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Chapter • Inhibitors of Hydrolases with an Acyl–Enzyme Intermediate
previr 23.51 and telaprevir 23.52, were approved in 2011
(. Fig.23.25). They were followed by avast number of
other inhibitors (such as simeprevir, sofosbuvir, asunaprevir, danoprevir, simeprevir, paritaprevir, vaniprevir,
and grazoprevir), some of which block the target enzyme
covalently and others as noncovalent peptidomimetics.
The assemblins, another group of serine peptidases,
have been found in herpes viruses. The enzyme from cytomegalovirus belongs to this group, as do those from
varicella-zoster virus and herpes simplex virus. These
proteases also use one serine and one histidine. An additional histidine forms the third amino acid in the triad.
Despite adifferent folding pattern, their triad ts very
well with the trypsin triad. Even the oxyanion hole is
present in these viral proteases.
The carboxy-serine peptidases (sedolisins) are another group folded analogously to subtilisin (Sect.14.7).
They have atriad of serine, glutamate, and aspartate.
Amember of this family was recently discovered on the
human cnl2 gene. Mutations in this gene cause severe
neurodegenerative diseases. This enzyme also contains
an oxyanion hole to which, interestingly, an aspartate
contributes. However, it is only in the protonated state
that this residue can act as ahydrogen bond donor and
negative charge stabilizer in the transition state. Since the
enzymes of this family are active in apH range of 3–5,
the requirement for protonation is met.
There may be many more cleavage enzymes that use
acatalytic serine to be discovered. It remains to be seen
which of the discovered peptidases will be selected for
pharmaceutical development. Their catalytic machinery shares the same spatial architecture in all examples.
Therefore, the general principles can be transferred between the individual members of the family.
23.8 Triads in All Variations:
Threonine as aNucleophile
In addition to serine, another amino acid carries an aliphatic OH group: threonine. This amino acid can also
be catalytically active in aprotease. The proteasome is
the cell’s central protein-shredding machine and cleaves
ubiquitin-labeled proteins into small oligopeptides of
3–20 amino acids. The ubiquitin tag itself is ahighly
conserved protein with 76amino acids. As acellular
shredding machine, the proteasome plays acentral role
in protein metabolism, cell growth, and cell death. It is,
therefore, an important target for the treatment of can-
cer. It is amultiprotease complex composed of more
than 30 proteins and is found in both the cytoplasm
and the nucleus (. Fig.23.26). The proteasome is constructed like alarge barrel with two lid regions that have
regulatory functions; these regions control the entry of
. Fig. 23.25 β-Lactamase-resistant
antibiotics of the penem and carbapenem
type. Imipenem 23.48 and meropenem
23.49 are derived from the carbapenem
type. The natural product clavulanic
acid 23.50 opens its lactam ring and
forms an acyl–enzyme complex with
the serine residue. Ahydrolysis-resistant
vinyl urethane analogue is formed by
arearrangement. Boceprevir 23.51 and
telaprevir 23.52 were the rst two drugs
approved as inhibitors of the hepatitisC
virus (HCV) NS3/4A serine protease
23
. • Triads in All Variations: Threonine as aNucleophile
. Fig. 23.26 The proteasome, acellular shredding machine, proteo-
lytically cleaves ubiquitinylated proteins selectively into small oligopeptides that have between 3 and 20amino acids. The crystal structure
of the 20S proteasome from yeast (subunits are shown in different colors) is shown on the left. Six of these units are inhibited by bortezomib
(yellow). The boronic acid derivative bortezomib 23.53 (right, gray)
substrates into the shredder. The catalytic sites of the
proteases, which have chymotrypsin-like, trypsin-like,
and peptidyl-glutamyl-peptide-like substrate specicity,
contain the threonine. The OH group of this threonine
acts as the nucleophile. An adjacent positively charged
lysine and abalancing aspartate enhance its nucleophilic
strength. Since the threonine is the rst amino acid at the
N-terminus, it also carries afree amino group. This group
serves as aproton acceptor in the mechanism. The nucleophilic center is complemented by two serine and one
aspartate residues, which contribute to the stabilization
of the transition state.
