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25
Chapter • Inhibitors of Hydrolyzing Metalloenzymes
. Fig. 25.21 Sildenal 25.69, vardenal 25.70, and tadalal 25.71 represent potent PDE 5inhibitors. The rst two compounds were developed
from phenyl-substituted purines such as 25.72, and modied to pyrazolopyrimidines such as 25.73 or imidazotriazenones such as 25.74
The market embraced Viagra® euphorically. By 2005,
more than 177 million prescriptions had been registered
in 120 countries around the world. In addition to PDE5,
PDE6 is also inhibited by sildenal, vardenal, and tadalal. This isoform is involved in visual processes, which
explains why the use of these drugs may be associated
with visual disturbances. Tadalal has better selectivity
against PDE6, but also inhibits PDE11 in addition to
PDE5. Sildenal and tadalal have another approved
clinical use: They are used in intensive care units to prevent and treat pulmonary hypertension in mechanically
ventilated patients.
Do PDE-5 inhibitors have another career? What
helps men also seems to give cut owers more stamina.
According to experiments conducted by Heribert
Warzecha at the Technical University of Darmstadt,
Germany, cut gerbera owers stay fresh longer when
Viagra® is added to the water in the vase! Aspirin®,
on the other hand, is less expensive and is also said
to keep cut owers fresh longer. Another study found
that hamsters were able to reset their circadian rhythms
faster when they had Viagra® in their blood. Ahigher
cGMP level apparently helps the internal clock to adapt
more easily to changes in external conditions. Whether
Viagra® also helps to overcome jet lag after long-distance travel faster remains to be proven. These examples
show that no drug is without side effects. These are often discovered only after some time in clinical trials or
after practical use.
25.9 What Zinc Can Do, Iron Can Too
What makes zinc so special that it preferentially occurs
in the catalytic center of so many hydrolyzing enzymes?
Zinc is an ion that is commonly found in biological systems. But so is an element like iron. Zinc exists as adoubly positively charged ion. This is also the case for other
ions such as Fe2+, Co2+, Ni2+, or Cu2+. Unlike the latter
elements, the zinc ion is not redox sensitive due to its lled
d-orbitals. When considering the reaction mechanism of
an ester or amide cleavage, apart from the coordination
properties, only the charge of the metal ion is critical. It
serves to polarize awater molecule that initiates nucleophilic attack on the carbonyl carbon atom of the ester
or amide to be cleaved. This task can also be performed
by other metal ions. In fact, under reductive conditions,
hydrolyzing enzymes can be found that have an iron ion
instead of azinc ion in the catalytic site.
New polypeptide chains synthesized in prokaryotes,
mitochondria, or plastids initially carry amethionine
substituted with aformyl group at the rst position of
the N-terminus. In other compartments of more complex
organisms, the same proteins are formed without these
formyl groups. In about one-third of all mature proteins,
the methionine is cleaved by amethionine aminopeptidase. In order for the formylated chains to undergo this
process, the formyl group must be removed. This is done
by peptide deformylases (PDFs). They carry an Fe2+ ion
in their catalytic center and are, therefore, very sensitive
to oxidation. It is only possible to exchange Ni2+ or Co2+,
but with adrastic loss of catalytic activity. On the other
hand, the exchange of iron for aZn2+ ion leads to acom-
0
1
0
1
0
1
0
1
. • Acetyl Group Cleavage Condenses Chromatin and Regulates Reading of Gene Segments: An Opportunity for Therapy?
stabilized by an NH of the main chain, aterminal carboxamide group of aglutamine, and the coordination
to the iron. The amino group of the bond to be cleaved
is bound to aglutamate by an H-bond. The polypeptide
chain is cleaved with simultaneous release of the N-ter-
minus. The remaining formate group leaves the metal ion
coordination site and dissociates from the enzyme. Two
water molecules take its place at the catalytic site. Inhibitors of this enzyme have hydroxamate groups to anchor
them to the iron ion. Since the natural peptide substrate
has amethionine in the
position, n-alkyl chains with
four or ve carbon atoms on inhibitors are ideal in the
same position. The
pocket is well formed in the PDFs,
but the surrounding pockets are not well characterized.
