Discovery of a potent protein kinase D inhibitor: insights in the binding mode of pyrazolo[3,4-d]pyrimidine analogues

Article abstract of DOI:10.1039/c6md00675b

In this study, we set out to rationally optimize PKD inhibitors based on the pyrazolo[3,4-d]pyrimidine scaffold. The lead compound for this study was 1-NM-PP1, which was previously found by us and others to inhibit PKD. In our screening we identified one compound (3-IN-PP1) displaying a 10-fold increase in potency over 1-NM-PP1, opening new possibilities for specific protein kinase inhibitors for kinases that show sensitivity towards pyrazolo[3,4-d]pyrimidine derived compounds. Interestingly the observed SAR was not in complete agreement with the commonly observed binding mode where the pyrazolo[3,4-d]pyrimidine compounds are bound in a similar fashion as PKD's natural ligand ATP. Therefore we suggest an alternate binding mode where the compounds are flipped 180 degrees. This possible alternate binding mode for pyrazolo[3,4-d]pyrimidine based compounds could pave the way for a new class of specific protein kinase inhibitors for kinases sensitive towards pyrazolo[3,4-d]pyrmidines.

Full text of DOI:10.1039/c6md00675b

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ARTICLE
Discovery of a potent protein kinase D inhibitor: insights in the
binding mode of pyrazolo[3,4-d]pyrimidine analogues‡
Klaas Verschueren,a† Mathias Cobbaut,b† Joachim Demaerel,a Lina Saadah,b Arnout RD Voet,c
Johan Van Lint,b Wim M. De Borggraeve*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
In this study, we set out to rationally optimize PKD inhibitors based on the pyrazolo[3,4-d]pyrimidine
scaffold. The lead compound for this study was 1-NM-PP1, which was previously found by us and others to
inhibit PKD. In our screening we identified one compound (3-IN-PP1) displaying a 10-fold increase in potency
over 1-NM-PP1, opening new possibilities for specific protein kinase inhibitors for kinases that show
sensitivity towards pyrazolo[3,4-d]pyrimidine derived compounds. Interestingly the observed SAR was not in
complete agreement with the commonly observed binding mode where the pyrazolo[3,4-d]pyrimidine
compounds are bound in a similar fashion as PKD’s natural ligand ATP. Therefore we suggest an alternate
binding mode where the compounds are flipped 180 degrees. This possible alternate binding mode for
pyrazolo[3,4-d]pyrimidine based compounds could pave the way for a new class of specific protein kinase
inhibitors for kinases sensitive towards pyrazolo[3,4-d]pyrmidines.
www.rsc.org/
functions in the context of various cancers. PKD1 is mainly
associated with tumor suppressive functions, since it decreases cell
motility and epithelial-mesenchymal transition (EMT).13 To this
Introduction
The Protein kinase D (PKD) family of serine/threonine kinases
belongs to the broader group of the Ca2+/calmodulin-dependent
protein kinases (CAMKs).1 PKDs have attracted a lot of attention
due to their crucial involvement in the pathogenesis of heart
hypertrophy2, different cancers, tumor angiogenesis3 and other
pathologies.4 The PKD family consists of 3 isoforms (PKD1/PKCµ,
PKD2 and PKD3/PKCυ) with a high degree of homology especially in
the catalytic domain.5 The activity of PKD is in many cases regulated
extent, PKD1 phosphorylates substrates involved in actin
remodeling, such as cortactin, the cofilin phosphatase slingshot 1L
(SSH1L) and PAK4 rendering tumor cells less mobile.14-18
Furthermore PKD1 inhibits EMT via phosphorylation of the Snail
transcription factor, resulting in decreased E-cadherin repression
and hence decreased cell-cell adhesion.19, 20 PKD1 also inhibits EMT
via β-catenin phosphorylation, reducing its nuclear functions.21 The
tumor-suppressive role of PKD1 is also reflected in its expression
level in various cancers. For example in androgen-independent
prostate cancer and pancreatic cancer PKD1 expression is
downregulated.22-24 In breast cancer and gastric cancer PKD1
by
a diacylglycerol (DAG) induced, protein kinase C (PKC)-
dependent mechanism wherein PKD is activated via direct
phosphorylation of two conserved activation loop serine residues
(Ser738 and Ser742) by PKC isoforms. Full, sustained PKD activation is
achieved by subsequent auto-phosphorylation.
