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26
Chapter • Transferase Inhibitors
. Fig. 26.5 Schematic representation of the ATP 26.1 recognition
site in kinases (so-called Traxler model). The adenine moiety is recognized at the hinge region by two parallel hydrogen bonds from the
peptide strand. Athird carbonyl group is available for interactions but
is not involved in ATP binding. Kinases with aglycine residue at this
position can switch an exposed acceptor function to adonor function
at this third position by ipping the adjacent amide bond (left). Next
to the ATP-binding site, the kinases open two differently composed
pockets, the so-called front and back pocket. The latter pocket is bordered by the gatekeeper residue. The residues in this pocket are not
involved in ATP binding. Spatially adjacent to this pocket is the phosphate binding site
. Fig. 26.6 Marketed products and development candidates of ATP-competitive kinase inhibitors 26.2–26.20; staurosporine 26.21 is anatural
product. All substances bind through hydrogen bonds to the peptide bonds in the hinge region of the kinases
. • Isosteric with ATP, and Selective Nonetheless?
its p-tolyl group has enough space in alarge pocket
gated by both athreonine and an adjacent cysteine
(. Fig.26.8). The combination of aThr and aCys resi due at these two positions has only been discovered in
three kinases in our genome. If areactive uoromethylene group is introduced, as in 26.22, this group can
react with the adjacent cysteine to form astable covalent
bond with the protein.
Another concept for the development of selective
inhibitors exploits the conformational adaptation of kinases. During their activation, kinases undergo several
steps on their way from an inactive to an active conformation (. Figs. 26.3 and26.9). Interestingly, kinases
show higher structural homology to one another in their
active states when they have bound ATP as the uniformly
identical substrate. Inhibitors that have ahigh afnity
for the active conformation are, therefore, less selective
than those that stabilize an inactive conformation. This
is because the differences in the inactive conformations
are much greater. Thus, the goal is to develop inhibitors that specically bind to an inactive state of akinase
(Sect.26.4).
Today, it is common practice to compile aso-called
inhibition or selectivity prole for development candidates
(. Fig.26.9). Their inhibition against alarge panel of kinases is measured in as many binding assays as possible.
The assay results are then plotted on afamily tree that
summarizes the structural relationships between kinases
from different subfamilies. The size and length of the
branches reect the degree of relationship between the
kinases. The level of inhibition of each kinase is represented by circles of varying sizes, with the larger circles representing greater inhibition (. Fig.26.10). It is
striking that many of the compounds from . Fig.26.6
have astrong effect on individual branches of the kinase
family tree. This suggests that the structural differences
within asubfamily described by such abranch are often
so small that no selectivity can be achieved with these
compounds. As mentioned above, there is functional
redundancy between kinases. If one kinase is blocked,
another may take over its function by upregulating its
expression. Therefore, it may be essential for asuccessful
drug therapy that not only one member of asubfamily is blocked, but that all members are equally affected.
. Fig. 26.7 The kinases p38α and p38β have threonine (Thr106, vi-
olet) as gatekeeper residues; asterically more demanding methionine
is in this position in the structurally related p38γ and p38δ kinases.
SB203580 26.3 binds with its p-uorophenyl group at the central imidazole ring in asmall niche next to the threonine (green surface, interior is blue). The activity is signicantly reduced on other kinases with
more voluminous amino acids in this position (Met, Gln) because of
steric conicts. (7 https://sn.pub/dTeold)
Chapter • Transferase Inhibitors
26
The natural product staurosporine (26.21, . Fig.26.6),
ahighly potent alkaloid of bacterial origin, is a promiscuous inhibitor of most kinases. It binds to kinases
in their active conformations. Sect.26.6 will show how
small modications of this lead structure can nevertheless result in highly selective inhibitors.
