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Protein kinase inhibition of clinically important staurosporine analogues

Osman A. B. S. M. Gani and Richard A. Engh*
Received 5th January 2010
First published as an Advance Article on the web 4th March 2010
DOI: 10.1039/b923848b

Covering: up to the end of 2009

The isolation in 1977 of the microbial alkaloid staurosporine inaugurated research into several
distinct series of related natural and synthetic compounds. This has especially included research into
applications as anticancer drugs, beginning with the observation of low nanomolar inhibition of
Published on 04 March 2010 on http://pubs.rsc.org | doi:10.1039/B923848B

protein kinases. At present, several staurosporine cognates are in advanced clinical trials as anticancer
agents, with the potential to join the 10 other protein kinase inhibitors now approved for clinical
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use. Staurosporine is a broadly selective and potent protein kinase inhibitor, with submicromolar
binding to the vast majority of the protein kinases tested, and binding most of them more tightly than
100 nM. Crystal structures have shown the extended buried surface area interactions between the
protein kinase adenine binding site and the extended aromatic plane of the inhibitor, together with
protein–saccharide interactions in the ribose binding site. Together with structures of closely related
analogues, there are now some 70 X-ray crystal structures in the Protein Data Bank that enable analysis
of target binding properties of the clinical compounds. In this manuscript we review the discovery of
these compounds, revisit crystal structures and review the observed interactions. These support the
interpretation of kinase selectivity profiles of staurosporine and its analogues, including midostaurin
(PKC412), for which a co-crystal structure is not yet available. Further, the mix of purely natural,
biosynthetically and chemically modified compounds described here offer insights into prospects and
strategies for drug discovery via bioprospecting.

Staurosporine: discovery, chemistry, and kinase
inhibition
Discovery
Staurosporine (STU, 1) was discovered in 1977 at the Kitasato
Institute in Japan while screening for microbial alkaloids.1 It was
isolated first from bacterium Streptomyces staurosporeus, and
subsequently from other actinomycetes.2 In the meantime, a total
of some 50 related indolocarbazole cognates have been isolated
from actinomycetes (including Streptomyces, Saccharothrix,
Lentzea, Lechevalieria, Nocardia, Nocardiopsis, Nonomuraea,
Actinomadura and Micromonospora), and cyanobacteria.3
Several STU analogues have also been isolated from marine
invertebrates, including sponges, tunicates, bryozoans and
mollusks.4,5 STU was first shown to possess antifungal and
hypotensive activity, but it attracted greater attention in 1986
when it was shown to be highly potent inhibitor of protein kinase
C (PKC) (IC50 ¼ 2.7 nM) and had a strong cytotoxic effect on
cancer cells.6 STU protein kinase inhibition is now known to be
broadly potent across the kinome,7 a property making STU itself
apparently too toxic for use as a drug. However, many STU
cognates have a more selective profile, and interest remains high
regarding the discovery or design of new cognate compounds,
both from natural sources and as synthetically generated
The Norwegian Structural Biology Center, Institute of Chemistry, University
of Tromsø, 9037 Tromsø, Norway. E-mail: Richard.engh@uit.no;
Tel: +47 77644073
variants. STU itself remains an essential research tool in kinase
inhibition studies, although often erroneously considered
a universal kinase inhibitor.
Chemistry
STU is a prototypical alkaloid of the indolocarbazole family.
Among the indole and carbazole ring fusion variants that form
this family, only the indolo[2,3-a]carbazole isomeric form is
known to occur naturally. It is present in many natural products
that show biological activities,8 although their biological
functions are largely unknown. The majority of the natural
indolocarbazoles isolated to now are derivatives of indolo[2,3-a]-
pyrrolo[3,4-c]carbazole ring, which is the 7-keto derivative of
K252c (2) (Fig. 1). The natural indolocarbazoles are closely
related to another group of clinically important compounds, the
bisindolylmaleimides, which lack the bond between the indole
moieties. Members of this group, including e.g. arcyriarubin A,
enzastaurin (3) and ruboxistaurin (4), possess substantial
flexibility relative to STU and the ‘closed’ indolo[2,3-a]carbazole
compounds. The structure of STU was first elucidated by single-
crystal X-ray analysis which showed the indolo[2,3-a]pyrrolo-
[3,4-c]carbazole ring, and the bridging of the two indole
nitrogens by glycosyl linkages9 (Fig. 1).
Early studies10–12 of staurosporine biosynsthesis identified
metabolic precursors and intermediates, and later studies13–15
discovered the gene cluster responsible for the process. In the
course of the biosynthesis of STU, the indolocarbazole ring is
derived from two tryptophan molecules, while the sugar ring is

