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Chapter • Inhibitors of Hydrolases with an Acyl–Enzyme Intermediate
23
to the original assumption, the naphthylsulfonyl group
swapped positions with the benzyl side chain.
The incorporation of anonproteinogenic amino acid
proved to be unfavorable from asynthetic point of view.
Therefore, other central building blocks that were more
readily accessible for synthesis were sought. This work
ultimately led to napsagatran 23.17, ahighly potent and
exceedingly selective compound. However, because it can
only be administered intravenously, it never found its way
into a marketed product, especially since argatroban,
amarketed product for intravenous use, was discovered
much earlier and was already available.
The search for small-molecule, orally available thrombin inhibitors occupied numerous large pharmaceutical
companies for many years. It took along time for AstraZeneca to launch the rst orally available thrombin inhibitor, ximelagatran (23.18, . Fig.23.13). The compound
is adouble prodrug of the active substance melagatran.
Its relationship to the original parent structures (e.g., the
tripeptide sequence d-Phe–Pro–Arg) is still quite obvious. The head group of the arginine residue was replaced
by abenzamidine, the ve-membered ring of the proline
was narrowed to afour-membered ring, and the terminal
benzyl group was shortened to acyclohexyl ring. The
N-terminus was substituted with amethylenecarboxylic
acid group. It proved to be extremely difcult to make the
thrombin inhibitors sufciently bioavailable and to maintain the required plasma levels for an acceptable period
of time. AstraZeneca, in collaboration with Bernd Clement’s group at the University of Kiel, Germany, pursued
adouble prodrug strategy to improve bioavailability: the
terminal acid function was masked as an ester, and the
benzamidine group was transformed into an N-hydroxy-
amidine. The drug, melagatran, is released in the body
by ubiquitous esterases and aset of three specic reductases. AstraZenecca withdrew ximelagatran (Exanta®)
after two years because of problems with liver toxicity
observed in asmall number of cases after weeks of use.
Many years of research into thrombin have nally led
to success at Boehringer Ingelheim as well. The substance
dabigatran (23.19, . Fig.23.13) was launched in spring
2008 for the prevention of stroke in patients with atrial
brillation. It also has abenzamidine anchor and apyridine group for the hydrophobic S3 pocket. Abenzimidazole moiety with an attached amide bond was chosen
as alinker between these groups. As with ximelagatran,
acarboxylic acid is attached to the N-terminus. Dabigatran has signicantly less peptide character than the
original lead structures. Adouble prodrug strategy was
also used for this compound to ensure adequate bioavailability. In addition to esterication of the acid group,
the amidine group was masked as acarbamoyl moiety.
The prodrug is called dabigatran (Pradaxa® in the United
States and Europe and Pradax® in Canada).
François Diederich’s group at the ETH in Zurich has
succeeded in developing athrombin inhibitor (23.20,
. Fig.23.13) that completely lacks the peptidomimetic
character. Accurate design in the binding pocket led to
an inhibitor with acentral tricyclic moiety, which was
readily prepared by a1,3-dipolar addition reaction. With
abenzamidine anchor for the S1 pocket and apiperonyl
moiety for the S3 pocket, this very rigid derivative entered
the realm of nanomolar inhibitors.
23.5 Design of Orally Available Low
Molecular Weight Elastase Inhibitors
Human leukocyte elastase is aserine protease released
in the lungs to destroy dead tissue and invading bacteria. The destructive potential of this enzyme is normally
controlled by anumber of endogenous inhibitors, such
as α1-protease inhibitor or leukocyte protease inhibitor.
When the balance between protease and inhibitor is
disturbed, for example, by genetic underexpression of
an inhibitor or by airborne toxins, elastase attacks even
healthy lung tissue. Cigarette smoke contains compounds
that oxidize an essential methionine side chain on the endogenous α1-protease inhibitor, thereby, deactivating the
inhibitor. The chronic destruction of cells in the alveoli
leads to alife-threatening disease: emphysema.
