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Chapter • Inhibitors of Hydrolases with an Acyl–Enzyme Intermediate
23
moved, still cleaves the peptide substrate more than 1000
times faster than the pure buffer solution! The remaining
substrate binding sites and the oxyanion hole, whose geometry and properties stabilize the tetrahedral transition
state, are responsible for this acceleration.
Now, it is certainly not difcult to destroy the binding site of an enzyme or its catalytic activity. It is more
difcult to intentionally alter its specicity or function.
The subtilisin mutants, in which the histidine has been replaced by an alanine, cleave substrates with the sequence
–Phe–Ala–X–Phe– (X= e.g., Ala or Gln) six orders of
magnitude slower than the unmodied subtilisin, with
one exception: A substrate with the sequence –Phe–
Ala–His–Phe– is cleaved only four orders of magnitude
slower. The histidine of the substrate takes over to some
extent the role of the histidine in the catalytic site! This
process is called substrate-supported catalysis. The conversion is still rather slow, but the specicity of this mutant is signicantly increased: The –Phe–Ala–His–Phe–
sequence is cleaved 200 times faster than any of the other
–Phe–Ala–X–Phe– sequences.
23.3 The S
Pocket of Serine Proteases
1
Determines Specificity
Proteases recognize polypeptide chains as substrates.
For this task, they use a series of more or less pronounced binding pockets on their surface, as described in
Sect.14.5. These are structurally and electronically complementary to the side chains of the corresponding residues in the substrate. As aresult, the polypeptide chain
of the substrate is immobilized on the surface in the vicinity of the catalytic site. Depending on the protease,
the crevices on the surface look very different. Surface
portions of four different serine proteases from the trypsin family are shown in . Fig.23.2. Acomparison of the
different serine proteases with different substrate specicities (. Fig.23.3) shows that particularly the structures
of the S1 pockets of these enzymes are different. The S1
pocket consists mainly of the sequence segments 189–195
and 214–220. There are signicant differences in the side
chains of the amino acid at positions189, 216, and 226.
In chymotrypsin, these are Ser 189, Gly 216, and Gly 226.
They adjust the depth and shape of this pocket in away
to accommodate the aromatic side chains of the amino
acids phenylalanine, tyrosine, and tryptophan. Accordingly, chymotrypsin preferentially cleaves peptide chains
after one of these three amino acids. Trypsin also has
adeep, spacious S1 pocket anked by Gly 216 and Gly
226. The negatively charged carboxylate group of Asp
189 at the bottom of the pocket is critical for the recognition of the long, positively charged side chains found
in the amino acids lysine and arginine of the substrate. In
elastase, the S1 pocket is formed by the amino acids Val
216 and Thr 226. This makes the pocket much smaller.
It can only accommodate amino acids with short hydrophobic side chains, such as alanine and valine. Amino
acids with large groups are no longer accommodated.
Amino acid 189, which is serine, is deeply buried.
The substrate specicity of the described serine pro-
teases is primarily achieved by recognition of the amino
acid in the P1 position. Even very small changes can alter
the substrate prole in this pocket. For example, trypsin
cleaves substrates after the two basic amino acids arginine and lysine, whereas thrombin cleaves exclusively
after the more basic amino acid arginine. The only difference in the S1 pocket is that thrombin has a sodium
ion next to the Asp 189 residue. Its positive charge attenuates the negative charge of the adjacent aspartate
residue, thus, reducing its polarizing effect. As aresult,
the more basic Arg residue continues to bind in apositively charged state, while for the less basic lysine, the
polarizing effect is no longer sufcient to induce protonation. This helps to discriminate between peptide chains
exhibiting Lys and Arg in P1 position (see also Sect.4.4,
. Fig.4.4). Pockets adjacent to S1 are also important for
substrate binding and selectivity. It is noteworthy that the
substrate-binding pockets of serine proteases that recognize the N-terminal part of the substrate (unprimed side,
S1–S4 pockets; Sect.14.5) are more pronounced. Pockets
on the unprimed side that anchor the C-terminal part
of the substrate are much less well developed. Since the
N-terminal cleavage product remains temporarily covalently bound to the protease as an acyl enzyme complex,
this part of the substrate is bound particularly selectively.