Millenium Pharmaceuticals, founded as an academic research institute, introduced bortezomib 23.53
(Velcade®) in 2006, the rst agent to block the threonine
protease function of the proteasome. Chemically, bortezomib is aboronic acid derivative (. Fig.23.26). The inhibitor reacts with the threonine of the catalytic triad to
form acovalent bond. In addition to this reactive group,
the molecule has adistinct peptide-like character. These
features allow the molecule to interact with the substrate
binding site in the proteasome.
reacts with the N-terminal Thr1 and forms a covalent boronic acid
ester complex. (7 https://sn.pub/eYXznx)
Another peptide analog, carlzomib, is in clinical trials. It carries aterminal α′β′-epoxy ketone. Upon inhibition, the threonine OH group nucleophilically attacks the
keto function of the inhibitor. The adjacent N-terminal
amino group then opens the epoxide ring. This results in
an irreversible covalent bond. Although the epoxy ketone is highly reactive, carlzomib is a highly selective
proteasome inhibitor. The proximity of the nucleophilic
Thr–OH of the rst residue in the sequence and the
N-terminal amino group is an unusual and exceptional
combination. However, it is an essential prerequisite for
the activation of this inhibitor.
The proteasome is an important target structure that
appears suitable for cancer treatment. More than 20different inhibitors are currently being developed. Bortezomib is used for the treatment of multiple myeloma, atype
of bone marrow cancer. This cancer is based on the malignant transformation of plasma cells, the physiological
function of which is to produce antibodies for immune
defense. Even though bortezomib cannot heal multiple myeloma, its use can extend the life of patients for
whom other therapies have failed. In multiple myeloma,
the plasma cells produce massive amounts of misfolded
Chapter • Inhibitors of Hydrolases with an Acyl–Enzyme Intermediate
proteins that must be digested by the proteasome. Therefore, these cells need a proteasome that functions correctly, otherwise apoptosis would be induced. However,
it is desirable to block the function of the proteasome in
order to utilize this mechanism for the degradation of degenerated cells. Such cells are signicantly more sensitive
to bortezomib therapy than normal cells. Some tumor
cells also activate atranscription factor, NF-κB, which
controls the proliferation and survival of the tumor cells.
The proteasome is critical for the activation of NF-κB
because it degrades an inhibitor of this transcription
factor that acts as akind of emergency brake on NF-κB.
Therefore, the inhibition of the proteasome serves to
keep NF-κB in its benign form, because its inhibiting
binding partner is no longer being degraded. Bortezomib
may also induce apoptosis of tumor cells by stabilizing
cyclin-dependent kinase inhibitors (Sect.26.2) and the
tumor suppressor p53.
Interestingly, aprotease has been discovered in bacteria that exists as a14mer and has aspatial structure reminiscent of the proteasome. The ClpP protein is aserine
protease involved in the degradation of cellular proteins
in bacteria. Treatment with amacrolide antibiotic can
cause its function to go out of control and degrade proteins in an unregulated manner. This leads to cell death
in the bacteria. The company Bayer recognized this principle and used it for an antibiotic therapy. The goal was
not to block the protease function of the ClpP protein,
but rather to promote its uncontrolled effects through
synthetic antibiotics.
. Table 23.4 Cysteine proteases with physiological impor-
tance (X=arbitrary amino acid). The 3D structures of all of
the listed enzymes are known
Enzyme Cleavage site Function or therapeutic
use
Papain –Val–X–X– Model botanical enzyme
from papaya
CathepsinsB, L,
K,M
Calpains –Lys–Ser– Stroke
Falcipain –Arg–Lys– Malaria
Cruzipain –Lys/Arg– Sleeping sickness
Caspases –Asp–X– Rheumatoid arthritis,
Picornavirus
3C-proteinase
SARS-main
proteinase
–Arg–X– Inammation
–Gly–X– Tumor metastasis
–Ser–X– Muscular dystrophy
–Tyr–X– Myocardial infarction
–Arg–Thr– Neuroprotection
–Tyr–Ala– Cataract
–Lys–X–
–Phe/Ala–
apoptosis, sepsis
–Gln–X– Viral infection
–Gln– Viral infection
Ser/Ala
23
23.9 Cysteine Proteases: Sulfur,
the Big Brother of Oxygen as
aNucleophile in the Triad
In addition to the OH group of serine and threonine,
the thiol group of cysteine is also capable of nucleophilic
hydrolytic attack on amide bonds. Enzymes using such
acysteine possess acatalytic triad analogous to serine
proteases and are called cysteine proteases. The rst pro-
tease of this family to be structurally studied in detail
was papain, which was isolated from the latex of papaya,
the fruit of the papaya tree (Carica papaya). Its triad
consists of anucleophilic cysteine, ahistidine, and an
asparagine. The asparagine assumes the role of aspartate
in serine protease. The catalytic mechanism is similar to
that of serine proteases. Even the oxyanion hole (Cys25
and Gln19) is found in proteases of the papain family.