One possible approach for the pharmaceutical treatment of this disease is the use of elastase inhibitors. Unlike thrombin, elastase does not have adeep, pronounced
S1 pocket with an acidic amino acid through which apolar contact can be made with apotential ligand. Elastase
only accepts substrates with small hydrophobic amino
acids such as valine (. Fig.23.3). If a large binding
contribution cannot be expected from occupying the S1
pocket, as in the case of thrombin, the catalytic serine
itself can be involved in the protein–ligand interaction
by forming areversible covalent bond with the inhibitor. Such aconcept was pursued at the former ICI (now
part of AstraZeneca), starting with a triuoromethyl
ketone R–COCF3 as a reversible covalently binding
serine protease inhibitor. Starting from the substrate se-
(. Fig.23.14) were found.
ICI200880 (23.22) proved to be an effective elastase
inhibitor in clinical trials, but it lacked oral bioavailability and had ashort biological half-life. The spatial
structure of the related inhibitor Ac–Ala–Pro–Val–CF3
complexed with elastase was determined. The main
interactions between elastase and the inhibitor are
shown in . Fig.23.15. The inhibitor binds to elastase
in aβ-pleated sheet conformation with two H-bonds to
Val 216 and one to Ser 214. The valine side chain lls
the S1 pocket and the carbonyl group binds covalently
as ahemiketal to the side chain of Ser 195. Research
has focused on nonpeptidic structures with functional
groups capable of forming the same interactions as the
peptidic inhibitors.
. • Serine Protease Inhibitors: Thrombin Was Just the Starting Point
. Fig. 23.14 Elastase inhibitors 23.21 and 23.22
(ICI200880) are substrate analogues. Compound
23.22 is ahighly active compound, but it is not
orally available
Based on the 3D structure of the protein–ligand
complex, pyridones were selected as the most promising
peptidomimetic replacements. The postulated binding
mode of the pyridone compared to that of the peptidic
inhibitors is shown in . Fig.23.15. Compounds of this
chemotype were synthesized at Zeneca (now AstraZeneca) and indeed proved to be very potent elastase inhibitors. Compound 23.23 (. Fig.23.16) binds to the protein
with Ki = 5.6 nM. However, this compound has several
unfavorable properties. It is not orally available and in-
hibits chymotrypsin (Ki = 60 nM) in addition to elastase.
Poor oral bioavailability was attributed to its excessive
lipophilicity (log P > 4) resulting in low water solubility.
The pyrimidone class, in which acarbon atom of the
heterocycle was replaced by anitrogen atom, appeared
to be synthetically simpler and, therefore, more variable.
Compound 23.24 is less lipophilic (log P = 2.1) than
23.23, ten times more water soluble, and orally available.
Its binding to elastase was found to be practically unchanged (Ki = 6.6 nM), whereas chymotrypsin inhibition
was much less pronounced (Ki = 1000 nM). Numerous
representatives of the new class of compounds were synthesized and tested for inhibitory activity and bioavailability. It has been shown that the potency of inhibition
and in vivo activity did not run in parallel. For example,
23.25 is ahighly potent elastase inhibitor in the enzyme
assay, but is not orally available. With an oral bioavailability of 60–90%, compound 23.26 (Ki = 100 nM)
proved to be optimal in the animal model. The crystal
structure with an analogous derivative 23.27, which has
only one additional sulfonamide group, conrmed the
expected binding mode (. Fig.23.17).
The Japanese company ONO Pharmaceuticals Co.
developed compound 23.28, which is derived from 23.26.
It has a1,3,4-oxadiazole ring instead of the triuoromethyl group on the ketone and an unsubstituted phenyl
ring on the pyrimidone. However, development of ONO6818 was discontinued in phaseII clinical trials due to
abnormally elevated liver enzyme levels. However, ONO
Pharmaceuticals has had success with ONO-5046, 23.29
which was developed under the name sivelestat (Elaspol®; . Fig. 23.16). This inhibitor reacts specically
. Fig. 23.15 Comparison of the binding mode of the elastase inhib-
itor Ac–Ala–Pro–Val–CF3 with the postulated binding mode of the
pyridone moiety (e.g., 23.23, . Fig.23.16). Both compounds should
be able to form adouble H-bond to Val216
with elastase and reversibly acylates the catalytic serine
residue.