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Fig. 1 Staurosporine and related analogues.
synthesized from glucose and methionine.16 However, the origin
of the nitrogen at the pyrrole[3,4-c] unit was not determined by
these experiments. Recent studies have catalogued 18 genes that
form a cluster for synthesis of STU (Fig. 2).3
Kinase inhibition
Recent kinase profiling studies7,17 show that the vast majority of
protein kinases tested are bound by STU with KD <3 mM, and
most with KD <100 nM. The highest affinity targets are generally
Ser/Thr protein kinases, but many tyrosine protein kinases,
including important drug targets, are also bound with high
affinity. The selectivity of STU is so broad that the kinases that
lack binding become particularly interesting. For example, the
profiling data agree that Her2/Erbb2 is bound only weakly, if at
all, by STU (see discussion below). While the broad selectivity of
Osman Gani obtained his PhD in
2007 from the University of
Tromsø, Norway. His PhD
thesis involved computational
docking and scoring methods in
the study of protein–ligand
interactions. In 2009 he began
postdoctoral studies at the
Norwegian Structural Biology
Centre at the same university.
Osman’s present research focus
is on the chemical determinants
of binding of ATP-dependent
Osman Gani enzymes and chemical library
design.
490 | Nat. Prod. Rep., 2010, 27, 489–498
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staurosporine can be linked to toxicities and disqualifies STU
itself as a potential drug, it does make STU a key starting point
for research into variants with more suitable target selectivity
profiles. Thus, the indolocarbazoles have been at the focus of
considerable research into their potential as chemotherapeutics
against cancer especially, but also e.g. against Alzheimer’s
disease, and other neurodegenerative disorders.18
Staurosporine analogues: biosynthesis and clinical use
STU diversification and clinical trials
The first STU analogues that were isolated from Streptomyces
were UCN-01 (5) and UCN-02 (6) (stereoisomers of 7-hydroxy
STU) in 1989.2 In 1993, another STU analogue (K252a, 7) was
also shown to inhibit in vitro phosphorylation of crude extracts
from Streptomyces griseus and Streptomyces sp. 16. Together
with rebeccamycin (8), these represent four major variants of the
g-lactam (or maleimide) ring with its key role in protein kinase
binding. Additional variants were produced by combinatorial
biosynthesis,19 utilizing natural diversity of metabolic pathways
in recombinant form for modification of bioactive substances.
Still other modifications are the result of chemical synthetic
methods. Ultimately, several STU analogues have advanced in
clinical trials, broadly subdivided into two subgroups based on
their target profiles. The first subgroup can be represented by
STU itself, and includes compounds that are potent inhibitors of
protein kinases. The second subgroup are substituted at the
maleimide nitrogen (of rebeccamycin-like compounds) and
therefore do not inhibit protein kinases, but instead are used as
potent stabilizers of DNA topoiomerase-I. Among the natural
and biosynthetically produced STU analogues, only UCN-01 (5)
has been tested for clinical use (now at phase II in the USA) for
various forms of cancers including pancreas, breast and
lymphoma.
Synthetic analogues involve mostly sugar ring substitution;
e.g. tetrahydrofuran instead of tetrahydropyran, and various
substitutions on the ring system (Table 1). A few synthetic
analogues are derived from substitutions on indolocarbazole
ring, combined with sugar ring substitution in e.g. CEP 1347 (9)
Richard Engh obtained his PhD
in 1987 in physical chemistry
from the University of Chicago
for time-resolved laser spectro-
scopic studies of protein
dynamics. After postdoctoral
research studies with Nobel
Laureate Robert Huber, he
directed collaborative research
in drug discovery between the
Max Planck Institute for