This is due to the function of the proteins. Awide range
of formylated substrates can be processed, meaning the
amino acid sequence after the formylmethionine is arbitrarily composed. Interestingly, thiorphan also inhibits
PDFs. This indicates that the thiol group can also coordinate to the iron atom. The benzyl group of the inhibitor
. Fig. 25.22 Crystal structure of actinonin 25.75 with the peptide
deformylase from Escherichia coli. The peptidic inhibitor binds to the
Fe2+ ion with its hydroxamate function. Its n-pentyl chain replaces the
methionine side chain in the natural substrate and lies in the deeply
buried
dines. (7 https://sn.pub/IzKrnZ)
pocket. The iron ion is bound to acysteine and two histi-
lls the
25.10 Acetyl Group Cleavage Condenses
pocket of the enzyme.
Chromatin and Regulates Reading
of Gene Segments: An Opportunity
for Therapy?
plete loss of enzymatic function in almost all PDFs. Pep-
tide deformylases are found in bacteria as well as in plant
plastids and some parasites. Initially, it was thought that
these enzymes were not present in humans, making them
an ideal target for antibacterial or antiparasitic therapy.
However, PDFs have been found in the mitochondria of
animals and humans. This must be taken into account
when developing antibiotics based on PDF inhibitors.
The potent inhibitor actinonin 25.75 (. Fig.25.22) not
only has antibacterial effects but also inhibits prolifera-
tion of human cells. This can lead to cytotoxic side ef-
fects, but can also be exploited for antineoplastic effects.
Moreover, these inhibitors are important as herbicides.
The iron ion is tetrahedrally coordinated by two his-
tidines and one cysteine in PDFs (. Fig.25.22). The
fourth position is occupied by awater molecule. The pKa
value of this water molecule is drastically shifted by the
direct coordination to the neighboring metal ion. It has
an increased nucleophilicity due to the thus facilitated
deprotonation. It presumably attacks the formyl peptide
group to be cleaved as ahydroxide ion. The mechanism
is very similar to that of proteases. The carbonyl carbon
of the cleaved formyl group assumes atetrahedral tran-
sition state. For this, the charge formed on the oxygen is
Sect.12.14 introduced enzymes involved in the epigenetic control of gene expression. Of particular note are
the histone deacetylases (HDACs), which remove an
acetyl group from the terminal nitrogen of an initially
acetylated lysine. This process restores the basic character of the terminal amino group of the lysine and the
released amino group is then positively charged. Histones
are important proteins in the cell nucleus around which
the DNA is coiled as if on aspool. Because of its many
phosphate groups, DNA is ahighly negatively charged
molecule. Histone proteins, in turn, are characterized by
numerous positively charged lysine and arginine residues
(. Fig.12.6). This allows them to form attractive electrostatic interactions with DNA. These interactions determine the efciency of unwinding, so the reading process
is highly dependent on the actual charge state of each
binding partner. If the lysines are acetylated, the positive
charges will be missing. If they are deacetylated, they
will become charged and the DNA more tightly bound
to the histones. As aresult, the structure of chromatin
in the nucleus is condensed. It is then less accessible for
transcription factors to read. This addition or removal
of acetyl groups inuences the process of reading specic
gene segments during gene regulation. Misregulation of
the DNA reading process can be the trigger for many
diseases, especially cancer. Therefore, manipulation of
deacetylation has been taken up as an option in tumor
therapy. First successes have already been achieved.
25
Chapter • Inhibitors of Hydrolyzing Metalloenzymes
. Fig. 25.23 The Streptomyces metabolite trichostatinA 25.76 was
the rst HDAC inhibitor to be discovered. As zinc-binding anchor,
this molecule has ahydroxamic acid followed by aslender bridge com-
parable to the side chain of lysine. It bridges to an aromatic moiety
that mimics the attached histone protein in the substrate. The inhibi-
tors vorinostat 25.77, tenostat 25.78, and tinostamustine 25.79 are
For example, ve compounds have been approved for
the treatment of certain cancers, and many more are in
clinical trials. However, because key enzymes such as
HDACs regulate gene expression in many processes in
our body, the side-effect prole of these approved drugs
is still rather unsatisfactory.
Our organism has a total of 18 enzymes of the
HDAC family. The family is subdivided into four classes.
Of these, 11are zinc-dependent metalloenzymes. They
cleave the acetyl group attached to the lysine residue
by an amide bond using water as anucleophile. They
thus follow amechanism analogous to that of the zinc
proteases. In the present case, one molecule of acetic
acid and one lysine residue are released; this means in
all cases the same substrate is converted in the cleavage
step. This suggests that selectivity towards individual
representatives of these enzymes is amajor challenge.