23.6 Serine Protease Inhibitors: Thrombin
Was Just the Starting Point
Factor Xa and factorVIIa precede thrombin in the coagulation cascade and are being investigated as targets for
antithrombotic agents. Both have an aspartic acid at the
bottom of their deep S
addition, anarrow and deep S3 pocket anked by aromatic amino acids (Tyr99, Trp 215, and Phe 174) is specic to factorXa. Therefore, this pocket is ideally suited
pocket, similar to thrombin. In
1
Chapter • Inhibitors of Hydrolases with an Acyl–Enzyme Intermediate
23
. Fig. 23.16 Development of orally available elastase inhibitors at
Zeneca. The original idea of replacing the Ala–Pro moiety with apyridone yielded 23.23. Later, pyrimidinones were mainly investigated. An
additional nitrogen atom was added to the heterocycle. Very potent
compounds (e.g., 23.25) are found in this class. Compound 23.26 has
the best in vivo properties. The p-uorophenyl group (in 23.26) or the
p-aminophenyl group (in 23.27) increases the lipophilic contact with
for aromatic groups on inhibitors. As mentioned above,
in the mid-1990s the dogma prevailed that the S1 pocket
of trypsin-like serine proteases could only accommodate
groups with abasic character. However, the binding of
chloro-substituted aromatic moieties to thrombin was
demonstrated at Merck & Co. in the USA. Such aromatic
moieties provided abreakthrough for factorXa inhibitors. Several research groups have been able to develop
highly potent inhibitors using chlorophenyl, chloronaphthyl, or chlorothiophene substituents as binders for the
S1 pocket. By placing additional substituents in the deep
the enzyme. The compound ONO-6818 23.28 was developed and advanced to clinical trials in Japan, where it was discontinued due to abnormally elevated liver values in treated patients. Another compound,
23.29, was clinically tested under the name sivelestat (ONO-4056).
These compounds specically transfer an acyl group to the catalytic
serine and reversibly block the enzyme
aromatic S3 pocket, inhibitors gained sufcient afnity to
bind to this protease in the single-digit nanomolar range
(. Fig.23.18). In addition to the development of compounds with achlorine-substituted aromatic moiety for
the S1 pocket, inhibitors with benzamidine substituents
for S1 have also been synthesized as factorXa inhibitors.
However, it has been much more difcult to achieve sufcient selectivity with abenzamidine moiety compared to
the other trypsin-like serine proteases. In addition, these
derivatives encounter similar problems as the thrombin
inhibitors in terms of lack of bioavailability. In Septem-
. • Serine Protease Inhibitors: Thrombin Was Just the Starting Point
. Fig. 23.17 Crystal structure of 23.27 (. Fig. 23.16) complexed
with elastase. The inhibitor forms two H-bonds to Val 216 and one
H-bond to Ser 214. Furthermore, the oxyanion hole is occupied by an
oxygen atom. (7 https://sn.pub/P2aZ)
ber 2008, Bayer launched the new factorXa inhibitor
rivaroxaban (Xarelto®, 23.30, . Figs.23.18 and23.19),
which places a chlorothiophene substituent in the S1
pocket.
Factor VIIa is found at the beginning of the extrinsic pathway of the coagulation cascade. This enzyme
also belongs to the family of trypsin-like serine proteases, for which specic inhibitors have been sought for
many years. In this case, the activation of the protease
is of interest. In cases of injury, blood comes into contact with tissue. When this happens, factor VIIa and
membrane-bound tissue factor can form acomplex that
causes aconformational change in the catalytic domain
of the protease. Apeptide segment adjacent to the catalytic center changes from an unfolded conformation to
ahelical structure. This leads to achange in the geometry
of the catalytic site. Only in the complexed state does
the protease have astructure that allows it to initiate the
coagulation cascade. Although numerous nanomolar
inhibitors are available, none of them has been able to
replace the basic P1 group at the aromatic ring.
The administration of a drug as a “direct” anticoagulant in the blood coagulation cascade always carries
the risk of life-threatening and uncontrollable bleeding.
For this reason, antidotes have recently been developed.
In the eld of factorXa, Andexanet alfa, arecombinant
human factor Xa enzyme variant, has been approved.
In its catalytic center, the essential serine has been replaced by an alanine. This reduces its catalytic activity
by several orders of magnitude (Sect.23.2). It is barely
able to cleave and activate prothrombin. In addition, its
γ-carboxyglutamic acid (Gla) domain has been removed.
