Study of a lipophilic captopril analogue binding to angiotensin I converting enzyme

Article abstract of DOI:10.1002/psc.1201

Human ACE is a central component of the renin-angiotensin system and a major therapeutic target for cardiovascular diseases. The somatic form of the enzyme (sACE) comprises two homologous metallopeptidase domains (N and C), each bearing a zinc active site with similar but distinct substrate and inhibitor specificities. In this study, we present the biological activity of silacaptopril, a silylated analogue of captopril, and its binding affinity towards ACE. Based on the recently determined crystal structures of both the ACE domains, a series of docking calculations were carried out in order to study the structural characteristics and the binding properties of silacaptopril and its analogues with ACE.

Full text of DOI:10.1002/psc.1201

Research Article
Received: 20 May 2009
Revised: 23 September 2009
Accepted: 6 October 2009
Published online in Wiley Interscience: 15 December 2009
(www.interscience.com) DOI 10.1002/psc.1201
Study of a lipophilic captopril analogue
binding to angiotensin I converting enzyme
Georgios A. Dalkas,a Damien Marchand,b Jean-Claude Galleyrand,b
Jean Martinez,b Georgios A. Spyroulias,a∗ Paul Cordopatisa
and Florine Cavelierb∗
Human ACE is a central component of the renin–angiotensin system and a major therapeutic target for cardiovascular diseases.
The somatic form of the enzyme (sACE) comprises two homologous metallopeptidase domains (N and C), each bearing a
zinc active site with similar but distinct substrate and inhibitor specificities. In this study, we present the biological activity
of silacaptopril, a silylated analogue of captopril, and its binding affinity towards ACE. Based on the recently determined
crystal structures of both the ACE domains, a series of docking calculations were carried out in order to study the structural
c
characteristics and the binding properties of silacaptopril and its analogues with ACE. Copyright ꢀ 2009 European Peptide
Society and John Wiley & Sons, Ltd.
Keywords: ACE; zinc metalloenzyme; silacaptopril; docking simulations
Introduction
Angiotensin I
Angiotensin II
The ACE, a zinc metalloenzyme, plays a fundamental role in
blood pressure regulation by converting the inactive decapeptide
angiotensin I to a potent vasopressor octapeptide angiotensin
II. Two isoforms of ACE transcribed from the same gene in a
tissue-specific manner [1]: the somatic form (sACE), which is found
in a variety of tissues, and the testicular form (tACE), which is
expressed in germinal cells exclusively. sACE is a 1277-residue type
I transmembrane glycoprotein with an ectodomain consisting of
two highly homologous domains (N and C domains). tACE is a
701-amino-acid isoform identical to the C domain of sACE [2],
except for the first 36 residues. Each domain contains an active
site bearing the characteristic HEXXH zinc-binding motif of Zn-
peptidases (zincins) [3].
The recent breakthrough in determining the high-resolution
crystal structures of human tACE (C domain of sACE) [4,5] and that
of the N domain of sACE [6], both in the absence and presence of
the potent inhibitor lisinopril, has renewed the interest in studying
their enzymatic activity at a molecular level and has provided a
structural basis for the design of domain-specific inhibitors [7,8].
DespitethestructuralhomologyofthetwodomainsofsACE,which
have ∼60% sequence identity, some notable differences between
the active sites were observed [6]. Consistent with the observed
chloride-dependent activation of ACE [9], only one chloride ion
was bound to the N domain as opposed to the two found in the
crystal structure of tACE [6,10].
ACE
CAPTOPRIL
Scheme 1. Role of ACE.
further enhance the importance of the hydrophobic interaction
of the proline moiety. Modifications of the proline ring-like
bicyclopropane or bicyclopentane have been performed [19].
Regarding our contribution, the proline was replaced with the
more lipophilic surrogate 4ꢁ4-dimethylsilaproline [20], in order
to increase the lipophilicity of captopril, while conserving the
inhibitory activity, since the conformational features of captopril
structural core will be preserved. The introduction of a di-
substituted silicon atom attenuates the hydrophilic character
of the molecule and could enhance hydrophobic interactions
with ACE. Additionally, three new analogues of di-substituted
silacaptopril have been designed and tested in silico for their
binding affinity and posing into the Zn catalytic site through
docking simulations.
