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Bibliography and Further Reading
H. D. Kleinert, S. H. Rosenberg, W. R. Baker etal., Discovery of a Pep-
tide-Based Renin Inhibitor with Oral Bioavailability and Efcacy,
Science, 257, 1940–1943 (1992)
Y. C. Li, Inhibition of Renin: An Updated Review of the Development
of Renin Inhibitors, Current Opinion in Investigational Drugs, 8,
750–757 (2007)
J. M. Wood etal., Structure-based Design of Aliskiren, a Novel Orally
Effective Renin Inhibitor, Biochem. Biophys. Res. Commun., 308,
698–705 (2003)
R. Güller etal., Piperidine-Renin Inhibitors Compounds with Im-
proved Physicochemical Properties, Bioorg. Med. Chem. Lett., 9,
1403–1408 (1999)
M. A. Navia etal., Three-dimensional structure of aspartyl protease
from human immunodeciency virus HIV-1, Nature, 337, 615–620
(1989)
A. Wlodawer etal., Conserved Folding in Retroviral Proteases: Crystal
Structure of a Synthetic HIV-1 Protease, Science, 245, 616–621
(1989)
J. V. N. Vara Prasad, K. S. Para, E. A. Lunney etal., Novel Series of
Achiral, Low Molecular Weight, and Potent HIV-1 Protease Inhib-
itors, J. Am. Chem. Soc., 116, 6989–6990 (1994)
J. P. Vacca etal., L-735,524: An Orally Bioavailable Human Immuno-
deciency Virus TypeI Protease Inhibitor, Proc. Natl. Acad. Sci.,
91, 4096–4100 (1994)
J. H. Condra, W. A. Schleif, O. M. Blahy etal., In Vivo-Emergence of
HIV-1 Variants Resistant to Multiple Protease Inhibitors, Nature,
374, 569–571 (1995)
D. J. Kempf etal., Pharmacokinetic enhancement of inhibitors of the
human immunodeciency virus protease by coadministration with
ritonavir, Antimicrob. Agents Chemother., 41, 654–660 (1997)
P. Y. S. Lam, P. K. Jadhav, C. J. Eyermann etal., Rational Design of
Potent, Bioavailable, Nonpeptide Cyclic Ureas as HIV Protease
Inhibitors, Science, 263, 380–384 (1994)
E. Specker, J. Boettcher, et al., Unexpected Novel Binding Mode of
Pyrrolidine-based Aspartyl Protease Inhibitors: Design, Synthe-
sis and Crystal Structure with HIV Protease, ChemMedChem, 1,
106–117 (2006)
A. Blum, J. Böttcher etal., Structure-Guided Design of C2-symmetric
HIV-1 Protease Inhibitors Based on a Pyrrolidine Scaffold, J. Med.
Chem., 51, 2078–2087 (2008)
K. Ersmark, B. Samuelsson, A. Hallberg, Plasmepsins as Potential Tar-
gets for New Antimalarial Therapy, Med. Res. Rev., 26, 626–666
(2006)
A. S. Nasamu etal., Plasmepsins IX and X are essential and druggable
mediators of malaria parasite egress and invasion, Science, 358,
518–522 (2017)
E. W. Baxter etal. 2-Amino-3,4-dihydroquinazolines as Inhibitors of
BACE-1 (β-Site APP Cleaving Enzyme): Use of Structure Based
Design to Convert a Micromolar Hit into a Nanomolar Lead, J.