Seven further HDACs are called sirtuins and they use
NAD+ as acofactor to transfer the acetyl group to the
C2 position of aribose sugar. Formally, HDACs are
transferases, but due to their analogous mechanism of
designed according to this principle. Meanwhile, atriuoromethyloxadiazole anchor (25.80) has been described as anew head group for zinc
coordination. The cyclopeptidic natural product romidepsin 25.81 is
a prodrug. Cleavage and reduction of its disulde bridge results in
thiol groups, of which the thiol group facing the viewer in the gure
more closely binds to the zinc ion of the HDACs
action, they should be mentioned here together with the
zinc hydrolases.
The development of selective inhibitors for these proteins has been the subject of intense research. It seems
easier to achieve selectivity against the individual classes
of HDACs, since small differences in the structure of the
catalytic centers can be exploited for inhibitor design.
It is more difcult to nd compounds that are selective
against the individual members of aclass. The majority
of known HDAC inhibitors have ahydroxamic acid as
the zinc-binding group (25.76–25.79, . Fig.25.23). Recently, atriuoromethyl oxadiazole (25.80) has also been
discovered for this task. In addition, the inhibitors have
amore or less slender hydrophobic bridge to which an
aromatic moiety is attached via apolar group (see 25.76).
An HDAC inhibitor with acompletely different structure is the cyclic peptide romidepsin 25.81. It was isolated
from aculture of Chromobacterium violaceum at Fujisawa Pharmaceutical Company in Tsukuba, Japan. This
cyclic peptide was shown to be highly cytotoxic against
several human cancer cell lines. It was characterized as
. • Synopsis
ahistone deacetylase inhibitor and acts as aprodrug.
Inside the cell, its disulde bond is reduced, releasing
azinc-binding thiol. This thiol then binds to the zinc ion
in the binding pocket of histone deacetylase and blocks
its activity.
Over the past 20years, more than 30HDAC inhibitors have entered clinical development, resulting in ve
approvals. Although the number of human HDACs is
limited, these enzymes perform very different cellular
functions. In addition to lysine deacetylation in various tissues, they interfere with fatty acid and polyamine
deacetylation and play arole in acetyl lysine recognition. Targeted and selective inhibition of these enzymes
is likely to be the key to successful therapeutic use of
HDAC inhibitors.
25.11 Synopsis
In metalloproteases, apositively charged metal ion,
-
usually azinc ion, activates acoordinated water mol-
ecule, which nucleophilically attacks the peptide bond
to be cleaved. Through expansion of its coordination
sphere, the zinc ion also polarizes the carbonyl group
of the amide bond to be cleaved, and an adjacent
glutamate residue helps in the transfer of protons.
Potent inhibitors exhibit appropriate functional
-
groups to coordinate the zinc ion efciently; they also
address the specicity pockets on the primed side that
recognize the C-terminal part of the substrate to be
cleaved.
Angiotensin-converting enzyme (ACE) transforms
-
angiotensinI toII by cleaving aC-terminal dipep-
tide. Rational design concepts resulted in dipeptide
mimetics with acarboxylate group at aproline-like
moiety and azinc-coordinating group at the opposite
end. Captopril was the rst compound introduced to
therapy; alarge number of ACE inhibitors followed.
The target protease ACE is composed of two slightly
-
different catalytic domains. Aside from angiotensinI,
ACE degrades other peptides such as the blood-pres-
sure-lowering bradykinin. Undesired side effects of
ACE inhibitors are related to this degradation. Be-
cause the two domains of ACE show different sub-
strate proles that can be translated into selective
inhibition, the possibility exists to develop domain-se-
lective active substances with efcient blood pressure
regulation properties that avoid the unwanted adverse
effects.
Matrix metalloproteases (MMPs) are alarge family
-
of structurally related neutral zinc endopeptidases.
They are involved in the construction and degrada-
tion of connective tissue. Several therapeutic indica-
tions have been proposed such as rheumatoid arthritis
or cancer.
The development of selective MMP inhibitors proved
-
to be extremely difcult. The adaptive nature of the
binding pockets of this protein class has proven to
be challenging, and the pronounced overlapping substrate proles are problematic because different members of the family can mutually take over the role of
the protease being inhibited.
Carbonic anhydrases are hydrolases that transform
-
carbon dioxide to bicarbonate. They catalyze important processes from respiration to CO2 transport, pH
homeostasis, electrolyte secretion, C1 building block
delivery, bone resorption and calcication, or tumor
growth.
Due to the narrow funnel-shaped architecture of the
-
enzyme with the catalytic zinc ion at the end, almost
all α-carbonic anhydrase inhibitors feature aterminal
sulfonamide group. Particularly diuretics and antiglaucoma agents, which reduce the internal eye pressure, have been brought to market.