expression
is
down-regulated
through
promoter
26
hypermethylation.25,
In contrast, PKD2 and PKD3 exert cancer
promoting properties, since they are necessary for the regulation of
genes and proteins involved in metastasis and invasion. For
example in pancreatic cancer cells, PKD2 regulates both the
expression and secretion of matrix metalloproteinases (MMPs)
7/9.24 In prostate cancer cells PKD2 and 3 also increase the
expression of MMP9 and the urokinase-type plasminogen activator
(uPA) via NF-κB and HDAC1.27 This results in breakdown of
extracellular matrix (ECM), consequently increasing cancer cell
invasiveness. Furthermore, ECM breakdown results in the release of
VEGF-A which is sequestered in the ECM upon secretion.24 In
pancreatic and gastric tumor cells, PKD2 has also been shown to
increase the angiogenic response to hypoxic conditions via its
actions in both the endothelial cell as well as the the cancer cell.28
PKD1 and 2 are both expressed in endothelial cells. However, there
are several indications that PKD2 may be more important. Firstly,
PKD plays a well-documented and crucial role in many important
cellular processes, including cell proliferation6, cell survival7, gene
expression8, protein trafficking9, 10, cell motility11 and immune
responses.12 These processes are known to play a key role in tumor
biology.4 Interestingly, the three isoforms have differential
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PKD2 is more abundantly expressed in HUVECs when compared to gatekeeper residue M627. Therefore, the commonly observed
PKD1.29 Only PKD2 knockdown was able to inhibit endothelial binding mode of 1-NM-PP1 was enforced in the binding site
proliferation, migration and tube formation in response to serum.29 according to the interactions and geometry observed in PDBs 3NCG
Azoitei et al. showed that PKD2 is highly expressed in a variety of and 3I7B.38, 41 In this model, shown in figure 2, a cavity aside from
gastrointestinal tumors.28 In the same study, they showed that gatekeeper residue Met-627 and flanked by residues K580, E598
depletion of PKD2 in pancreatic tumors inhibited tumor driven and V625 was introduced that accommodates the naphthalene
blood vessel formation in a model for angiogenesis, as well as in group. From this second model it appeared that modification of this
orthotopic pancreatic cancer xenografts. Interestingly, mice lacking naphthalene group at the 3-position of 1-NM-PP1 could lead to
PKD1 die in utero, whilst mice lacking PKD2 are phenotypically improved potency and specificity. This hypothesis urged us to start
normal and viable, indicating that PKD2 is not strictly required for modifying the 3-position and to initiate a SAR study, via possible
the functioning of normal cells.30 These combined studies indicate modifications such as varying the aromatic substituent at the 3-
that PKD2 is a crucial mediator of the angiogenic response being position and creation of longer chain lengths in between the core
both needed in the tumor endothelial cell (for proliferation, scaffold and the aromatic substituent.
migration and tube formation) as well as in the tumor cell (for
expression and secretion of angiogenic factors). Therefore, PKD2 is
an interesting target kinase to inhibit tumor development and
aggressiveness. Several PKD inhibitors have recently been described
by various authors, both ATP-competitive active site, and non-
competitive, presumably allosteric site inhibitors.
noteworthy examples are: an indolocarbazole Gö697631 (a dual
PKC/PKD inhibitor), 2,6-
benzoxoloazepinolone CID75567332
naphthyridine and pyridyl inhibitors33, 34, 3,5-diarylazoles35 and a
pyrazine benzamide36
Some
a
,
.
Fig. 2 Model of the kinase domain of PKD2 with a blow up of 1-NM-PP1
Herein we describe the exploration of a novel PKD inhibitory
chemotype based on 1-naphtylmethyl-pyrazolo[3,4-d]pyrimidine (1-
NM-PP1, figure 1). 1-NM-PP1 is a bulky analog of the Src inhibitor
PP1. It was originally designed to be active only towards kinases
where the large so-called ‘gatekeeper’ residue is substituted with a
smaller amino-acid, such as alanine, via site-directed mutagenesis.37
The pyrazolo[3,4-d]pyrimidine scaffold was also applied in ‘bumped’
kinase inhibitors of CDPKs found in plants and ciliates but claimed
not to be active in humans.38 In our search for PKD2 inhibitors, we
screened the Calbiochem kinase inhibitor library, during which we
identified 1-NM-PP1 as a potent PKD inhibitor. Other reports also
identified this compound as a lead inhibitor in a PKD screening.39
bound into the ATP binding site.