Med. Chem., 50, 4261–4264 (2007)
M. E. Kennedy etal., The BACE-1 inhibitor verubecestat (MK-8931)
reduces CNS β-amyloid in animal models and in Alzheimer’s dis-
ease patients, Sci. Transl. Med. 8, 363ra150 (2016)
Barber, J. (2018). Merck & Co. terminates Phase III study of
verubecestat in prodromal Alzheimer’s disease. Retrieved from
https://www.merck.com/news/merck-announces-discontinuation-
of-apecs-study-evaluating-verubecestat-mk-8931-for-the-
treatment-of-people-with-prodromal-alzheimers-disease/ (Last
accessed Nov. 21, 2024)
Inhibitors of Hydrolyzing
Metalloenzymes
Contents
25.1 Structure of Zinc Metalloproteases – 428
25.2 Key Step in the Design of Metalloprotease
Inhibitors: Binding to the Zinc Ion – 429
25.3 Thermolysin: Tailored Design of Enzyme Inhibitors – 431
25.4 Captopril, aMetalloprotease Inhibitor for
Hypertension Therapy – 432
25.5 Finally the Crystal Structure of ACE: Does aSuccess
Story Have to Be Rewritten? – 434
25.6 Inhibitors of Matrix Metalloproteases: An Approach
to Treat Cancer and Rheumatoid Arthritis? – 436
25.7 Carbonic Anhydrases: Catalysts of aSimple
but Essential Reaction – 440
25.8 A Case for Two: Zinc and Magnesium in the Catalytic
Centers of Phosphodiesterases – 444
25.9 What Zinc Can Do, Iron Can Too – 446
25.10 Acetyl Group Cleavage Condenses Chromatin
and Regulates Reading of Gene Segments:
An Opportunity for Therapy? – 447
25.11 Synopsis – 449
Bibliography and Further Reading – 450
© The Author(s), under exclusive license to Springer-Verlag GmbH, DE, part of Springer Nature 2024
G. Klebe, Drug Design, https://doi.org/10.1007/978-3-662-68998-1_25
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Chapter • Inhibitors of Hydrolyzing Metalloenzymes
25
Ametal ion in the catalytic site is required for the function of another important class of enzymes that cleave
peptide and ester bonds. By coordinating the metal ion,
these enzymes activate awater molecule for nucleophilic
attack of the bond to be cleaved. The water molecule
undergoes adrastic change in its pKa value in this state.
Zinc is by far the most commonly used metal ion in these
enzymes, but iron, cadmium, cobalt or manganese are
also found. The presence of ametal ion is essential for
the activity of the protease or esterase. If the metal ion
is removed from the enzyme by the addition of astrong
complexing reagent, such as β-mercaptoethanol or ethylenediaminetetraacetic acid (EDTA), catalytic activity
will no longer be observed.
Many therapeutically relevant enzymes are metal-
loproteases. The rst to be mentioned are the zinc proteases, most notably angiotensin-converting enzyme
(ACE). ACE inhibitors have been used for many years
in the treatment of hypertension. In recent years, other
metalloproteases have been identied as potential drug
targets. These include endothelin-converting enzyme,
neutral endopeptidases, and matrix metalloproteases
(MMP; . Table25.1). Other groups of important zinc
enzymes include carbonic anhydrases, zinc-containing
β-lactamases, and phosphodiesterases. From the group
of transferases, histone deacetylases follow the same
mechanism to cleave an amide bond.
25.1 Structure of Zinc Metalloproteases
In 1967, William Lipscomb determined the 3D structure
of the rst zinc protease for the digestive enzyme carbo-
peptidaseA. The zinc ion required for enzyme activity
is complexed to two His and one Glu side chains. The
fourth coordination site is occupied by awater molecule. An additional glutamate is located near the zinc
ion. The same amino acids are responsible for zinc binding in many other metalloproteases. The presence of the
amino acid sequence His–Glu–X–X–His (where Xis any
amino acid) is characteristic of most known zinc proteases. For example, it is found in collagenase, thermolysin,
neutral endopeptidase 24.11, and endothelin-converting
enzyme (. Table25.2). The discovery of this amino acid
sequence in the primary sequence of anew protein is
astrong indication that it is azinc protease. In metal
loproteases or carbonic anhydrases, zinc is complexed
by three histidine residues. Here, too, the fourth site is
occupied by awater molecule.