Phosphodiesterases (PDEs) are a small family of
-
metalloenzymes that hydrolyze the intracellularly
formed second messenger cAMP and cGMP. They
are broadly distributed in different tissues and regulate many important processes.
Inhibitors of PDE5 such as sildenal, originally de-
-
veloped for the treatment of angina pectoris, proved
to be agents to stimulate penile erections via the inhibition of cGMP degradation.
Under reductive conditions, iron (II) ions can be
-
found instead of zinc ions in the catalytic center
of hydrolyzing enzymes. In peptide deformylases,
the iron ion takes asimilar role as the zinc ion and
helps to remove the formyl group that is found at the
rst position of the N-terminus of anewly formed
polypeptide chain in prokaryotes, mitochondria, or
plastids. Inhibitors of peptide deformylases are either
potential antibiotics or can be exploited for antineoplastic effects.
Histone deacetylases (HDACs) remove the acetyl
-
group from the terminal nitrogen of an acetylated
lysine, thereby, restoring the basic character of the
terminal amino group and its positive charge. Histone
proteins serve to efciently coil the negatively charged
DNA in the nucleus. The enhanced electrostatic interactions with the charged lysine residues strengthen
DNA binding. As aresult, transcription factors cannot efciently read the gene information on the DNA.
Gene expression is, thus, repressed.
HDAC inhibitors attempt to interfere with the read-
-
ing of DNA in the case of misregulation of gene expression by inhibiting lysine deacetylation and, thus,
regulating gene expression. HDAC inhibitors can,
therefore, be used in many diseases, but especially for
cancer treatment.
Chapter • Inhibitors of Hydrolyzing Metalloenzymes
25
Bibliography and Further Reading
General Literature
A. Fersht, Enzyme Structure and Mechanism, W. H. Freeman, New
York, p. 416 ff. (1985)
B. W. Matthews, Structural Basis of the Action of Thermolysin and
Related Zinc Peptidases, Acc. Chem. Res., 21, 333–340 (1988)
D. H. Rich, Peptidase Inhibitors, in: Enzymes & Other Molecular Tar-
gets, P. G. Sammes, Eds., Vol. 2: Comprehensive Medicinal Chem-
istry, C. Hansch, P. G. Sammes and J. B. Taylor, Eds., Pergamon
Press, Oxford, p. 391–441 (1990)
R. P. Becket, A. H. Davidson, A. H. Drummond, P. Huxley and M.
Whittaker, Recent Advances in Matrix Metalloproteinase Inhibitor
Research, Drug Discov. Today, 1, 16–26 (1996)
B. Türk, Targeting Proteases: Sucesses, Failures and Future Prospects,
Nature Reviews Drug Discov., 5, 785–799 (2006)
T. Fischer, N. Senn, R. Riedl, Design and Structural Evolution of Ma-
trix Metalloproteinase Inhibitors, Chem. Europ. J., 25, 7960–7980
(2019)
Special Literature
T. A. Steitz, M. L. Ludwig, F. A. Quiocho, W. N. Lipscomb, The Struc-
ture of Carboxypeptidase A, J. Biol. Chem., 242, 4662–4668 (1967)
I. Bertini, V. Calderone, M. Fragai, C. Luchinat, M. Maletta, and K. J.
Yeo, Snapshots of the Reaction Mechanism of Matrix Metallopro-
teinases, Angew. Chem. Int. Ed., 45, 7952–7955 (2006)
B. P. Morgan, D. R. Holland, B. W. Matthews and P.A. Bartlett, Struc-
ture-Based Design of an Inhibitor of the Zinc Peptidase Thermol-
ysin, J. Am. Chem. Soc., 116, 3251–3260 (1994)
S. H Ferreira, A bradykinin-potentiating factor (bpf) present in the
venom of Bothrops jararaca, Br. J. Pharmacol. Chemother., 24,
163–169 (1965)
D. W. Cushman, H. S. Cheung, E. F. Sabo and M. A. Ondetti, De-
sign of Potent Competitive Inhibitors of Angiotensin-Converting
Enzyme. Carboxyalkanoyl and Mercaptoalkanoyl Amino Acids,
Biochemistry, 16, 5484–5491 (1977)
K. R. Acharya, E. D. Sturrock, J. F. Riordan and M. R. W. Ehlers,
ACE Revisited: A New Target for Structure-based Drug Design,
Nat. Rev. Drug Discov., 2, 891–902 (2003)
S. R. Bertenshaw etal., Thiol and Hydroxamic Acid Containing Inhib-
itors of Endothelin Converting Enzyme, Bioorg. & Med. Chem.