Results and discussion
The SAR study contains variations in 3 different regions as depicted
in figure 3. Region 4 should remain untouched due to clashes with
the gatekeeper when the free amine is functionalized.
'Region 4'
NH2
'Region 1'
R1
N
N
N
R3
N
'Region 3'
'Region 2'
R2
NH2
Fig 3: Interesting regions in the SAR study of the pyrazolo[3,4-d]pyrimidine
N
based inhibitors.
N
N
N
1
The study began with the variation of region 1(R1). For this the 3-
position of the core pyrazolo[3,4-d]pyrimidine scaffold was varied
using different aromatic moieties. Direct functionalization was
achieved based on a synthesis by Todorovic et al. and is depicted in
scheme 1.42 Starting from (ethoxymethylene)malononitrile 2 a first
cyclisation was performed using tert-butylhydrazine hydrochloride
to obtain pyrazole 3. A second cyclisation was performed using
formamide to obtain the core scaffold 4. A bromination in aqueous
environment provided compound 5.
Fig. 1 1-NM-PP1 compound
The lack of activity of 1-NM-PP1 against the majority of the kinome,
combined with its activity against PKD, offers some opportunities to
develop a compound with selectivity towards the PKD family.
Hence, we considered this compound a valid starting point to
create analogues, and for exploring the SAR aiming to design a
highly specific PKD inhibitor with pharmaceutical potential. To guide
the design, a structural model of 1-NM-PP1 inside the PKD2 kinase
domain was constructed using MOE (CCG, Montreal, Canada)
employing PDB 3PFQ (PKC), 1KOA (Twitchin Kinase fragment) and
3NOY (IspG), which were identified by Phyre2 as suitable templates
for modeling the PKD2 kinase domain.40 In this first model, 1-NM-
PP1 cannot be accommodated in its typical binding mode, similar to
its natural ligand ATP, as the naphthalene group clashes with the
NH2
NH2
Br
NC
O
CN
CN
(i)
(ii)
(iii)
N
N
N
N
N
N
H2N
N
N
N
N
2
3
4
5
(95%)
(93%)
(95%)
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Scheme 1 Synthesis of bromo precursor 5, based on Todorovic et al.
Reagents and conditions: (i) tert-butylhydrazine hydrochloride, Et3N, EtOH
(ii) formamide, 150°C (iii) Br2, H2O.42
Region 3 was varied using a third synthetic approach to introduce
variations at the 6-position. Based on the results from molecular
modelling, it was hypothesized that an extra interaction (hydrogen
donor) with the hinge region could increase compound activity. For
this reason a synthetic approach was used based on a procedure by
Verheijen et al. depicted in scheme 4.43 Starting from barbituric
acid, a Villsmeier-Haack reaction provided 2,4,6-trichloro-5-formyl-
pyrimidine 8. Cyclisation with tert-butyl hydrazine hydrochloride
yielded compound 9. The latter was used in two subsequent
aromatic substitution reactions with ammonium hydroxide and n-
butylamine, respectively. Bromination provided the starting
compound 12 which was further functionalized using Suzuki
coupling reactions.
Variation at the 3-position was achieved by performing Suzuki-
coupling reactions with compound 5 as is depicted in Scheme 2. 12
compounds shown in scheme 2B were tested for activity against
PKD2.
Scheme 2 Suzuki reaction conditions to obtain variation at the 3-position
(A). List of all variations synthesized (B).
Analogues containing a methylene linker between the core scaffold
and the aromatic moeity at the 3-position was based on a
procedure by Bishop et al. as depicted in scheme 3.37 The synthetic
procedure is more elaborate as the functionality is introduced at
the very start of the synthesis. The acid is converted to the acid
chloride and then reacted with malononitrile under basic
conditions. Methylation of the intermediate followed by treatment
with the appropriate hydrazine provides the pyrazole intermediate.
The last step is the formation of the pyrimidine ring system using
formamide at 150°C. Region 2 was varied using methylhydrazine
instead of tert-butylhydrazine (compounds7d, 7j, 7l, 7m).
Scheme 4 Synthesis based on Verheijen et al. Reagents and conditions: (i)
POCl3, DMF (ii) tert-butyl hydrazine hydrochloride, Et3N, MeOH (iii) 25% aq.
