doi:10.1016/j.jmb.2007.01.004
J. Mol. Biol. (2007) 367, 882–894
A Structural Comparison of Inhibitor Binding to
PKB, PKA and PKA-PKB Chimera
Thomas G. Davies 1 ⁎, Marcel L. Verdonk 1 , Brent Graham 1
Susanne Saalau-Bethell 1 , Christopher C. F. Hamlett 1 , Tatiana McHardy 2
Ian Collins 2 , Michelle D. Garrett 2 , Paul Workman 2 , Steven J. Woodhead 1
Harren Jhoti 1 and David Barford 3
1
Astex Therapeutics Ltd,
436 Cambridge Science Park,
Milton Road, Cambridge,
CB4 0QA, UK
2
Cancer Research UK Centre
for Cancer Therapeutics,
Haddow Laboratories,
The Institute of Cancer
Research, 15 Cotswold Road,
Sutton, SM2 5NG, UK
3
Section of Structural Biology,
Chester Beatty Laboratories,
The Institute of Cancer
Research, 237 Fulham Road,
London SW3 6JB, UK
*Corresponding author
Although the crystal structure of the anti-cancer target protein kinase B
(PKBβ/Akt-2) has been useful in guiding inhibitor design, the closely
related kinase PKA has generally been used as a structural mimic due to its
facile crystallization with a range of ligands. The use of PKB-inhibitor
crystallography would bring important benefits, including a more rigorous
understanding of factors dictating PKA/PKB selectivity, and the opportunity to validate the utility of PKA-based surrogates. We present a “backsoaking” method for obtaining PKBβ-ligand crystal structures, and provide
a structural comparison of inhibitor binding to PKB, PKA, and PKA-PKB
chimera. One inhibitor presented here exhibits no PKB/PKA selectivity, and
the compound adopts a similar binding mode in all three systems. By
contrast, the PKB-selective inhibitor A-443654 adopts a conformation in
PKB and PKA-PKB that differs from that with PKA. We provide a structural
explanation for this difference, and highlight the ability of PKA-PKB to
mimic the true PKB binding mode in this case.
© 2007 Elsevier Ltd. All rights reserved.
Keywords: kinase; inhibitor; structure-based drug design; cancer; Akt
Introduction
Protein kinase B (PKB/Akt) is a key component of
the PI3K kinase pathway, which is responsible for
cell-proliferation and survival.1–4 It acts as a central
node on the pathway, phosphorylating a large
number of downstream proteins, including GSK3β,
FKHRL1, BAD and TSC2, and thereby regulating cell
growth, protein translation, apoptosis, and cell-cycle
progression.
Numerous hormones, growth and survival factors
trigger the activation of PKB via PI3K-mediated
generation of the second messenger PtdIns(3,4,5)P3
(PIP3). The interaction of PIP3 with the PH-domain
of PKB localises the enzyme to two membrane
associated kinases, PDK1 and rictor-mTOR, 5,6
responsible for phosphorylation of PKB on its
activation segment and hydrophobic motif, respectively. These two phosphorylation events cooperate
Abbreviations used: PKA, B, protein kinase A, B.
E-mail address of the corresponding author:
[email protected]
to activate PKB some 1000-fold. Down-regulation of
PKB is achieved via a combination of PIP3 dephosphorylation by the lipid phosphatase PTEN (thus
antagonising PI3K), and PP2A-mediated dephosphorylation of PKB.
PKB is increasingly being recognized as an
important therapeutic target for the treatment of
malignancy, due to the large number of human cancers in which the PI3K pathway is disregulated.1,7–11
Deletion or mutation of the tumour suppressor gene
PTEN is found in several common human tumours,
and leads to constitutive PKB activity, with continuous high levels of signalling through the PI3K
pathway. In addition, mutation and amplification of
the PI3K kinase, PIK3CA, and amplification of the
three PKB isoforms, have been observed in ovarian,
prostate and cervical tumours, amongst others.12,13
Most recently, somatic mutations of PKBβ have also
been linked to colorectal cancers.14
Blockade of PKB-mediated signalling can be
achieved by disrupting the phosphorylation, and
hence the activation, of the enzyme by inhibition of
upstream kinases such as PI3K, mTOR and PDK1, or
through inhibiting the association of the enzyme's
0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.
883
Inhibitor Binding to PKB, PKA and PKA-PKB Chimera
PH domain with phosphatidylinositols.15–18 One of
the most active area of research, however, is the
development of inhibitors that target the ATP-site of
the PKB catalytic domain, and several chemical
series are currently in pre-clinical development.19–25
In 2002, the three-dimensional structure of active,
phosphorylated PKBβ (Akt-2) was solved26 (in
complex with the nucleotide analogue AMP-PNP),
and has given important insights into the design of
novel inhibitors targeted towards the ATP-binding
site. However, to date, all detailed structure-based
accounts of PKB-inhibitor development described in
the literature have employed the closely related
kinase, PKA, as a surrogate system due to its facile
co-crystallization and soakability with a range of
ligands.19,20,22–28 PKA is highly homologous to PKB,
sharing approximately 45% sequence identity with
the kinase domain, and this rises to approximately
80% within the ATP site, with only three key amino
acid differences within the cleft itself.