However, its afnity for factorXa inhibitors such as rivaroxaban or apixaban is virtually unchanged. It effectively removes these substances from the bloodstream.
Boehringer-Ingelheim has taken adifferent approach in
the case of thrombin. The company has developed the
antibody idarucizumab (Sect.32.3). In an emergency sit-
uation, it cancels out the anticoagulant effect of dabigatran 23.19 by capturing the active substance.
In addition to the serine proteases of the coagulation
cascade, other proteases in this family have been selected
for drug development. Drug design for these target enzymes has beneted greatly from the experience gained
with thrombin inhibitors. The lessons learned from
thrombin inhibitors are well transferable to the specic
conditions of these proteins. Tryptase, urokinase, and
matriptase belong to this family. Tryptase inhibitors are
being investigated for the treatment of asthma, and the
other two are targets for potential cancer therapeutics.
Chapter • Inhibitors of Hydrolases with an Acyl–Enzyme Intermediate
. Fig. 23.18 Four potent
inhibitors of factorXa.
The rst three bind with
achloroaromatic group
in the S1 pocket of the
enzyme. Rivaroxaban
23.30 was launched by
Bayer in 2008 as the rst
orally available anticoagulant. Betrixaban 23.31
(Portola), edoxaban 23.32
(Daiichi Sankyo), and
apixaban 23.33 (Bristol-Myers-Squibb) are
three other compounds
on the market. Apixaban
binds with subnanomolar
afnity to with amethoxy-substituted aromatic
ring in the S1 pocket of
factorXa
23
Tryptase is atetramer with four trypsin-like catalytic
sites. These sites are separated by several angstroms. To
develop selective inhibitors, compounds were designed
that carry two benzamidine-like anchor groups and
abridge long enough to connect them. In this way, two
of the four sites in the tetrameric tryptase molecule are
blocked simultaneously. The disadvantage of this design
concept is that the developed inhibitors are very large.
They are well above the molecular weight limit of 600
Da that should not be exceeded for good bioavailability.
The protease furin also belongs to the serine protease family, but adopts the folding of the subtilisin family (Sect.14.8). It is involved in the maturation of proproteins. It cleaves the envelope proteins of viruses to
convert them into their active form. Its involvement in
the “arming” of viruses has even been reported in the
tabloid press: On August28, 2003, Germany’s tabloid
newspaper BILD-Zeitung called furin “the world’s most
brutal protein,” which “turns epidemics into adeadly
threat to humans and acts like adetonator on abomb.”
Furin and other closely related subtilases cleave certain
basic tetrapeptide sequences at the C-terminus: Arg–X–
(Arg/Lys)–Arg–. Many glycoproteins of lipid-enveloped
viruses are cleaved and activated at this recognition sequence. An example is the highly pathogenic avian inuenza viruses, which contain such cleavage sequences in
hemagglutinin, one of the surface glycoproteins. Whether
the viruses can be activated depends on the availability of
the omnipresent furin, and this is aprerequisite for the
high pathogenic potential of the avian inuenza viruses.
Furthermore, other genetic combinations or requirements
must be met to transform these viruses into dangerous
pathogens for animals and humans. Inhibitors of furin
could prevent this activation of the viruses. This concept
for the development of new antiviral agents is, therefore,
being intensively pursued. Furin has also been implicated
in the treatment of other diseases such as cancer and cystic brosis. Recently, the nanomolar, highly selective and
cell-permeable inhibitor BOS-318 (23.34, . Fig. 23.20)
was successfully developed to treat cystic brosis. Surprisingly, the crystal structure with the inhibitor bound shows
arearrangement in the catalytic center that has never been
observed before. BOS-318 does not interact with the polar
amino acids in the highly charged S1 pocket. This pocket
remains unoccupied and is lled with water molecules.
Instead, there is aip of Trp 254, which in thrombin and
factorXa corresponds to Trp 215 at the bottom of the
S3 pocket (. Figs. 23.10 and 23.19). As aresult, the S1
pocket is largely occluded. The vacant hydrophobic position of Trp 254 is now occupied by the dichlorophenyl
substituent of BOS-318. Once again, serendipity was the
godfather of this most surprising design result.