These structural features dene what apotential competitive inhibitor of a serine protease should look like:
It is crucial that the S1 pocket is lled as well as possible.
The chemical composition of the parts of the inhibitor
that bind in this region must be complementary to the S1
pocket. In some cases, lling the S1 pocket alone is sufcient to generate aselective serine protease inhibitor with
respectable binding afnity. For example, in 1967, Marcos Mares-Guia and Elliott Shaw described small-molecule trypsin inhibitors with micromolar binding afnity
that occupied only the S1 pocket. It is not difcult to
see that all molecules 23.1–23.4 in . Fig.23.4 mimic the
basic amino acids arginine or lysine in the P1 position of
the substrate.
Arst approach to the design of serine protease inhibitors could be based on the search for asuitable group
to occupy the S1 pocket, which could then be coupled to
achemically reactive group that binds to the catalytic
serine. The various groups described in the literature for
this purpose are summarized in . Table23.2. Natural
products also follow this principle. The macrocyclic pentapeptide thrombin inhibitor cyclotheonamideA from the
marine sponge Theonella sp. contains an α-keto function
next to an amide bond. As the X-ray structure shows, this
ketone group forms atetrahedral hemiacetal structure
with the OH group of the catalytic serine (. Fig.23.5).
ab
cd
. • The S Pocket of Serine Proteases Determines Specicity
. Fig. 23.2 The surfaces of the trypsin-like serine proteases tryp-
sin, thrombin, factorVIIa, and factorXa show astrong indentation
in the area of the catalytic center. To highlight this surface pattern,
the color of the surface changes from blue to green to red as the
depth of the indentation increases. In the indentations, the exposed
physicochemical properties determine the substrate selectivity of
the proteases. The preferred cleavage sequences are indicated in the
If the sequence of the peptide substrate of the serine
protease is known, the N-terminal amino acid preceding
the cleavage site can be coupled with one of the groups
from . Table23.2 to produce acompound that is most
likely to be an inhibitor. An example of this is the elastase inhibitor N-(methylsuccinyl)–Ala–Ala–Pro–Val–
CF3 (23.21 in . Fig.23.14), which is derived from the
substrate sequence Pro–Val. In favorable cases, the P1
equivalent alone is sufcient, for example, in the tryp-
structures, where XXX represents any amino acid at that position.
(7 https://sn.pub/KPYo7E)
sin and thrombin inhibitors 23.5 and 23.6 (. Fig.23.4).
However, the usually high chemical reactivity of the
functional groups in covalently bound serine protease in-
hibitors, which is necessary to interact with the catalytically active serine, can be problematic. Because of their
reactivity, such groups can also react undesirably with
serine residues of other enzymes, causing side effects. The
design of highly potent and selective inhibitors requires
at least the occupancy of the S2, S3, and S4 pockets. An-
Chapter • Inhibitors of Hydrolases with an Acyl–Enzyme Intermediate
23
. Fig. 23.3 Comparison of the S1 pockets of chymotrypsin, trypsin,
and elastase. The binding pocket of chymotrypsin is tailored for large,
lipophilic side chains. The S1 pocket of trypsin binds amino acids with
positively charged side chains through its negatively charged Asp 189
. Fig. 23.4 The molecules 23.1–23.4 that bind in the S1 pocket of
trypsin are micromolar inhibitors. All of these molecules contain
astrongly basic group that is protonated under physiological conditions; therefore, a positive charge is available to form asalt bridge to
other structural feature common to all serine proteases
should also be mentioned: Their substrates are bound
to the peptide backbone by two antiparallel hydrogen
bonds. This orientation of the two hydrogen bonds results in apleated sheet-like geometry. In the majority of
designed inhibitors, an attempt was made to mimic this
pattern of hydrogen bonding (. Fig.23.6).
23.4 Seeking Small-Molecule Thrombin
Inhibitors
The serine protease thrombin plays acentral role in the
control of blood coagulation. Thrombin is at the end
of acomplex, highly regulated cascade of serine proteases. Injury to the arterial vasculature causes membrane-bound tissue factor outside the vessel to contact
the serine protease precursor factorVII in the blood.