∗
Correspondence to: Georgios A. Spyroulias, Department of Pharmacy, Univer-
sity of Patras, Panepistimioupoli – Rion, GR-26504, Greece.
E-mail: G.A.Spyroulias@upatras.gr
Drug design based on peptide structure–activity relationships
is an area of great importance [11]. The knowledge of an
enzyme belonging to a defined family and the analogy with
other proteins is often used to assist the design of new potential
inhibitors [12–14]. Captopril, a competitive inhibitor of ACE, is
currently used as an oral anti-hypertensive agent (Scheme 1)
[15–18]. Taking into consideration that the captopril’s methyl
group (Scheme 2) contributes to the inhibitory potency, possibly
via a hydrophobic interaction with the enzyme, we aim to
∗
´
Florine Cavelier, IBMM, UMR CNRS-UM1-UM2 5247, Universite Mont-
pellier 2, Place Eugene Bataillon, 34095 Montpellier cedex, France.
`
E-mail: florine@univ-montp2.fr
a
Department of Pharmacy, University of Patras, Panepistimioupoli – Rion, GR-
26504, Greece
´
`
b
IBMM, UMR CNRS-UM1-UM2 5247, Universite Montpellier 2, Place Eugene
Bataillon, 34095 Montpellier cedex, France
c
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Copyright ꢀ 2009 European Peptide Society and John Wiley & Sons, Ltd.

DALKAS ET AL.
O
CO2H
O
CO2H
HS
N
N
HS
Si
Silacaptopril
2
Captopril
1
O
O
CO2H
CO2H
N
HS
N
HS
Si
Si
Analogue
CO2H
4
Analogue
3
O
N
HS
Si
Analogue
5
Scheme 2. Structure of captopril and analogues.
Materials and Methods
RMN1H (CDCl3): (2 diastereoisomers) δ = 0.29 and 0.31 (2 s, 12 H,
2 × Si(CH3)2), 1.15–1.40 (m, 4 H, 2 × CHaCH2Si), 1.25 and 1.28 (2d
J = 6.4 Hz, 6.7 Hz, 6 H, 2×CH3CHCO), 2.89and3.022dJ = 13.2 Hz,
13.3 Hz, 1 H, NCHHSi), 3.04 and 3.06 (2d J = 13.3 Hz, 13.2 Hz, 1 H,
NCHHSi), 2.25–2.40 (m, 4 H, BrCH2), 3.65–3.75 (m, 2 H, CH3CHCO),
3.72 and 3.73 (2 s, 6 H, 2 × OCH3), 5.00–5.10 (2dd J = 4 Hz, 9.8 Hz,
2 H, CHα).
Synthesis
3-bromo-2-methylpropionic acid 6
HBr gas was bubbled for 2 h in a solution of metacrylic acid (5 ml,
58 mM) at room temperature. The reaction mixture was purified
by Kugelrhor distillation under reduced pressure to afford the title
compound as a colourless oil; (70%) Rf: 0.75 (CHCl3/MeOH/AcOH
120/10/5) m.p. 106–108 ◦C/15 mbar; RMN1H (CDCl3) δ = 1.35 (d
J = 7 Hz, 3H, CH3), 3.00 (m, 1H, CH), 3.60 (m, 2 H, CH2Br).
Ac-Silacaptopril-OMe (R/S, R) 9
Compound8(200 mg,0.621 mM)wasdissolvedinacetone(1.2 ml),
and potassium thioacetate (142 mg, 1.242 mM) was added. The
reaction mixture was refluxed overnight. After removal of acetone,
the resulting residue was purified by flash chromatography on
silica gel (84%).
Compound 8
A
mixture of 3-bromo-2-methylpropionic acid 6 (177 mg,
1.066 mM) containing two drops of DMF in CH2Cl2 was stirred.
Oxalyl chloride (140 µl, 1.5 eq, 1.6 mm) was added slowly and
stirring at room temperature was continued until gas evolution
substantiallyslowed.Anadditionalportionofoxalylchloride(26 µl,
1/3 eq, 0.3 mm) was added, and when gas evolution ceased, the
mixturewasconcentratedinvacuo.Twice,portionsofcyclohexane
(2 ml) were added and evaporated in vacuo.