In the early 1990s, an interesting observation was made
that the incretin hormones GIP and GLP-1 (glucagon-like
peptide-1), which stimulate the pancreas to release insulin
after ameal, are substrates for dipeptidylaminopeptidaseIV
(DPPIV). They are rapidly degraded by this serine aminopeptidase. Since incretins were already interesting candidates for diabetes therapy, the idea immediately arose
that inhibition of DPPIV could be used as aprinciple for
the treatment of type 2 diabetes (noninsulin-dependent
diabetes). The membrane-bound protease cleaves dipeptides from its substrate when aprolyl or alanyl group is
in the second position from the N-terminus. Vildagliptin
(Galvus®) 23.35 and saxagliptin 23.36 (Onglyza®) both
use aproline-related cyanopyrrolidine (. Fig.23.20) that
. • Serine Protease Inhibitors: Thrombin Was Just the Starting Point
. Fig. 23.19 Crystal structure of rivaroxaban 23.30 (. Fig.23.18) in
factor Xa. The inhibitor’s chlorothiophene group binds in the deep
S1 pocket, at the end of which Tyr 228 and Asp 189 are found. The
chlorine atom forms interactions with the aromatic ring. The central
phenyl ring and the terminal lactam ring of the inhibitor are found
in the S3 pocket, which is enclosed by the three aromatic groups of
Tyr99, Phe 174, and Trp 215. (7 https://sn.pub/q86HYM)
can reversibly, covalently bind to the catalytic serine. Sitagliptin (Januvia®) 23.37 is another compound available
for the treatment of type2 diabetes. It blocks the protease without covalently binding to the catalytic serine.
Recently, mimetics of the aforementioned incretin hormone GLP-1 have been introduced into diabetes therapy.
In type2 diabetics, GLP-1 is released at lower levels, which
reduces the glucose-lowering effect. GLP-1 receptor agonists, such as semaglutide, mimic the action of GLP-1
by binding to aGPCR (Sect.29.1). Chemically, they are
slightly modied from the biologically active human oligopeptide GLP-1, which consists of 31amino acids. The
amino acid substitutions make the peptide more resistant
to DPP IV protease degradation, and the addition of an
18-carbon fatty acid moiety via a short polyethylene glycol
(PEG) linker results in binding to human serum albumin.
This provides a human half-life of approximately 7 days
and further steric protection from proteolytic degradation
and rapid renal ltration. The peptides stimulate insulin
secretion and reduce glucagon release, thereby delaying
gastric emptying and reducing appetite. Semaglutide
(Ozempic®), rst introduced for the treatment of diabetes, was approved by the FDA in June 2021 as high-dose
injectable semaglutide (Wegovy®) for long-term weight
management in adults. The drug is currently being heralded and touted as anew way to treat obesity. Novo Nordisk, the company that developed the peptide, has become
in 2024 the most valuable publicly traded company in Europe and is even generating remarkable economic growth
in Denmark. The fat-away shot seems to make the dream
of easy weight loss come true for many overweight people.
Meanwhile, Eli Lilly and Company have launched asecond drug with asimilar application, tirzepatide (Mounjaro®), another modied oligopeptide.
Chapter • Inhibitors of Hydrolases with an Acyl–Enzyme Intermediate
. Fig. 23.20 BOS-318 23.34 is
apotent and highly selective furin
inhibitor with asurprising binding
mode due to an unexpected protein
rearrangement. Vildagliptin 23.35,
saxagliptin 23.36, and sitagliptin
23.37 are inhibitors of the serine
aminopeptidase DPPIV for the
treatment of type2 diabetes
23
Certainly, more serine proteases will be discovered
and validated as putative drug targets in the coming
years. However, the eld is increasingly beneting from
the experience already gained with individual members
of this protein family. When it comes to identifying new
targets, this experience is helpful in quickly nding suitable lead structures as astarting point.