There is evidence that the transition state is structurally
similar to the acyl–enzyme intermediate. Attempts have
been made to replace serine with cysteine in trypsin. The
substrate binding properties (K
same, but the catalytic rate of the reaction decreased by
ve orders of magnitude. Although the structures are
geometrically almost unchanged, the experiment shows
) remained virtually the
m
that the difference between serine and cysteine proteases
is more complicated than asimple exchange of sulfur
for oxygen. The ne-tuning of structural and electronic
properties is the key. Unlike the trypsin-like serine proteases, the nucleophilic cysteine exists as apreformed ion
pair with its neighbor histidine.
Three families of cysteine proteases that are import-
ant targets for drug therapy have been characterized
(. Table23.4). The rst group is derived from papain
and includes the cathepsins. They are proteases involved
in the degradation of extracellular matrix proteins and
the basal membrane. Inhibiting their function opens up
awide range of therapeutic possibilities, for example, in
inammation, tumor metastasis, bone resorption, muscle
atrophy, or myocardial infarction. Another group is the
calcium-dependent calpains, whose hydrolytic domain is
folded very similar to that of papain. They are found in
many cells and have different functions. Calpains occur
in higher concentrations at sites of cell damage, such as
after traumatic brain injury, stroke, or during the formation of cataracts in the eye. Calpains appear to be
regulatory enzymes. For example, they reduce blood ow
through blood vessels after injury to limit blood loss.
Unfortunately, this natural protective function leads to
the contrary situation during astroke: activation of cal-
0
4
. • Cysteine Proteases: Sulfur, the Big Brother of Oxygen as aNucleophile in the Triad
pains reduces blood ow and parts of the brain become
ischemic. Destruction of affected brain cells is the result.
Specic inhibitors could counteract the over-functioning
of calpains. Cysteine proteases of the papain family have
also been discovered in parasites. Inhibition of cruzipain
could be aconcept for the treatment of sleeping sickness.
Falcipain, which is used by the malaria parasite to digest
hemoglobin, is apromising target enzyme for malaria
therapy.
The second large family of cysteine proteases are the
caspases. These are involved in the control of apoptosis,
or programmed cell death. When acell is damaged beyond the ability of natural cellular repair mechanisms
to restore it to its normal state, caspases are activated
to induce apoptosis. Dysregulation of apoptosis leads to
various pathological conditions associated with cancer,
immune dysregulation, or neurodegenerative disorders.
Inhibitors of different caspases have potential as neuroprotective agents, as active substances for cancer treatment or for the treatment of rheumatoid arthritis.
The third family includes the viral 3C proteases found
in picornaviruses (human rhinovirus, poliomyelitis, or
hepatitis viruses) or coronaviruses (SARS). These viral
proteases process the primary polypeptide chain and produce the specic viral proteins during maturation. Inhibitors of these proteases represent aconcept for antiviral
chemotherapy.
Aspecial feature of papain-type proteases is the stereochemistry of the nucleophilic attack. In contrast to
other serine and cysteine proteases, the attack occurs
from the opposite side, the so-called Si face. The S1
pocket in papain is not prominent and the P1 group of
the substrate is oriented away from the protein. In contrast, all neighboring pockets are much more prominent.