The residue was dissolved in THF (3 ml), then a solution of (L)-
silaprolinemethylesterhydrochloride7(200 mg, 0.954 mM)inTHF
(3 ml) and propylene oxide (267 ml, 3.82 mM) were added and the
reaction was stirred for 3 h at 50 ◦C in a sealed system. The solvent
wasremovedandtheresiduepurifiedbyflashchromatographyon
silica gel. The relevant fractions yielded the expected compound
8 as an oil (87%).
Rf: 0.4 (AcOEt/hexane 3/7);
SM–ESI +: 318–320 [M+H]+;
RMN1H (CDCl3): (2 diastereoisomers) δ = 0.21 and 0.24 (2 s,
12 H, 2×Si(CH3)2), 1.10–1.30 (m, 4 H, 2×CHaCH2Si), 1.15 and 1.20
(2d J = 6.4 Hz, 6.7 Hz, 6 H, 2 × CH3CHCO), 2.27 and 2.29 (2 s, 6 H,
CH3COS), 2.78–3.19 (m, 10 H, SCH2, CH3CHCO, NCH2Si), 3.65 and
3.67 (2 s, 6 H, 2×OCH3), 4.90–5.00 (2dd J = 4 Hz, 9.8 Hz, 2 H, CHa).
Silacaptopril (R/S,R) 2
The fully protected silacaptopril 9 (100 mg, 0.315 mM) was
dissolved in degassed solvents (THF/H2O : 10/1) (10 ml). Lithium
hydroxide (20.2 mg, 2.523 mM) was then added and the reaction
was monitored by HPLC. When all starting materials disappeared,
12N HCl was added and the aqueous phase was extracted with
Rf: 0.3 (AcOEt/hexane 3/7);
SM–ESI +: 322–324 [M+H]+, 643–647 [2 M+H]+;
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J. Pept. Sci. 2010; 16: 91–97

BINDING STUDIES OF SILACAPTOPRIL
EtOAc. The combined organic phases were dried over MgSO4,
filtered and concentrated in vacuo to afford the title compound in
85% yield.
partialchargeswereassignedtoallproteinatoms, whereasforzinc
and chloride ions formal charges and van der Waals parameters
from the AMBER database were assigned. The sulfhydryl groups
were set to be deprotonated, depending on their coordination
to the catalytic zinc of ACE. The grid maps were centred on the
ligand’s binding site, with 81 × 81 × 81 grid points of 0.25 Å
spacing. The Lamarckian genetic algorithm was employed with
the following parameters: a population size of 250 individuals; a
maximum number of 5 × 106 energy evaluations and a maximum
numberof27 000generations;anelitismvalueof1;amutationrate
of 0.02 and a crossover rate of 0.80 [27]. For all the calculations,
100 docking rounds were performed with step sizes of 0.2 Å
for translations and 5◦ for orientations and torsions. Docked
conformations were clustered with a 0.5 Å tolerance for the root
mean square positional deviation. The protein–ligand complexes
were visually inspected with AutoDockTools.
Rf: 0.6 (CHCl3/MeOH/AcOH 120/10/5)
SM–ESI +: 262–264 [M+H]+;
RMN1H (CDCl3): (2 diastereoisomers) δ = 0.32–0.38 (m, 12 H,
2 × Si(CH3)2), 1.20–1.30 (m, 8 H, 2 × CH3CHCO and 2 × HSCH2),
1.40–1.70 (m, 4 H, CHaCH2Si), 2.85–3.20 (m, 10 H, SCH2 and
CH3CHCO and NCH2Si), 5.05–5.20 (m, 2 H, CHa), 6.00–6.70 (bs,
2 H, CO2H).
Biological Assays
Lyophilized ACE was restored in distilled water and stored at
−80 ◦C until tested. The substrate was the tripeptide Hip-His-Leu-
OH. ACE activity was measured by using the fluorimetric method
of Santos et al. with slight modifications [21].
Briefly, the enzymatic hydrolysis of the substrate (3.5 × 10−4 M)
was carried out for 1 h at 37 ◦C in 50 mM Tris HCl pH 8.3 containing
NaCl 0.3 M (final volume 100 µl) [22] in the presence of 0.005U
of ACE (2.2 ng). These conditions provided constant velocity and
optimal enzymatic activity.