This use of PKA, either as wild-type,20,22–25 or as a
mutant PKA, in which ATP-site residues that differ
from PKB are mutated to form a “PKA-PKB
chimera”,19,27 has provided a useful approach, and
allowed the development of inhibitors with low
nanomolar potency. However, there is a clear
requirement to validate the general applicability of
such surrogate systems, particularly for the development of molecules with a PKB versus PKA
selective profile. A comparison with PKB-ligand
structural information would allow such an analysis
to be carried out, and would also give a more
rigorous and complete understanding of the molecular recognition with this important enzyme.
Despite extensive screening for novel conditions,
neither apo-crystallization of PKBβ nor co-crystallization with inhibitors other than AMP-PNP was
successful. We have therefore developed a “backsoaking” method for obtaining high resolution,
phosphorylated PKBβ-inhibitor crystal structures,
which has allowed us to fully exploit PKB structural
information within the drug discovery process. To
illustrate the use of this method, we present the
structures of two contrasting inhibitors (Figure 1)
Figure 1. Chemical structures of 1 (isoquinoline
sulphonamide) and 2 (A-443654).
bound to PKB, and provide a comparison of their
binding modes with both PKA and PKA-PKB.
The isoquinoline sulphonamide inhibitor 1 is
derived from a series that exhibits little or no
PKB/PKA selectivity (IC50(PKA) = 0.17 μM, IC50
(PKBβ) = 0.23 μM25), whereas the indazole-based
inhibitor 2 (A-443654) exhibits approximately 40fold selectivity for PKB ((Ki(PKA) = 6.3 nM, Ki
(PKBα) = 160 pM24). This use of PKB-inhibitor
crystallography allows us to present a detailed
comparison of the binding of selective and nonselective inhibitors in each system for the first
time.
Results
PKB-inhibitor structural overview and
comparison with PKA
PKBβ-inhibitor co-crystals with compounds 1
and 2 were obtained using a back-soaking protocol
(see Materials and Methods), X-ray diffraction data
were collected, and the structures were refined to
1.8 Å and 2.3 Å resolution, respectively. The
overall protein structure is in each case essentially
identical to that observed for the published structure
of activated PKB in complex with the nucleotide
analogue AMP-PNP.26 PKB adopts the classical bilobal kinase fold, with a predominantly β-sheet
containing N-terminal domain, and a mainly αhelical C-terminal domain linked by a short polypeptide strand known as the “hinge”. The inhibitors
presented here bind in the kinase's ATP-site, which is
located in the deep cleft between the N and Cterminal domains (Figure 2).
As for the initial PKBβ-AMP-PNP structure
determination, the protein construct used here
contains a PRK2-derived mimic of the phosphorylated hydrophobic motif (the “PIFtide” sequence) at
its C terminus, which binds to a hydrophobic pocket
on the N-terminal lobe.26,29 The combination of this
stabilizing engagement of the C-terminal tail with
the protein, and the phosphorylation of the kinase's
activation loop at position Thr309, leads to an
ordered and catalytically competent active site.
Similarly, the PKB structures presented here also
contain a substrate peptide derived from the kinase
GSK3β that faciliates crystallization.26
The overall fold of the closely related kinase,
PKA is very similar to that of PKB,27,29 although
there are differences at the N terminus of PKA,
which contains an additional 18 residue α-helix
(Figure 3(a)). Superposition of the two kinase
structures reveals that they possess nearly identical
ATP-sites, with just three residue differences within
the active site itself (Figure 3(b)). Specifically,
Val123, which is located in the hinge region is
substituted by Ala232 in PKB, Val104 at the base of
the pocket is substituted by Thr213, and Leu173 is
substituted by Met282. Other residues differing
between PKA and PKB in the vicinity of the ATP
884
Inhibitor Binding to PKB, PKA and PKA-PKB Chimera
Figure 2. Cartoon representation of PKB with 1 bound at the ATP site. The surface of the inhibitor is depicted as a
green surface, and the GSK3β peptide is shown in yellow.
site are generally considered too remote to significantly influence the binding of inhibitors with
“drug-like” molecular weight, or are orientated
with their side-chains away from the cleft itself.
The PKA-PKB chimera structures presented here
contain these three mutations, and additionally, a
fourth mutation, Gln181→Lys, in the vicinity of the
kinase hinge. Although not directly part of the activesite, the PKA-residue Gln181 was found to have the
potential for perturbation of ligand binding when
combined with mutation of the nearby residue
Val123→Ala. 27 This residue has therefore been
commonly mutated in PKA-PKB chimeric systems
described in the literature.19,27 The details of the
binding of each inhibitor to PKB are described in
detail below, along with a comparison of their binding
to PKA and PKA-PKB.