In the body, zinc exists as adoubly positively charged
cation, Zn2+. This positive charge is used by the enzyme
for amide cleavage. Ivano Bertini’s group at the University
of Florence, Italy, was able to determine the high-resolution structures of the uncomplexed and product-inhibited metalloprotease MMP-12. These structures allow the
following mechanism to be deduced: In the uncomplexed
. Table 25.1 Function and preferred cleavage sites of some
metalloproteases
Enzyme Cleavage site Function
Thermolysin
Carboxypeptidase
ACE Phe–His,
NEP 24.11 Phe–Leu,
ECE Trp–Val Transforms big endothelin
Collagenase
Stromelysin
ACE angiotensin-converting enzyme, NEP neutral endopepti-
dase, ECE endothelin-converting enzyme
X–Ala, X–
Val, X–Ile
X–Tyr, X–
Phe
Phe–Leu,
Pro–Phe
Cys–Phe
Gly–Leu,
Gly–Ile
Gly–Leu,
Gly–Ile
Bacterial protease
Digestion
Transforms angiotensinI into
angiotensinII, which increases
blood pressure
Multifunctional (cleaves enkephalin, among others)
into endothelin, which increases blood pressure
Tissue remodeling
Tissue remodeling
metalloprotease, the zinc ion is octahedrally coordinated
by three water molecules in addition to three amino acid
residues (His or Glu). One of the water molecules forms
an additional hydrogen bond to aneighboring glutamate.
This residue, Glu 219 in MMPs, Glu 270 in carboxypeptidase, and Glu 143 in thermolysin, also polarizes the water molecule. Therefore, this water is likely to be present
as an OH− ion (. Fig.25.1). The peptide substrate diffuses into the binding pocket and displaces the other two
water molecules from the zinc ion. The remaining water
molecule, polarized by glutamate, attacks the carbonyl
group of the amide bond of the substrate to be cleaved.
The substrate is held in place by hydrogen bonds to the
peptide backbone at the C-terminal side. Ageminal diol
structure is formed at the reaction site, which is stabilized
by the now pentacoordinated zinc ion. The actual cleavage of the amide bond is achieved and the two product
molecules initially remain in the vicinity of the zinc. The
glutamate residue presumably assumes the role of the
-
proton transfer agent in this step. The cleavage product
of the former N-terminus is coordinated to the zinc ion
by an oxygen atom of the newly formed carboxylic acid
function (. Fig.25.2). However, it does not form any
further hydrogen bonds to the protein. On the other
hand, the cleavage product of the C-terminus forms four
hydrogen bonds to the main chain of the enzyme, and its
side-chain binds in the
pocket. The newly formed
free amino group initially remains in the vicinity of the
zinc ion. It probably exists in an uncharged state next to
the zinc ion. Then, the cleavage product originating from
abc
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1
0
2
. • Key Step in the Design of Metalloprotease Inhibitors: Binding to the Zinc Ion
. Table 25.2 Characteristic amino acid sequences His–Glu–
X–X–His in the active site of different metalloproteases
Enzyme Position Amino acids
Thermolysin
NEP 24.11 583–587 His Glu Ile Thr His
ECE 590–594 His Glu Leu Thr His
Astacin 92–96 His Glu Leu Met His
Collagenase 201–205 His Glu Phe Gly His
Stromelysin 201–205 His Glu Ile Gly His
NEP neutral endopeptidase, ECE endothelin-converting
enzyme
142–146 His Glu Leu Tyr His
the N-terminus leaves the catalytic site. It is presumably
displaced by water, which takes its place next to the zinc
ion. Finally, the C-terminal product leaves the binding
pocket.
The three-dimensional structures of many zinc proteases have meanwhile been solved, including those of
angiotensin-converting enzyme and many of the interesting matrix metalloproteases, such as collagenases, gelatinases, and stromelysin (. Table25.1). Four subfamilies
of carbonic anhydrases are known. The therapeutically
most important ones are the α-carbonic anhydrases,
which fulll important tasks in many organs and for
which numerous drugs have been developed.
25.2 Key Step in the Design
of Metalloprotease Inhibitors:
Binding to the Zinc Ion
The zinc ion plays akey role in the catalytic mechanism.
The known spatial structures of metalloprotease inhibitor complexes show that almost all highly potent inhib-
itors contain functional groups that bind directly to the
zinc ion. If these groups are omitted, the binding afnity will decrease signicantly in most cases. Therefore,
the rst step in the design of new inhibitors must be to
search for functional groups that bind particularly well
to the Zn2+ ion. Various groups have been described in
the literature and are summarized in . Fig.25.3. Phosphonamides –PO2NH–, phosphonates –PO2O–, and
phosphinates –PO2CH2– can all be considered as transition state analogues of the enzyme reaction. In fact,
afew potent metalloprotease inhibitors are known, such
as the natural product phosphoramidon 25.1, which
contain such agroup. The relative binding strength of
various groups has been studied for carboxypeptidaseA
(. Table25.3). Similarly, various zinc-binding groups
were tested for endothelin-converting enzyme. The results of these studies are shown in . Table25.4.