The enzyme reaction was stopped by adding 400 µl of 2N NaOH
and 3 ml of distilled water. A measured quantity of 0.1 ml of 1%
(w/v) 1,2-benzenedicarboxaldehyde in methanol was added in the
alkalized mixture. After exactly 4 min, 0.2 ml of 6N HCl was added.
The fluorescence of the mixtures was measured with excitation at
365 nm and emission at 495 nm.
Results and Discussion
Consideringthattheprolineofthecaptoprilfitsintoahydrophobic
binding pocket of the active site, it was supposed that increasing
the hydrophobicity of this ligand moiety could improve its binding
affinity. A few years ago, we designed a lipophilic analogue
of proline, the silaproline [20], which exhibits an enhanced
hydrophobicity and conserves structural feature [28–30]. We
synthesized a silylated analogue of captopril 1, the silacaptopril
2, with a dimethylsilyl group replacing a methylene, which could
improve hydrophobic interactions with ACE.
Computational Methods
Synthesis
Structure preparation
The 3-bromo-2-methylpropionic acid 6, which is easily obtained
by hydrobromation of methacrylic acid [31], was condensed with
the methyl ester of silaproline 7. Several coupling conditions
have been tested, varying the reagents, the base and the solvent.
Neither of coupling reagents classically employed in peptide
synthesis (BOP, DCC/DMAP, DCC/HOBT, EDCI) gave satisfying
results whatever bases and solvents were used. The intermediate
8 was finally obtained in 87% yield by activation of 6 with oxalyl
chloride followed by coupling with the silaproline derivative 7
in the presence of propylene oxide. Subsequently, the bromide
was substituted by potassium thioacetate in refluxing acetone to
afford 9 in 84% yield. A final treatment with lithium hydroxide
in a mixture of THF and water allowed the deprotection of both
methyl ester and acetate to provide the silacaptopril 2 in 85% yield
(Scheme 3).
The crystal structure coordinates of ACE were obtained from the
Protein Data Bank (PDB): PDB ID code 1UZF [4], for the C domain
(tACE) and PDB ID code 2C6N [6], for the N domain of sACE.
All crystallographic water molecules, bound inhibitors and other
heteroatoms with the exception of zinc and chloride ions were
removed. Missing hydrogen atoms and heavy atoms were added
usingtheXLEaPmoduleofAMBER9[23].Themissingresidueswere
added by manual placement of their Cα atoms and subsequent
automatic modelling of the remaining atoms using XLEaP. The
protonation state of ionizable side chains was predicted by (i) the
programme H++ using a continuum electrostatic model based
on the Poisson–Boltzmann method [24] and (ii) visual inspection
of all histidine residues to identify hydrogen-bonding networks
with neighbouring residues. The system was initially relaxed with
500 steps of energy minimization using the steepest descent
method and positional restraints with a harmonic force constant
of 50 kcal/mol/Å2 on all heavy atoms except those not determined
in the X-ray structure. The generalized Born implicit solvation
model GBHCT [25] was employed with a 16 Å cut-off for the non-
bonded interactions. Ligands displayed in Scheme 2 were also
treated with the united-atom approximation, and were prepared
with the OpenEye suite of programmes using the AM1-BCC partial
charge distribution [26].
Inhibitory Effect for ACE Activity
Inhibitory activities of captopril (0.1 nM–1 µM) and silacapto-
pril derivatives were determined at different concentrations
(0.1 nM–0.1 mM) by using the optimized fluorimetric assay previ-
ously described (see Section Materials and Methods). ACE activity
without addition of captopril or silacaptopril in the reaction mix-
ture was set as 100%.
Captopril and silacaptopril (R/S,R) were both able to inhibit ACE
activity completely (Figure 1). The evaluated IC50 of captopril (6.3
nM) was highly coherent with data reported previously in the
literature. Silacaptopril (R/S,R) with IC50 value of 43 nM exhibited
a lower inhibitory effect for ACE, compared to captopril.
The replacement of proline by silaproline did not affect
the inhibition significantly, possibly indicating that hydrophobic
Docking of the ligands
AutoDock 3.05 [27] was used for the docking calculations and
AutoDockTools was used for visual inspection of the docking
results. Protein and ligands were treated with the united-atom
approximation by merging all non-polar hydrogens. Kollman
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DALKAS ET AL.