Interestingly, some of the pockets on the C-terminal side
(the primed side, 0–
structured. This can be exploited in the design of potential inhibitors. Papain prefers substrates with hydrophobic P2 and P3 groups. An aspartate is recognized as
aP1 group by caspases of the second folding family. For
these reasons, many inhibitors developed for caspases
carry afunctional group with acarboxylic acid group or
acorresponding mimetic at this position. The interaction
with the thiol group of the catalytic cysteine is crucial
for the binding of cysteine protease inhibitors to their
target enzyme. Interestingly, many of the inhibitors developed attempt to involve the sulfur atom in acovalent
bond. Reversible and irreversible head groups have been
developed for this purpose. The inhibitor leupeptin 23.54
(. Fig.23.27) is anatural product that has aterminal
aldehyde function. This group reacts with the thiol group
of cysteine to form ahemithioacetal. Leupeptin binds
with high afnity to many members of the papain family. In addition to the aldehyde head group, many other
functionalities (so-called warheads) that can be used to
inhibit cysteine proteases (. Fig.23.27) are known. Such
) of cysteine proteases are highly
irreversible inhibitors have been developed for viral proteases and have aMichael acceptor group (e.g., 23.55).
This reactive group forms an irreversible bond with cysteine and permanently shuts down the enzyme. Attempts
have been made to develop inhibitors for cathepsins, calpains, and caspases that can form areversible bond to the
thiol group. Most of these structures are derived from aldehydes or ketones (23.56–23.59). From achemical point
of view, Vertex’s caspase inhibitor 23.58 is interesting. It
combines in acyclic structure an aspartate-like side chain
for the S1 pocket of the enzyme and acapped aldehyde
function in the form of acyclic acetal. The aldehyde is
released as the active compound from this prodrug.
An example of the successful development of
acysteine protease inhibitor in record time is the new
COVID-19 inhibitor nirmatrelvir (Paxlovid®). When
the coronavirus pandemic hit the world in late 2019,
there was no drug or vaccine available to protect against
the virus. With lightning speed, many concepts were
brought to market, shattering all previously predicted
drug development timelines. In less than ayear, the rst
vector-based or mRNA vaccines (Sect.32.4) were available. Just one year later, the rst orally available SARS-
CoV-2 inhibitor, nirmatrelvir, developed at Pzer, was
approved. Like many viruses, SARS-CoV-2 possesses
alarge protease (SARS-CoV-2 M
the cleavage of mature peptide chains into virus-specic
proteins. It belongs to the class of cysteine proteases. It
specically cleaves the polypeptide chain at 11sites following aP1-Gln residue. No human cysteine protease
cuts after this amino acid. The perspective to develop
asufciently selective inhibitor was, therefore, promising. The Pzer team was fortunate that they did not to
have to start from scratch. In 2002, when the rst SARS
virus infection shocked the world, Pzer had already developed the potent but still very substrate-like inhibitor
23.60 (. Fig.23.28). This inhibitor binds covalently to
the substrate by forming acovalent bond to the catalytic
cysteine (green) via an α-hydroxymethyl ketone. By the
time Pzer had the compound in hand, however, the 2002
SARS outbreak had largely subsided. The compound
was not pursued further. Since the main protease of the
new 2019 SARS-CoV-2 virus is virtually identical in the
catalytic center, 23.60 also inhibits this protease. This set
the stage for optimization into an orally available compound in March 2020. The peptidic nature of 23.60 had
to be removed, as did the excessive polarity of the many
H-bond donors. Pzer initially used anitrile and abenzothiazole-2-yl ketone in parallel as covalent anchors
(. Fig.23.27, groupsd andh). The nitrile group eventually prevailed (circled in green). A6,6-dimethyl-3-azabicyclohexane was used as amimetic for the central
P2-leucine (red). The P3 indole moiety was replaced by
branched acyclic groups, with amethanesulfonamide and
atriuoroacetamide being the favorites. In the end, the
uorinated substituent proved superior (blue). Less than
pro
) that is required for
a
b
c
d
e
f
g
h
i
Chapter • Inhibitors of Hydrolases with an Acyl–Enzyme Intermediate
. Fig. 23.27 In addition to the aldehyde head group(a), as found in
the natural product leupeptin 23.54, many other functionalities(b–i)
have been developed. They bind reversibly or irreversibly to the catalytic cysteine (reactive site is shown in red), thereby, blocking cysteine
proteases. Irreversible inhibitors such as 23.55 that have aMichael acceptor group are available for viral proteases. The two aldehydes 23.56
six months after optimization began, 23.61 was ready
for toxicological testing and in-depth clinical trials as
ananomolar inhibitor with good oral bioavailability and
antiviral activity. By the end of 2021, 23.61 could be introduced into therapy under the trade name Paxlovid®.