Over the past decade, the scale of protein kinase
research has exploded. More than 5000 kinase crystal
structures can now be found in the public PDB database
(Sect.15.3). In addition, many thousands of inhibitor
binding data have been published. More than 90kinase
inhibitors have been approved, almost all for cancer therapy. Mechanistically, our knowledge has expanded so
much that it sometimes seems difcult to keep track of the
different inhibition mechanisms. On the one hand, one
distinguishes between the above-mentioned “DFG-in”
and “DFG-out” binding of akinase. They block the active or inactive state of akinase. They are called typeI
and typeII inhibitors. The transition from the inactive
to the active conformation, which is accompanied by
arearrangement of the DFG loop, causes the formation
of the so-called regulatory backbone (R-spine) in many
kinases. Like aspine, the R-spine forms astacked packing of hydrophobic amino acids (. Fig. 26.3, brown).
This packing loses its shape in the inactive state. In the
active state, however, additional binding of ATP forms
asecond spine of stacked amino acids called the C-spine
(. Fig.26.3, purple). Here, the adenine ring is included
in the stack. In addition to typesI andII, there are also
typeIII inhibitors, which bind noncompetitively to ATP.
They leave the hinge region unoccupied and are more
likely to be found in the back pocket. TypeIV inhibitors
bind allosterically in apocket far from the ATP binding
site. TypeV inhibitors are bivalent inhibitors that block
two binding sites of a kinase simultaneously. TypeVI
inhibitors are covalent kinase inhibitors.
26.4 Gleevec
®
: Success Stories Breed
Copycats!
Well into the 1980s, drug development for cancer therapy
focused almost exclusively on processes that interfered
with DNA synthesis or cell division. This led to the development of antimetabolites, alkylating compounds, microtubule disruptors, and inhibitors of DNA synthesis.
These strategies attempt to attack target cells with very
high division rates, such as cancer cells. The disadvantage of this type of chemotherapy is the massive side
effects that severely limit the quality of life of the treated
patients. In 1960, Peter Nowell and David Hungerford
were the rst to recognize that chronic myeloid leukemia is
caused by aspecic genetic defect. This defect causes approximately 15% of all leukemia cases. Chronic myeloid
leukemia is the second most common form of chronic
leukemia and is caused by asevere proliferation of white
blood cells, particularly granulocytes. Areciprocal translocation between chromosomes9 and22 results in the
shortening of chromosome22. This is termed the Phil-
adelphia chromosome. The result of this exchange is the
so-called BCR-ABL fusion gene, which encodes aprotein
with constitutionally activated tyrosine kinase activity.
This protein belongs to the group of receptor tyrosine
kinases (Sect.29.8) and plays an important role in the
regulation of cell growth. Uncontrolled proliferation is
the result of unregulated activation and the cell becomes
atumor cell. It has been shown in other leukemia models that this gene is responsible for causing this type of
cancer. Therefore, it seemed that the increased kinase
activity as aresult of the misregulated gene was responsible for the disease. It should be possible to intervene in
this overregulation with apharmaceutical therapy. As
aresult, Sandoz initiated aprogram to develop selective
inhibitors of ABL tyrosine kinases.
. Fig. 26.8 With its p-tolyl
group, 26.22 achieves selective
binding to the p90-ribosomal S6
kinase because it nds asufciently large niche next to the threonine
gatekeeper residue. This places the
adjacent uoromethylene group
near acysteine residue with which
the inhibitor can then react. In
this way, astrong covalent bond
is formed with the kinase. The
necessary arrangement of Thr and
Cys residues has been discovered
in three kinases in our genome, so
that 26.22 achieves high selectivity
for kinases with this amino acid
composition in the back pocket
. • Gleevec®: Success Stories Breed Copycats!
. Fig. 26.9 Kinases go through multiple
conformations during their activation
from an inactive (red) to an active (green)
state. The shown ATP molecule binds
to the active conformation. For this,
acomplete loop, the so-called DFG loop,
of the protein (inactive form, violet)
moves from an inwards oriented geometry
into an exposed orientation (yellow, see
arrow). At the same time the binding
site for ATP is rendered accessible and
the substrate (blue) can bind. Interestingly, kinases possess great structural
homology among themselves in this state.
Therefore, inhibitors that bind with high
afnity to the active conformation are
less selective than inhibitors that block
the inactive conformation of the kinase.