O
CO2Me
1) oxalyl chloride
CO2H
Br
N
Br
2) propylene oxide
HCl,Sip-OMe 7
THF / 50 °C / 3h
8
6
Si
87%
CH3COS-K+
(CH3)2CO / reflux
84%
O
CO2H
O
CO2Me
LiOH
THF - H2O
N
HS
N
AcS
Si
85%
Si
2
9
Scheme 3. Synthesis of silacaptopril.
Figure 1. Inhibitory effect of captopril and silacaptopril on ACE activity.
interactionsarenotessentialforthebindingofsilacaptopriltoACE.
In addition, steric hindrance, which has increased significantly,
might hamper binding site approach. Docking calculations can
probe the binding properties of silacaptopril and its analogues.
Active Site Nomenclature
In order to illustrate the enzyme active site and subsites of
proteases that are susceptible to accommodating the pep-
tide or substrate group, Schechter and Berger introduced in
1967 [32] a nomenclature using as an example the proto-
type of the papain family of cysteine peptidases. According
to this nomenclature, the active site of the protease is di-
vided into different subsites that may come into contact
with the substrate. The protease subsites interacting with the
N-terminus of the substrate are numbered S1 –Sn (non-primed
sites), whereas tꢁhose that interact with the C-terminus are num-
Figure 2. (A) Stereo representation of the cACE–captopril complex. The
X-ray captopril (PDB ID 1UZF) and the simulated captopril are shown in
pink and green, respectively. (B, C) Stereo representation of cACE and
nACE in complex with captopril (green) and silacaptopril (pink). In A–C,
the catalytic residues’ carbon atoms are shown in orange and zinc as silver
sphere.
prediction of small molecule or short peptide binding modes
into the Zn-containing catalytic sites of the enzyme. The binding
subsites S1, S1ꢁ and S2ꢁ are well characterized, and the differences
at the active sites between the two domains of ACE are
documented [6]. Based on the structure models of ACE, efforts
were focused on the generation of the protein–ligand complexes
throughdockingsimulationapproaches.Thereferencecompound
captopril, (2R)-1-[(2S)-2-methyl-3-sulfanyl-propanoyl]pyrrolidine-
2-carboxylic acid, exhibits a remarkable preference for binding
to the active site of both ACE domains. The compounds used
ꢁ
bered as S1 –Sn (primed sites). Accordingly, the position of the
residues/groups of the substrate/inhibitor relative to the scissile
ꢁ
ꢁ
bond is denoted as P1 –Pn, and P1 –Pn , respectively.
ACE-captopril Simulated Docking Interactions
The recently reported crystal structures of human tACE [4]
(C domain of sACE) and N domain of sACE [6] allowed the
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J. Pept. Sci. 2010; 16: 91–97

BINDING STUDIES OF SILACAPTOPRIL
captopril, Val380 displays favourable hydrophobic interactions
with the proline moiety of silacaptopril, probably due to the two
coordinating methyl groups of Si. These additional interactions of
Val380 might contribute to the better binding energies of cACE
with silacaptopril (Table 1). This is probably due to the limited
accuracy of the AutoDock scoring function, which has a residual
error of 2.18 kcal/mol [27]. The predicted binding affinities of the
lowest energy docked conformations, using the LGA method and
the new empirical free energy function, were within the standard
residual error of the force field for captopril and silacaptopril.
Therefore, a straightforward comparison of the two ligands based
exclusively on their predicted binding energy could not explain
which one is a more potent inhibitor of ACE, and thus more
experimental data are needed.