Biochemistry (Martinsried) and
pharmaceutical companies
Richard Engh Boehringer Mannheim GmbH
and Roche. In 2006 he became
professor of chemistry at the University of Tromsø and the
Norwegian Centre for Structural Biology.
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Fig. 2 Biosynthetic pathway of staurosporine in Streptomyces sp. TP-A0274.19 The genes associated in synthetic steps are shown (dTDP ¼ deoxy-
thymidine-50 -diphosphate).
(Table 1). The compounds now in clinical trials have passed pre-
clinical testing, in part because the substitutions have improved
selectivity profiles. These substitutions were made at a time when
little specific structural information was available, and large-
scale protein kinase profiling was not possible. Now that the
relevant technologies and information is becoming readily
available, optimization efforts will be greatly facilitated.
Structural determinants of binding selectivity
Staurosporine binding to kinase domains
A key aim in the design of kinase inhibitors is to achieve a high
degree of selectivity against a chosen (set of) kinase target(s). The
prediction of the effects of modifications on binding strengths is,
however, only partially possible, and the most successful
approaches have involved parallel synthesis and inhibition
profiling. While the necessary degree of selectivity for low
toxicity cannot be simply stated,20 testing inhibitors against
panels of hundreds of kinases now provides data for this type of
evaluation, while assisting the choice of new potential drugs.
The first structures of STU in protein kinase complexes were
determined with PKA21 and CDK2.22 To date, about 40 3D
coordinate sets of protein kinase domains co-crystallized with
STU have been deposited in the Protein Data Bank (PDB). These
structures show a nearly universal common binding mode of
STU to the ATP binding site, which is formed where the N- and
C-terminal folding domains of the kinase are linked by a hinge
segment (Fig. 3a). In short, two conserved hydrogen bonds
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anchor the lactam or maleimide amide moiety to the hinge
region. Both hydrogen bonds involve backbone atoms (CO and
NH) of two hinge residues, (one of which is highly conserved as
glutamic acid). Other hydrogen bonds are observed involving the
methylamino nitrogen of glycosidic ring: one to the backbone of
a catalytic loop residue,23,24 and the other to a polar side-chain
from a residue C-terminal to the hinge.25
Thus, the polar interactions of STU are not mediated by side-
chains of the target protein kinase binding pocket, but by the
highly conserved polar groups of the hinge main-chain atoms.
On the other hand, hydrophobic interactions are dominated by
side-chain interactions, involving residues that are less conserved
(but still must enable adenosine binding) and that are subject to
induced fit structural rearrangements. Only one side-chain
hydrogen bond exists: it anchors the methylamino nitrogen of
glycosidic ring. These interactions determine in part the
selectivity profiles across active conformation kinases of the
kinome (Fig. 3b and 3c).
The sugar ring in STU is perpendicular to the indolocarbazole
plane and forms a boat conformation predicted to be the bio-
logically active form.26 It also occupies the space in the ATP
pocket that is bound by the ribose in the ATP/ADP bound forms
of the kinase.
The broad aromatic planar structure of the indolocarbazole
plane is sandwiched by hydrophobic residues from the N-lobe
and C-lobe (Fig. 3b). Besides the van der Waals interactions
between the side-chains of these residues to the aromatic plane of
STU, several CH–p interactions are also formed between the CH
moieties of these residues and the conjugated plane of STU.25
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Table 1 Synthetically modified staurosporine analogues in clinical trials (USA). Only compounds in phase II/III trials are shown