Structures of inhibitor 1
(isoquinoline-sulfonamide) bound to PKB,
PKA and PKA-PKB
Following the back-soaking procedure, clear
electron density for compound 1 was observed in
the binding site of PKB, revealing a complete
displacement of the co-crystallized ligand AMPPNP (Figure 4(a)). The structure of 1 bound to PKB
reveals an overall ligand orientation that is very
similar to that previously observed with PKA25
(PDB code 2c1a), as might be predicted for a non-
Inhibitor Binding to PKB, PKA and PKA-PKB Chimera
885
Figure 3. Superposition of PKA and PKB structures. (a) Overview of PKA and PKB folds. PKA-1 (PDB code 2c1a) and
PKB-1 are shown as cartoon representations in blue and red, respectively. The additional helix present in PKA is visible on
the left-hand side of the Figure. (b) Detail of PKA and PKB active sites. The three active site differences discussed in the
text are highlighted (PKB numbering), and compound 1 is shown as a green surface bound to PKB.
PKA/PKB selective compound (Figure 4(b)). In both
structures, the isoquinoline binds in the adenine site
of the ATP cleft, and is sandwiched between
hydrophobic side-chains forming the top and
bottom of the ATP-binding cleft, namely Val57 and
Leu173 in PKA, and Val166 and Met282 in PKB. In
both kinases, a single hydrogen bond is formed
between the heteroaromatic nitrogen of the isoquinoline and a backbone amide on the kinase hinge
(Val123 in PKA and Ala232 in PKB), an interaction
that is highly conserved in kinase-inhibitor recognition. A “CH…O”-type interaction is formed in each
kinase between a hinge backbone carbonyl (Glu120
in PKA and Glu230 in PKB) and the inhibitor's
isoquinoline C-1 hydrogen, and the fused phenyl
ring of the heterocycle is oriented towards the
“gatekeeper” methionine (Met229 in PKB, Met120
in PKA). In both PKA and PKB, no close protein
contacts are observed with the sulfonamide oxygen
atoms of the ligand, and the group adopts a
rotational conformation placing the S–N bond
approximately orthogonal to the plane of the
aromatic ring. However, in PKB, the sulphonamide
nitrogen is observed to donate a hydrogen bond to
Wat309, forming a bridging interaction with the
carboxylate oxygen of Glu236. In complex with
PKA, the sulphonamide nitrogen is positioned to
potentially form a hydrogen bond directly with
Glu127, although the geometry is sub-optimal
(rNH…Oε2 = 3.3 Å, ∠ Oε2..NH..S = 100°).
The flexible, seven-atom chain between the sulfonamide and the terminal aromatic ring adopts a
similar conformation in both kinases, and orientates
the ligand's terminal 4-chlorophenyl ring towards
the glycine-rich loop, where it occupies a hydrophobic pocket that is conserved between PKA and
PKB. In PKA, this pocket is formed by the side-chains
of Leu74 and the hydrophobic portion of Lys72, and
by the face of the glycine loop in the region of Gly52
and Gly55. Similarly, in PKB, the pocket is formed by
Leu183, Lys181, Gly161 and Gly164.
Despite the similar presentation of the chlorophenyl to this pocket, subtle changes in the precise
conformation of the seven-atom chain leads to
slightly different interactions being formed for the
inhibitor's ether oxygen, and the secondary amine.
In PKA, the ether oxygen is orientated towards the
back of the ATP-cleft, and forms no direct interactions with the protein. In PKB, the oxygen is
orientated towards the front of the cleft, and is in a
suitable geometry to accept a hydrogen bond from
the side-chain Arg6 of the substrate GSK3β peptide
(rNH2…O = 3.0 Å). However, the contribution of this
interaction to the observed affinity for PKB is likely
to be small due to the solvent-exposed nature of this
site.
The protonated amine nitrogen occupies the
ribose binding site of PKA, and is involved in saltbridging and hydrogen-bonding interactions with
the carboxylates of Asp184, the backbone carbonyl
of Glu120 and the side-chain carboxamide of
Asn171. Electrostatic interactions of basic functionalities with this “acidic pocket” are believed to be
important for potent inhibition of both kinases,25
and consistent with this, the structure of 1 in
complex with PKB shows similar molecular recognition with hydrogen bonding and electrostatic interactions to Asp293 and Glu279.
886
Inhibitor Binding to PKB, PKA and PKA-PKB Chimera
Figure 4. Inhibitor 1 binding to PKA, PKB and PKA-PKB. (a) Structure of PKB-1 in the ATP site. The final 2mFo–DFc
electron density for inhibitor, contoured at 1σ is shown in blue. (b) Superposition of PKA-1 (grey) and PKB-1 (yellow) in
the region of ATP-site. Selected residues are labelled using the PKB-structure numbering. (c) Structure of PKA-PKB-1 in
the ATP site. The final 2mFo–DFc electron density for inhibitor, contoured at 1σ is shown in blue. (d) Superposition of
PKA-1 (grey) and PKA-PKB-1 (cyan). Selected residues are labelled using the PKA-structure numbering. The PKA-1
complex used for these Figures is the previously determined structure 2c1a.