Table 1. Docking results for each ligand in complex with ACE
cACE domain
nACE domain
Mean
docked
energy
Mean
binding
energy
Mean
docked
energy
Mean
binding
energy
Ligand
(kcal/mol)
(kcal/mol)
(kcal/mol)
(kcal/mol)
Captopril
−10.47
−10.50
−9.24
−9.69
−10.38
−9.21
−9.11
−12.02
−10.54
−11.00
−11.10
−8.23
−11.94
−10.49
−10.88
−11.04
Silacaptopril
Analogue 3
Analogue 4
Analogue 5
−9.66
−9.60
−10.22
−10.11
in this study are captopril 1 [15,18] and silacaptopril 2 (N-R-
3-mercapto-2-methylpropanoyl-L-silaproline), which are derived
from the replacement of the proline of captopril with 4ꢁ4-
dimethylsilaproline [20] (Scheme 2). Variation on silicon atom
substitution has been introduced to evaluate the importance
of steric hindrance at these positions (analogues 3, 4 and 5,
Scheme 2). AutoDock was able to predict the conformation of
captopril bound to cACE within 1.22 Å root mean square deviation
(Figure 2A) as the top ranked solution with the lowest binding free
energy, indicating the ability of the method to predict a proper
conformation. All analogues exhibited binding energies ranging
from −8.0 to −11.0 kcal/mol, with silacaptopril exhibiting the
lowest docking energy in both ACE domains (Table 1). In addition,
docking results reveal that all the compounds adopted similar
orientations into the ACE ligand binding site.
ACE N Domain (nACE) Contacts with Captopril
and Silacaptopril
Similarly, docking simulation approaches were performed in order
to examine the interactions of nACE residues with both captopril
and silacaptopril. Several key interactions indicative of positioning
and binding of the ligands into the binding site of nACE domain
are summarized in Table 2. The zinc ion is coordinated by the
sulfhydryl group (2.31 and 2.44 Å for captopril and silacaptopril,
respectively), as in the case of cACE with captopril. It is noteworthy
that captopril and silacaptopril orient one oxygen of the proline
moiety carboxylate group in a way that favours interactions with
Gln259, Lys489 and Tyr498, similarly to the cACE domain and
residues Gln281, Lys511 and Tyr520 (Figure 2C). Furthermore, the
central carbonyl group of both captopril and silacaptopril forms
two hydrogen bonds with histidines His331 and His491.
Several C domain active site residues differ in the corresponding
N domain sequence. An important difference is the alteration of
Val380 to Thr358. The central methyl group of captopril displays
van der Waals contacts with Thr358 (nACE), in contrast with Val380
of cACE, which does not exhibit strong interactions with captopril.
This could be attributed to the fact that the side-chain of Val380 is
distant from the central methyl group.
ACECDomain(cACE)ContactswithCaptoprilandSilacaptopril
Structural analysis of the cACE–captopril X-ray complex reveals
the important ACE residues for interaction with captopril and
for accommodation of the ligand into the binding site [16].
The major interactions between cACE residues and captopril are
summarized in Table 2. Captopril’s deprotonated sulfhydryl group
interacts directly with the catalytic Zn2+ ion (2.32 Å), the carbonyl
group between the sulfhydryl group, and the terminal proline is
positioned by two strong hydrogen bonds from the two histidines
(His 513 and His 353) and one oxygen of the proline moiety
carboxylate group is held by interactions with Tyr 520, Gln 281
and Lys 511.
We have carried out docking simulation approaches in order
to examine the interactions of cACE residues with silacaptopril.
Analysis of the docking simulations data reveals that the
orientation of the most favoured docking conformation of
silacaptopril in the binding site of cACE is rather similar to that
of captopril (Figure 2B), with a similar strong interaction between
the catalytic Zn2+ ion and the sulfhydryl group of silacaptopril
(2.25 Å). In addition, similar to captopril, electrostatic interactions
are formed between the central carbonyl group of silacaptopril
and the histidines His353 and His513. Similarly, an oxygen atom
of the proline moiety carboxylate group exhibits electrostatic
interactions with Gln281, Lys511 and Tyr520.
Ontheotherhand, themethylgroupsof4ꢁ4-dimethylsilaproline
exhibit favourable hydrophobic contacts with Val380 (cACE)
and Thr358 (nACE). One of the methyl groups of 4ꢁ4-
dimethylsilaproline displays additional favourable hydrophobic
interactions with Phe505, a contact that is not observed in
the nACE–captopril complex. These contacts in the simulated
complex of cACE–silacaptopril might be crucial for the lowest
binding/docking energy. In addition, several ꢁhydrophobic and
ꢁ
van der Waals contacts are present in the S1 and S2 subsites
of nACE, comprising residues Thr358, Phe435, Phe505, Tyr501,
His331, His361 and Ala332 (Table 2).