Inhibitor Indication Status Chemical structure

Midostaurin Acute myeloid Phase II (with

(PKC412) leukemia (AML) cytarabine + daunorubicin)
Systemic myeodysplastic Phase II
syndrome
Mast cell leukemia
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Lestaurtinib Myelofibrosis Phase II

(CEP-701) AML
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Polycythemia vera
Psoriasis
Prostate cancer

CP-751 Adenosquamous cell cancer Phase III (with Erlotinib)

AML Phase II
Non-small-cell lung cancer
Head and neck cancer
Large cell cancer
Squamous cell cancer

CEP-1347 Parkinson’s disease Phase II/III

Edotecarin Glioblastoma Phase III

Stomach cancer Phase II
Breast cancer
Tumor metastasis

Becatecarin Breast cancer Phase II

Ovarian cancer
Colorectal cancer
Brain and CNS tumors
Lymphoma
Neuroblastoma
Retinoblastoma
Sarcoma

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Fig. 3 Three-dimensional structure of protein kinase domain (PDB
1STC) in complex with STU. a) STU binding cleft of kinase domain is
shown in a surface representation. Several structural elements that are
relevant to STU binding are labelled. b) Kinase domain residues in close
contact with STU are shown. c) Target–STU hydrogen bonds are shown.
One of the most conserved interactions is between the backbone
amide of the first glycine of GxGxxG motif and the ether oxygen
of the glycosidic ring of STU.27 This interaction results from an
induced fit of the Gly-rich loop upon STU binding.
A recent study of 20 of the published coordinate sets of kinase
domains in complex with STU has shown that the binding
affinity of STU for a specific kinase is especially correlated with
two factors: size of the gatekeeper residue, and the distance
between the first Gly of the GxGxxG motif and the Asp of the
DFG loop.28 This distance measures the closure of the N-lobe
and the C-lobe. The study also indicated that binding affinity of
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STU increases with the increase of the number of hydrogen
bonds between the methylamino nitrogen of glycosidic ring with
surrounding residues.
Anomalous binding
Among the published kinase domains in complex with STU, one
coordinate set (PDB 3CBL29) has shown two STU molecules that
are not bound at the ATP binding site but bind anomalously
between two monomers of the tyrosine protein kinase FES (from
feline sarcoma) in crystal packing contact.29 Besides STU binding
in the ATP site, one STU molecule is docked at the hydrophobic
groove which is on the reverse side to the ATP binding pocket,
i.e., the sheet formed by b4 and b5 separates the two pockets.
A hird STU molecule is docked at a surface produced by the SH2
and kinase domain. Whether these binding interactions of STU
result from crystallographic artefacts or from inclusion of an
SH2 domain in the crystallization of kinase domain require
further investigation. However, a trimer of Abl kinase domains
(PDB ID 2HZ430) includes two monomers that have unoccupied
ATP sites but are bound instead by a modified staurosporine
(see below) at a site similar to that seen in 3CBL, sandwiched in
a crystal contact position. This demonstrates the potential of
preferential non-ATP site binding, possibly as a function of
protein–protein interactions. Whether this occurs among physi-
ological complexes remains to be shown.
Despite the broad kinase selectivity of STU, several kinases are
apparently only weakly inhibited by the compound. These
kinases are scattered throughout the kinome tree,7 but TK and
TKL families have the highest density of these STU-uninhibited
kinases. Some of these STU-uninhibited kinases are known to be
medically important kinases. For example Her2/Erbb2 is known
to be overexpressed in 20–30% breast cancer patients, and this
overexpression is a marker of tumour aggressiveness and
responsiveness to therapy.31 Despite substantial sequence simi-
larity in two tyrosine kinases (EGFR and HER2), EGFR
inhibited by STU at low nM concentrations, but HER2 remains
unbound even at 105 nM STU concentration. The only difference
between EGFR and HER2 kinases that contact the ATP site is
a substitution of Cys to Ser in the loop between hC and b4. This
loop has been recognized as part of a ‘‘molecular brake’’ mech-
anism that links the hinge with helix C motions, and is implicated
in cancer resistance mutations.32 Although the single substitution
does not explain how staurosporine binding might be abrogated
in HER2, additional substitutions in the loop, including the
introduction of two glycine residues, do hint at significant
changes to the ‘‘molecular brake mechanism’’ and the possibility
of a structural reorganization that at least in the in vitro assays
alters the ATP binding site.
Staursoporine analogues binding to kinase domains
At present, there are coordinate sets of 10 ‘closed’ STU
analogues that have been deposited in the PDB, in complex with
nearly 50 protein kinase domains. Some of these are DNA-top-