23.7 Serine, aFavored Nucleophile
in Degrading Enzymes
Serine peptidases use the OH group of an endogenous
serine as an attacking nucleophile. The adjacent histidine
residue mediates the temporary proton transfer and the
aspartate compensates for the intermediate charge on the
imidazole ring of the histidine. Aspecial feature is the
temporary covalent bond between the N-terminal part of
the substrate and the enzyme. Many other hydrolytically
cleaving enzymes use an analogous principle. Esterases
and lipases also have acatalytic triad. Occasionally, these
enzymes exchange an aspartate for a glutamate. The
neurotransmitter acetylcholine acts on many synapses
in the vegetative nervous system and is involved in the
transmission of nerve impulses. It binds to the nicotinic
acetylcholine receptor and activates this ion channel
(Sect.30.4). Acetylcholine must be removed to limit the
duration of the transmission process and to reset the receptor to its starting point. An imbalance in this nerve
impulse transmission system leads to acute and chronic
movement disorders. Acetylcholinesterase is responsible
for the degradation of acetylcholine. Inhibitors of this
enzyme are used to treat Parkinson’s and Alzheimer’s
disease. Reversible binding inhibitors such as donepezil,
rivastigmine, or galantamine exert their therapeutic effect
by reducing acetylcholine degradation in the brain.
Acetylcholinesterase has a catalytic triad of ser-
ine, histidine, and glutamate. Acetylcholine (3.46,
. Fig.3.10) is cleaved by the enzyme by transferring its
acetyl group to the catalytic serine; hydrolysis slowly releases acetic acid from the esterase. The drug (S)-rivastig-
mine 23.38 (. Fig.23.21) is also attacked by the catalytic
serine and its carbamoyl group is transferred. Due to the
increased stability of the carbamoyl enzyme complex,
the esterase is subsequently deacylated very slowly and
regenerated for the next transformation. This is equivalent to inhibiting the target enzyme for several hours.
The suppressed degradation of acetylcholine leads to
permanent excitation with muscle contraction and subsequent paralysis. Victims die from respiratory and cardiac inhibition. Cholinesterase inhibitors are, therefore,
used as insecticides. Active ingredients such as paraoxon
23.39 (. Fig.23.21), parathion (E605) 23.40, propoxur
23.41, or malathion 23.42 contain phosphoric acid esters
or thioesters that are virtually irreversibly transferred to
the catalytic serine. Because of this inhibition, acetylcholine increases to lethal concentrations in insects. In intelligence conicts, the Novichok nerve toxins developed
in Russia have gained sad notoriety. They also belong to
this group of agents and carry auorine atom (X = F)
on the phosphorus atom.
Analogous to esterases, lipases also hydrolyze ester
bonds. The catalytic triad consists of aserine, histidine,
and aspartate or glutamate. Pancreatic lipase cleaves triglycerides during the digestion of fats. Inhibitors of this
intestinal enzyme are used to treat obesity. The result is
asignicantly reduced absorption of fats and their degradation products. Orlistat (Xenecal®, 23.43; . Fig.23.22),
asynthetic hydrogenation product of the natural product
lipstatin, has avery long aliphatic side chain and areactive β-lactone ring in its core. Serine in the catalytic site
of lipase attacks the carbonyl group of the lactone ring
. • Serine, aFavored Nucleophile in Degrading Enzymes
. Fig. 23.21 (S)-Rivastigmine 23.38
transfers acarbamoyl group to the catalytic
serine in the binding pocket of acetylcholine
esterase and blocks its function because the
carbamoyl–esterase complex decomposes very
slowly. The acetylcholine esterase inhibitors
paraoxon 23.39, parathion 23.40, propoxur
23.41, or malathion 23.42 are phosphoric
acids, thiophosphoric acids, or carbamic esters
and are used as insecticides. They also react
with the catalytic serine and form astable
covalent bond
and opens the strained ring by transformation into astabilized acyl–enzyme complex. Once blocked, the enzyme
is no longer able to break down triglycerides, resulting in
areduced ability to extract calories from food.
Lipases are often used for the kinetic resolution of
racemates. This is usually achieved by enzymatically converting aracemic mixture of esters in which one of the
two forms reacts faster than the other. An example was
described in Sect.5.4 where the lipase was used not only
to hydrolyze but also to form anew amide bond. For
this, the intermediate acyl–enzyme complex cannot be
exposed to awater molecule as anucleophile, but acompound with afree amino group must be available. This
transformation produces anew amide bond. Bacteria use
such atranspeptidase reaction to build their cell wall. This
cell wall has acompletely different composition than the
cell wall in humans. Therefore, the enzymes used to synthesize the cell wall are bacteria-specic and particularly
suited as atarget for adrug therapy with few side effects.