cACE and nACE Catalytic Site Interactions with the Silacapto-
pril Analogues
As far as the contacts of silacaptopril analogues (Scheme 2) with
cACE and nACE are concerned, the docking simulations display
some minor contact differences with the interactions that are
observed between cACE/nACE and silacaptopril. Specifically, the
central carbonyl group of analogue 3 is being oriented to an
opposite way regarding the orientation of silacaptopril in the
binding site of cACE. Furthermore, the ethyl groups of silicon
exhibit favourable hydrophobic contacts with residues Phe457
and Phe527 but, opposed to silacaptopril, the ethyl group does
Furthermore, S1ꢁ andS2ꢁ subsitesofcACEdomainaccommodate
the proline analogue moiety of silacaptopril into a hydrophobic
cage comprising residues Val380, Phe457, Phe527, Tyr523 and
Ala354 (Table 2). The hydrophobic and van der Waals interactions
between cACE–captopril and cACE–silacaptopril are similar, with
one exception. It is interesting to note that, in contrast to
c
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DALKAS ET AL.
Table 2. cACE and nACE residues that exhibit major electrostatic and hydrophobic interactions with captopril and silacaptopril
cACE domain nACE domain
Electrostatic
interactions
Hydrophobic
interactions
Electrostatic
interactions
Hydrophobic
interactions
Captopril
Gln281, Lys511, Tyr520,
His513, His353
Tyr523, Phe457, Tyr523
Gln259, Lys489, Tyr498,
His331, His491
Thr358, Tyr501, Ala332,
His331, His361
Silacaptopril
Gln281, Lys511, Tyr520,
His513, His353
Tyr523, Val380, Phe527,
Tyr523
Gln259, Lys489, Tyr498,
His331, His491
Thr358, Tyr501, Phe505,
Ala332, His331, His361
not exhibit hydrophobic interactions with Val380 due to the
different orientation.
5 Watermeyer JM, Sewell BT, Schwager SL, Natesh R, Corradi HR,
Acharya KR, Sturrock ED. Structure of testis ACE glycosylation
mutants and evidence for conserved domain movement.
Biochemistry 2006; 45: 12654–12663.
6 Corradi HR, Schwager SL, Nchinda AT, Sturrock ED, Acharya KR.
Crystal structure of the N domain of human somatic angiotensin
I-converting enzyme provides a structural basis for domain-specific
inhibitor design. J. Mol. Biol. 2006; 357: 964–974.
7 Redelinghuys P, Nchinda AT, Sturrock ED. Development of domain-
selective angiotensin I-converting enzyme inhibitors. Ann. N Y Acad.
Sci. 2005; 1056: 160–175.
8 Spyroulias GA, Cordopatis P. Current inhibition concepts of zinc
metallopeptidases involved in blood pressure regulation. Curr.
Enzyme Inhibit. 2005; 1: 29–42.
Concerning analogue 4, the only observed differences are the
stronger hydrophobic interactions of the methyl/ethyl groups
of silicon displayed with Tyr523 and Phe527 respectively, and the
prolinemoietyvanderWaalsinteractionswithPhe457.Asfarasthe
analogue 5 is concerned, the differences of the most energetically
favourable conformation with regard to the silacaptopril are the
hydrophobic interactions of the silicon ethyl group with Ala354
and Val380, the proline van der Waals interactions with His353 and
the central methyl group hydrophobic interactions with Phe527.
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converting enzyme: dependence on nature of substrate.
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Conclusions
In the present study, docking simulations were used to analyze
interactions between the ACE active site and silacaptopril as
well as interactions between ACE and silacaptopril analogues,
in order to predict the structural factors that might contribute
significantly to their binding. It should be noted that the choice
of the most favoured docking conformations of captopril and
silacaptopril for binding in the catalytic site of ACE was ultimately
dictated by its final binding energy and the orientation of
the ligand. Data analysis reveals that silacaptopril/analogues
exhibited affinity in the same range as captopril and displayed
great similarity regarding the orientation of captopril in complex
with ACE. Thus the substitution of the proline moiety with
silaproline did not affect considerably the inhibition properties
of the parent molecule, although the silacaptopril exhibits
slightly higher IC50 than captopril. Nevertheless, the silacaptopril
analogue may benefit from the presence of silaproline by
exhibiting increased biodisponibility and resistance towards
enzyme degradation.
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