Overall, despite the minor differences in interactions between PKB and PKA discussed above, the
binding mode of the non-selective compound 1 is
very similar in both systems. In addition, the binding
mode of 1 with PKA-PKB (Figure 4(c) and (d)) is
essentially identical to that observed with wild-type
PKA, and so for this compound the use of either PKA
or PKA-PKB chimera as surrogates for PKB is likely
to be valid.
Structure of inhibitor 2 (A-443654) bound to PKB
and PKA
The structure of 2 in complex with PKA was
published recently,24 and revealed its binding mode
within the ATP site. In order to confirm the binding
mode, and to allow a full comparative analysis with
PKB, we have re-solved this PKA structure in
addition to the structure of 2 with PKB (Figure 5(a)
and (b)). Inhibitor 2 exhibits selectivity for PKBα
(Akt-1) over PKA,24 and thus provides a useful tool
molecule to compare in both structural systems. The
PKB crystallography described here is for the βisoform (Akt-2), and although the ATP sites of α and
β-isoforms are identical it is possible that the use of a
PKBβ structure to discuss potency versus PKBα
could be misleading. To address this, compound 2
was assayed for its ability to inhibit the kinase
activity of the PKBβ-PIFtide construct used for the
crystallography, as well as for PKA. These results
(IC50 (PKA) = 27 nM, IC50 (PKB) = 0.5 nM) confirm
that the selectivity is also observed for our construct.
The structures of 2 bound to PKA and PKB are
superposed and presented in Figure 5(c), and key
non-covalent interactions are summarized schematically in Figure 5(d). The methyl-indazole adopts an
identical binding mode in both kinases, forming
bidentate hydrogen bonding interactions with the
Inhibitor Binding to PKB, PKA and PKA-PKB Chimera
887
Figure 5. Inhibitor 2 binding to PKA and PKB. (a) PKA-2 and (b) PKB-2 (in the region of the ATP site). Final 2mFo–
DFc electron density for the inhibitors is contoured at 1σ and shown in blue. (c) Superposition of PKA-2 (grey) and
PKB-2 (yellow). Residues are labelled using PKB numbering. (d) Schematic diagram showing binding of 2 to PKA and
PKB. Key non-covalent interactions are depicted as broken lines. The alternative positions of the indole ring are shown
by shading: PKA (light grey) and PKB (black). (e) Surface representation of PKB with compound 2 bound. The surface
was coloured by lipophilicity in AstexViewer46 using the method described by Gaillard et al.,53 with red/pink
representing the most lipophilic regions, and blue/green the least lipophilic. The putative methyl-aromatic interaction
discussed in the text is shown as a broken line. (f) Overlay of surfaces for PKA (grey) and PKB (yellow) with
compound 2 bound. The indole group of 2 packs with the side-chain of Met282, but would leave a cavity in PKA due
to the substitution by leucine at this point in the active site.
888
hinge in both cases: in PKB, one nitrogen of the
indazole accepts a hydrogen bond from the backbone nitrogen of Ala232 (Val123 in PKA), and the
other nitrogen donates a hydrogen bond to Glu230
(Glu121 in PKA). The methyl of the indazole is
orientated towards the front of the cleft, where it
packs against the AGC-kinase-specific side-chain
Phe439 (Phe327 in PKA). The pyridine “spacer” also
adopts a similar position when bound to both PKA
and PKB, placing the pyridine nitrogen in the
vicinity of the conserved catalytic lysine (Lys181 in
PKB, and Lys72 in PKA), and in the case of PKA,
potentially accepting a charged hydrogen bond
from this residue. In PKB, subtle changes in the
positioning of the lysine side-chain lead to suboptimal geometry for a formal hydrogen bond
(rN…N = 3.5 Å, ∠CE..NZ..N = 78°), although at this
resolution, such interpretation should be treated
with care. Regardless, the contribution to the overall
affinity is likely to be similar in both PKA and PKB
due to the charged, and hence longer range nature of
this interaction.
In contrast to the structures observed for inhibitor
1, the conformation of the remainder of inhibitor 2 is
very different when bound to PKB, compared to
PKA. A combination of sp3-sp3 and sp3-sp2 torsions
in the ether-linker leads to a different binding mode
for the indole, although the key pharmacophoric
electrostatic interaction of the primary amine in the
region of the “acidic pocket” (Asp293/Asp283)
remains similar. In complex with PKA, and in
agreement with previous observations,24 the indole
ring is directed towards the glycine loop, packing
against the hydrophobic face of the β-strands in the
region of Gly52 and Gly55, and adopting a binding
mode reminiscent of that observed for 1 bound to
both PKA and PKB. In contrast, the structure with
PKB shows that the inhibitor adopts a different
conformation in which the indole is directed
towards the front of the ATP-binding site. This
“folded” or “U-shaped” conformation brings the
indole ring close to the methyl group on the indazole
(rmethyl…indole = 4.0 Å), and places the indole ring in a
new hydrophobic pocket formed by the side-chains
of Met282, Phe439 and Val166, and the face of the
glycine loop near Gly159. The indolinic nitrogen is
also in the vicinity of the side-chain of Glu236,
although the relatively long distance to the carboxylate oxygen atoms (rN…Oε2 = 3.5 Å), evidence for
disorder in the glutamate side-chain, and the
solvent-exposed nature of this site suggest that any
hydrogen-bonding interaction is likely to be weak.
