Optimization of a Dibenzodiazepine Hit to a Potent and Selective Allosteric PAK1 Inhibitor

Article abstract of DOI:10.1021/acsmedchemlett.5b00102

The discovery of inhibitors targeting novel allosteric kinase sites is very challenging. Such compounds, however, once identified could offer exquisite levels of selectivity across the kinome. Herein we report our structure-based optimization strategy of

Full text of DOI:10.1021/acsmedchemlett.5b00102

Letter
pubs.acs.org/acsmedchemlett
Optimization of a Dibenzodiazepine Hit to a Potent and Selective
Allosteric PAK1 Inhibitor
Alexei S. Karpov,*,† Payman Amiri,‡,§ Cornelia Bellamacina,‡ Marie-Helene Bellance,†
Werner Breitenstein,† Dylan Daniel,‡,§ Regis Denay,† Doriano Fabbro,†,∥ Cesar Fernandez,† Inga Galuba,†
Stephanie Guerro-Lagasse,† Sascha Gutmann,† Linda Hinh,‡ Wolfgang Jahnke,† Julia Klopp,† Albert Lai,‡
Mika K. Lindvall,‡ Sylvia Ma,‡ Henrik Mobitz,† Sabina Pecchi,‡ Gabriele Rummel,† Kevin Shoemaker,‡
̈
Joerg Trappe,† Charles Voliva,‡ Sandra W. Cowan-Jacob,† and Andreas L. Marzinzik†
†Novartis Institutes for BioMedical Research, Novartis Campus, CH-4056 Basel, Switzerland
‡Novartis Institutes for BioMedical Research, 5300 Chiron Way, Emeryville, California 94608, United States
S
* Supporting Information
ABSTRACT: The discovery of inhibitors targeting novel allosteric kinase sites is very challenging. Such compounds, however,
once identified could offer exquisite levels of selectivity across the kinome. Herein we report our structure-based optimization
strategy of a dibenzodiazepine hit 1, discovered in a fragment-based screen, yielding highly potent and selective inhibitors of
PAK1 such as 2 and 3. Compound 2 was cocrystallized with PAK1 to confirm binding to an allosteric site and to reveal novel key
interactions. Compound 3 modulated PAK1 at the cellular level and due to its selectivity enabled valuable research to interrogate
biological functions of the PAK1 kinase.
KEYWORDS: PAK1, p21 activated kinase, allosteric inhibitor, dibenzodiazepine, tool compound
affects chromatin and nuclear signaling, and promotes hormone
AKs or p21-activated kinases are a family of serine/
Pthreonine protein kinases that can be divided into two independence.10 Unfortunately, despite enormous interest in
further elucidating PAK1 biology there is still no potent and
selective inhibitor targeting exclusively the PAK1 isoform that
would be suitable as a tool compound. There have been several
pan-PAK inhibitors reported to date.11 Most of them such as
PF-3758309,12 FRAX486, FRAX597,13,14 and compound 415
bind in the active site (Figure 1). Despite targeting the same
site, they exhibit different levels of selectivity across PAK
isoforms and across the kinome in general. For example, PF-
3758309 is a pan-PAK inhibitor of both group I and group II
isoforms, while FRAXs are highly selective for group I, and
compound 4 displays group II selectivity (Table 1).
subgroups: group I consisting of PAK1−3 and group II
consisting of PAK4−6.1 They are effectors of Rac/Cdc42
GTPases and play an important role in cell proliferation,
survival, motility, and angiogenesis.2 Group I PAKs are directly
activated upon binding of GTPases, while the activity of group
II PAKs is independent of GTPase binding.3 Most PAK
isoforms (in particular PAK1 and PAK4) have attracted a lot of
interest and are considered as promising oncology targets
because of their overexpression, amplification, and/or activation
in many cancers.4,5 In addition, there are reports suggesting that
group I PAK inhibitors have potential in treating Alzheimer and
Huntington diseases6 and FXS (fragile X syndrome).7 PAK1 is
one of the most studied family members, and there are reports
implicating dysregulation of PAK1 in breast and squamous
NSCLC cancers8 and upregulation of PAK1 in pancreatic
cancers.9 It is also described that PAK1 regulates cytoskeletal
signaling, promotes oncogenic transformation and survival,
However, due to the fact that they target the highly
conserved ATP site, selectivity over other kinases remained
Received: March 10, 2015
Accepted: May 22, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.5b00102
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Letter
indirect competition due to incompatibility of ATP binding
with the DFG-out binding conformation of our allosteric hit.