The precursor is activated to factorVIIa and initiates
the coagulation cascade. Along the cascade, various factors are released, which are activated from their zymogen
form by proteases from the previous step. Finally, the
cascade leads to the release of von Willebrand factor,
which binds to platelets and initiates blood clot formation. In addition to extrinsic activation, there is also an
intrinsic coagulation pathway. It is initiated by reduced
residue. Because of the spatial lling of the side chains of Thr 216 and
Val 226, elastase has arelatively small S1 pocket and, therefore, binds
small hydrophobic amino acids such as alanine and valine
the negatively charged side chain of Asp 189. The thrombin inhibitors
23.5 and 23.6 contain an additional functional group that can form
acovalent bond to the catalytically active serine
blood ow or pathologically altered vasculature. In this
case, the coagulation cascade is initiated to form aplatelet aggregate, which is then stabilized by abrin network.
FactorX is found in one of the nal steps where the two
pathways merge. All of these steps involve serine proteases, which are potential targets for drug therapy. To
date, development efforts have focused on the enzymes
thrombin, factorXa, and factorVIIa. This has already
led to development candidates and marketed products
for the rst two.
Thrombin converts inactive brinogen into reactive brin. Together with aggregated platelets, it forms
apolymer in which the different blood cells are trapped.
Athrombus is formed, which is further cross-linked and
stabilized by transglutaminase factorXIII (Sect.23.8).
This is an essential protective mechanism of the body to
ensure wound closure. In certain diseases or situations,
such as after surgery, heart attack, or to prevent stroke in
patients with atrial brillation, it is necessary to reduce
the blood’s ability to clot. For this reason, there is great
interest in the development of selective and, above all,
orally available inhibitors of the coagulation cascade.
Thrombin cleaves brinogen between the amino acids
arginine and glycine. This sequence served as the starting
point for the development of the rst synthetic thrombin
inhibitors, which therefore contained either an Arg or
0
3
0
3
0
1
. • Seeking Small-Molecule Thrombin Inhibitors
an Arg-analogous moiety. In this section, three different
approaches to the development of thrombin inhibitors
will be presented: substrate analogues, benzamidine analogues, and signicantly modied structural analogues.
One approach for the design of thrombin inhibitors
is provided by the P3…
substrate sequence Gly–Val–
Arg–Gly–Pro–Arg of brinogen. In the early 1970s,
the Japanese group of Hamao Umezawa found that
peptide aldehydes with C-terminal arginine residues
isolated from bacteria were potent inhibitors of some
trypsin-like serine proteases. The tripeptide aldehydes
studied by Sándor Bajusz were derived from amino acids
P3–P1 or
–
, i.e., the three amino acids “before” and
“after” the cleavage site. The relative binding afnities of
some peptide aldehydes are summarized in . Table23.3.
Interestingly, the direct comparison of Gly–Val–Arg–H
and Gly–Pro–Arg–H shows that aproline at the P2 position inhibits thrombin about nine times more strongly.
The introduction of phenylalanine instead of glycine
in the P3 position leads to an additional signicant in-
. Table 23.2 Reactive groups that can covalently react with
the catalytically active serine
Inhibitor type Functional group
Irreversible Chloromethylketone –COCH
Sulfonyluoride –SO2F
a
Ester
Boronic acid
Reversible Aldehyde –CHO
Ketone –COR (R=Alkyl,
Triuoromethylketone –COCF
α-Ketocarboxylic acid
α-Ketoamide
α-Ketoester
a
Reversible as well as irreversible examples are known.
a
–COOR
–B(OR)
Aryl)
–COCOOH
–COCONHR
–COCOOR
Cl
2
2
3
. Fig. 23.5 Crystal structure of the inhibitor cyclotheonamide A
with thrombin. The inhibitor forms acovalent bond to the catalytic
serine with its α-keto group to form ahemiketal structure. The now
negatively charged oxygen is stabilized by two hydrogen bonds in an
oxyanion hole. (
7 https://sn.pub/zJhqBx)
d
d
d
Chapter • Inhibitors of Hydrolases with an Acyl–Enzyme Intermediate
23
. Fig. 23.6 General binding mode of a peptide chain that is to be
cleaved (gray carbon atoms) in the catalytic site of a serine protease.