Lett., 3, 1953–1958 (1993)
J. Hu, P. E. van den Steen, Q.-X. A. Sang and G. Opdenakker, Matrix
Metalloproteinase Inhibitors as Therapy for Inammatory and
Vascular Diseases, Nat. Rev. Drug Discov. 6, 480–498 (2007)
H. Matter and M. Schudok, Recent Advances in the Design of Matrix
Metalloprotease Inhibitors, Curr. Opin. Drug Discov. Devel. 7,
513–535, (2004)
N. Borkakoti, F. K. Winkler, D. H. Williams, A. D’Arcy, M. J. Broad-
hurst, P. A. Brown, W. H. Johnson and E. J. Murray, Structure of
the Catalytic Domain of Human Fibroblast Collagenase Com-
plexed with an Inhibitor, Nature Struct. Biol., 1, 106–110 (1994)
J. R. Porter, N. R. Beeley, B. A. Boyce etal., Potent and Selective In-
hibitors of Gelatinase-A, 1. Hydroxamic Acid Derivatives, Bioorg.
& Med. Chem. Lett., 4, 2741–2746 (1994)
C. T. Supuran and A. Scozzafava, Carbonic Anhydrase Inhibitors and
Their Therapeutic Potential, Expert Opin. Ther. Pat., 10, 575–600
(2000)
J. J. Baldwin, G. S. Ponticello, P. S. Anderson etal., Thienothiopy-
ran-2-sulfonamides: Novel Topically Active Carbonic Anhydrase
Inhibitors for the Treatment of Glaucoma, J. Med. Chem., 32,
2510–2513 (1989)
J. Greer, J. W. Erickson, J. J. Baldwin, M. D. Varney, Perspective Ap-
plication of the Three-Dimensional Structures of Protein Target
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1035–1054 (1984)
A. Weber, A. Casini, A. Heine, D. Kuhn, C.T. Supuran, A. Scozza-
fava, G. Klebe, Unexpected Nanomolar Inhibition of Carbonic
Anhydrase by COX-2-Selective Celecoxib: New Pharmacological
Opportunities due to Related Binding Site Recognition, J. Med.
Chem., 47, 550–557 (2004)
K. Köhler, A. Hillebrecht, A. Innocenti, A. Heine, C. T. Supuran, G.
Klebe, Saccharin, a Potent Inhibitor of Carbonic Anhydrases:
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7841–7843 (2007)
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tential Applications, Nat. Rev. Drug Discov., 1, 674–682 (2002)
C. T. Supuran, A. Mastrolorenzo, G. Barbaro and A. Scozzafava,
Phosphodiesterase 5 Inhibitors—Drug Design and Differentiation
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Transferase Inhibitors
Contents
26.1 The Kinase “Gold Rush” – 452
26.2 Structure of Protein Kinases: More than 500
Variations with Similar Geometry – 453
26.3 Isosteric with ATP, and Selective Nonetheless? – 454
26.4 Gleevec
26.5 Tracing Selectivity: The Bump-and-Hole Method – 462
26.6 Metals Teach Kinase Inhibitors Selectivity – 464
26.7 Phosphatases: Reversal Switch to Activate
and Inactivate Proteins – 466
26.8 Inhibitors of PTP-1B: Treatment for
Diabetes and Obesity? – 468
26.9 Molecular Glue Inhibits the Release of
Phosphatase Activity – 472
26.10 Inhibitors of Catechol-O-Methyltransferase – 473
26.11 Blocking the Transfer of Farnesyl and Geranyl Anchors – 477
26.12 Synopsis – 480
Bibliography and Further Reading – 481
®
: Success Stories Breed Copycats! – 458
© 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_26
26
Chapter • Transferase Inhibitors
In the late 1970s, it became clear that proteins are not
only translated and synthesized in ribosomes, but can
also undergo postsynthesis modications. In addition
to glycosylation, phosphate groups are attached to hydroxyl functions on serine, threonine, and tyrosine residues. Later it was recognized that even histidine can be
phosphorylated. It was also shown that the degree of
phosphorylation of a protein can change dramatically
over time in the cell. Cell proliferation was found to be
strongly dependent on these changes. It became obvious
to associate phosphorylation with intracellular signaling processes. ATP was identied as the source of the
transferred phosphate groups. However, the bonds between the phosphate groups of ATP cannot be easily
transferred to an amino acid. This reaction is kinetically
too slow in aqueous solution. Nature has, therefore,
developed efcient catalysts for this task: protein kinases. Similarly, the cleavage of aphosphate group from
aphosphorylated amino acid is avery slow process. This
process also requires efcient enzymes, the phosphatases.