oisomerase-1 inhibitors, such as edotecarcin (10) and becatecarin
(11), but most are protein kinase inhibitors. We focus our
discussion on the latter category. The STU analogous kinase
inhibitors can be grouped with respect to differences: 1) in the
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indolocarbazole ring at the hinge, 2) at peripheral ring positions,
and 3) at the sugar moiety. The web-based servers CEMC,33
aligning multiple protein structures based on Ca coordinate
distances, and Multiband,34 recognizing binding patterns and
spatial arrangements of physico-chemical properties common to
a set of protein structures were used to analyse these structures.
Table 2 shows the structural and sequence alignment of several
kinases discussed here.
Hinge binding variants
UCN-01. UCN-01 (5) is undergoing clincal trials for cancer
treatment as a kinase inhibitor. It has been found to show strong
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inhibition of CHK1 (Ki ¼ 5.6 nM),35 PDK1 (IC50 ¼ 5.0 nM),36
and PKCb37 (IC50 ¼ 10 nM). It has also been found to inhibit
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Table 2 Structural alignment of selected protein kinases that bind STU
analogues
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CDKs (Ki: CDK1 95 nM, CDK2 30 nM, and CDK4 3.6 mM),
and other isoforms of PKCs below IC50 ¼ 1 mM.
CHK1 is a Ser/Thr kinase that plays important roles in DNA
damage response, including G2/M cell cycle control. UCN-01 is
found to abrogate the G2/M checkpoint which is induced by
DNA damaging agent.38 PDK1 (3-phosphoinositide-dependent
protein kinase-1) plays key roles in insulin and growth factor
signalling through activation of a number of AGC kinase family
members, including protein kinase B.
Specificity tests against a panel of 29 kinases show that
UCN-01 exhibits a kinase inhibition profile significantly distinct
from STU, but still rather broad.36,39,40 The comparison of X-ray
structures between the PDK1-STU and PDK1-UCN-01 shows
that the hydrophobic interactions of central heterocyclic moiety
of STU/UCN-01 with kinase residues are the same. Also, the
hydrogen bonds to the heterocyclic and the sugar moiety are
conserved. However, the 7-hydroxy group of UCN-01 forms an
additional hydrogen bond with the side-chain of Thr 222
(Fig. 4a). An ordered water molecule is also shown to contact the
7-hydroxy group. This water molecule is buried in a hydrophobic
pocket lined by Val143, Leu212, and Thr222 in the PDK1-STU
complex. Similar hydrogen bonds are observed in CHK1-UCN-
01 complex.35 Here, however, two water molecules form
hydrogen bonds with 7-hydroxy group (PDB 1NVQ).
Fig. 4 Binding of UCN-01 (2) to protein kinases. a) Superposition of
PDK1 bound to STU (white, 1OKY) and UCN-01 (pink, 1OKZ). The
additional alcohol group of UCN-01 does not shift the overall binding
position, and makes hydrogen bonds (yellow) to T222 and a water
molecule. b) Superposition of UCN-01 from PDK1 (white, 1OKZ) and
PDK2 (purple, 1PKD). T222 in PDK1 corresponds to A144 in PDK2,
which cannot stabilize UCN-01 binding with a hydrogen bond. The
hydrogen bonding interactions in PDK1 are shown in yellow.
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The kinase inhibition profiles show eleven kinases to be bound
equally by STU and UCN-01. Of these, five kinases have
a residue equivalent to Thr222 in PDK1, and two (PKB and
PKC) have Thr at the Val143 position of PDK1, thus all may
have hydrogen bonds to the 7-hydroxy group.
Five of nine kinases with weaker inhibition of UCN-01
compared with STU lack residues that can form a hydrogen
bond with the 7-hydroxy group. Among the others, PKA and
MAPKAP-K2 do have a threonine at this position, but also have
a bulkier methionine at the PDK1-Leu159 position that hinders
the hydrogen bond between the 7-hydroxy group and Thr.
Molecular modelling studies show that an extra 7-hydroxy group
at STU generates a steric clash with Met120 (the ‘gatekeeper’
residue) in PKA.21 Thus, the size of residues lining the 7-hydroxy
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group contribute to the binding specificity. For example, PDK1-
Val143 is another residue which is replaced by Leu in mitogen-
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activated protein kinases, which may explain the lack of
inhibition of these kinases by UCN-01.
Several features shown by the X-ray crystal structure of
CDK2-UCN-01(PDB 1PKD) explain the relatively lower affinity
of UCN-01 for CDK2, compared with CHK1 and PDK1. The
greatest difference is the absence of a hydrogen bonding residue
for the 7-hydroxy group of UCN-01. CDK2 has an alanine (144)
at the PDK1-Thr222 position (Fig. 4b). Also, the presence of the
gatekeeper residue (Phe80 replaces Leu159 in PDK1) may hinder
the binding of the 7-hydroxy group. Overall, the position of the
7-hydroxy group in CDK2 creates an unfavorable hydrophilic–