Remarkable variability has been observed in the binding potency of functional groups interacting with the
Zn2+ ion. Attenuated partial charges on the zinc ion and
on the anchor group are probably responsible for this
effect. The zinc ion itself can be found in very different
local environments (i.e., [3×His] or [2×His & 1×Glu] or
[1×His & 1×Glu & 1×Cys]). Obviously, thiol groups, –
SH, and hydroxamic acids, –CONHOH, are particularly
well suited to contribute to astrong binding of the metalloprotease. The latter group binds as abidentate ligand
to the zinc ion. Carboxylic acids and ketones bind to the
zinc ion more weakly than the above groups. Nevertheless, acids are of particular interest because acids in the
form of esters are often used as orally available prodrugs
(Sect.9.2). In contrast to phosphinates and phosphonic
acids, phosphonamides are chemically not very stable
and are, therefore, not the rst choice in the development
of anew drug. On the other hand, sulfonamides are excellent zinc anchors, especially for carbonic anhydrases.
How might potential drug candidates for metalloproteases be designed? Acomparison of known crystal
structures (e.g., MMP-12, . Fig.25.2) shows that the
. Fig. 25.1 Mechanism of peptide cleavage by a metalloprotease.
The peptide substrate binds with its P
corresponding specicity pockets of the protease. The amide group
to be cleaved is found between the zinc ion and awater molecule (or
OH−), which is polarized by the acid group of the neighboring glu-
, P1,
2
, and
residues in the
tamate residue(a). This water molecule nucleophilically attacks the
carbonyl carbon atom to form atetrahedral transition state. The zinc
ion is temporarily pentacoordinated and stabilizes the negative charge
of the newly formed geminal diol structure(b). The transition state
collapses with release of both cleavage products(c)
25
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Chapter • Inhibitors of Hydrolyzing Metalloenzymes
. Fig. 25.2 A crystal structure of MMP-12 with both cleavagepro-
ducts has been determined (cf. . Fig.25.1c). The cleavage product
of the former N-terminus (left, light-red carbon atoms) coordinates
with its newly formed carboxylic acid function through an oxygen
atom to the zinc ion, but it does not form hydrogen bonds to the
enzyme itself. The cleavage product originating from the C-terminus (right, light-green carbon atoms) forms four H-bonds to the
main chain of the protein and binds with its
residue in the deeply
binding pockets in these proteins are much better dened
on the primed side of the cleavage site. Therefore, inhibitor design must focus on the
and its adjacent pockets on the primed side. Nevertheless, it has been shown
that the occupancy of the S1 and S2 pockets can be very
important to obtain inhibitors with adequate selectivity.
The importance of the
pocket is well illustrated
by the model protease thermolysin. The enzyme cleaves
peptide chains before aLeu, Ile, or Phe residue at the
position. By inserting the side chain of these residues
into the
pocket, the substrate binds to the enzyme.
When analogous ligands are examined that carry instead
of an aliphatic leucine side chain, the four-carbon shorter
hydrogen atom of aglycine residue at
, the binding constant drops by afactor of 41,000! The enzyme achieves
this dramatic afnity differentiation by placing the side
formed
and the water molecule that is bound simultaneously to the zinc ion.
(7 https://sn.pub/b8uUPw)
chains in an
pocket. The released amino group coordinates to Glu 219
pocket that is virtually anhydrous in the
uncomplexed state. This saves the cost of desolvation of
the pocket, and the binding of only four additional aliphatic carbon atoms results in this increase in afnity,
which accounts for the essential part of the binding afnity (Sect.4.6).