. Fig.23.28 shows the crystal structure of 23.61 with
the protease.
and 23.57 are development substances for the inhibition of calpains;
23.58 and 23.59 are caspase inhibitors. Compound 23.58 is aprodrug
that releases an aspartate-like P1 side chain upon ring opening and
forms ahemithioacetal with the protein through its newly generated
aldehyde function
Another group of enzymes that actually belong to
the transferase family, but follow acysteine protease-like
mechanism, are the transglutaminases. Nine isozymes
have been discovered in our genome. They are composed of four domains and contain acatalytic domain
consisting of aCys–His–Asp triad. Their function is the
posttranslational modication of proteins (Chap. 26),
23
. • Cysteine Proteases: Sulfur, the Big Brother of Oxygen as aNucleophile in the Triad
. Fig. 23.28 Crystallographically determined binding mode of nir-
matrelvir 23.61 to the SARS-Cov-2 M
molar inhibitor was optimized for stability and oral bioavailability
from apreviously discovered peptide analog 23.60 via several design
steps. Nirmatrelvir binds covalently via its nitrile group to the catalytic
Cys 145 and occupies the specicity pockets S1, S2, and S3 with its side
pro
major protease. The nano-
meaning they modify proteins after they have been synthesized in the ribosome. For example, they can deaminate glutamine residues to glutamate. In addition, they
catalyze the cross-linking of chain strands on proteins by
the transaminase reaction. For this purpose, the terminal
amino group of alysine is coupled to aglutamate residue to form an isopeptide bond. This results in aproteolytically stable cross-linking, so that transglutaminases
can be compared to a“biological glue.” The reaction is
analogous to that of cysteine proteases. Anucleophilic
cysteine rst forms an acyl enzyme with the glutamine
of the substrate with loss of ammonia, which is cleaved
in the next step by the reactive lysine. Aprotein crosslink is formed. Transaminases have many functions in the
body, the most important of which is to stabilize tissue
proteins. In the blood coagulation cascade, the transglu-
chains. The oxyanion hole is formed by the NH groups of Gly 143 and
Cys 145. (7 https://sn.pub/hNPere)
taminase factorXIII stabilizes the initially formed clot by
cross-linking (Sect.23.4). Therefore, factorXIII inhibitors may be potent anticoagulants. Other transglutaminases are also being investigated as potential targets for
drug development. Transglutaminase-2 (TG2) plays an
important role in celiac disease, aform of gluten intolerance. Patients with this disease are sensitive to gluten,
an adhesive protein found in many grain products. They
develop inammation in the mucous membranes of the
small intestine, leading to the destruction of intestinal
epithelial cells and severely limiting their ability to absorb
nutrients from food. TG2 inhibitors may be atherapeutic approach. Inhibitors for transglutaminases can be
developed using analogous principles to those used for
cysteine protease inhibitors.
Chapter • Inhibitors of Hydrolases with an Acyl–Enzyme Intermediate
23
23.10 Synopsis
Serine proteases belong to the class of hydrolyzing en-
-
zymes that cleave amide or ester bonds. Depending on
where they cleave apeptide chain, they are classied
as amino-, carboxy-, or endopeptidases.
Three amino acids, aserine, ahistidine, and an as-
-
partic acid that reside at quite distant positions in the
sequence, are folded in characteristic proximity to one
another. The hydroxyl oxygen atom of the serine nucleophilically attacks the carbonyl carbon atom of the
scissile peptide bond. Its nucleophilicity is enhanced
by an H-bond to an adjacent imidazole moiety of
ahistidine.
The histidine accepts aproton from the nucleophilic
-
serine OH group and is, thereby, transposed into
apositively charged state. The neighboring aspartate
residue compensates for the positive charge. The simultaneously created negative charge on the former
carbonyl oxygen is stabilized by NH functions in the
H-bond-donating oxyanion hole. Simultaneously, the
carbon atom of the cleaving amide bond rearranges
to atetrahedral geometry.
Upon release of the N-terminal part of the peptide
-
substrate, the C-terminal part remains covalently
bound as an acyl–enzyme complex. This is nally
degraded via asimilar mechanism that uses awater
molecule as anucleophile.