Thus, protein phosphorylation is areversible process that
can be “switched” in either direction by the above named
enzyme classes (. Fig.26.1). Although these enzymes
catalyze very general reactions, their substrate recognition is highly specic. This is the only way to precisely
control signal transduction processes and switch protein
function on and off.
These examples do not exhaust the range of posttrans-
lational modications. Each newly synthesized protein
carries an N-formylmethionine at its N-terminus. This
formyl group is rst cleaved by adeformylase (Sect.25.9)
before amethionine aminopeptidase removes the methionine residue from the peptide chain of many proteins.
The attachment of sugar residues (glycosylation) not only
improves the solubility and proteolytic stability of the
protein, but also serves to label proteins with recognition
characteristics that are crucial for signal transduction
and intracellular transport processes. Sugar residues are
particularly important for cell–cell recognition and interactions with the extracellular matrix (Sect.31.3). The
transglutaminases, which posttranslationally crosslink
proteins by forming isopeptide bonds through glutamate
and lysine side chains, were discussed in Sect.23.9. They
are also transferases, although they are mechanistically
closer to cysteine proteases and were, therefore, discussed
in Chap.23. Transferases can also transfer alkyl groups.
For one family of transferases, it is amethyl group that
will be used to modify residues. Others transfer aprenyl
group, the terpene anchor of which can be used to immobilize proteins at amembrane (Sect.26.11). Lysine and
arginine residues can be acetylated or the acetyl group
can be removed. The latter process requires cleavage of
an amide bond. This is done by histone deacetylases,
which are mechanistically analogous to zinc proteases
(Sect.25.10). Finally, ubiquitin and SUMO (small ubiquitin-like modier) should be mentioned. Ubiquitin is
. Fig. 26.1 The posttranslational phosphorylation of proteins is
critical for the regulation of intracellular signaling processes, for example, cellular reproduction is highly dependent on these processes.
Aphosphate group ( ) is transferred from ATP (green) to the hydroxyl function of aserine, threonine, or tyrosine. This task of switching
on protein function is performed by kinases. Conversely, phosphate
groups can be removed from aphosphorylated amino acid. This step
turns off the protein function. This simplied picture of switching
on and off will be used in the context of this book for cellular tasks.
Ultimately, however, the consequences of the transfer of aphosphate
group for the function of acell must be considered in detail in each
individual case. The picture is often more complex than the simple
switching on and off of afunction
apolypeptide chain that marks proteins for proteolytic
degradation in the proteasome (Sect.23.8). SUMO is
also asmall protein that can be attached to proteins and
inuences processes in the cell nucleus.
26.1 The Kinase “Gold Rush”
In case of disease, it sounds very attractive to target
enzymes that act as switches in signaling cascades. In
Sect.12.4, kinases were identied as enzymes that are
often involved in disease processes. In eukaryotes, approximately 30% of all proteins are reversibly phosphorylated. The addition of ahighly charged phosphate
group changes the electrostatic properties of the protein,
which can lead to conformational rearrangements and,
thus, the formation of new binding sites. The design of
kinase inhibitors initially focused almost exclusively on
the competitive displacement of ATP from its binding
site. However, ATP is not the only substrate used by kinases. This molecule is the most important energy transfer system in cellular metabolism. Many cofactors use
ATP as abuilding block to perform their cellular functions. There are approximately 2000 proteins in the human genome that use ATP as asubstrate in various ways.
The intracellular concentration is very high at 0.01 M. In
. • Structure of Protein Kinases: More than Variations with Similar Geometry
total, the physiological turnover of ATP in an adult is
75 kg per day! Given this situation, it is reasonable to ask
how specically and selectively abinding site of aparticular kinase can be blocked by an inhibitor: the same
substrate, ATP, is converted by each of these enzymes,
and its cellular concentration is very high. The problem
is further complicated by the fact that Nature has built
redundancy into many of these processes as afailsafe. If
one signal transduction pathway is removed, asimilar
pathway can take its place by producing more of its own
phosphorylated proteins. In this way, they help to correct
the decit caused by the blocked function. Is this particularly true for signaling cascades that use many different
structurally similar kinases and phosphatases to transmit
information? Until the early 1990s, all these problems
were considered so complex and intractable that anyone
who tried to develop selective kinase inhibitors as drugs
was considered crazy. Since then, the tables have been
completely turned. Today, a pharmaceutical company
that is not working on multiple kinase projects is considered backward and not innovative! No other protein
family has been investigated with so much fervor. What
caused this change of heart that led to apharmaceutical
“kinase gold rush”?
