixed competitive and non-competitive type of inhibition toward ACE
(18, 19).
The most potent ACE2 inhibitor (DX600) identified had a
Ki of 2.8 nM. It is remarkable that a
peptide inhibitor with a Ki in the low single digit
nanomolar range can be extracted from the selection of first generation
peptide libraries, demonstrating the utility of such well constrained
peptide libraries for the rapid identification of high affinity enzyme
inhibitors. If more potent inhibitors with Ki values
at subnanomolar concentrations are needed, affinity maturation by
peptide optimization through soft randomization can be conducted
(20).
Although small molecule ACE2 inhibitors have been recently
synthesized (21), the peptides discovered here are the first ACE2-specific peptide inhibitors. These inhibitors should be useful for
in vivo studies to elucidate ACE2 function. In fact, one of the inhibitors, DX512, which is the first one synthesized and tested to
be a very strong inhibitor, has been studied in spontaneously hypertensive rats.2 Upon intravenous bolus injection in the
awake rats, the peptide inhibitor DX512, but not the control peptide
DX510 (with no inhibitory effect on ACE2), caused a
dose-dependent depressor response characterized by an
initial transient fall in mean arterial pressure lasting about 1-2 min
at the lower doses and about 6 min in duration at the 3 mg/kg dose
level, with the maximal average depressor response at 70.5 ± 4.6 mm Hg from an average mean arterial pressure of 155 ± 10 mm Hg.
The depressor response was also accompanied by transient tachycardia.
The in vivo demonstration of the antihypertensive effect of
the ACE2 inhibitor is not consistent with the recent findings from the
knock-out mice study showing that disruption of ACE2 in mice does not
alter blood pressure and renal function (16). Further investigation
including inhibition studies with ACE2-specific inhibitors will be
needed to elucidate the physiological roles of ACE2 in blood pressure mediation.
In summary, potent ACE2 peptide inhibitors with the lowest
Ki in the low single digit nanomolar range were
discovered by selecting constrained peptide libraries. These inhibitors
exhibited a mixed competitive and non-competitive type of inhibition.
They were stable inhibitors not hydrolyzed by ACE2 and were specific to
ACE2. These specific ACE2 inhibitors can be used in in vivo studies to elucidate the physiological functions of ACE2 in
cardiovascular regulation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Insert and flanking sequences in constrained loop libraries
20 °C. Peptide concentrations were quantified by extinction coefficient.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
6 to
10
5 at the first round to 10
2 to
10
1 by the third round.
2. ELISA of selected phage
isolates (n = 144) from the linear libraries,
however, showed no binders.
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Fig. 1.
Sequence cluster analysis of ACE2 peptide
binders. Peptide sequences from positive ELISA isolates were
aligned to search for motif sequences. Shown here are peptide sequences
comprising 10 motifs. Representative peptide sequences are listed in
each motif. The motif sequences in each cluster are in boldface
type. The template sequence is shown at the top of each
cluster. The DX numbers at the right sides of the sequences are the
names of the peptides synthesized, and the annotation within
parentheses indicates the inhibitory activity of each peptide, same as
described in Table II: , no inhibition; +, weak inhibition; ++,
moderate inhibition; +++, strong inhibition; +++, very strong
inhibition.
), weak (+), moderate (++), strong (+++), and very
strong (++++) inhibition, respectively (Table
II). Peptides from the first group show
no inhibition on ACE2 activity at a peptide concentration of 100 µM. Some of these peptides were tested at concentrations
up to 2 mM, and yet showed no inhibition on ACE2 (data not
shown). Peptides from the second group showed weak inhibition on ACE2,
exhibiting 20-60% inhibition at 100 µM. These peptides
could have Ki values no lower than about 50 µM. Peptides from the third group showed moderate
inhibition, exhibiting about 80% inhibition at 100 µM;
these peptides could have Ki of ~25
µM. Peptides from the fourth group showed strong
inhibition, exhibiting about 99% inhibition at 100 µM;
Ki could be around 1 µM. Peptides from
the fifth group showed very strong inhibition, exhibiting complete
inhibition at 100 µM; Ki could be <1
µM. Peptides with strong and very strong inhibition were
further analyzed to determine their Ki values. The
strong inhibitors have Ki values ranging from 0.38 to 1.7 µM, whereas the very strong inhibitors have
Ki values ranging from 3 to 139 nM
(Table II).
Sequences of synthesized peptides and their effects on ACE2 activity
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Fig. 2.
Ki determination of DX600
peptide using ACE2 assays with the synthetic substrate. DX600, at
concentrations ranging from 5 to 73 nM, was preincubated
with 7 nM ACE2. The substrate M-2195 was added at
concentrations ranging from 12 to 50 µM. A,
Dixon plot. Filled squares, 12.6 µM substrate
(M-2195); open squares, 15.2 µM; filled
triangles, 17.7 µM; ×, 20.2 µM; open triangles, 22.8 µM;
filled circles, 27.8 µM; and open
circles, 45.6 µM. B, Dixon secondary
plot. The slope at each substrate concentration in A was
plotted against the reciprocal substrate concentration. Data were
fitted to a linear regression (y = mx + b, where m = Km/(Ki × Vmax) = 1.7077, b = 0.0089). Km (20.6 µM) and
Vmax (4.3 farads/s) were obtained by a fit of
the data in the absence of inhibitor to the Michaelis-Menten equation
by nonlinear regression analysis. Ki was calculated
to be 2.8 nM from the equation Ki = Km/(Vmax × m) (17).