The residues involved can be different; in particular,
-
the nucleophilic serine can be replaced by athreonine
or cysteine. The corresponding enzymes are named
threonine and cysteine proteases.
The peptide chain to be cleaved is primarily recog-
-
nized in small binding pockets on the protease surface
that accommodate the amino acid side chains on the
C-terminal end adjacent to the cleavage site. Their
composition determines the chemical building blocks
required for inhibitor design to develop highly potent
ligands for the protease.
Anumber of warhead groups are known to either
-
reversibly or irreversibly block the catalytic serine,
threonine, or cysteine residue.
The major contribution to binding afnity and li-
-
gand specicity is achieved through binding to the S1
pocket next to the cleavage site.
Blood coagulation is ahighly regulated cascade of
-
serine proteases. Potent inhibitors for antithrombotic
therapy have been developed for thrombin and factorXa, which participate in the last steps of the cascade.
Whereas thrombin and factorXa exhibit deep and
-
well-structured S1 pockets, elastase exhibits aat S1
pocket. Binding to this pocket contributes much less
to the overall afnity of an inhibitor for this protease
and the developed compounds all involve the catalytic
serine in areversible covalent attachment.
To block lipases or transpeptidases, irreversible inhi-
-
bition is achieved by covalent bond formation with
the catalytic serine. The covalent bond is formed by
ring opening of areactive highly strained lactone or
lactam ring. The latter principle is used by the peni-
cillins and cephalosporins. Astable acyl form of the
enzyme, which does not allow further conversions in
the catalytic center, results. Penicillins and cephalo-
sporins use this principle of inhibition.
The β-lactamases, which are structurally closely re-
-
lated to the transpeptidases, hydrolyze the acyl–en-
zyme form produced by the penicillins and cephalo-
sporins. They orient apolar glutamate residue into
the catalytic center, thereby, stabilizing awater mol-
ecule in an optimal position for nucleophilic attack.
In transpeptidases, this site is inaccessible to water
molecules due to hydrophobic residues. Lactamase
inhibitors break this resistance by blocking the water
position with apolar side chain.
Many cysteine proteases are found in bacteria, para-
-
sites, and viruses. By replacing the OH group with an
SH group, the transition state in cysteine proteases
appears to be closer to the acyl–enzyme form and the
thiol group is presumably present in adeprotonated
state. Typically, ahead group that is reversibly or ir-
reversibly covalently bound to the sulfur is used as
astrategy to inhibit these enzymes.
The transglutaminases follow avery similar enzyme
-
mechanism as the cysteine proteases. However, in-
stead of cleaving apeptide bond in the main chain,
they form an isopeptide bond between the terminal
amino group of alysine and the carboxylate group of
aglutamate. Because these bonds cause across-link-
ing between different segments of the polypeptide
chain, they can be compared to biological glue that
make proteins more stable.
Bibliography and Further Reading
General Literature
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of protease-directed drugs, Curr. Opin. Struct. Biol., 16, 769–775
(2006)
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S. D. Kimball, Challenges in the Development of Orally Bioavailable
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• Bibliography and Further Reading
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Aspartic Protease Inhibitors
Contents
24.1 Structure and Function of Aspartic Proteases – 404
24.2 Design of Renin Inhibitors – 405
24.3 Design of Substrate Analogue HIV Protease Inhibitors – 411
24.4 Structure-Based Design of Nonpeptidic
HIV Protease Inhibitors – 413
24.5 The Development of Resistance Against
HIV Protease Inhibitors – 416
24.6 A Basic Nitrogen as aPartner for the Aspartic
Acids of the Catalytic Dyad – 418
24.7 Other Targets from the Family of Aspartic Proteases – 423
24.8 Synopsis – 423
Bibliography and Further Reading – 424
© 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_24
Chapter • Aspartic Protease Inhibitors
24
Aspartic (also aspartyl) proteases also cleave peptide
bonds, but by adifferent mechanism. They owe their
name to the presence of two aspartates, which determine
the catalytic mechanism. To attack the peptide bond to
be cleaved, they use awater molecule as anucleophile,
which they polarize in asuitable manner with the two aspartate residues. At the same time, these groups stabilize
the transition state, balance the charges, and transfer protons. The digestive enzyme pepsin was the rst member
of this class of enzymes to be intensively studied. It is active at strongly acidic pH conditions between values of1
and5. The rst 3D structure of this aspartic protease was
determined in the early 1970s in the group of Alexander
Fedorov. The aspartic protease family is relatively small
in the human genome; it contains 15members. Some
important aspartic proteases are listed in . Table24.1.
