From PIM1 to PI3Kδ via GSK3β: Target Hopping through the Kinome

Article abstract of DOI:10.1021/acsmedchemlett.7b00296

Selective inhibitors of phosphoinositide 3-kinase delta are of interest for the treatment of inflammatory diseases. Initial optimization of a 3-substituted indazole hit compound targeting the kinase PIM1 focused on improving selectivity over GSK3β through consideration of differences in the ATP binding pockets. Continued kinase cross-screening showed PI3Kδ activity in a series of 4,6-disubstituted indazole compounds, and subsequent structure-activity relationship exploration led to the discovery of an indole-containing lead compound as a potent PI3Kδ inhibitor with selectivity over the other PI3K isoforms.

Full text of DOI:10.1021/acsmedchemlett.7b00296

Letter
pubs.acs.org/acsmedchemlett
From PIM1 to PI3Kδ via GSK3β: Target Hopping through the Kinome
Zoe A. Henley,*,† Benjamin D. Bax,§,Δ Laura M. Inglesby,‡ Aurelie Champigny,‡ Simon Gaines,‡,○
́
̈
Paul Faulder,‡,◊ Joelle Le,§ Daniel A. Thomas,# Yoshiaki Washio,‡ and Ian R. Baldwin*,‡
†Refractory Respiratory Inflammation DPU, GlaxoSmithKline R&D, Gunnels Wood Road, Stevenage, SG1 2NY, U.K.
#
§
‡Molecular Discovery Research, Biological Sciences, and Computational Chemistry, Platform Technology & Science,
GlaxoSmithKline R&D, Gunnels Wood Road, Stevenage, SG1 2NY, U.K.
S
* Supporting Information
ABSTRACT: Selective inhibitors of phosphoinositide 3-kinase
delta are of interest for the treatment of inflammatory diseases.
Initial optimization of a 3-substituted indazole hit compound
targeting the kinase PIM1 focused on improving selectivity over
GSK3β through consideration of differences in the ATP binding
pockets. Continued kinase cross-screening showed PI3Kδ activity in
a series of 4,6-disubstituted indazole compounds, and subsequent
structure−activity relationship exploration led to the discovery of an
indole-containing lead compound as a potent PI3Kδ inhibitor with
selectivity over the other PI3K isoforms.
KEYWORDS: Phosphoinositide-3-kinase delta inhibitor, PI3Kδ inhibitor, kinase cross-screening
inases play a pivotal role in nearly every aspect of cellular
function and represent an important class of biological
The PIM (provirus insertion site of Moloney murine
leukemia virus) family of serine/threonine protein kinases is
represented by PIM1, PIM2, and PIM3, which are all highly
conserved in vertebrates.6 PIM proteins have major functions in
cell survival, proliferation, and differentiation in response to
growth factors and cytokines,7 and PIM1 was the first PIM
kinase discovered to have a key role as a proto-oncogene.8 The
overexpression of PIM1 has been found in a wide range of
human tumors, including B-cell lymphomas and various types
of human leukemia as well as solid tumor cancers.9 Therefore,
the development of PIM1 inhibitors as new anticancer drugs is
of considerable interest.10
K
targets in the development of novel drugs.1 Kinases modify
substrates by chemically adding a phosphate group from
adenosine triphosphate (ATP), and this phosphorylation event
controls many cellular processes, making them attractive
therapeutic targets.2
Identification of starting hits for biological targets can be
based on approaches such as high throughput screening (HTS)
for diversity or structure-based drug design (SBDD) as a
focused design approach,3 but in a target class such as kinases
an efficient way of finding hits is from focused or knowledge-
based screening. Screening a focused set containing compounds
that have previously been shown to have activity against kinases
has proven to be a highly successful strategy in discovering hits
against new kinases.4 This approach predominantly targets
small molecules that bind to the ATP-binding pocket, which is
well-conserved across the whole kinome.5 Therefore, a resultant
issue in translating hits into candidates and medicines has been
selectivity over other undesired kinase targets, thus avoiding
unwanted side effects.
Extensive in-house kinase cross-screening is routine within
GSK and ensures any unexpected compound activities against
off-targets are identified. This extensive cross-profiling has the
benefit of providing unexpected opportunities for other targets.
This letter discusses how, by considering a hit compound for
PIM1 and issues of selectivity over GSK3β, kinase cross-
screening and subsequent structure−activity relationship (SAR)
exploration led to the discovery of a selective lead series for
PI3Kδ.