hydrophobic contact. Further, UCN-01 is not seen to form
hydrogen bonds with its methylamino group in the sugar moiety.
In contrast, the side-chain of the CHK1/PDK1 Glu residue
following the hinge region forms hydrogen bonds with nitrogen
of the methylamino group. The Asp in the equivalent position in
CDK2 forms a hydrogen bond with main-chain amide group.
Finally, differences exist in the Gly-rich loop, which is more open
in CDK2 than in CHK1.35
Despite the clear correlations, the sequence- and structure-
based analyses of UCN-01 binding described above do not
simply explain the inhibition profiles of several tyrosine kinases
such as Lck, Csk, AMPK and SGK1. Both SGK1 and MSK1
show same sequence in the 7-hydroxy group binding pocket.
However, SGK1 is most strongly inhibited by UCN-01, while
MSK1 is most strongly inhibited by STU.
Peripheral ring variants
3-Hydroxystaurosporine (3OH-STU, 12). This natural deriv-
ative of STU was isolated from flatworm Pseudoceros sp., and
has shown potent antiproliferative effects in leukemia cell lines.41
The X-ray structure of 3OH-STU in complex with Pak1(p21-
activated kinase, PDB 2HY8) shows a binding mode similar to
that of STU. The Pak family of kinases plays a prominent role in
cell proliferation, cell survival, and cell motility, e.g. by blocking
intrinsic apoptopic signals via a Pak1–Raf1–BAD pathway. The
X-ray structure shows that the 3-hydroxy group forms
a hydrogen bond with the carbonyl oxygen of the Leu134
backbone. The nitrogen atom of the hinge Leu134 backbone also
forms the canonical hinge hydrogen bond with the carbonyl of
the 3OH-STU lactam amide. The nitrogen atom of Gly 350
backbone and oxygen atom of the Phe246 backbone are 3.7 and
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 away from the 3-hydroxy oxygen, respectively. This
3.5 A
network of electrostatic interactions contributes to the binding of
3OH-STU. Pak1 was also cocrystallized with octahedral
ruthenium compounds which are synthetic STU-derived
compounds.42 These structures show a similar binding mode of
the 3-hydroxy group attached to the indole ring (PDB 3FY0
and 3FXZ).
Tetrahydrostaurosporine (4H-STU, 13). 4H-STU is derived
from STU and differs in that one indole ring has been partially
hydrogenated to break planarity and reduce the extent of
aromaticity. One consequence is increased solubility.43 This
compound has proven to be a useful probe for kinase inhibition
studies. 4H-STU has been cocrystallized with several kinase
domains such as active Abl kinase,30 focal adhesion kinase,44 and
Jak3 kinase.43 These structures show that 4H-STU binds to the
active form of kinases similar to binding by STU. However, and
as described above, complexation with ABL kinase (PDB 2HZ4)
shows anomalous binding, with two of three subunits bound
preferentially at the back surface of the ATP binding site. The
Gly-rich loops of these subunits were found to be disordered.
7-Ketoindolo[2,3-a]carbazole compounds. The PDB contains
one structure of a protein kinase complexed with an inhibitor
containing the 7-keto variant of the hinge binder having
a ‘closed’ indolo[2,3-a]carbazole ring. These inhibitors include
e.g. rebeccamycin (8) and its aglycone cognate represented by
arcyriaflavin A, which is planar and possesses two fold
symmetry. More structures are available of bis-indolyl maleimide
inhibitors, such as ruboxistaurin (4) (PDB 2JIJ),45 showing
details of the hinge binding.46 Bis-indolylmaleimide (BIM)
structures were originally discovered as protein kinase C (PKC)
inhibitors,47 but later efforts to develop BIM derivatives made
use of the disruption of planarity of the indolocarbazole ring to
achieve selectivity and potency against therapeutically important
kinases. For example, ruboxistaurin (4) selectively inhibits PKCb
(IC50 ¼ 4.7 nM for PKCb1 and 5.9 nM for PKCb2).48 Thus,
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‘‘controlled’’ deviation from the more rigid planarity of the
indolocarbazole system targets the subtle differences among the
ATP binding sites with ‘‘designed’’ conformational flexibility of
the bound inhibitors.49,50
Sugar ring variants
Midostaurin (PKC412, 14). Midostaurin (N-benzoyl-STU) is
now under investigation as an oral multitargeted kinase inhibitor
that targets especially FLT3, a receptor tyrosine kinase that is
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activated by mutation in about one-third of AML patients.51,52
PKC412 also inhibits several other molecular targets that are
thought to be involved in the pathogenesis of acute myeloid
leukemia (AML) such as VEGFR-2, c-KIT, and PDGFR.
There is no experimental structure showing binding of
PKC412 to a target kinase. Modelling staurosporine-like inter-
actions at the hinge places the additional benzoyl group near
residues Glu127, Glu170, and Asn171 (PKA numbering). The
kinase inhibition profile data from Karaman et al.7 allows
a comparison of STU and PKC412 binding (Fig. 5); the