Despite being a diverse and privileged scaffold among many
receptors and targets, dibenzodiazepines are not particularly
well-known as kinase inhibitors. There are only a few reports
describing specific structural analogues such as dibenzodiaze-
pine-ones as Chk1 inhibitors24 and amino-pyrimido-diazepines
as BMK1 inhibitors.25 In these examples the compounds bind
to the kinases in the active site and interact with the hinge
either through the amide function present on the tricyclic core
or via a fused amino-pyrimidine ring.
We reasoned that compounds 1 and 5 could serve as highly
attractive starting points with the goal of improving biochemical
potency while retaining exquisite kinase selectivity.
Figure 1. Known PAK inhibitors.
The synthesis of optimized PAK1 inhibitors is described in
the Supporting Information. All compounds were first tested
for potency in the primary biochemical assay using a more
sensitive dephosphorylated form of the PAK1 protein. Their
selectivity against other PAK isoforms was assessed at
DiscoveRx Corporation (San Diego, CA, United States).
Both compounds 1 and 5, despite being very selective across
a panel of kinases, turned out to modulate several GPCRs in
the nanomolar range. For example compound 1 exhibited
inhibition of the histamine receptor H1 (IC50 = 1 nM) and the
muscarinic receptor M1 (IC50 = 6 nM). Therefore, mitigation
of GPCR liability was one of the key aspects of the optimization
process. Our optimization began with probing the hydrophobic
region in the northern part of the molecule. We introduced a
bulkier and more hydrophobic ethyl substituent on the aniline
nitrogen to result in inhibitor 11 (Table 2). This modification
led to a 3-fold improvement in potency. Compound 11 was
cocrystallized with PAK1 (Figure 3). The X-ray crystal
structure of PAK1 and 11 confirmed an allosteric binding
mode in the DFG-backpocket adjacent to the ATP binding site
beside the gatekeeper (Met344) and beneath helix αC. The
DFG-motif and helix αC are displaced from the active
conformation (PDB code 1YHV) and resemble the auto-
inhibited inactive state of PAK1 (PDB code 1F3M). The
binding is characterized by shape complementarity, a halogen
carbonyl interaction (Thr406), and a salt bridge of the
piperazine moiety with the catalytic amino acid Glu315 from
helix αC. The additional ethyl group of 11 causes a marked
shift of the N-terminal lobe of the kinase compared to the
structure of the original hit 1. Space is created between the two
methionine side chains and Val328 by a further shift of the C-
helix outward and a rotation of the beta-sheet domain down
over the ATP site, along with a reordering of the DFG motif
and activation loop, burying the ligand.
hard to achieve. Only the recently published compound 4,
which extends into the back pocket from the ATP site, has been
shown to be specific and can be considered as a good tool to
study PAK group II biology. In general, allosteric kinase
inhibitors are expected and have been shown to possess
excellent selectivity across the kinome.16 The field is rapidly
growing and recent examples of allosteric inhibitors of Akt1,17
LIMK2,18 and RIP119 have been reported. Not surprisingly,
there have also been efforts directed toward finding allosteric
PAK1 inhibitors. For example, screening of a small library of
compounds against full-length PAK1 resulted in the discovery
of IPA-3, which binds to PAK1 in the regulatory domain and
prevents its activation by Cdc42.20,21
It inhibits PAK1 with moderate potency (IC50 2.5 μM), is
noncompetitive with ATP, and was found to be selective over
group II PAKs and other kinases (Figure 1, Table 1). However,
it lacks sufficient potency, and further optimization is required
to develop it into a compound to explore PAK1 biology on a
cellular level.
Dibenzodiazepines, such as 1 and 5, discovered in a
fragment-based screen, were found to bind to PAK1 kinase in
a novel allosteric site adjacent to the ATP binding site with the
DFG loop adopting the “out” conformation (Table 2, Figure
2).
Typical for the DFG-out binders targeting the inactive
conformation of the enzyme, such as type II kinase inhibitors,
compounds 1 and 5 were found to be more active in the PAK1
dephosphorylated assay (compound 1, PAK1 phos/dephos
IC50 = 54/12 μM; compound 5, PAK1 phos/dephos IC50 = 9/
0.9 μM). Compounds 1 and 5 are usually referred to as type III
allosteric inhibitors and are distinguished from type II inhibitors
in that the ATP site is occluded (Figure 2) and canonical
interactions with the hinge region are not formed. Several
allosteric ligands binding to a similar site have been previously
reported for other kinases (e.g., IGF1R,22 FAK,23 LIMK2,18
etc.). Interestingly, we observed a shift in IC50 upon increase of
the ATP concentration for compound 5 (IC50 1, 6, and >25 μM
at 1.5, 15, and 150 μM ATP concentration, respectively),
suggesting that it may be ATP-competitive. This is likely an
To relieve the conformational strain evident in the X-ray
structure of PAK1 with compound 11, we designed a series of
amine derivatives with a preorganized bent conformation. To
our surprise, the Boc-intermediate 13 maintained activity
despite lacking the charged amine (and the associated salt
Table 1. PAK IC50 for Known Inhibitors
Compound
PAK1 IC50 (nM)
PAK2 IC50 (nM)
PAK3 IC50 (nM)
PAK4 IC50 (nM)
PAK5 IC50 (nM)
18.1 5.1
PAK6 IC50 (nM)
17.1 5.3
PF-3758309
FRAX486
FRAX597
4
13.7 1.8
8.3
190
39.5
12.8
970
99
18.7 6.6
779
55.3
19.3
>10000
7.7
>10000
7.5
5420
2500
126
36
IPA-3
>10000
>10000
>10000
B
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a
Table 2. SAR of PAK1 Inhibitors; Selectivity vs. Other PAK Isoforms
a
IC50 values represent means of 2−4 measurements. Individual data points in each experiment were within 2-fold range with each other.