24.1 Structure and Function of Aspartic
Proteases
Pepsin preferentially cleaves peptides containing hydrophobic residues to the right and left of the cleavage site.
Its spatial structure shows that two catalytically active
aspartic acid residues are located side by side with ashort
distance to each other. One of these residues has an unusually low pKa of1.5. The other aspartic acid residue
has ahigher pKa of4.7. Thus, under the low pH conditions in the stomach, one of the side chains in the catalytic site is apparently protonated, while the other is not.
This difference is crucial for the catalytic mechanism. In
other aspartic proteases that function at higher pH values, acomparable difference is observed between the two
groups. It is the local environment that determines the
pKa values (Sect.4.4). On the other hand, the two aspartic acid residues are so close to each other that they can
no longer be considered independent. The two aspartates
behave like acoupled system, similar to adicarboxylic
acid; they are practically adiprotic acid (. Table24.2).
Here, the relative distance between the two acid groups
determines the magnitude of the pKa difference.
The mechanism of peptide cleavage by aspartic prote-
ases is shown in . Fig.24.1. The cleavage of the amide
bond occurs by nucleophilic attack of awater molecule
on the carbonyl carbon atom. The deprotonated aspartate polarizes this water molecule. At the same time, the
protonated aspartate forms an H-bond to the carbonyl
group of the amide bond to be cleaved. This polarizes
the C=O bond and facilitates nucleophilic attack on the
carbon atom. The reaction proceeds through atetrahedral transition state in which the oxygen atom of the
nucleophilic water forms abond to the carbonyl atom. At
the same time, the proton is transferred from the water to
the deprotonated aspartate. One approach to developing
aspartate protease inhibitors is to mimic the transient, un-
stable geminal diol transition state with astable molecule.
Hydroxyl compounds (. Fig.24.2), but also α-ketoamides and phosphinates can be used for this purpose.
To gain access to the substrate, the protease must
open amobile ap along the conversion pathway. In
. Fig.24.3, the upper part of these tunnels is clipped
off. The blue areas on the surface indicate the regions
into which the protein directs hydrogen bond-forming
groups. In the center, the two catalytic aspartate residues
are hidden beneath the blue region. Adjacent are regions
. Table 24.2 pKa values of afew dicarboxylic acids
a
. Table 24.1 A few aspartic proteases and the preferred site
for enzymatic cleavage
Enzyme Cleavage site Function
Pepsin Phe–Phe, Leu–Phe,
etc.
Renin Leu–Val, Leu–Leu Blood pressure regu-
CathepsinD Phe–Phe, Leu–
Leu, etc.
β-Secretase
Chymosin Phe–Met Milk curdling
HIV protease
Plasmepsin Phe–Leu Hemoglobin digestion
Met–Asp, Leu–
Asp
Phe–Pro, Tyr–Pro,
Phe–Tyr, Leu–Phe,
Phe–Leu, Met–
Met, Leu–Ala
Digestion
lation
Tissue degradation
Proteolytic degradation of membrane
proteins
Virus replication
Dicarboxylic acid
HOOC–(CH2)n–
COOH
n = 0 1.46 4.40 1.40
n = 1 2.83 5.85 2.60
n = 2 4.17 5.64 3.82
n = 3 4.33 5.52 4.95
n = 8 4.55 5.52 10.00
Z-HOOC–
CH=CH–COOH
E-HOOC–
CH=CH–COOH
1,2-C
1,3-C6H4(COOH)23.62 4.60 4.93
1,4-C
a
pKa = 4.76; C6H5COOH pKa = 4.22
(COOH)22.96 5.40 3.14
6H4
(COOH)23.54 4.46 5.71
6H4
Reference values: HCOOH pKa = 3.77; CH3COOH
pKa1 pKa2 HOOC–
COOH
distance(Å)
1.90 6.50 3.14
3.00 4.50 3.80