Initial screening of a focused kinase compound set against
PIM1 identified indazole compound 1 as a moderately potent
inhibitor with a pIC50 of 6.3 (Figure 1).11
Compound 1 was as an attractive hit with a potential to
rapidly expand SAR. However, selectivity data from cross-
screening against a panel of in-house kinases revealed a
moderate selectivity profile, with inhibition of GSK3β being
one of the most significant off-target activities (Figure 2).
GSK3β is a multifunctional serine/threonine protein kinase,
which regulates a wide range of cellular processes, involving
various undesirable signaling pathways.12,13 ALK5 and AurA are
serine/threonine kinases that were also inhibited by compound
1, and this would also need to be addressed; however, with X-
Received: July 20, 2017
Accepted: September 5, 2017
© XXXX American Chemical Society
A
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Figure 1. Initial indazole hit compound 1.
Figure 2. Selectivity profile of compound 1. *Asterisks designate a less
than significant value was observed on at least one occasion.
Figure 4. X-ray cocrystal structure of compound 2 with GSK3β.
Images generated using PyMol.
Scheme 1. Synthetic Route for Compounds 8a, 8b, 8d, 8e,
a
and 8f
Figure 3. X-ray cocrystal structure of compound 1 in PIM1 with
PIM1. Images generated using PyMol.
ray crystallography available in-house for GSK3β, our initial
SAR exploration sought to improve selectivity for PIM1 over
GSK3β.
Cocrystallization of compound 1 with PIM1 demonstrated
that the proton-donor center N1−H of the indazole forms a
hydrogen bond with Glu121 (Figure 3).
a
Reagents and conditions: (a) 3,4-dihydropyran, TFA; (b) boronic
acid/ester, Pd(dppf)Cl2, aq. NaHCO3, IPA, 150 °C, microwave; (c)
H2, Pd/C, EtOAc; (d) acid chloride, DIPEA, DCM or acid, HATU,
DIPEA, DMF; (e) 4 M HCl in dioxane, MeOH. See Table 1 for range
of substituents.
The greater affinity of compound 1 for GSK3β over PIM1
can be rationalized through consideration of the hinge
interactions that are made upon binding to the different
kinases. A commonly conserved H-bonding interaction to
inhibitors in the hinge region of the ATP site of kinases is that
of an NH on the protein backbone interacting with an acceptor
motif on the inhibitor. PIM1 is unusual because the presence of
a proline residue (Pro123, Figure 3) in the hinge region
prevents the formation of this hydrogen bond.
A cocrystal structure of a close analogue of compound 1 with
GSK3β confirmed that the two commonly conserved hydrogen
bonding interactions are made with Asp133 and Val135 (Figure
4).14 The accessible carbonyl group of Val135 in fact enables a
B
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Table 1. SAR of 4,6-Disubstituted Indazoles
Figure 5. Close-up of the hinge and back pocket region of compound
8f docked in PI3Kδ (magenta) and overlay with compound 2
cocrystallized in GSK3β (green).
We looked to alter the PIM1/GSK3β selectivity profile in
favor of PIM1 by disrupting the additional H-bonding
interaction between Val135 and the amido-NH observed in
GSK3β (Figure 4) by moving the amido-substituent to the
indazole 4-position.
To access the desired 4,6-disubstituted indazoles, a routine
synthesis was employed that began with conversion of the
commercially available 4-nitro-6-bromoindazole (3) to the
corresponding THP-protected indazole intermediate 4 (general
synthesis shown in Scheme 1). Installation of the 6-aryl groups
under standard palladium-catalyzed cross-coupling conditions
with the required boronic acid or boronate ester yielded
compounds of structure 5 (Scheme 1). The THP-protected
phenolic boronic acids were employed when introducing
phenol-substituted aromatic groups (see Supporting Informa-
tion). Reduction of the nitro group afforded aromatic amine
intermediates of structure 6, which allowed the introduction of
different amide substituents by coupling with the corresponding
acid chlorides. Subsequent THP-deprotection gave the target
compounds 8a, 8b, 8d, 8e, and 8f (see Table 1 for the range of
substituents). Compound 8c was synthesized using an
analogous route with SEM-protection of the indazole (see
Supporting Information).
*
Data is mean of at least three separate test occasions unless stated.
a
b
Data from in-house PIM1 assay, n = 1. Tested <4.6 on one occasion.
c
d
Data from in-house PIM1 assay. Tested <4.6 on two occasions.