Nevertheless, methylation of the indole nitrogen in a
related compound has been associated with decreased affinity for PKB,22 and the relatively close
distance from the nitrogen to Glu236 could provide
a rationale for this. Interestingly, previous attempts
to explain this effect in the context of the PKA
structure as a surrogate for PKB had proved
difficult,22 suggesting that this compound might
also adopt a conformation similar to 2 when bound
to PKB, or that the lower energy conformation
observed for 2 is disallowed by the additional
Inhibitor Binding to PKB, PKA and PKA-PKB Chimera
methyl group. In PKA, the indolinic methyl of
such a compound would be expected to be directed
into a large solvent filled cavity, with little or no
expected effect on the overall affinity.
In contrast to the extended conformation of the
glycine loop for PKA-1, the glycine loop in the PKB2 structure adopts a new conformation in which
Phe163 at the tip of the loop folds into the ATP-site
(Figure 5(c)). Of note is that the phenyl ring of
Phe163 now occupies the hydrophobic pocket filled
by the indole of compound 2 in the PKA structure,
and packs closely on top of the inhibitor's etherlinker, thus potentially contributing to the highly
favourable binding of 2 to PKB. The glycine-rich
loop is well known to be a flexible element in
kinases, and it is likely that in apo-PKB this position
is one of many approximately isoenergetic conformations that can be adopted by the protein.
The difference in observed inhibitor conformation
with PKA and PKB appears to arise due to one of the
three key residue differences between the kinase
active sites: namely the presence of methionine in
the place of leucine at the base of the PKB cleft at
position 282. In the PKB structure, the indole ring
packs closely on top of the terminal ε-methyl of
Met282 (rCε…face ∼ 3.7 Å), forming a highly favourable lipophilic contact, and potentially resulting in
an increased hydrophobic effect compared to its
occupation of the glycine loop pocket. Indeed, an
analysis of surface hydrophobicity within the ATPcleft of PKB reveals that the indole binds close to a
lipophilic hotspot on the protein (Figure 5(e)). It is
possible therefore that the difference in the observed
binding of 2 to PKB could be due in part to this more
favourable lipophilic interaction, which could not be
formed in PKA. In PKA, substitution by a branched
amino acid, leucine, at this point means that despite
its apolar nature, the residue is not positioned as
optimally as methionine for hydrophobic packing
with the inhibitor (Figure 5(f)). In fact, modelling
suggests that if the inhibitor did adopt this PKBbinding mode with PKA, an empty lipophilic cavity
would be formed in this region, which is expected to
be energetically unfavourable. As a result, 2 binds to
PKA with its indole occupying the hydrophobic
pocket in the glycine loop in a similar fashion to the
chlorophenyl of compound 1.
It is interesting to observe that in PKB, inhibitor 2
is positioned such that the ε-methyl group of Met282
is located at the centroid of the “phenyl” portion of
the indole, with the Cε→centroid vector almost
perpendicular to the plane of the ring (angle to
normal ∼7°). A search of the Cambridge Structural
Database (CSD)30 for methyl…aromatic contacts
revealed that this geometry is strongly preferred,
indicating the possibility of a specific interaction
between the methyl hydrogen atoms and the face of
the indole ring in addition to van der Waals contacts.
Polar interactions with aromatic systems have been
documented,31 and although generally weak, such
CH…π interactions are likely to be more favourable
in electron-rich systems such as indole. As described
previously for the purely lipophilic contact,
889
Inhibitor Binding to PKB, PKA and PKA-PKB Chimera
substitution of Met282 by leucine in PKA is likely to
remove the potential for this interaction, with the
orientation of leucine's terminal methyl hydrogen
atoms being unsuitable for interaction with the face
of the inhibitor's indole ring.
Taken together, these observations suggest that
the difference in binding mode, and the difference in
potency of 2 for PKB compared to PKA arise
predominantly as a result of the Leu-Met substitution, which allows the inhibitor to adopt a conformation where it can form increased lipophilic
contacts with the enzyme. In addition to these
intermolecular considerations, the “folded” nature
of this conformation leads to the favourable intramolecular burial of approximately 50 Å2 of apolar
surface within the inhibitor itself.
Interestingly, the PKB/PKA selectivity observed
for 2 is lost in a related compound in which the
indole is substituted for phenyl.24 Simple modelling
suggests that for this inhibitor, the phenyl ring
would not be able to adopt a position in which it can
form such favourable hydrophobic packing with
Met282. It is possible therefore that this compound
will adopt a similar binding mode in both PKA and
PKB in which the phenyl interacts with the glycine
loop. Clearly the Met282 region is a very “SAR-rich”
part of PKB, with the free energy of binding being
very sensitive to changes in both the residues
forming this subsite, and to the structure of this
part of the inhibitor.