(7 https://sn.pub/XWoK8O)
The search for protein kinaseC (PKC) inhibitors
began in the 1980s. Phenylaminopyrimidine (26.23,
. Fig.26.11) was identied as agood lead structure in
ascreening campaign. The compound was derivatized
(i.e., 26.24) and initially optimized as aPKC inhibitor.
It was found that the introduction of amethyl group
at position6 (i.e., 26.26) completely reversed the kinase
inhibition. This “magic” methyl group inuences the
conformation between the central aromatic ring systems,
which are coupled by an amino group. In the binding
mode observed with ABL tyrosine kinase, the inhibitor
adopts an extended conformation and the methyl group
contributes to atwisted arrangement between the two
ring systems.
Compound 26.26 proved to be ideal for inhibiting
members of this family of tyrosine kinases. Initially, this
derivative had insufcient oral bioavailability and water
solubility. Therefore, an attempt was made to improve
these properties by introducing polar groups such as an
N-methylpiperazine group. Compound 26.5 proved to
be optimal; it passed all phases of clinical trials and was
launched in 2001 as imatinib (Gleevec®). The compound
selectively blocks the BCR-ABL receptor tyrosine kinase
and prevents the phosphorylation of its substrate proteins. It was later discovered that other kinases, namely
the related c-Kit and PDGF receptor kinase, are also
inhibited.
Why has imatinib been such asuccess story? First of
all, the development of this inhibitor represented acom-
pletely new approach to cancer therapy. After all, it was
treating acancer variant with aselective therapy. The
drug showed very few side effects. However, treatment
with this compound is not cheap. It quickly became
ablockbuster for Novartis, generating annual sales of
more than abillion euros. In terms of both therapy and
sales, such asuccess story is highly stimulating for the
26
Chapter • Transferase Inhibitors
. Fig. 26.10 Inhibition prole of the inhibitors 26.2–26.21 that were
shown in . Fig.26.6 for 113 different kinases. The size of the red circle
quanties the strength of the inhibition. The data are shown on the
kinase family tree. In this diagram, the branching and the length of the
individual branches denote the degree of amino acid sequence similarity between protein kinases, grouped into families. The longer the distance in the dendrogram is, the smaller the degree of relatedness. The
natural product staurosporine 26.21 is alargely unselective inhibitor,
eld of kinase research. Success stories breed copycats!
The initial pessimism about selectivity problems and
kinase redundancy seemed to have blown over. But experience has shown how difcult it is to write asimilar
success story. As mentioned, in the meantime, more than
90kinase inhibitors for different indications (mostly cancer therapy) have been introduced to the market. There
are also imatinib successors (see below), but no other
compound has been able to achieve asimilar economic
and therapeutic success.
Binding of imatinib to the kinase stabilizes an inactive conformation of the enzyme. The DFG loop, which
is critical for the catalytic mechanism, remains in an out-
whereas 26.9 and 26.15 inhibit afew kinases very selectively. TKnon-
receptor tyrosine kinase, RTKreceptor tyrosine kinase, TKLtyrosine
kinase-like kinase, CKcasein kinase family, PKAprotein-kinase-like
family, CAMK calcium/calmodulin-like kinase, CDK cyclin-dependent kinase, MAPKmitogen-activated kinase, CLKCDK-like kinase.
(From M.A. Fabian etal. 2005, with kind permission from the author
and publisher)
wards-facing conformation (. Figs.26.9 and26.12). The
N-methylpiperazine group of the inhibitor, originally
introduced to improve solubility, occupies a position
that would be occupied by this loop in the active state.