The amide bond to be cleaved is highlighted in yellow. The substrate’s
P1 (light blue) and P2 groups (green) are shown with asurface; they
bind in the S1 and S2 pockets of the protein. Two antiparallel-oriented
hydrogen bonds (green) are formed to the main chain. The H-bonds to
the oxyanion hole are in purple, and the direction of the nucleophilic
crease in binding. d-Amino acids in the P3 position were
then investigated. Surprisingly, these led to a dramatic
improvement in binding afnity. This result was not expected considering that the substrate sequence from P5 to
. Table 23.3 Relative binding afnity of tripeptide alde-
hydes on thrombin. Arg–H is for the aldehyde that was obtained by reducing the carboxylic acid of arginine. The larger
the value of the relative inhibition, the stronger the inhibitor
binds to thrombin
Peptide Relative inhibition
Gly–Val–Arg–H 1
Gly–Pro–Arg–H 9
Phe–Pro–Arg–H 57
-Ala–Pro–Arg–H 469
-Val–Pro–Arg–H 1273
-Phe–Pro–Arg–H 7370
attack of the Ser 195 oxygen on the carbonyl carbon is indicated in
violet blue. (7 https://sn.pub/yKx9nH)
P3 Gly–Gly–Gly–Val–Arg contains only achiral glycine
residues without lipophilic side chains that can hardly
form interactions corresponding to the d-Phe side chain.
At the time of the work described above, the spatial
structure of thrombin had not yet been determined. Wolfram Bode and Milton Stubbs at the Max Planck Institute of Biochemistry in Martinsried, Germany, solved
the structure of athrombin complex with achemically
activated brinopeptide, Gly–Asp–Phe–Leu–Ala–Glu–
Gly–Gly–Val–Arg–CH2Cl. This peptide corresponds
to the N-terminal portion from P11 to P1 that thrombin cleaves from brinogen. Comparison of this structure with that of d-Phe–Pro–Arg–chloromethylketone
(. Fig.23.7) provided an explanation for the surprising
structure–reactivity relationship found by Sándor Bajusz. The S3 pocket is lled by both ligands, in the case
of the brinopeptide by the side chains of leucine and
phenylalanine at positions P
and P9. The peptide forms
8
aβ-turn that allows the amino acids in this sequence to
be positioned in the S3 pocket. The same pocket is ac-
. • Seeking Small-Molecule Thrombin Inhibitors
. Fig. 23.7 Comparison of the binding mode of the irreversibly
binding thrombin inhibitors d-Phe–Pro–Arg–CH2Cl (dark-red carbon
atoms) with that of the brinopeptide derivative (gray carbon atoms).
Both inhibitors bind with an arginine side chain in the S1 pocket. The
S2 pocket is occupied by avaline side chain of the brinopeptide. Its
additional peptide chain is folded back so that the Leu and Phe side
chains in positions P8 and P9 are oriented into the lipophilic S3 bind-
cessed by the tripeptide through the side chain of the
d-amino acid at the P
.
3
The compound d-Phe–Pro–Arg–H, synthesized by
Bajusz, is ahigh-afnity thrombin inhibitor (Ki = 75 nM).
However, the compound proved to be chemically unstable. This problem could be solved by N-methylation of
the free NH2 group. N-Methyl-d-Phe–Pro–Arg–H 23.7
(Gyki14766, Efegatran, . Fig.23.8) is chemically stable.