To further quantify the differences in non-covalent
interactions formed between compound 2 and PKA
and PKB, the structures of the protein-ligand
complexes were analysed with the empirical scoring
function Chemscore,32 which is widely used in
docking algorithms. Such an analysis gives an
estimate of the overall binding affinity, but more
importantly for the purposes of this article, a
breakdown of the relative contributions arising
from different intermolecular terms such as hydrogen bonding and lipophilic interactions. The results,
which are presented in Table 1, show that whilst the
hydrogen bonding terms for 2 bound to PKA and
PKB are similar, the lipophilic term is significantly
more favourable for PKB-2 (ΔΔGlipo(PKA→PKB) =
−7.38 kJ mol−1), in agreement with the observations
presented above. As a result of this change in
lipophilic score, compound 2 is predicted to bind
more favourably to PKB than PKA, in agreement
with experiment.
Table 1. Chemscore parameters obtained from scoring of
PKA-2 and PKB-2 structures
Complex
PKA-2
PKB-2
ΔG°total/
kJ mol−1
ΔGhbond/
kJ mol−1
ΔGlipo/
kJ mol−1
−44.31a
−49.56a
−13.53
−11.42
−29.86
−37.24
a
The total predicted free energy of binding, ΔG°total, also
contains a constant term ΔGo (−5.48 kJ mol−1)32 and a term
which accounts for the freezing of ligand rotatable bonds, ΔGrot
(4.56 kJ mol−1 for compound 2).
Structure of compound 2 bound to PKA-PKB
The structure of 2 bound to PKA-PKB was also
determined (Figure 6(a)). In contrast to the binding
mode of compound 2 with wild-type PKA, the
conformation observed with the PKA-PKB chimera
is essentially identical to that with PKB. The
inhibitor adopts the same U-shaped conformation
discussed above, and forms similar non-covalent
interactions in both enzymes (Figure 6(b)). The
indole ring of 2 packs against the side-chain of
Met173 (analogous to Met282 in PKB), and the
phenyl ring of Phe54 at the tip of the glycine-loop is
observed to fold into the ATP-cleft as discussed for
PKB above. This is an important result, as it
provides a level of validation in the ability of a
chimeric system to correctly mimic the binding of a
PKB versus PKA selective compound. In addition,
this observation further supports the hypothesis that
the PKB-bound conformation results from the
specific presence of the Met282 side-chain (as
opposed to being a consequence of a gross difference
in overall PKB properties such as flexibility).
Discussion
Over the last few years, PKB has increasingly
become recognized as a potential target for the
treatment of malignancy due to its central position
in a network of biochemical pathways that regulate
cellular growth and proliferation. The determination
of the structure of activated PKBβ complexed with
AMP-PNP was an important step towards using a
structure-based approach to PKB inhibitor development, but design studies presented in the literature
have until now utilized the experimentally more
amenable kinase, PKA, as a surrogate. This has
proved to be a useful approach, and in particular,
PKA-PKB chimeric structures have provided important insights into molecular recognition with these
kinases. Nevertheless, the use of PKB protein-ligand
data would be highly beneficial in understanding
more subtle structure-activity relationships, as well
as offering the potential to investigate the validity of
PKA-based mimics.
The method we have presented here for obtaining
PKBβ-ligand co-crystals, has allowed the first
comparison of inhibitor binding to both PKBβ, and
to the model systems PKA and PKA-PKB. The
structures of PKA, PKA-PKB and PKB bound to a
non-selective inhibitor 1 revealed similar binding
modes in each case, and the formation of energetically equivalent interactions. It is possible therefore,
that for compounds with a non-selective profile,
both PKA and PKA-PKB are useful surrogates. In
contrast, structures of the selective compound 2
revealed that the inhibitor adopts different conformations in PKB and PKA, but that the PKB-conformation
is also adopted when bound to PKA-PKB. This altered
binding mode appears to arise due to additional
favourable lipophilic interactions, which can occur as
a result of a single amino acid substitution within the
890
Inhibitor Binding to PKB, PKA and PKA-PKB Chimera
Figure 6. Inhibitor 2 binding to PKB and PKA-PKB. (a) PKA-PKB-2 in the region of the ATP-site. Final 2mFo–DFc
electron density for the inhibitor is contoured at 1σ and shown in blue. (b) Superposition of PKB-2 (yellow) and PKAPKB-2 (cyan), with selected residues labelled using PKB numbering. The inhibitor adopts a similar binding mode in
both systems.
891
Inhibitor Binding to PKB, PKA and PKA-PKB Chimera
enzymes' ATP clefts. This difference in binding
between PKA and PKB is useful in rationalizing the
observed selectivity, but extrapolating the binding
mode directly from wild-type PKA to PKB in this
instance could be potentially misleading for further
cycles of structure-based design.
The pragmatic use of more easily produced and/
or crystallized proteins as mimics for less amenable
family homologues has been widely employed both
for kinases 33 and other enzymes (e.g. ACE/
carboxypeptidase,34 trypsin/factor Xa35). This methodology has provided information useful for
inhibitor development, and indeed, the close structural similarity between members of the kinase
superfamily has been the basis of “chemogenomic”
approaches,36 in which information gleaned from
the study of one protein is directly extrapolated to
close homologues by sequence comparisons alone.