26.2 Structure of Protein Kinases:
More than 500 Variations
with Similar Geometry
Protein kinases are one of the largest target families in the
human genome. More than 530 protein kinases switch on
and off the most diverse signaling pathways in our body
and convert proteins from inactive to active states. They
are related to one another to varying degrees by their
sequence and structure and are grouped into subfamilies
according to afamily tree (Sect.26.3). Kinases can also
be regulated by other binding partners. Allosteric binding
sites and second messengers are known to be involved in
the regulation of kinase function. Inhibitory or activating proteins (e.g., cyclins) control kinase activation by
complexing with the kinase domains. Autophosphorylation of kinases exerts an important inuence on their
conformation and the correct positioning of the catalytic
residues for the transfer of the γ-phosphate group of ATP
to the amino acids serine, threonine, tyrosine, or histidine
(. Fig.26.2). The conserved architecture of protein kinases is shown in . Fig.26.3. The N-terminal domain
is composed of ve β-pleated sheets. The C-terminal domain is predominantly α-helical and contains the substrate binding site. The two domains are connected by
the hinge region. This contains the recognition motif for
the adenosine moiety of ATP. The ribose moiety and the
triphosphate group are bound in acleft between the two
domains and are coordinated by amagnesium ion, which
is essential for the transfer mechanism. The activating
loop with the DFG (Asp–Phe–Gly) and APE (Ala–Pro–
Glu) motifs adjacent to the catalytic site is also important
for the mechanism.
The structure determination of acAMP-dependent
kinase with a bound ADP and aluminum triuoride
molecule provided more detailed information about the
putative reaction mechanism. The AlF3 molecule, resembling ATP’s γ-phosphate group, is located between the
β-phosphate group of ADP and the serine residue of
the substrate peptide chain. Both were cocrystallized as
acomplex with the enzyme. In addition, two magnesium
ions are found in the binding pocket. Detailed information about the mechanism of phosphate transfer could
be derived from these structural ndings (. Fig.26.4).
Asp 184 of the DFG loop coordinates one of the two
Mg2+ ions that bring the three ATP phosphate groups
into the correct position for the reaction. The substrate’s
serine OH to be phosphorylated nucleophilically attacks
the terminal γ-phosphate group. The phosphate group
is then transferred forming atrigonal bipyramidal phosphorus intermediate. The adjacent Asp 166 polarizes
the nucleophilic serine OH group and accepts its proton
during the reaction. The positively charged residues Lys
168 of the kinase and Arg18 of the substrate act as stabilizers. In addition, the two magnesium ions compensate
for the negative charges on the phosphate groups. The aromatic rings of Phe54 and Phe 187 shield the transition
state from the aqueous environment.
Basically, there are three strategies to inhibit kinases:
blocking peptide substrate binding, displacing ATP from
the binding site, or modulating an allosteric regulation
site (see below). In the rst case, the formation of the
protein–protein contact must be prevented because kinases recognize and bind other proteins as substrates.
Inhibition of such contacts is considered to be extremely
difcult due to the size of the interaction surface that is
formed, especially if it is to be achieved with asmall molecule (Sects.10.6 and29.8). In the second case, the focus
is on the competitive displacement of ATP from the binding site. But is such aconcept doomed to failure, given
the many structurally similar kinases, the high concen-
. Fig. 26.2 Kinases transfer phosphate groups (red) to the
hydroxyl function of serine, threonine, or tyrosine (black, peptide
strand is blue)
26
Chapter • Transferase Inhibitors
. Fig. 26.3 The catalytic domains of all kinases share the same
architecture. The N-terminal domain (yellow) is composed of ve
β-pleated sheets and helices, whereas the C-terminal domain (red) is
predominantly made up of α-helical segments. Both domains are connected by the hinge region (green). They contain the recognition motif
for the adenosine moiety of ATP (molecule in the center). The terminal phosphate groups are oriented near the substrate chain (blue),
which is symbolized here by asegment of its polymer chain. It carries
the Ser, Thr, or Tyr in close proximity to the phosphate groups to be
transferred. Kinases are conformationally highly dynamic. A long
loop containing the so-called “DFG” motif (D = Asp, F = Phe,
G=Gly) rearranges upon kinase activation. It forms the hydrophobic
R-spine (brown). The simultaneous insertion of the at adenine building block of ATP forms the C-spine (purple). Both structural elements
are crucial for the activation of the kinase together with the conformational change of the loop from “DFG-out” to “DFG-in” (see also
. Fig.26.9). (7 https://sn.pub/3M5Pl5)
tration of ATP in the cell, and the many other proteins
that use ATP as asubstrate? Some kinases are regulated
allosterically. Here, the third strategy offers apossibility
to interfere with the regulatory function of the kinases
via the allosteric binding sites (Sect.26.4).