Consequently, this group is crucial for the binding mode
adopted by 26.5. Astructural comparison of the kinase
in complex with imatinib 26.5 and tetrahydrostaurosporine 26.27 (. Fig.26.13) is shown in . Fig.26.12. The
latter inhibitor stabilizes the enzyme in its active conformation. The DFG loop takes acompletely different
course, resulting in the DFG sequence motif being directed inwards. The magic methyl group at the 6-position of the central phenyl ring of 26.5 forces this ring to
. • Gleevec®: Success Stories Breed Copycats!
be perpendicular to the adjacent pyrimidine ring. This
geometry allows favorable hydrophobic contacts with
the gatekeeper residue Thr 315, and ahydrogen bond is
formed between the NH group connecting the two rings
and the hydroxyl group of this threonine. The combination of optimal interaction with Thr 315 and strong
binding to an inactive conformation of the protein provides the selectivity advantage of imatinib. c-Kit is the
only other kinase for which imatinib has apronounced
afnity. This is explained by the high sequence homology
of this kinase with BCR-ABL kinase in the DFG loop
and in the ATP-binding region. In both cases, the gatekeeper residue is threonine.
In the meantime, cases of resistance to imatinib have
developed. The observed mutations desensitize the kinase
to imatinib inhibition. To date, approximately 30mutations have been described. They are the result of single
base pair exchanges in the genetic code (Sect.12.11) and
have evolved from several cell populations in which the
exchanges occurred by chance or were inuenced by oxidative damage to the DNA. These variants have become
established under the selective pressure of imatinib block-
ade. The most commonly observed resistance mutation
is caused by an exchange of the gatekeeper residue Thr
315 for isoleucine. Due to the larger size of the exchanged
amino acid, the inhibitory effects of imatinib fail. In addition, hydrogen bonds can no longer be formed. The
afnity decreases from Ki = 85 nM to 10 μM. In the
hinge region, Phe 317 forms aromatic contacts with the
pyridine ring of the inhibitor. Mutation of this residue
to aleucine leads to aloss of aromatic interactions and
reduces the binding afnity by afactor of three. Most of
the other observed mutations are rationalized by shifting
the conformation of the kinase more towards the active
conformation. Consequently, the selective advantage of
imatinib due to its strong binding to the inactive conformation becomes adisadvantage in terms of suscep-
tibility to resistance mutations. Novartis has introduced
afollow-up to imatinib, the structurally similar nilotinib
(Tasigna®) 26.28 (. Fig.26.13), which has an improved
resistance prole. With the exception of the Thr 315
→Ile mutation, it shows good afnity for all the resistance-conferring exchanges described and stabilizes the
inactive conformation of the kinase. Nilotinib, with its
modied side chain containing atriuoromethyl-substituted aromatic ring and an imidazole motif, ts better
into the preformed binding pocket and achieves ahigher
binding afnity. The afnity advantage is thought to account for its reduced susceptibility to resistance, as small
shifts from the inactive to the active conformation are
better tolerated. Another compound, dasatinib (Sprycel®) 26.29 from Bristol-Myers Squibb, may circumvent
the observed resistance to imatinib. It has acompletely
different mode of binding to the BCR-ABL kinase. For
example, it also binds to kinases of the Scr family (afamily of tyrosine kinases on the kinase phylogenetic tree
that phosphorylates many cellular cytosolic, nuclear, and
membrane proteins).
The native ABL kinase is posttranslationally modied with amyristic acid residue at its N-terminal glycine
residue. The addition of this fatty acid plays an important role in the self-regulation of this kinase. The fatty
acid residue occupies what is known as the myristoyl
pocket on the catalytic domain and stabilizes the entire
complex that it forms in aclosed, inactive conformation. This regulatory mechanism is lost in the genetically modied BCR-ABL kinase. The N-terminal region
in the fusion protein is replaced by afragment of the
BCR protein, leaving the BCR-ABL kinase permanently
active. It has been shown that the myristoyl pocket on
BCR-ABL kinase can be occupied by small molecules
that weakly inhibit its activity. Subsequently, afragment
search was initiated at Novartis using the NMR method
(Sect.7.8). To further optimize the fragment hits found,
. Fig. 26.11 By starting with the PKC kinase inhibition screening hit 26.23, multiple development steps afforded imatinib26.5
Chapter • Transferase Inhibitors
26
aconformation-sensitive assay using NMR spectroscopy
was developed. This allowed testing whether structurally enlarged and more potent binding ligands actually
stabilize the closed inactive conformation of the kinase. The result of this optimization was the inhibitor
asciminib 26.30 (. Fig.26.12), which highly selectively
binds with subnanomolar potency. It mimics the function of the fatty acid residue in the myristoyl pocket and
allosterically stabilizes the global inactive state of the
kinase. Most importantly, its binding is unaffected by
the development of resistance due to substitutions in the
ATP-binding pocket, such as the gatekeeper residue. As
aresult, asciminib therapy is still effective in patients who
have already developed massive resistance to ATP-competitive inhibitors. Using the crystal structure depicted in
. Fig.26.12, it has been shown that the ATP-competi-
tive inhibitor nilotinib 26.28 and the allosteric inhibitor
asciminib 26.30 can bind simultaneously. Therefore, it is
expected that an appropriate combination of both drugs
with different inhibitory mechanisms will signicantly
impede the emergence of resistance mutations.