2 value, the standard statistical measure of the
closeness of the fit, is less than 0.4 (data not shown), indicating a
close fit. Representative sensorgrams of DX600 and DX512 are shown in
Fig. 3. The on-rates
(kon), off-rates (koff),
and Kd values of all six very strong inhibitors
(DX512, DX513, DX599, DX600, DX601, and DX602) are listed in Table II.
The kon values of all six inhibitors were in the
order of 104 to 105
M
1 s
1; the
koff values were in the order of
10
2 to 10
4 s
1, and the
Kd values (Kd = kon/koff) ranged from 10.8 to 170 nM (Table II). DX600 had the slowest off-rate
(4.6 × 10
4) and the lowest Kd
(10.8 nM), which was consistent with it being the most
potent inhibitor with a Ki of 2.8 nM. The Kd values of the other inhibitors were close to
their respective Ki values (Table II).
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Fig. 3.
Kd determination of DX600
and DX512 peptides. The binding affinities of the very strong
inhibitors were analyzed by BIAcore as described under "Experimental
Procedures." Shown here are representative sensorgrams of DX600
(A) and DX512 (B). The data (response units,
RU) were background corrected and plotted against time
(seconds). The wavy lines depict actual data, and the
solid lines depict fitted data. DX600 was assayed at 100, 50, 25, and 12.5 nM, corresponding to respective curves
from top to bottom. DX512 was assayed at 500, 250, 125, and 62.5 nM, corresponding to respective curves
from top to bottom.
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Fig. 4.
DX600 and DX512 peptides were stable ACE2
inhibitors. DX600 (A) and DX512 (B), at both
low and high concentrations, were each incubated with ACE2 (20 nM) for 10 min or 20 h at room temperature prior to
the addition of M-2195. The relative ACE2 activity was plotted against
the peptide concentration. Open square, 10 min; filled
square, 20 h.
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Fig. 5.
DX600 was a specific inhibitor to ACE2.
A, effects of DX600 on ACE2 and ACE activity. Increasing
concentrations of DX600 peptides were incubated with ACE2 (20 nM) or ACE (7.5 nM) prior to the addition of
the substrate M-2195 (50 µM). B, effects of
ACE peptide inhibitor teprotide on ACE2 and ACE activity. Increasing
concentrations of teprotide were incubated with ACE2 (20 nM) or ACE (7.5 nM) prior to the addition of
the substrate M-2195. The relative enzymatic activity was plotted
against the peptide concentration. ACE2, filled circle; ACE,
open circle.
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Fig. 6.
Ki determination of DX600
peptide using ACE2 assays with the natural substrate Ang I. DX600,
at concentrations ranging from 0 to 12.5 nM, was
preincubated with 7 nM ACE2. The substrate Ang I was added
at concentrations ranging from 40 to 160 µM.
A, Dixon plot. Filled squares, 40 µM substrate (Ang I); open squares, 60 µM; filled triangles, 80 µM;
×, 100 µM; open triangles,
120 µM; and filled circles, 160 µM. B, Dixon secondary plot. The slope at each
substrate concentration in A was plotted against the
reciprocal substrate concentration. Data were fitted to a linear
regression (y = mx + b, where
m = Km/ (Ki × Vmax) = 23.041, b = 0.039). Km (145.52 µM) and
Vmax (2.26 farads/s) were obtained by a fit of
the data in the absence of inhibitor to the Michaelis-Menten equation
by nonlinear regression analysis. Ki was calculated
to be 2.8 nM from the equation Ki = Km/(Vmax × m)
(17).
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Fig. 7.
DX600 but not ACE inhibitors potently
inhibited ACE2 activity toward its natural substrate Ang I. DX600
at concentrations ranging from 0 to 1 µM, teprotide, or
captopril at concentrations ranging from 0 to 1 mM was
incubated with 10 nM ACE2 prior to the addition of the
substrate Ang I (60 µM). ACE2 activities were measured as
described under "Experimental Procedures." The relative ACE2
activity was plotted against the peptide concentration. A,
DX600. The IC50 was ~8 nM. B,
teprotide. The IC50 was over 700 µM.
C, captopril. The IC50 was well over 1 mM.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank James Beltzer and Fayelle Whelihan for their input and helpful discussion.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Division of Research, Dyax Corp., 300 Technology Square, Cambridge, MA 02139. Tel.: 617-250-5742; Fax: 617-225-2501; E-mail: lhuang@dyax.com.
Published, JBC Papers in Press, February 26, 2003, DOI 10.1074/jbc.M212934200
2 T. J. Parry, R. Tallarida, R. Schulingkamp, D. Keleti, L. Huang, L. Sekut, R. Smith, V. Albert, N. Nguyen, D. Chinchilla, D. Parmelee, Y. Li, H. Lin, S. Strawn, J. Porter, M. C. Barber, M. Valmonte, T. Coleman, S. Ruben, and I. Sanyal, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: ACE, angiotensin-converting enzyme; Ang I, angiotensin I; Ang II, angiotensin II; ACE2, angiotensin-converting enzyme homolog; Ang-(1-9), angiotensin-(1-9); Ang-(1-7), angiotensin-(1-7); vgDNA, variegated DNA; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; Me2SO, dimethyl sulfoxide.
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