The nal step in cell wall biosynthesis is the cross-link-
ing of the peptidoglycan strands. The terminal amino
group of a pentaglycine chain attacks between two dalanine residues of another peptide unit. The d-Ala–d-
Ala bond is cleaved and anew peptide bond is formed
between d-Ala and glycine. This cross-linking is mediated
by aglycopeptide transpeptidase. It has acatalytic machinery very similar to that of serine proteases. In addi-
tion to acatalytic serine, the reaction center also contains
alysine and aglutamate, as well as an oxyanion hole.
Penicillins 23.44 and cephalosporins 23.45 (. Fig.23.23)
inhibit these transpeptidases. They have aspatial structure analogous to the d-Ala–d-Ala dipeptide and are,
therefore, recognized as “false” substrates (. Fig.23.23).
The β-lactam ring is opened by the attacking catalytic
serine, resulting in an irreversible covalent bond to the
enzyme. Cross-linking of the glycan strands is prevented
and the newly synthesized cell wall does not achieve sufcient stability. It cannot withstand the osmotic pressure
of the cell contents and the bacterial cell is killed.
Of the rst penicillins 23.44 discovered by Alexander
Fleming (Sect.2.4), only benzyl and phenoxymethylpenicillin are still of clinical importance (. Fig.23.23). The
substituents on the 6-amino group of penicilloic acid
have been exchanged to improve pharmacokinetics, spectrum of activity, and acid stability. Electronegative atoms
on the α-carbon of the acyl function increase stability to
acid-catalyzed degradation and contribute to improved
oral bioavailability.
Bacteria rapidly develop resistance to penicillins. They
use lactamases, which are enzymes structurally related to
transpeptidases. Four classes of lactamases are known,
three of which have a catalytic serine in the active site.
Another class belongs to the zinc-dependent metalloenzymes (Chap.25). The catalytic serine of β-lactamases is
Chapter • Inhibitors of Hydrolases with an Acyl–Enzyme Intermediate
also acylated by penicillins and related cephalosporins
(. Fig.23.23). Up to this step, the mechanism of transpeptidases and β-lactamases is identical. However, the
transpeptidases form very stable acyl enzymes, whereas
the covalent intermediate of the β-lactamases is rapidly
hydrolyzed. The antibiotic designed to inactivate the transpeptidase is, therefore, rendered inactive. β-Lactamases
are probably descendants of the transpeptidases. They are
widespread in Nature and have evolved as aresult of com-
. Fig. 23.22 Orlistat (Xenical®) 23.43 is a synthetic hydrogenation
product of the natural product lipstatin, which has two additional
double bonds. It has areactive β-lactone ring that reacts with the catalytic serine in the catalytic site of pancreatic lipase to form an acyl–
enzyme complex with ring opening. The enzyme’s function is then
blocked
petition between bacteria and molds. The resistance gene
for β-lactamases is easily transferred between bacteria be-
cause the information is stored on an extrachromosomal
plasmid. Such plasmids are transferred very rapidly.
How do β-lactamases differ from transpeptidases in
that they are able to rapidly dispose of the covalently
bound ring-opened penicillin? The release requires hydrolytic cleavage from the protein. This requires awellplaced water molecule in the active site that can initiate
the nucleophilic attack on the acyl–enzyme species. Although the spatial architecture of transpeptidases and
β-lactamases is very similar, there is little sequence identity. It has been suggested that both types of enzymes
originated from acommon ancestor. Thus, by targeted
mutagenesis, it was possible to endow atranspeptidase
with the hydrolyzing properties of alactamase!