26.5 Tracing Selectivity:
The Bump-and-Hole Method
The properties of a cell are controlled by acomplex
network of interwoven signaling pathways. Kinases are
regulators of such information cascades. Because of the
complexity of these networks, it is extremely difcult to
isolate the individual signaling pathways and to tease
apart the role of individual kinases. This task is further
complicated by the overlapping substrate specicities of
the kinases. Therefore, methods have been developed to
dissect these signaling pathways using appropriate chemical probes and genetic techniques. In principle, these
techniques are not limited to kinases; they can also be
used to analyze the functional properties of individual
members of other protein families. The structural differences between kinases that allow the design of selective
inhibitors have been highlighted in detail in Sect.26.2.
The gatekeeper residue occupies a key position. The
size and polarity of this residue varies from kinase to
kinase. Since the gatekeeper residue is not involved in
. Fig. 26.12 LeftSuperimposed crystal structures of imatinib 26.5
and tetrahydrostaurosporine 26.27 (. Fig. 26.13) with the active
(green) and inactive forms (red) of BCR-ABL receptor tyrosine ki-
nase, respectively. Center A section magnication (gray outline) of
the left gure is displayed. While 26.5 blocks the inactive form of
the kinase (DFG-out loop, purple), the nonselective inhibitor 26.27
binds to the active conformation (DFG-in loop, yellow). With the socalled magic methyl group, imatinib faces the gatekeeper residue Thr
315, and the amino group between the two rings forms ahydrogen
bond to its OH group. Right The crystal structure of the ternary com-
plex of BCR-ABL kinase with the ATP-competitive inhibitor nilotinib 26.28 (green/blue surface) and the allosteric inhibitor asciminib
26.30 (light blue/purple surface) is shown. Both bind to the enzyme
in its inactive form. Their binding sites are more than 20 Å apart.
7
https://sn.pub/zvNzKE)
(
. • Tracing Selectivity: The Bump-and-Hole Method
. Fig. 26.13 Nilotinib 26.28, which has aresistance-breaking pro-
le, was developed as afollow-up compound for imatinib 26.5. This
compound binds with almost the same binding mode, but with stronger afnity to the BCR-ABL kinase. Dasatinib 26.29, which was developed at Bristol-Myers Squibb, also binds to this kinase, but adopts
ATP binding, the substrate ATP binds almost identically
to all kinases with the same afnity. If the back pocket
is enlarged by replacing aparticular gatekeeper residue
with an amino acid with asmaller side chain (e.g., Thr
→Gly), the modied kinase variant can recognize amod-
ied ATP with an attached side chain (i.e., 26.30) and use
this ATP surrogate as the phosphorylation reagent for the
protein substrate (. Fig.26.14). This concept has been
colorfully termed the “bump-and-hole” method. Aligand
that is too large and would create asteric conict with
the protein (bump) can be converted into awell-tting
ligand if acorresponding hole is made on the side of
the protein.