NH4OH, dioxane (iv) n-butylamine (v) Br2, DCM (vi) Pd(PPh3)4, K2CO3,
THF/H2O 2/1 (A).43 List of all variations synthesized (B).
Another final synthetic procedure was to verify if adding an extra
hydrogen acceptor functionality in the aromatic moiety at the 3-
position would improve potency. Scheme 5 shows the synthesis of
the 3-IN-PP1 (18), containing an NH hydrogen donor in the form of
an indole. Starting from the commercially available indole,
bromination at the 3-position was achieved using pyridinium
tribromide in pyridine. Protection of the free NH-functionality with
benzenesulfonyl chloride and formation of the boronate ester with
bis(pinacolato)diboron followed by suzuki coupling and
deprotection of the indole yielded the desired product 3-
indolylmethyl-pyrazolo[3,4-d]pyrimidine (3-IN-PP1).
O
O
O
A
(i)
(ii)
(iii)
CN
R1
R1
OH
R1
Cl
CN
N
NH2
R1
NC
R3
O
(v)
(iv)
CN
N
N
R1
H2
N
N
N
N
CN
R2
7
R2
B
Cl
Cl
Br
7e R2 =tBu
7a R2 =tBu 7b R2 =tBu
7c
R2 =tBu
7d R2 =Me
O
O
Br
B
Br
Cl
(i)
(ii)
(iii)
R1
Br
Cl
N
N
N
H
Cl
N
H
Cl
SO2
SO2
16
14
15
76%
7h
7f
7g
R2 =tBu
R2 =tBu
R2 =tBu
7i R2 =tBu
7j
89%
59%
R2 =Me
SO2
H
N
N
NH2
NH2
NH2
Br
N
7k R2 =tBu
7l R2 =Me
1 R2 =tBu
7m R2 =Me
(iv)
(v)
N
N
N
N
N
N
N
N
N
N
17
74%
N
Scheme 3 Synthesis based on Bishop et al. Reagents and Conditions: (i)
SOCl2, reflux (ii) malononitrile, NaH, dry THF (iii) Me2SO4, NaHCO3,
dioxane/H2O (iv) tert-butyl/methylhydrazine hydrochloride, Et3N, EtOH (v)
formamide, 150°C (A).37 List of all variations synthesized (B).
5
18
73%
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Scheme 5 Synthethic procedure of the 3-IN-PP1 compound. Reagents and
conditions: (i) pyridinium tribromide, pyridine, rt (ii) benzenesulfonyl
chloride, TBAHS, 50% aq-NaOH, toluene, rt (iii) bis(pinacolato)diboron,
Pd(dppf)Cl2, KOAc, DMF, 80°C (iv) Pd(PPh3)2Cl2, Na2CO3, dioxane/H2O 4/1,
reflux (v) TBAF, THF,reflux
provide an extra hydrogen acceptor to interact with the hinge
region of PKD. For this reason n-butyl amine was added at the 6-
position of the pyrazolo[3,4-d]pyrimidine scaffold. The long
alkylchain should not hinder due to it pointing towards the outside
of the binding pocket. However these compounds displayed
All 35 described analogues were characterized in vitro via an initial strongly reduced activity (13h, IC50 of 8.169µM) when compared to
screening at 1µM against PKD2. This revealed some unexpected 1-NM-PP1. Although all previous analogues did not result in an
properties (for complete data see Supplementary Information). increase in potency when compared to 1-NM-PP1, compound 18 (3-
Based on the results of the initial screening, a subset of compounds IN-PP1) showed strong PKD2 inhibition with an IC50 of 0.033µM, or
was selected in order to construct a structure-activity relationship about a 10 fold increase in potency compared to 1-NM-PP1. Thus
(SAR) based on IC50 values. (see table 1 for IC50 values).
the addition of a hydrogen donor at the 3-substituent seems to
increase potency by a substantial amount. The addition of a
hydrophobic benzothiophene on the other hand seemed to reduce
activity (6l, IC50 of 2.206 µM). The findings described above are
summarized in figure 3.
Table 1
Residual PKD2 activity at 1µM and corresponding IC50 values in µM shown
for 1-NM-PP1 and 13 analogues.
% PKD 2 activity
IC50 (µM)
remaining (1µM)
Compound
(average ±SD)
1-NM-PP1 (1)
23 ±1.7
35 ±1.3
34 ±0.4
41 ±2.6
101 ±8.3
55 ±2.4
83 ±6.4
43 ±6.6
76 ±2.9
93 ±5.8
64 ±3.9
89 ±6.2
8 ±0.8
0.398
0.449
0.403
0.451
>50
7a
Fig. 3 Summary of the SAR for the provided compounds.