Jörg Stürzebecher and Fritz Marquardt took adifferent route. They pursued the goal of managing inhibition without acovalent attachment. Their approach
was based on the nding that in addition to trypsin
(Ki = 18 μM), benzamidine 23.1 (. Fig.23.4, Sect.23.3)
also inhibits thrombin (Ki = 220 μM). The combination
ing pocket. In the case of d-Phe–Pro–Arg–CH2Cl, the phenyl ring of
d-Phe is also located in this pocket. (7 https://sn.pub/MG65ha)
of the benzamidine group with areactive group from
. Table23.2 yielded potent thrombin inhibitors. The
rst low molecular weight thrombin inhibitor to be
clinically tested in the 1970s was p-amidinophenylpyru-
vic acid 23.5 (. Fig.23.4, Sect.23.3). The compound
proved to be efcacious, but its selectivity was unsatisfactory. The simple benzamidine derivatives 23.8 and 23.9
(. Fig.23.8) are further typical representatives with micromolar afnity for thrombin, but without selectivity
compared to trypsin.
The coupling of the benzamidine groups with
a peptide structure brought signicant improvement.
Nα-(β-naphthylsulfonylglycyl)-d,l-p-amidinophenylalan-
ylpiperidide 23.10 (NAPAP, . Fig.23.9) was the result
Chapter • Inhibitors of Hydrolases with an Acyl–Enzyme Intermediate
. Fig. 23.8 The inhibitor 23.7 (Gyki14766, efega-
tran) contains an aldehyde group that binds reversibly
to Ser 195. Compounds 23.8 and 23.9 are simple
derivatives of benzamidine that noncovalently inhibit
the enzyme
of amore than 10-year-long systematic search for potent and selective thrombin inhibitors. NAPAP was the
most potent representative of the class of low molecular
weight thrombin inhibitors (Ki = 6 nM) for along time,
but it has only modest selectivity over trypsin.
In 1989, Wolfram Bode solved the crystal structure of
thrombin with abound inhibitor. The structure was determined rst with the irreversible inhibitor d-Phe–Pro–
Arg–CH2Cl and then with NAPAP. The 3D structure of
the thrombin–NAPAP complex is shown in . Fig.23.10.
The racemic form was used for cocrystallization. The
result that p-amidinophenylalanine binds to thrombin
as ad-amino acid was rather surprising. The substrate
consists only of l-amino acids, so it was expected that
p-amidinophenylalanine would also bind in the l-con-
guration.
. Fig. 23.9 The thrombin inhibitors NAPAP
23.10, CRC220 23.11, the latter was developed at the former Behringwerke, and 23.12
which was derived from 23.10. The two latter
compounds have distinctly better afnity to
thrombin and improved selectivity relative to
trypsin. The IC50 values for 23.10 and 23.12
are given for the racemates. Inhibitor 23.11 was
measured as an enantiopure compound
The groups of the ligand that form polar interactions
with the protein can be deduced directly from the crystal structure. For NAPAP, these are the glycine moiety
in the center of the molecule (two hydrogen bonds to
the peptide backbone) and the amidinium group in the
S1 pocket. Omitting the positively charged amidinium
group results in aloss of binding afnity because the
salt bridge to Asp 189 cannot be formed. However, later
work showed that chloro-substituted aromatic rings can
also bind into the S1 pocket and form ahydrophobic interaction with Tyr 228. At the same time, they displace
awater molecule from the S1 pocket (. Fig.4.7). Today,
an arsenal of building blocks that can be used as arginine side chain mimics to ll the S1 pocket of thrombin
(. Fig.23.11) are available.
23
. • Seeking Small-Molecule Thrombin Inhibitors
. Fig. 23.10 Structure of the thrombin–NAPAP complex. The most
important interactions are outlined on the left side. The positively
charged benzamidine group occupies the S1 pocket and forms asalt
bridge to the negatively charged side chain of Asp 189. Two hydrogen
bonds are formed to the amino acid Gly 216. The piperidyl and naph-
With its naphthyl and piperidyl side chains, NAPAP
largely lls the lipophilic S3 pocket and the spatially
limited S2 pocket (. Fig.23.10). However, it appears
that even larger substituents could t into the S3 pocket.
Aweakness of NAPAP was its lack of selectivity compared to the digestive enzyme trypsin. Fortunately, the
structures of NAPAP complexed with thrombin and also
with trypsin are known (. Fig.23.12). A comparison
of the 3D structures shows that there is asignicant difference in the binding mode between the two enzymes
in the S3 pocket, resulting in a180° ipped orientation
of the naphthyl group with respect to the bond to sul-
fur. In thrombin, the S3 pocket is more pronounced and
surrounded by several lipophilic amino acid side chains.