Tested <4.6 on three occasions. GSK3β data was generated at
e
f
Reaction Biology Corporation.23 Tested <4.6 on four occasions. n =
g
h
i
1. n = 2.
a
Scheme 2. Synthesis of Compounds 8g and 8h
A shortened synthesis from commercially available 4-amino-
6-bromoindazole (9), where amide formation was carried out
as the first step before introduction of the required aryl groups
via palladium-catalyzed cross-coupling, was followed for the
synthesis of compounds 8g and 8h (Scheme 2, see Table 1 for
range of substituents).
Consideration of the residue differences in the hinge region
of PIM1 and GSK3β quickly led to the design of compound 8a.
The initial data for compound 8a was encouraging, with GSK3β
potency reduced but PIM1 activity maintained (Table 1). The
introduction of a picolinamide substituent at the indazole 4-
position in compound 8b showed a slight increase in PIM1
activity, while a corresponding increase in potency for GSK3β
was not observed (Table 1).
a
Reagents and conditions: (a) 2-pyridinecarbonyl chloride hydro-
chloride, DIPEA, DCM; (b) boronic acid, Pd(dppf)Cl2, Na2CO3, 1,4-
dioxane, H2O. See Table 1 for the range of substituents.
donor−acceptor−donor interaction between the amido-inda-
zole ring and the protein (Figure 4), which is not possible with
PIM1.
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target for respiratory diseases such as asthma18 and chronic
obstructive pulmonary disease (COPD).19 PI3Kγ is also
enriched in leukocytes and has been targeted for the treatment
of inflammatory disease,20 whereas the α and β isoforms are
ubiquitously expressed and the α and β murine knockout
phenotypes are embryonically lethal.21 This demonstrates the
potential requirement for isoform selectivity when trying to
treat chronic disease.
Compound 8a was the first in the series to show significant
PI3Kδ activity (pIC50 = 5.2) compared with the original hit
compound 1, which was inactive. The increased potency for
PI3Kδ over the other class 1 PI3K isoforms was a key
observation, as this was the first compound to show this
selectivity profile from many compound series screened in-
house against the PI3Ks. These initial results from kinase cross-
screening and recognition of the therapeutic potential of
selective PI3Kδ inhibitors led to a shift in target emphasis from
PIM1 to PI3Kδ for this series of compounds.
A significant increase in PI3Kδ potency was observed for the
picolinamide compound 8b, and subsequent SAR exploration
has shown that the internal hydrogen bond was key to
maintaining the planarity of the amide leading to stabilization of
the preferred binding conformation to PI3Kδ, which was
confirmed through crystallography as described by Down et
al.22
Removal of the phenolic hydroxyl group in compound 8c
caused a reduction in potency against PIM1, GSK3β, and
PI3Kδ, but moving the phenol from the para- to the meta-
position in compounds 8d and 8e was more detrimental to
PIM1 activity than to that of GSK3β and PI3Kδ.
Replacement of the phenol with a bioisoteric indole moeity
in compound 8f resulted in reduced activities for PIM1 and
GSK3β but increased activity for PI3Kδ. Combining the
previously recognized 4-position picolinamide with 6-position
indole moieties led to compounds 8g and 8h, where the 4-
substituted indole displayed much more potent PI3Kδ
inhibition than the 5-substituted analogue. Significantly, there
was a continued lack of activity of the 4,6-disubstituted indazole
compounds against the other PI3K isoforms.
Due to the lack of a suitable crystal structure of PI3Kδ at this
time, a homology model was built to aid the rationalization of
the SAR.24 The model suggested that the indazole nitrogen
engaged in the hinge-binding interaction but that the core was
shifted significantly when compared with the indazole binding
in GSK3β and PIM1 (overlay of PI3Kδ homology model with
GSK3β cocrystal structure shown in Figure 5). This movement
is hypothesized to be due to changes in the gatekeeper residue
and nearby back pocket residues between the three kinases.
The gatekeeper residue is a single residue in the ATP pocket
that has been shown to control sensitivity to small molecule
kinase inhibitors since it influences the size and shape of the
back cavity that is not occupied by ATP.5 The PI3Kδ Ile825
gatekeeper residue (PIM1 = Leu120, GSK3β = Leu132) and
Tyr813 residue (PIM1 = Ile104, GSK3β = Val110) extend
further into the back pocket than the corresponding residues of
PIM1 and GSK3β. The movement of the indazole core, to
avoid clashing with the aryl substituent of Tyr813, orients the
back pocket substituent such that a key H-bond interaction
with Asp787 can be made, and this is achieved when the indole
substituent is substituted at the 4-position (Figure 5). This
differs from the para-phenol substituents that are preferred for
PIM1 and GSK3β, which give the optimal orientation for an H-
Figure 6. Kinase selectivity profiles of compounds 8a, 8b, and 8g.