Implicit to this, however, is the assumption that
inhibitor binding modes remain constant between
the superfamily members. A recent comparison of
Gleevec (imatinib) binding to the kinases Syk and
c-abl, showed that this is not always the case,37 and
this is also borne out by the differences in binding
of 2 to wild-type PKA and PKB presented here.
The more sophisticated approach involves the use
of chimeric protein systems, which improve further
the similarity between an experimentally recalcitrant target protein and its closest homologue. The
PKA-PKB chimera presented here reproduces the
PKB binding mode accurately for both inhibitors 1
and 2, and thereby provides a level of validation in
these particular cases. Nevertheless, it should be
noted that subtle differences between the PKA-PKB
and PKB crystallization conditions (concentration of
salts and differing buffer pH, for example) might
occasionally have the potential to induce differences
in binding modes for certain inhibitor chemotypes;
and in these cases the definition of what is the
physiologically valid structure may become less
obvious. Additional factors such as the use of
truncated constructs in crystallography, the phosphorylation state of the system, as well as the
method for obtaining the protein-ligand complex
(i.e. soaking versus co-crystallization38–40) may also
have an influence in some cases.
Despite the accurate reproduction of PKB binding
modes by PKA-PKB described here, there also
remain inherent concerns about the general applicability of such a surrogate system for addressing
cases where more subtle differences in ligand
binding between PKB and PKA are responsible for
driving selectivity. A molecular understanding of
PKA/PKB selectivity is desirable as perturbation of
PKA activity has been found to be both oncogenic
and tumour-suppressing, dependent on tissue type,
cAMP levels and duration of PKA activation.41,42
The use of PKA-based structures as PKB surrogates
potentially exacerbates the problem of rationally
addressing PKA/PKB cross-reactivity during inhibitor design, and so in the absence of more extensive
validation, PKB itself should represent the system of
choice for studying selective compounds.
The availability of a methodology for the generation of phosphorylated, PKBβ-inhibitor crystal
structures therefore represents an important step
forward for the development of small molecule
therapeutics in this area, and will hopefully permit a
more rigorous understanding of molecular recognition in this enzyme.
Materials and Methods
PKA crystallography
The alpha catalytic subunit of bovine PKA was expressed, purified and crystallized with reference to
previously described protocols.28,43 Hanging drops containing 17 mg ml−1 of tetra-phospho PKA, 25 mM MesBisTris (pH 6.5), 75 mM LiCl, 0.1 mM EDTA, 1 mM DTT,
1.5 mM octanoyl-N-methylglucamide and 1 mM PKI(5-24)
were equilibrated at 4 °C against 15% (v/v) methanol.
Crystals of apo PKA appeared overnight, and were
soaked for approximately 18 h at 4 °C in a solution
containing 10% (w/v) PEG 400, 25 mM Mes-BisTris (pH
6.5), 0.1 mM EDTA, 1 mM DTT and 10 mM of inhibitor.
The crystals were briefly immersed in a cryoprotectant
containing 22.5% MPD, 25 mM Mes-BisTris (pH 6.5),
0.1 mM EDTA and 1 mM DTT, before plunge-freezing into
liquid nitrogen.
X-ray diffraction data were collected in-house using a
Rigaku-MSC Jupiter CCD mounted on an RU-H3R
rotating anode generator. Data were integrated and scaled
using D*Trek44 before input into an in-house developed
procedure for automated molecular replacement, refinement and ligand fitting.45 The starting model used for
refinement was the previously solved PKA structure
1yds,28 with ligand and water molecules removed. After
ligand fitting, subsequent cycles of model adjustment and
refinement were carried out using AstexViewer46 and
REFMAC5.47
PKA-PKB crystallography
A clone with four mutations (V104T, V123A, L173M,
Q181K) introduced into the cDNA of the alpha catalytic
subunit of bovine PKA wild-type was generated using a
QuikChange Site-Directed Mutagenesis Kit (Stratagene)
following the supplier's protocols. Purification, crystallization and structure solution was carried out as described
for the wild-type PKA above.
PKB crystallography
Active human β-PIFtide PKB was expressed and purified with reference to previously described procedures.26,29 PKB was concentrated to approximately
10 mg ml−1 in a buffer containing 10 mM Tris-HCl (pH 7.5),
300 mM NaCl and 2 mM DTT, and AMP-PNP-MnCl2 and
GSK3β peptide were added to concentrations of 5 mM
and 0.6 mM, respectively. The resulting PKB-AMP-PNPpeptide complex was crystallized by the hanging drop
method from a mother liquor containing 15–20% PEG
10,000 and 1 mM DTT. Crystals of PKB-AMP-PNPpeptide appeared overnight, and were transferred to a
soak solution containing 22% (w/v) PEG 10,000, 100 mM
Hepes (pH 7.5), 0.5 mM EDTA and 1 mM inhibitor.