Fig. 5 Comparison of staurosporine and midostaurin binding strengths across the kinome. Data were taken from Karaman et al.7 The disk sizes
represent the strength of STU binding (the Dlog KD values in the key are relative to micromolar binding). The colors represent the Dlog KD effect of the
removal of a benzoyl group from midostaurin. Red and orange colors indicate that the benzoyl group weakens binding by 4–5 orders of magnitude
(the kinome tree is reproduced by courtesy of Cell Signaling Technology, Inc. (http://www.cellsignal.com).

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benzoylation generally decreases binding by two orders of
magnitude but the distribution of the effect is not homogeneous
across the kinome. The greatest weakening occurs with kinases of
the STE family, while some kinases such as MLK1, MLK3, and
JAK3 are uniquely unaffected by the change, retaining low nM
binding.
K252a. K252a (7), a natural STU analogue, is a potent
inhibitor of a number of Ser/Thr kinases and the several receptor
tyrosine kinases.53 K252a has been cocrystallized with the
tyrosine kinase domain of hepatocyte growth factor receptor
encoded by protooncogene c-Met,54 and MEK1 kinase.55 c-Met
signalling is important for embryonic development and tumori-
genesis, especially in the context of invasive and metastatic
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phenotypes.56 MEK1 is a member of the highly conserved
mitogen-activated protein kinase cascade signalling pathway.
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K252a was found to bind (Kd ¼ 10 nM) and stabilize the acti-
vation loop of c-Met with a binding mode analogous to STU-ki-
nase complexes. A notable interaction of K252a to c-Met is the
presence of Pro1158 residue at the hinge region, the backbone of
which forms hydrogen bond with the lactam of K252a. The
presence of Pro at this position may restrict the opening of the
N- and C-lobe, even after binding of K252a. In PKA and CDK2,
STU binding is accompanied by a relative opening of the N- and
C-lobe. Another important feature is that the furanose moiety of
the compound forms three additional hydrogen bonds with the
surrounding residues, supplementing the conserved hydrogen
bonds located at the hinge region. In the MEK1-K252a complex,
an unusual feature was the presence of magnesium ion, which was
located at a negatively charged polar pocket formed by Asp208
and Asp190 close to the ATP binding site.
KT5720 (15). KT5720 is a semisynthetic derivative of K252a
(4), and a selective inhibitor of PKA (IC50 ¼ 56 nM).57 Although
it is a K252a derivative, KT5720 does not inhibit PKC. KT5720
is widely used to examine PKA in various cellular events such as
cell adhesion and regulation of transcription by cyclic AMP-
dependent response element binding protein (CREB).58 KT520
has recently been cocrystallized with a Ser/Thr kinase PknB
receptor from Mycobaterium tuberculosis (PDB 3F69).59
Combined hinge binding and sugar ring variation
SB-218078 (UCM 16). CHK1 was cocrystallized in complex
with SB-218078.35 In contrast to STU, it has a maleimide group
in place of lactam, and a tetrahydrofuran ring which does not
have the methylamine group. SB-218078 forms only two
conserved hydrogen bonds in the hinge region of CHK1, and
inhibits CHK1 with Ki ¼ 15 nM (compared to 5.6 and 7.8 nM
This journal is ª The Royal Society of Chemistry 2010
View Article Online
with UCN-01 and STU respectively). But SB-218078 is a potent
(Ki ¼ 5.6 nM) inhibitor of CDK2. CDK4 is also relatively
strongly (Ki ¼ 16 nM) inhibited by SB-218078, compared to STU
and UCN-01. Understanding the selectively and interactions
of SB-218078 with kinases is more complex than other STU-
analogues.35
Conclusion
A great many lessons are to be learned from staurosporine and its
analogues. The time between initial discovery and current clinical
trials has spanned a period of intense technological advances;
drug discovery efforts now are high-throughput, targeted, and
information-driven in ways hardly imaginable in 1977. These
developments promise to greatly accelerate discovery and opti-
mization. The body of data related to staurosporine and its
analogues reveals much in the way of mechanism and will
certainly enhance structure-based design and in general the
predictivity of new efforts. On the other hand, the data also
demonstrates the enormous amount of complexity, diversity, and
unpredictability of target binding properties. The physiological
responses to drugs represents still greater complexity. Drug
discovery efforts should continue to improve in the foreseeable
future, combining rational and screening efforts across technol-
ogies in bioprospecting, combinatorial biosynthesis, parallel
synthesis, screening, and targeted design.
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