*Asterisks designate a less than significant value was observed on at
least one occasion.
Continued kinase profiling also highlighted some interesting
SARs against the PI3Ks. The phosphoinositide 3-kinases
(PI3Ks) are a family of enzymes that are central to signaling
involving intracellular lipids.15 The Class 1 PI3K family includes
phosphoinositide 3-kinase delta (PI3Kδ) and the closely related
isoforms α, β, and γ, which convert phosphatidylinositol-4,5-
bisphosphate to phosphatidylinositol-3,4,5-trisphosphate in
vivo.16 This leads to downstream signaling cascades that
control cell growth, cell cycle entry, and cell survival. PI3Kδ
inhibition is of interest for oncology indications, with the
approval of GS-1101 (idelalisib) recently reported for the
treatment of patients with relapsed chronic lymphoid leukemia
and non-Hodgkin lymphoma.17 Since PI3Kδ is primarily
expressed in leukocytes and plays a key role in immune
signaling and inflammatory processes, it is also an attractive
D
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bond interaction with a glutamic acid residue in both kinases
(Glu97 of GSK3β shown in Figure 5).
manuscript. All authors have given approval to the final version
of the manuscript.
Since the compounds were not expected to extend into areas
of the protein where there are significant residue differences
between the PI3K isoforms, the PI3K selectivity profile was
unexpected. Subsequent SAR investigation showed that the
PI3K activities were greatly affected by the back pocket
substituent.21 Since the residues are conserved in this region, it
suggested a more complex mechanism of achieving selectivity
than could be explained by modeling. Potentially, a subtle
change in the extended H-bonding network could be
responsible for the isoform selectivity observed.25
In addition to the highly encouraging selective PI3Kδ
inhibitory characteristics of compound 8g, in-house kinase
cross-screening indicated a good overall kinase selectivity
profile compared with the initial 4-substituted indazole
compounds 8a and 8b (Figure 6). This identified the indole-
substituted indazole as a potentially novel PI3Kδ selective
template. ALK5 remained the only kinase where moderate
selectivity (<10-fold) was observed and would need to be
addressed by further optimization.22
In summary, continued kinase cross-screening of indazole
compounds focused on PIM1 antagonism identified the 4,6-
disubstituted indazole series to be a novel, potent, and selective
class of PI3Kδ inhibitors. The promising selectivity profile of
compound 8g for PI3Kδ, specifically against the other PI3K
isoforms, led to its further development and enabled the
discovery of potent PI3Kδ inhibitors with therapeutic
potential.22
Notes
The authors declare the following competing financial
interest(s): All authors were employees of GlaxoSmithKline
at the time the work was carried out.
ACKNOWLEDGMENTS
■
The authors would like to thank Luke Carter, Maire Convery,
and Lisa Shewchuk-Chapman for protein production and
crystallography assistance; Geoffrey Cutler, Yvonne Joseph, and
Sarah Smith for assay support; and Andrew Perrett for synthetic
contributions.
ABBREVIATIONS
■
AKT1, v-Akt murine thymoma viral oncogene homologue 1;
ALK5, activin receptor-like kinase 5; AurA, Aurora kinase A; B-
Raf, v-Raf murine sarcoma viral oncogene homologue B;
COPD, chronic obstructive pulmonary disease; dppf, 1,1′-
bis(diphenylphosphino)ferrocene; EGFR, epidermal growth
factor receptor; GSK3β, glycogen synthase kinase 3 beta;
HATU, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo-
[4,5-b]pyridinium 3-oxid hexafluorophosphate; IKK2, inhibitor
of nuclear factor kappa-B kinase subunit beta; ITK, interleukin-
2-inducible T-cell kinase; IPA, isopropyl alcohol; JNK1, c-Jun
N-terminal kinase 1; MK2, mitogen-activated protein kinase-
activated protein kinase-2; p38α, p38-mitogen-activated protein
kinase alpha; PAK1, p21-activated kinase 1; PI3K, phosphoi-
nositide 3-kinase; PIM, provirus insertion site of Moloney
murine leukemia virus; PLK1, polo-like kinase 1; ROCK1, Rho-
associated protein kinase 1; SEM, 2-(trimethylsilyl)-
ethoxymethyl; SYK, spleen tyrosine kinase
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.7b00296.
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ORCID
Zoe A. Henley: 0000-0001-6686-3210
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carried out computational modeling and molecular design. D.T.
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