1-NA-PP1 (6i)
7b
We also investigated if these compounds, especially 3-IN-PP1 (18),
also showed similar activity profiles in a cellular setting. Therefore
we used a cellular assay in which we were able to specifically follow
PKD mediated phosphorylation of cortactin, a known PKD substrate,
at Ser-298.15 Probing for substrate phosphorylation has advantages
compared to other readouts used for PKD activity such as C-
terminal auto-phosphorylation, which has been shown to be a very
efficient event, quite resistant to inhibition.44 Activation loop Ser-
738/742 phosphorylation is also an unfavorable readout for PKD
activity, since it has been shown to be unexpectedly upregulated
upon inhibition.45 Cortactin phosphorylation at Ser-298 in our assay
was PKD-specific, since co-transfection of PKD and cortactin was
necessary to produce an adequate signal (data not shown). We
selected four compounds for further validation in a cellular assay
against PKD2 (fig. 4). Based on in silico predictions, 13h was
expected to show increased potency compared to 1-NM-PP1 due to
its extra interaction with the hinge region, but this was not the case
(IC50 of 8.169µM) which is supported by its negligible cellular
activity. Both 1-NM-PP1 and 3-IN-PP1 (18) display dose-dependent
inhibition of PKD2 in our cellular system. As is mirrored in our in
vitro assays, 3-IN-PP1 (18) showed highly increased potency
compared to 1-NM-PP1, with almost full inhibition at 40 µM (85%
inhibition compared to 50% for 1-NM-PP1). In contrast, compound
6l, containing a more hydrophobic benzothiophene, did not show
cellular activity as was expected from its in vitro potency.
7i
7c
0.814
4.841
0.535
9.488
>50
7d
7e
6a
7k
7m
4.657
8.169
0.033
2.206
13h
3-IN-PP1 (18)
6l
53 ±7.1
In our SAR study, 1-NM-PP1 is used as a reference to compare these
results with (1, IC50 of 0.398µM). The IC50 we observed is higher
than previously reported by the Wang group (IC50 of 0.133µM). This
is likely due to differences in assay setup and differences in enzyme
preparation (expression system and activation of PKD, see
Supplementary Information for more information). When we
change the substituent on the 3-position in region 1 (fig. 3) of the
pyrazolo[3,4-d]pyrimidine scaffold to a benzyl group (7a, IC50 of
0.449 µM), activity is slightly decreased compared to 1-NM-PP1.
Deletion of the benzylic carbon resulted in a similar inhibition
towards PKD2 for 6i (IC50 of 0.403 µM). Follow-up modification of
the phenyl group with electron donating and electron withdrawing
groups slightlyl reduced activity (e.g. 7b, IC50 of 0.451µM). Further
chain elongation of the methylene linker completely abolished
activity (7i, IC50 of > 50µM). Via these analoguess it was established
that an optimum is reached when using benzylic substituents at the
3-position. The N-substituent in region 2 was also varied between
tert-butyl and methyl (7c, 7d, 7i, 7j, 7k, 7l, 1, 7m). These results
39
along with the paper from Tandon et al. suggest that a tert-butyl
group seemed to be most suitable for this position, since we
observed a loss in potency upon changing a tert-butyl (7c, IC50 of
0.814 µM) to a methyl group (7d, IC50 of 4.841 µM). It was also
hypothesized that the addition of an extra NH-functionality could
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aminoglycoside phosphotransferase, the overall protein fold is
identical to the protein kinase active domain architecture. This
supports the possibility of the proposed alternate binding
mode. Further exploration of region 2 and 4 is now highly
recommended, since the free amine in the alternate binding
mode is not blocked by the gatekeeper residue and the alkyl
substituent at the 1-position now points towards
a
hydrophobic pocket. In addition, earlier described ‘bumped’
kinase inhibitors (BKI’s) may need to take PKD side effects into
account.38, 47
Fig. 4 The effect of 1-NM-PP1 analogues on PKD mediated phosphorylation
of cortactin at Ser-298. HEK293T cells co-transfected with FLAG-PRKD2 and
myc-CTTN were pretreated with the indicated compounds for 30 min prior
to stimulation with 500nM Phorbol 12,13-dibutyrate (PDB) for 15 min.