Of course, the technique is not limited to the phosphorylation of substrates. It can also be used to design specic inhibitors. In the research group of Kevan
Shokat, formerly at Princeton and later at UCSF in
San Francisco, USA, protein kinases were modied by
replacing the gatekeeper residue with aglycine or alanine (. Fig.26.15). Because of this enlargement of the
back pocket, the mutated kinase variant became highly
sensitive to inhibition by 26.31 and 26.32, which only
weakly inhibit the wild type. This observation in an in
vitro assay was later translated to in vivo conditions. The
researchers used the baker’s yeast Saccharomyces cere-
visiae as amodel organism. The yeast genome encodes
120 kinases, many of which are related to mammalian
kinase families. One such case is the yeast cell division
an entirely different binding mode. Tetrahydrostaurosporine 26.27 is
anonselective inhibitor that binds to the active form of the kinase.
Asciminib 26.30 was developed as the rst allosteric inhibitor of BCRABL kinase and blocks the myristoyl pocket with subnanomolar afnity (. Fig.26.12)
control protein28 (Cdc28), member of the class of cyclin-dependent kinases (CDKs). It plays an important
role in yeast reproduction and controls specic phases
of the cell cycle. It shares 62% sequence identity with
acomparable human enzyme, CDK2. To demonstrate
the high specicity of the inhibitors 26.31 and 26.32 for
the mutated kinase variant, the altered protein had to be
incorporated into the yeast genome. This was done using retroviral methods established in molecular genetics
(Sect.12.15). Finally, it had to be shown that the cells of
the genetically modied yeast showed normal growth.
Only a20% longer replication time was observed. Next,
the inhibitor 26.32 was added to the cells of the wild-type
yeast and the genetically modied yeast. The cell growth
of the wild-type yeast remained unaffected, except at
an inhibitor concentration above 50 μM, where alonger replication time was observed. On the other hand,
the yeast with the modied cdc28 gene showed astrong
dependence on 26.32 under in vivo conditions. At concentrations as low as 50–100 nM, growth was reduced
by 50%; at 500 nM, growth was completely arrested. Apparently, the inhibitor blocks cells at the premitotic step
(cell nucleus division during cell replication), because the
phenotype of these inhibited cells seemed very similar
to those in which the mitotic cyclins (proteins with akey
function in cell cycle control) were knocked out.
This method can be used to study individual processes
in the cell cycle and, in particular, to determine the phase
26
Chapter • Transferase Inhibitors
. Fig. 26.14 In the context of the bump-and-hole method, the back
pocket of a kinase is enlarged by exchanging the gatekeeper residue
(yellow surface patch) for smaller amino acids (e.g., Thr →Gly). The
at which aspecic inhibitor intervenes. This information
is crucial for the development of atherapeutically effective drug. However, at the beginning of aproject, there
are usually no sufciently selective inhibitors available to
allow this targeted study to proceed. Furthermore, this
problem is particularly acute when many proteins with
high homology are found in the cell. The bump-and-hole
method, acombined chemical–genetic technique, allows
aspecic therapeutic validation of the biological relevance
of the target protein as well as the optimization of the
inhibitor class intended for development in amodel organism in an early phase of the project.
26.6 Metals Teach Kinase Inhibitors
Selectivity
Metals and metal ions play an important role in biological systems, especially as catalytic centers. But can they
also perform other tasks and support the design of inhibitors? In this section, we will discuss such an example.
Zinc and calcium ions can contribute to the cross-linking
and stabilization of proteins by acting as multidentate
ligands (cf. zinc nger proteins, Sect.28.2). Magnesium
altered kinase (right) can then recognize achemically modied ATP
26.30 with an enlarged side chain, which can subsequently be used as
aphosphorylating reagent for the protein substrate
ions often serve as akind of charge buffer to counteract
the electrostatic contribution of the strongly negatively
charged phosphate groups. As described in Sect.26.2,
they are involved in the phosphate transfer mechanism
from ATP to the hydroxyl groups of Ser, Thr, or Tyr. In
rare cases, metal ions serve as part of aligand that binds
to the biomolecule. One example is magnesium ions,
which are so tightly coordinated to the β-hydroxyketo
group of tetracycline 26.33 (. Fig.26.16) that they remain bound to the ribosome or the tet repressor during
complex formation (Sect.32.6). Another example is cisplatin 26.34, which, through asubstitution reaction on
platinum, induces cross-linking in the adjacent base pairs
of the DNA strands; this renders the DNA unreadable in
the replication process (Sect.14.9, . Fig.14.20).