In trypsin, the upper end of this pocket is open and not
spatially restricted. Obviously, its structuring is not necessary in the largely unspecic digestive enzyme. There-
fore, the selectivity can be increased by occupying the
S3 pocket of thrombin as optimally as possible. Acloser
look at the thrombin–NAPAP complex reveals that an
additional methoxy substituent on the naphthyl ring
should be suitable for increasing selectivity. Indeed, in-
thyl groups together occupy the two large lipophilic pockets S2 and S3.
(7 https://sn.pub/CPfw8M)
hibitor 23.12 binds 600 times more strongly to thrombin
than to trypsin.
The compound CRC220 (23.11, . Fig.23.9), which
lls the hydrophobic S3 pocket much better than NAPAP,
was developed at the former Behringwerke in Marburg,
Germany. Because of this improved lling, CRC220 inhibits thrombin almost 200 times more effectively than
trypsin.
Researchers at Hoffmann-La Roche took adifferent
approach to the search for thrombin inhibitors. First,
they focused on the optimal lling of the S1 pocket. Benzamidine was known to be aweak thrombin inhibitor that
occupies the S1 pocket. However, it has the disadvantage
of binding more strongly to trypsin (. Fig.23.4). The
researchers in Basel were, therefore, looking for asmall
molecule that would bind more strongly to thrombin
than to trypsin. More than 200 small molecules of typical fragment size were tested in this narrow search. Structures were only selected if their functional groups could
interact with the negatively charged side chain of Asp
189. Guanidines, amidines, and amines were screened.
N-Amidinopiperidine (23.13, . Fig.23.13) was identied
as an interesting lead structure. In contrast to benzami-
Chapter • Inhibitors of Hydrolases with an Acyl–Enzyme Intermediate
23
. Fig. 23.11 Numerous building blocks have been developed that bind as amimetic for arginine in the S1 pocket of thrombin
. Fig. 23.12 Comparison of the 3D structures of trypsin (left)
and thrombin (right), each complexed with NAPAP. The active site
in thrombin is further narrowed by an additional loop from above.
The depth of the pocket is, once again, color-coded (see . Fig.23.2).
(7 https://sn.pub/f6r1mg)
. • Seeking Small-Molecule Thrombin Inhibitors
. Fig. 23.13 One approach to the structure-based design of throm-
bin inhibitors began with 23.13 in the S1 pocket. Compound 23.14
was derived from this lead structure. Its docking into the active site
of thrombin let to the idea for the synthesis of 23.15. Systematic variation of the side chain R yielded compounds with better binding afnity such as 23.16 and 23.17. The compound was extensively tested
in the clinic under the name napsagatran. The compound melagatran
dine, amidinopiperidine binds more strongly to thrombin
(Ki = 150 μM) than to trypsin (Ki = 300 μM). Systematic
derivatization yielded 23.14, amoderately active throm-
bin inhibitor (Ki = 0.48 μM). Based on the structural
model with the protease, it seemed obvious that replac-
ing the glycine moiety with ad-amino acid, e.g., d-Phe,
should ll alipophilic pocket and lead to asignicant
increase in afnity. The compound was quickly prepared
from AstraZeneca was introduced as the double prodrug ximelagatran
23.18 as the rst orally available thrombin inhibitor on the market.
It is derived from the tripeptide sequence d-Phe–Pro–Arg. Another
orally available inhibitor, dabigatran 23.19, was launched to market by
Boehringer Ingelheim. The tricyclic inhibitor 23.20, which was developed at the ETH in Zurich, does not possess apeptide character at all.
Argatroban is approved for intravenous use
and tested. Indeed, 23.15 bound ten times more strongly
to thrombin. Other d-amino acids were then explored
and the afnity was increased further. The high selectivity against trypsin was also encouraging; 23.16 binds
840 times more strongly to thrombin than to trypsin. The
surprise came when the 3D structure of 23.14 complexed
with thrombin was determined: The compound binds differently in the binding pocket than predicted! Contrary