The choice of suitable groups to place in such
apocket is determined by the chemical composition of
the pocket. In addition, the inhibitors must have asuitable head group to coordinate with the zinc ion, as described above. From the mechanism of peptide cleavage
discussed in the previous section, it is easy to understand
why the binding pockets on the unprimed side are less well
established. After peptide cleavage, apeptide with aterminal acid function is formed on this side. Such afunction is itself a good coordination anchor for the zinc
. • Thermolysin: Tailored Design of Enzyme Inhibitors
. Fig. 25.3 Functional groups of metalloprotease inhibitors that are
often used to bind to the zinc ion. Hydroxamic acids and thiols (upper
left) in particular lead to highly potent inhibitors. Aphosphoramide
group is found in the natural product phosphoramidon 25.1 that the
inhibitor uses to coordinate to the zinc ion. It inhibits thermolysin in
the nanomolar range
. Table 25.3 Binding of phenylpropionic acids 25.2 to
carboxypeptidase. The strongest binding was found with the
thiol derivative
R Ki (nM)
H 6200
COOH 450
CH
2
CH2S(=NH)2CH
OP(=O)(OH)
CH
SH 11
2
. Table 25.4 Inhibition of the endothelin-converting
enzyme by tryptophan derivatives 25.3. The hydroxamic acid
(R=CONHOH) as well as thiol compounds have much better
afnity than the carboxylic acid derivatives
3
2
250
140
ion. If the N-terminal end of the cleaved peptide were
to be bound in astrongly pronounced pocket on the unprimed side, self-inhibition of the protease would result.
Normally, this feature is not of interest. However, if the
cleaved N-terminus has alow afnity for the protease,
this type of inhibition will become important at high
product concentrations. This may be adesirable regula-
tory feedback mechanism of Nature to temporarily slow
down enzymatic turnover.
25.3 Thermolysin: Tailored Design
of Enzyme Inhibitors
Thermolysin is a bacterial zinc protease of no therapeutic importance. Nevertheless, the 3D structures of
thermolysin complexed with alarge number of different
inhibitors have been determined. The inuence of many
elementary factors on the strength of the protein–ligand
interaction could be studied with this protease. Therefore, this enzyme is well suited for the study of 3D structure–activity relationships. Furthermore, its high stability
makes it arobust object for experimental studies, and the
3D structure of thermolysin has been repeatedly used as
areference for model building of other related metalloproteases.
One of the central assumptions of structure-based
drug design is the idea that the binding afnity of aligand can be improved if the receptor-bound conformation
can be embedded in arigid scaffold (“pre-organization”).
This working hypothesis was investigated in Paul Bartlett’s group using thermolysin inhibitors as an example.
R Ki (μM)
CONHOH 24
SH 12
CH
2
COOH > 100
CH
COOH > 100
2
The 3D structure of the complex of Cbz–GlyP–Leu–Leu
25.4 (Ki = 9 nM, . Fig.25.4) complexed with thermol-
ysin served as astarting point. The peptidic inhibitor
binds in aconformation similar to aβ-turn. Therefore,
the design of a macrocyclic ligand that stabilizes this
turn conformation seemed possible. The analysis of the
3D structure of this inhibitor with thermolysin revealed
essential interactions. The Bartlett group then sought
arigid structural element to form ascaffold in which the
conformation of the two leucine side chains remained
unchanged. Chromane 25.5 (. Fig. 25.4) was chosen.
The additional methyl group on the ring had to be added
for synthetic reasons.
A comparison of the binding constants of com-
pounds 25.5 and 25.7 shows that the rigidication caused
25
Chapter • Inhibitors of Hydrolyzing Metalloenzymes
by the chromane group increased the binding afnity by
afactor of50. This corresponds to an energetic gain of
about 10 kJ/mol. The X-ray structure analysis of the
macrocyclic ligand25.5 shows that it binds as expected.
Both leucine side chains and the main chain atoms are
found in the same position as in Cbz–GlyP–Leu–Leu
(25.4, . Fig.25.5). Certainly, the gain in binding energy
is not only aresult of the ligand becoming more rigid.
The direct interaction of the chromane group with the
enzyme also contributes to the afnity. The aim of the
synthesis of 25.6 was to differentiate between the two effects of rigidication and the afnity gain from the chromane moiety. Compound 25.6 binds 20-times weaker to
thermolysin than 25.5. However, the 3D structure shows
that the open-chain inhibitor binds to the enzyme in
a different conformation. This is another example of
how structures that are thought to be very similar do
not necessarily bind in the same way!