Only afew amino acid substitutions were necessary. It
is mainly the hydrophobic amino acids such as phenylalanine and tryptophan that protect the acyl–enzyme complex from hydrolysis in the transpeptidase. They prevent
the transpeptidase from accepting awater molecule at the
critical position for nucleophilic attack. In contrast, polar amino acids such as glutamic acid (. Fig.23.24, Glu
166) are found at the same positions in the lactamases. In
contrast to the hydrophobic amino acids of transpeptidase, they anchor and activate the water molecule in the
correct orientation for nucleophilic attack on the acyl–
enzyme complex in lactamases. As aresult, the covalent
complex with the penicillin cleavage product that was
. Fig. 23.23 In the last step of the bacterial cell
wall synthesis, aglycopeptide transpeptidase cleaves
the bond between two d-Ala–d-Ala groups and forms
anew bond between d-Ala and aglycine in apeptidoglycan strand. Lactam antibiotics of the penicillin
(23.44) or cephalosporin type (23.45) can block this
step. The penicillin scaffold (green) is reminiscent of
the d-Ala–d-Ala group (orange) and is bound analo-
gously by the enzyme. An irreversible inhibition of the
transpeptidase is achieved by anucleophilic opening
of the lactam ring with the help of the catalytic serine.
(7 https://sn.pub/sQuPJA)
23
. • Serine, aFavored Nucleophile in Degrading Enzymes
formed by ring opening in the lactamases is hydrolyzed,
whereas it remains stable in the transpeptidases.
How can this lactamase-caused resistance and degradation of penicillins be stopped? Unsubstituted penicilloic acid 23.46 is rapidly cleaved by TEM-1β-lactamase
(. Fig.23.24). Based on structural considerations, it was
suggested that ahydroxymethyl group should be added at
the 6-position. This group should be in the exact position
where the water molecule would start its nucleophilic attack on the acyl–enzyme form. Indeed, derivative 23.47
inactivates TEM-1β-lactamase. In the subsequently determined crystal structure, awater molecule was detected
near the CH2OH group, but it is too far away to successfully hydrolyze the acyl enzyme. The hydroxyl group,
therefore, blocks the attack of awater molecule on the
ester carbonyl group of the acyl enzyme.
The incorporation of such a hydroxymethyl group
has been accomplished in important β-lactamase-resis-
tant β-lactams such as imipenem 23.48 or meropenem
23.49 (. Fig. 23.25). β-Lactamases can also be irreversibly inhibited. If such an inhibitor is administered
with apenicillin, the degradation of the penicillin by the
lactamase is blocked, and it is available to inhibit the
transpeptidase. The natural product clavulanic acid 23.50
forms an acyl–enzyme complex upon the opening of its
lactam ring. By rearrangement avinylogous urethane is
formed that is resistant to hydrolysis.
With these examples, the spectrum of enzymes that
use aserine as anucleophile is far from exhausted. Viruses need cleavage enzymes. They have to cleave the
polypeptide chains synthesized by the infected cell according to their own specications into functional viral
proteins. Viruses either use proteases of the infected host
cell (e.g., furin) or they use their own viral proteases. Since
the correct function of the latter enzymes is essential for
the maturation of new viruses and is also virus-specic,
these proteases are privileged targets for drug development. Peptidases with acatalytic serine as well as acysteine (Sect.23.9) are recognized. As we will see in Sect.24.3,
an aspartic protease serves other viruses. In recent years,
inhibitors of the viral NS3/4A serine protease of the hep-
atitisC virus (HCV) have been successfully developed. An
estimated 170 million people worldwide are infected with
this virus. It is transmitted through contact with infected
blood. If left untreated, chronic HCV infection can lead
to serious liver diseases, including cirrhosis and hepatocellular carcinoma. The NS3/4A protease is responsible
for the selective cleavage of the initial polypeptide chain
into the individual viral proteins (NS4A, NS4B, NS5A,
and NS5B). The rst two inhibitors of this enzyme, boce-
. Fig. 23.24 Unsubstituted penicilloic acid 23.46 is quickly cleaved
by TEM-1β-lactamase (left). By adding ahydroxymethyl group to the
6-position of 23.47, acompound is obtained that forms ahydrolytically stable acyl–enzyme complex with the enzyme (right). Anew crystal
structure was determined with this compound. The hydroxyl group is
found at the position where the water molecule (orange sphere) starts
its nucleophilic attack on the acyl–enzyme intermediate (left, lower
part of the image, modeled structure taking the coordinates from the
crystal structure of the complex with 23.47). The hydrophobic amino
acids such as phenylalanine and tryptophan are found at positions 166
and 170 of the transpeptidases, which are structurally related to the
β-lactamases. (7 https://sn.pub/0wjVve)