In fact, metal ions can be incorporated into drugs for
very different purposes. Typically, carbon is the architectural element in drugs. However, its coordination geom-
etry is rather boring. It is limited to linear, trigonal-planar, and tetrahedral geometries. Astereocenter can occur
when four different substituents are on the tetrahedron
(Sect.5.2); this gives the possibility of two stereoisomers. Metals are much more exciting in this respect. By
expanding their coordination sphere, they have amuch
abc
. • Metals Teach Kinase Inhibitors Selectivity
de
. Fig. 26.15 aThe wild type of akinase activates aprotein substrate
by transferring aphosphate group. bWhen apotent inhibitor is added, phosphorylation is inhibited. cExchanging agatekeeper residue
for asmaller amino acid such as glycine does not change the catalytic
activity of this mutated kinase variant. dWhen an inhibitor that has
an enlarged substituent to ll the pocket next to the gatekeeper residue
greater variety of coordination geometries at their disposal. An octahedral center with only six different substituents yields 30stereoisomers! Initially, any medicinal
chemist would balk at the idea of incorporating metals
as structural centers into adrug molecule. The risk that
such centers might impart undesirable toxic properties
to the compounds seems too great. However, if one considers metals that only form bonds with coordination
partners that are inert to substitution, this argument
seems less valid. Ruthenium fullls these requirements
for inert behavior very well. Why not use the advantages
of a much more exciting coordination chemistry to
construct acompletely different molecular geometry to
generate an alternative pharmacophore pattern in avery
small space? The goal is to use the metal center as ascaf
fold and not as apartner to interact with the biomolecule. This concept, which seems unusual at rst glance,
has been pursued by Eric Meggers and his research group
at the University of Marburg in Germany. In Sect.26.3,
staurosporine 26.21 was presented as alargely unselective inhibitor of almost all kinases. This indolocarbazole
alkaloid has amolecular building block that resembles
acarbohydrate and occupies aposition comparable to
that of the ribose ring in ATP (. Fig.26.16). On the
other hand, the molecular architecture of staurosporine
is added to awild-type kinase, it can barely bind to the wild-type due
to steric conicts. eThis inhibitor could, however, block the mutated
kinase variant with the enlarged pocket. The two inhibitors 26.31 and
26.32 hardly block the wild type at all, but they are able to efciently
inhibit the kinase variant with the enlarged binding pocket due to the
modied gatekeeper residue
suggests ascaffold for achelating ligand. If the sugar
moiety is replaced by ametal center, avariety of novel
and interesting scaffolds can be generated. Considering
hexacoordinated metal ions, four additional coordination sites are available for further substitution.
Derivatives such as 26.35 were synthesized in the
group of Eric Meggers (. Fig.26.16). They turned out
to be highly potent kinase inhibitors. Interestingly, and
unlike staurosporine, they have clearly graded selectivity
proles. Even complexes with cyclopentadiene groups, in
which the ve-membered ring covers three coordination
positions, could be synthesized. Compound 26.36 was
found to be selective for GSK-3 and PIM-1 kinases in
an inhibition study with 57different kinases. Compared
-
to staurosporine (IC50 = 40 nM), 26.36 is ten times more
potent (IC50 = 3 nM) at these kinases. The metal-free coordination ligand26.37 (IC50 = 50 μM) and the N-methyl
compound 26.38 (IC50 > 300 μM) were almost inactive.
Acrystal structure of the R-stereoisomer with PIM-1 was
determined (. Fig.26.17). The structure largely agrees
with the geometry of the staurosporine complex. The ruthenium complex with its carbonyl group is oriented opposite to aβ-strand in the kinase fold located above the
ATP binding site. The cyclopentadiene group replaces
the lower part of the sugar-like moiety in staurosporine.