26.3 Isosteric with ATP, and Selective
Nonetheless?
Adetailed analysis of the binding sites for ATP in alarge
number of kinases revealed asurprising and promising
picture: there are indeed unoccupied regions near the
ATP recognition site that differ from kinase to kinase!
Two hydrophobic regions open up, one deep inside the
kinase and asecond on the opposite side towards the
surface (. Fig.26.5). The aminopyrimidine ring of the
adenine forms two adjacent hydrogen bonds to the peptide main chain in the hinge region of the kinase. Athird
interaction site on the polymer chain remains unused by
ATP 26.1, but may be involved in an interaction with
the H-bond donor function of aligand. The design of
ATP-competitive kinase inhibitors has uncovered many
interaction motifs that address the hinge region. These
have been incorporated into many clinically tested kinase inhibitors (26.2–26.21, . Fig.26.6). The ubiquitous
H-bonding pattern of the hinge region, which is present
in all kinases, makes it difcult to endow ligands with
selectivity. However, certain MAP kinases (mitogen-ac-
tivated protein kinases, signal transduction pathways in
cell differentiation, cell growth, and cell death) offer the
opportunity to design inhibitors with interesting selectiv
ity associated with aconformational change in the hinge
region. The orientation of the amide bond in this region
is rotated, so that an H-bond donor function rather than
an acceptor function is directed towards the bound ligands (. Fig.26.5). The ipping of the amide bond is
possible in these kinases because of the presence of agly-
cine residue in the adjacent position. Glycine lacks aside
chain at its Cα atom. Therefore, this residue has access
to amuch larger conformational space. Inhibitors with
adihydroquinazolinone scaffold mimicking the adenine
motif of ATP can trigger this conformational ip. In the
altered protein conformation, they can selectively bind
to kinases that carry aglycine at this position in their
sequences. If an amino acid with aside chain is present at
this position, as in most other kinases, the rearrangement
cannot be induced. Inhibitors that require this conformational ip to produce the specic H-bonding pattern with
the hinge region will, therefore, bind only with reduced
afnity to the latter kinases. In these cases, the required
conformational rearrangement of the main chain is not
possible for steric reasons.
The occupancy of the hydrophobic pockets on either
side of the adenine-binding site (. Fig.26.5) is agenerally applicable approach to design kinase inhibitors with
sufcient selectivity. The pocket located deep within the
protein (the so-called back pocket) has amino acids in its
front part that can have very different properties in different kinases. These are called gatekeeper residues. This
residue is present in all kinases, but can vary greatly in
size. It can be asmall amino acid such as serine, valine,
or threonine, or abulky residue such as phenylalanine,
-
. • Isosteric with ATP, and Selective Nonetheless?
. Fig. 26.4
nase with abound ADP and aluminum triuoride as atransition-state
mimetic, the reaction steps of the phosphate group transfer from ATP
(red) to the serine residue of the substrate (blue) can be modeled. Asp
184 from the DFG loop is coordinated to the β- and γ-phosphate groups
of ATP via amagnesium ion. An additional Mg2+ helps to position the
three phosphate groups correctly. Ser21, which is to be phosphorylated,
nucleophilically attacks the terminal γ-phosphate group, and aphosphorus atom is transferred with formation of a trigonal bipyramidal
Based on the crystal structure of acAMP-dependent ki-
methionine, or tryptophan. For example, athreonine
is found in the gatekeeper position in p38α and p38β
kinases. Amuch larger methionine residue is found in
the same position in the structurally similar p38γ and
p38δ kinases (. Fig.26.7). Compound SB203580 26.3
has ap-uorophenyl group at the 5-position of its indole
ring. The steric demand of this group is sufcient to just
intermediate. The neighboring Asp 166 takes the proton from the hydroxyl group of Ser21 during this reaction step. At the same time, the
positively charged residues Lys 168 of the kinase and Arg 18 of the
substrate stabilize this intermediate. (7 https://sn.pub/ag9Vm2)
allow its accommodation in the binding pocket next to
the threonine. On the other hand, amethionine in this
position requires such an amount of space that there is
not enough room for the p-uorophenyl group and the
afnity of 26.3 drops signicantly.
In asimilar way, 26.22 benets from abinding ad-
vantage on the p90 ribosomal S6 kinase (RSK) because