Figure 3. Overlay of the X-ray structures of PAK1 (cyan and green,
respectively) with 1 (cyan) and 11 (yellow) binding in the allosteric
DFG-out pocket between the gatekeeper Met344, helix αC (right),
and the DFG motif (Asp407 and Phe408 are shown). Key residues are
shown in stick form. Hydrogen bonds between PAK1, water molecules
and 11 are shown as dashed red lines.
Figure 2. X-ray structure of 1 (green) binding in the novel allosteric
site of PAK1.
carbonyl is engaged in a water-mediated hydrogen bond
network with Lys299. Finally, the optimization focused on
improving the solubility of compound 2 (<0.004 mM at pH
6.8). This was achieved by replacing the tert-Bu group by the
less hydrophobic i-Pr substituent. In addition, incorporation of
two fluorine atoms to the ethyl group resulted in a slight
improvement of the biochemical potency. Our optimization
bridge interaction with Glu315). Introduction of the hydrogen
bond donor in the urea analogue 2 (Table 2) led to a further
10-fold improvement in potency. The X-ray cocrystal structure
of PAK1 and ligand 2 (Figure 4) showed that the urea NH
indeed interacts with Glu315, positioning the tertiary butyl
group in a hydrophobic groove above the tricycle. The urea
C
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3 had a biochemical PAK1 Kd of 7 nM and a PAK2 Kd of 400
nM. Selectivity over PAK2 was not anticipated at the beginning
of the project since it is known that PAK1 and PAK2 share 93%
of homology in the kinase domain.1 Such excellent selectivity
over PAK2 is hard to rationalize (our hypothesis is explained in
the Supporting Information). It was pleasing to see that
omitting the basic nitrogen resulted in a very clean profile in
the off-target panel (22 targets with all IC50s > 10 μM, for a list
of off-targets see Supporting Information). Compound 3
possessed good physicochemical properties (solubility 0.055
mM at pH 6.8, HT logP 5.5, high PAMPA permeability cFA,
calculated fraction absorbed = 99%, high Caco2 permeability
Papp A-B 42.8 × 10−8 cm/s, and efflux ratio 0.55). In addition
compound 3 did not display significant inhibition of CYP450s
(13.2 μM for 3A4 the midazolam site, >20 μM for 2D6 and
16.7 μM for 2C9). Only relatively poor stability of compound 3
in rat liver microsomes (RLM) would limit its application for in
vivo studies (t1/2 in RLM 3.5 min); however, our primary goal
was to study PAK1 biology on the cellular level.
Having identified 3 as a potent and selective PAK1 inhibitor,
we decided to validate the cellular potency in cell lines where
both PAK1 and PAK2 are activated as measured by the
autophosphorylation of Ser144 on PAK1 and Ser141 on PAK2.
These phosphorylation sites prevent the autoinhibition by the
p21-binding domain (PBD) of PAK1 and PAK2.26,27 Given
that there is no known downstream substrate of PAK1 that
differs from PAK2, we believe that measuring PAK1 and PAK2
autophosphorylation is the best estimation of cellular potency
of PAK1 and PAK2.
Figure 4. Overlay of the X-ray structures of PAK1 (magenta and
green, respectively) with the allosteric ligands 2 (yellow) and 11
(green) binding in the DFG-out pocket between the gatekeeper
(Met344), helix αC (right), and the DFG motif (Asp407 and Phe408
are shown). Key residues are shown in stick form. Hydrogen bonds
between PAK1, water molecules, and 2 are shown as dashed red lines.
Several water molecules were omitted for clarity as were the hydrogen
bonds between water molecules and the protein.
efforts resulted in compound 3, which exhibited significantly
improved aqueous solubility (0.055 mM at pH 6.8; >1 mM at
pH 4.0). Compound 3 showed excellent activity in biochemical
assays and an exceptional selectivity profile against other known
kinases (Figure 5).