25.4 Captopril, aMetalloprotease
Inhibitor for Hypertension Therapy
Angiotensin-converting enzyme (ACE) converts the decapeptide angiotensin I to the octapeptide angiotensinII by cleaving off the C-terminal dipeptide His–Leu
(. Fig.24.5, Sect.24.2). The release of this octapeptide
leads to an increase in blood pressure. In addition, ACE
catalyzes the degradation of the blood-pressure-lowering nonapeptide bradykinin to inactive peptides, thereby,
also indirectly increasing blood pressure. This means that
ACE inhibition can simultaneously prevent blood pressure increases by blocking multiple mechanisms. In 1965,
Sergio Henrique Ferreira and John Robert Vane isolated
apeptide mixture from the venom of asnake, Bothrops
jararaca (the South American pit viper), that prolonged
the blood-pressure-lowering effects of bradykinin by inhibiting aprotease that degrades bradykinin in the body.
This peptide (originally called bradykinin potentiating
peptide, BPP) was also shown to inhibit the conversion of
angiotensinI to angiotensinII. Several structurally related
peptides were identied. The most active was the teprotide
Pyr–Trp–Pro–Arg–Pro–Gln–Ile–Pro–Pro (Pyr=pyroglutamic acid). This nonapeptide was synthesized by Miguel
Ondetti at Squibb. Teprotide is apotent ACE inhibitor
with abinding constant of Ki = 100 nM. In clinical trials,
the compound has been shown to be antihypertensive not
only in animal models but also in humans. However, being
apeptide, teprotide is not orally bioavailable and is, therefore, not suitable as adrug. Despite this observation, the
studies demonstrated that an ACE inhibitor is an interesting compound for the treatment of hypertension. Further
investigations showed that even dipeptides such as Val–Trp
(Ki = 1.8 μM) and Ala–Pro (Ki = 230 μM) inhibit ACE,
although more weakly than the nonapeptide teprotide.
. Fig. 25.4 The development of cyclic thermolysin inhibitors based
on the open-chained inhibitor Cbz–GlyP–Leu–Leu 25.4. The cyclic inhibitor 25.5 binds 50-fold more strongly to thermolysin than the openchain compound 25.7. Compound 25.6 also contains the chromane
scaffold, but the conformation is not enforced by aring closure
The decisive breakthrough was the hypothesis of Miguel Ondetti and David Cushman that ACE was structurally similar to the metalloprotease carboxypeptidaseA,
which had been extensively studied. Lipscomb had shortly
before determined the 3D structure of this enzyme. In addition, benzylsuccinic acid was known to be an extraordi-
narily potent inhibitor of carboxypeptidaseA given its
small molecular size (. Fig.25.6). Abinding mode was
postulated for this molecule involving interactions with
the enzyme that were also experienced by the two products
of substrate hydrolysis (. Fig.25.7). Ondetti and Cushman applied this concept to ACE. Whereas carboxypeptidaseA cleaves off the last amino acid of apeptide, ACE
cleaves off a dipeptide. This means that asuccinic acid
derivative substituted with an adequate additional amino
acid should result in apotent ACE inhibitor (. Fig.25.8).
Following the observation that proline as the C-ter-
minal amino acid of peptidic ACE inhibitors yielded
good results, carboxyalkanoylprolines were rst evaluated as possible ACE inhibitors (. Fig.25.9). Succi-
noyl-l-proline (25.8) was the rst compound synthesized
in this project at Squibb. As hoped, it proved to be an
ACE inhibitor, but with an afnity only in the micromolar range (IC50 = 300 μM). Replacing the proline moiety
with another amino acid did not improve binding: proline was already the optimal amino acid. Next, the length
of the acid side chain was optimized. Glutaryl-l-proline
(25.9) proved to be the best representative with amoderate improvement in binding (IC50 = 70 μM). The intro-
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. • Captopril, aMetalloprotease Inhibitor for Hypertension Therapy
433
25
. Fig. 25.5 The 3D structure of a complex of thermolysin and
Cbz–GlyP–Leu–Leu 25.4 (gray carbon atoms). The leucine side chain
(right) adjacent to a phosphate group occupies the deep
which faces the interior of the protein; the second leucine residue is in
the shallow
group is oriented in the S1 pocket (left). The macrocyclic inhibitor 25.5
(green carbon atoms) with the chromane scaffold locks the conformation of 25.4 and analogously places the leucine side chains in
pocket, which is open to the protein surface. The Cbz
pocket,
and
duction of amethyl group in the side chain (25.10 and
25.11) resulted in astrong increase of the binding afnity by afactor of 15. Finally, replacing the carboxylate
with athiol group (25.12 and 25.13) provided the breakthrough with an order of magnitude increase in potency.