During a 2 h back-soak, the AMP-PNP was observed to
892
Inhibitor Binding to PKB, PKA and PKA-PKB Chimera
Table 2. X-ray data collection and refinement statistics
Data collection
Space group
Cell dimensions
a, b, c (Å)
α, β, γ (°)
Resolution (Å)
Rmerge
I/σI
Completeness (%)
Redundancy
Refinement
Resolution (Å)
No. reflections
Rwork / Rfree
No. atoms
Protein + peptide
Inhibitor
Water
B-factors (Å2)
Protein + peptide
Ligand
Water
r.m.s deviations
Bond lengths (Å)
Bond angles (°)
PKB-1
PKB-2
PKA-2
PKA-PKB-1
PKA-PKB-2
P212121
P212121
P212121
P212121
P212121
45.26, 60.70, 132.57
90, 90, 90
1.80 (1.90–1.80)
5.5 (17.4)
6.4 (2.4)
95.4 (81.5)
2.7 (2.4)
44.93, 60.99, 124.96
90, 90, 90
2.30 (2.42–2.30)
7.7 (33.7)
6.5 (2.0)
94.8 (93.7)
2.6 (2.6)
72.54, 75.06, 80.13
90, 90, 90
2.00 (2.10–2.00)
6.1 (40.0)
9.2 (1.8)
98.7 (94.6)
3.3 (2.8)
72.86, 75.56, 80.10
90, 90, 90
2.15 (2.23–2.15)
7.5 (36.3)
8.5 (2.6)
99.3 (99.6)
4.0 (4.0)
72.81, 74.79, 80.36
90, 90, 90
2.08 (2.15–2.08)
5.1 (23.2)
10.8 (3.1)
93.6 (87.1)
2.3 (2.2)
55–1.80
31,145
17.7/21.3
55–2.30
14,109
19.3/25.2
36–2.00
28,286
20.8/27.2
43.9–2.15
23,262
20.0/25.9
35–2.08
24,115
20.6/27.6
2666
30
328
2684
30
105
2947
30
326
2935
30
296
2935
30
308
24
27
38
48
36
38
36
32
42
36
40
41
27
19
32
0.007
1.1
0.013
1.4
0.013
1.3
0.013
1.3
0.016
1.5
be fully displaced by the new inhibitor of interest. The
crystals were subsequently briefly immersed in a
cryoprotectant containing 22% PEG 10,000, 100 mM
Hepes (pH 7.5) and 22% ethylene glycol, before plungefreezing into liquid nitrogen.
X-ray diffraction data were collected on beamline ID29 at the ESRF using an ADSC Q210 detector. Data were
integrated using MOSFLM,48 before input into an inhouse developed procedure for automatic scaling,
molecular replacement, refinement and ligand fitting.45
The starting model used for the refinements was the
previous solved PKB structure, 1okl26 with ligand and
water removed. After ligand fitting, subsequent cycles of
model adjustment and refinement were carried out using
AstexViewer46 and REFMAC5.47 As observed previously,26 residues 450–466 in the PKB N-terminal
domain are disordered, and have not been built in the
structures described here. In addition, there appears to
be additional flexibility in the N-terminal domain of
PKB-2, compared to PKB-1, as judged by weak electron
density and high B-factors for some solvent-exposed
side-chains, and the protein backbone in the region
residues 445–449. Data collection and refinement statistics for all structures presented in this paper are given in
Table 2.
the ligand position and torsional degrees of freedom to
relax during the SIMPLEX optimisation.
Bioassay of compound 2
Details of the β-PIFtide PKB assay have been described.25 The PKA assay follows a similar protocol, but
uses a nine residue PKA-specific peptide substrate
(GRTGRRNSI) and 40 μM ATP.
Chemistry
The syntheses of compounds 1 and 2 have been
described.25,52
Protein Data bank accession codes
Coordinates and structure factors for the complexes
described here have been deposited with the RCSB Protein
Data Bank49 with the following accession codes: 2jds
(PKA-2), 2jdo (PKB-1), 2jdr (PKB-2), 2jdt (PKA-PKB-1) and
2jdv (PKA-PKB-2).
Analysis of protein-ligand complexes with Chemscore
Protein and ligand structures were prepared for the
PKA-2 and PKB-2 complexes described here, ensuring that
all bond types and protonation states are correct. The
X-ray binding mode of each ligand was then scored
against its native protein structure, using the Chemscore
scoring function32 in the protein-ligand docking program
GOLD.50 This was done using the “Local scoring”
protocol described previously,51 which only optimises
terminal groups on protein and ligand during the
searching part of the docking algorithm, and then allows
Acknowledgements
The authors acknowledge Andrew Sharff for
assistance with PKB protein purification, Wendy
Blakemore for assistance with the PKA-PKB chimera
crystallography and Lisa Seavers for the bioassay
results. This work was funded in part by Cancer
Research UK [CUK] Programme Grant C309/
A2187.
Inhibitor Binding to PKB, PKA and PKA-PKB Chimera
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Edited by R. Huber
(Received 7 November 2006; received in revised form 20 December 2006; accepted 3 January 2007)
Available online 9 January 2007