Phosphorylation of cortactin was followed via western blot using a home-
made pSer-298 antibody and subsequently reprobed for total cortactin
(anti-c-Myc 9E10) and PKD (Anti-FLAG M2). Quantification of pSer-298/c-
Myc signals of three independent experiments is shown in the graph below.
Fig.
5 The top row represents the binding mode of representative
compounds according to the binding mode of 1-NM-PP1 as observed in
classic kinase structures. The bottom row represents an alternate secondary
binding mode which is in better agreement with the observed SAR.
Anticipated from our initial modelling results, the analogues
showed some unexpected results. Docking a diverse set of
structurally different analogues (1, 18, 6l, 13h shown in fig. 5)
to the first PKD2 model, without the binding mode of 1-NM-
PP1 enforced, revealed a predominant binding mode different
from the commonly observed binding mode where the
pyrazolo[3,4-d]pyrimidine compounds are bound in a similar
fashion when compared to PKD’s natural ligand ATP. In this
alternate binding mode, the pyrazolo[3,4-d]pyrimidine
compound is flipped 180 degrees where the gatekeeper
residue does not require an induced fit to accommodate the
phenyl or benzyl substituent (see figure 5). This alternate
binding mode is in better agreement with the observed SAR
and explains the lack of influence on the activity by the
different decorations on the now solvent exposed phenyl and
benzyl substituents. It also explains the 10-fold increase in
activity of 3-IN-PP1 (18), since the NH-hydrogen is stabilized by
a Glu636 residue in the alternate binding mode as depicted in
figure 5. For this reason 6l is destabilized since its free electron
pair of the sulfur atom clashes with this hydrophilic
environment. Compound 6l was expected to be more potent in
the classical binding mode, since the 3-position points towards
a hydrophobic pocket, supporting the hypothesis of this
alternate binding mode. Also the complete lack of activity of
13h can be explained as the butyl tail now clashes with the
gatekeeper residue, while it is completely solvent exposed in
the first binding mode. All crystal structures of 1-NM-PP1
derivatives (PDBs 2WEI, 3I7B, 3MWU, 3N51, 3NCG, 3SVV,
3MA6, 4LGG, 4QOX) exhibit an identical binding mode for the
purine ring isostere in protein kinase structures in accordance
with the binding mode of ATP. Interestingly, this inversed
alternate binding mode is already observed in one structure
(PDB 4GKI).46 While this protein is not a protein kinase, but an
Conclusions
In summary, we rationally explored the pyrazolo[3,4-d]pyrimidine
structure to find potential novel PKD2 inhibitors with enhanced
inhibitory properties. We identified 3-IN-PP1 as a potent PKD
inhibitor, displaying a 10-fold increase in in vitro potency (IC50 of
±33nM) when compared to 1-NM-PP1. In combination with its
remarkable in cellulo potency, 3-IN-PP1 is a very promising target
for further optimization studies. This compound along with other
pyrazolo[3,4-d]pyrimidine analogues studied in this paper, exposed
some interesting trends as shown in scheme 5. These trends point
towards an alternate binding mode where the pyrazolo[3,4-
d]pyrimidine is flipped 180 degrees in comparison with the normal
binding mode (figure 5). This new binding mode for pyrazolo[3,4-
d]pyrimidine compounds means that further structure based
optimization may open up a completely new class of specific kinase
inhibitors for kinases that show sensitivity towards pyrazolo[3,4-
d]pyrimidine derived inhibitors.
Acknowledgements
WMDB acknowledges KU Leuven for financial support to the
lab via project OT/14/067. JD thanks FWO Vlaanderen for an
Aspirant Fellowship. JVL acknowledges support of the EU-FP7
Project “Lungtarget” (nr 259770) and the FWO-Fonds voor
Wetenschappelijk Onderzoek Vlaanderen (proj. nr G.0816.11.
MC is supported by a fellowship of the Agency for Innovation
by Science and Technology in Flanders (IWT) (grant no.
111101)
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Herein we report the synthesis of pyrazolo[3,4-d]pyrimidine analogues of 1-NM-PP1 and the
identification of 3-IN-PP1 as a new potent PKD inhibitor suggesting an alternate binding
mode to PKD.
H
N
NH2
N
NH2
N
N
N
N
N
N
N
1-NM-PP1
PKD2 IC50 ±330nM
3-IN-PP1
PKD2 IC50 ±33nM