Several pancreatic cancer cell lines with mutated KRAS were
shown by us to express high levels of activated PAK1 and PAK2
as measured by PAK1/2 phosphorylation on Ser144/141. One
of them, Su86.86, was particularly sensitive to PAK1/2 shRNA
mediated growth inhibition and was used as our cell line of
choice for any downstream signaling analysis. As shown in
Figure 6, incubation of 3 at increasing concentrations inhibited
both PAK1 and PAK2 autophosphorylation.
Figure 6. Target modulation of PAK1/2 by 3 in the Su86.86 cell line.
Consistent with the observation that 3 is a more potent
inhibitor of PAK1 than PAK2 measured biochemically using in
vitro kinase assays and reflected in the biophysical binding Kd
measurement (Table 2), the compound demonstrates higher
inhibitory activity on PAK1 autophosphorylation than on
PAK2.
Using compound 3, we attempted to demonstrate inhibition
of phosphorylation of the downstream substrate MEK1 Ser289.
However, since both PAK1 and PAK2 mediate MEK1
phosphorylation, compound 3 is not sufficiently potent to
inhibit this phosphorylation event at concentrations up to 2−6
Figure 5. Selectivity profile of compound 3 assessed at DiscoveRx.
Image generated using TREEspot Software Tool and reprinted with
permission from KINOMEscan, a division of DiscoveRx Corporation.
It demonstrated a selectivity score (S10-score) of 0.003 when
tested at 10 μM against 442 kinases in the KinomeScan
competition binding assay (DiscoveRx Corporation, San Diego,
United States). In contrast, the reference inhibitor PF-3758309
showed a selectivity score of 0.111 (hit 10 out of 96 kinases,
screened at 1 μM, data not shown). The optimized compound
D
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μM because PAK2 is not completely inhibited. We do observe
significant inhibition of pMEK Ser289 with compound 3 at 6−
20 μM, the lowest tested concentrations that show complete
and unambiguous inhibition of both pPAK1 and pPAK2.
Consistent with this observation, compound 3 inhibited
proliferation of Su86.86 cell line only above a concentration
of 2 μM. In contrast, by applying a mixture of compound 3 and
PAK2 shRNA, we achieved inhibition of downstream signaling
and cell proliferation at a significantly lower 0.21 μM
concentration (Supporting Information). Thus, for cell lines
that are dependent upon and express both isoforms, we
hypothesize that a dual PAK1/2 inhibitor would be necessary
for efficient inhibition of cell proliferation. Attempts to identify
a cancer cell line (and cancer indications) that are dependent
upon and express only PAK1 were not successful.
In summary, we have developed a potent and selective PAK1
inhibitor. Due to its allosteric binding mode, compound 3
demonstrates high selectivity for inhibition of PAK1 over other
PAK isoforms and the kinome in general. In addition, the lack
of activity on GPCRs combined with favorable physicochemical
properties make it an excellent research compound to study
biological functions of PAK1 on the cellular level. Furthermore,
we have demonstrated that this compound shows target
modulation of PAK1 in cells but does not result in inhibition
of cell proliferation. Further studies concerning biological
consequences of PAK1 inhibition will be reported later.
guanosine 5′-triphosphatase; H1, the histamine H1 receptor;
M1, the muscarinic M1 receptor; PAK, p-21-activated kinase
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ASSOCIATED CONTENT
* Supporting Information
■
S
Complete experimental procedures, biochemical assays, crys-
tallization, structure determination, rationale explaining selec-
tivity over PAK1, inhibition of cell proliferation, and safety
panel assays. The Supporting Information is available free of
charge on the ACS Publications website at DOI: 10.1021/
acsmedchemlett.5b00102.
Accession Codes
The coordinates for the PAK1:1, PAK1:2, and PAK1:11
complexes have been deposited in the Protein Data Bank with
accession codes 4ZLO, 4ZJJ, and 4ZJI, respectively.
AUTHOR INFORMATION
Corresponding Author
*Phone: + 41613246904. E-mail: alexei.karpov@novartis.com.
■
Present Addresses
§Patronus Therapeutics, Inc., San Francisco, California 94549,
United States.
∥PIQUR Therapeutics AG, Hohe Winde-Strasse 120, 4059
Basel, Switzerland.
Author Contributions
The manuscript was written through contributions of all
authors.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Christian Guenat, Francis Roll, and Odile Decoret
for analytical support, and we are grateful to Alex Bussenault
and Guillaume Camus for their excellent technical assistance.
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ABBREVIATIONS
■
ATP, adenosine 5′-triphosphate; DFG, aspartate-phenylalanine-
glycine loop; GPCR, G-protein coupled receptor; GTPase,
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