The compound SQ14225, 25.13 d-2-methyl-3-mercaptopropanoyl-l-proline binds to ACE with Ki = 1.7 nM and
is orally available. SQ14225 has been marketed for many
years under the name captopril and has proven itself as
an effective treatment for hypertension. Because lowering
blood pressure signicantly reduces the workload on the
heart, captopril has also been used successfully in the
treatment of congestive heart failure.
The compounds shown in . Fig.25.10 demonstrate
that both afree SH group and afree carboxylate group
are necessary for the strong binding of captopril to ACE.
Esterication of the carboxyl group in 25.14 or S-methyl-
ation in 25.15 leads to adramatic loss of afnity, as does
replacement of the amide group with a–CH2CH2– group
in 25.16 to 25.17. Because of their susceptibility to oxidation, thiol groups are not very popular functional groups
for drugs. Therefore, other anchor groups were sought.
Although this inhibitor leaves the S1 pocket completely unoccupied, it
binds to thermolysin more strongly than the open-chain compound
25.4. (7 https://sn.pub/fXOSRm)
.
. Fig. 25.6
cinate complex. Acarboxylate group binds to the zinc ion and the other
forms achelate-like salt bridge to the arginine side chain of Arg 145.
The phenyl group lls alipophilic pocket.
The crystal structure of the carboxypeptidase–benzylsuc-
(7 https://sn.pub/yXJLB4)
25
Chapter • Inhibitors of Hydrolyzing Metalloenzymes
. Fig. 25.7 Comparison of the binding mode of the inhibitor ben-
zylsuccinic acid and the peptidic substrate to carboxypeptidaseA. The
inhibitor forms the same interaction to the enzyme as the substrate.
The amide group to be cleaved was replaced by acarboxylate group
. Fig. 25.8 The development of ACE inhibitors: a comparison of
the substrates with the inhibitors that were investigated by Ondetti and
Cushman. In the initially investigated structure, the amide bond to be
cleaved is replaced by acarboxylate group
In the meantime, awhole range of effective ACE in-
hibitors have become available (. Fig.25.11); 17products have found their way into clinical trials. Of particular note is enalapril 25.18 from Merck & Co. Like
most other marketed products, with the exception of
lisinopril, it is administered as aprodrug to increase oral
availability (Sect.9.2). As with other ethyl esters, it is
rapidly converted in the body to its biologically active
form, enalaprilat, the anion of the free acid. Interestingly,
lisinopril 25.19 is sufciently bioavailable even without
esterication of the acid group. In addition to the acid
function, the ligand also has abasic nitrogen that is protonated under physiological conditions. This makes lisinopril zwitterionic. At short distances, the two opposite
charges cancel each other out so that the molecule has
amore hydrophobic character with respect to the local
environment (Sect.19.4). Both enalapril and lisinopril
have amuch longer plasma half-life than captopril.
25.5 Finally the Crystal Structure of ACE:
Does aSuccess Story Have
to Be Rewritten?
In his seminal publication in 1977 on the design of captopril, David Cushman once again highlighted the importance of structural models for the work at Squibb:
. Fig. 25.9 Binding of ACE inhibitors. The rationally designed lead
structure 25.8 is optimized stepwise. The introduction of a methyl
group in the side chain to give 25.10 as well as the replacement of the
carboxylate group with athiol are crucial for the increase in afnity.
The result was captopril25.13
“The studies described above exemplify the great heu-
»
ristic value of an active-site model in the design of in-
hibitors, even when such amodel is ahypothetical one.
Only when suitable information on substrate specicity
and mechanism of action of an enzyme is available can
one make areasonable working hypothesis with regard
to complementary functionality needed in an inhibitor.”
Could he have dreamed that it would take another
25years for this structure to be available? In 2003, Edward Sturrock’s group in Cape Town, South Africa,
completed the structure determination. Could it conrm
the previously proposed model? Not all details of the
predicted binding modes for the inhibitors were correct,
but the structure provided critical insights that reinvigorated the eld of ACE inhibitor research. The human
enzyme is highly glycosylated. It consists of 1227 amino
acids in an extracellular domain and is anchored to the
cell membrane by 28additional residues. Interestingly, it
has two catalytic domains, aphenomenon that is rarely
seen in enzymes and has its origin in gene duplication.
The N-terminal domain contains 612 residues and the
C-terminal domain 650 residues. The two domains are
60% identical. Both domains are catalytically active and
0
1
0
2
0
2
0
2
0
2
. • Finally the Crystal Structure of ACE: Does aSuccess Story Have to Be Rewritten?
. Fig. 25.10 A free thiol and carboxylate function are necessary for
binding to ACE. Esterication of the acid group of 25.12 (. Fig.25.9)
to give 25.14 reduced the binding afnity by almost two orders of
magnitude. The S-methylation of 25.12 gives 25.15, which has abinding afnity that is reduced by a factor of 20,000. Compound 25.17
contains merely the thiol and the carboxylate group. These two groups
alone are just enough to achieve detectable binding
their catalytic sites differ by only afew amino acids. Nevertheless, adifference in selectivity for potential ligands
is to be expected. In addition, the C-domain is highly
dependent on the local chloride concentration, whereas
the N-domain is much less dependent. In addition to
this so-called somatic form (s-ACE), there is also atestis
form (t-ACE), which is 701 amino acids long and consists
of asingle domain. Except for the rst 36residues, it is
almost identical to the C-domain of the somatic form.
The structure with bound lisinopril 25.19 (. Fig.25.12)
was determined with this latter form. The inhibitor binds
with its central acid group to the zinc ion. Its phenethyl
group is located in the S1 pocket. The lysine-like group is
in the
moiety binds with its acidic group in
pocket and interacts with Glu 162. The proline
to Lys 511 and
Tyr 520.
It was then interesting to model the differences
in the N- and C-domains of the s-ACE based on the
t-ACE structure and to prepare the proteins by mutagenesis. Both domains bind lisinopril with very similar
afnity, whereby the ligand leaves the S2 pocket unoccupied (. Table25.5). The N-domain has in the S1 and
S2 pocket Tyr 396, Asn 494, and Thr 496. In the C-do-
main, aphenylalanine, aserine, and avaline are found at
these positions. In addition, there is an asparagine in this
pocket in the N-domain that limits access to the S2 pocket
by glycosylation. Therefore, it is not surprising that keto-ACE 25.29 with its bulky benzamido group interacts
much better with the C-domain. Two other compounds
are known, RXP407 25.30 and RXP A380 25.31, which
bind to the two domains with a1000-fold difference in
selectivity. They are derived from phosphinic acids. Because the zinc-binding group is in the center of the mole-
. Fig. 25.11 Examples of ACE inhibitors that are used in therapy
cule, these inhibitors can occupy all four pockets from S2
to
well. RXP A380 has amuch larger moiety for the S2
pocket. In addition, this molecule has an indole moiety
in the
interactions in
position that can undergo stronger hydrophobic
. At this position, the C-domain has an
advantage over the N-domain: Instead of aserine, there
is ahydrophobic valine at position 379, which results in
stronger binding of the inhibitor to the C-domain.
What might be the benet of a domain-specic inhibition of ACE? The enzyme not only converts angiotensinI to angiotensinII, but it also metabolically degrades bradykinin, which lowers blood pressure. ACE
is also thought to be involved in the cleavage of other
signal peptides. ACE inhibitors are generally well tolerated by patients. However, some adverse effects have
been described. For example, many patients develop an