Potent and selective inhibitors of CDPK1 from T. Gondii and C. Parvum based on a 5-aminopyrazole-4-carboxamide scaffold

Article abstract of DOI:10.1021/ml400315s

5-Aminopyrazole-4-carboxamide was used as an alternative scaffold to substitute for the pyrazolopyrimidine of a known bumped kinase inhibitor to create selective inhibitors of calcium-dependent protein kinase-1 from both Toxoplasma gondii and Cryptosporidium parvum. Compounds with low nanomolar inhibitory potencies against the target enzymes were obtained. The most selective inhibitors also exhibited submicromolar activities in T. gondii cell proliferation assays and were shown to be nontoxic to mammalian cells.

Full text of DOI:10.1021/ml400315s

Letter
pubs.acs.org/acsmedchemlett
Potent and Selective Inhibitors of CDPK1 from T. gondii and C.
parvum Based on a 5‑Aminopyrazole-4-carboxamide Scaffold
Zhongsheng Zhang,† Kayode K. Ojo,‡ RamaSubbaRao Vidadala,§ Wenlin Huang,† Jennifer A. Geiger,∥
Suzanne Scheele,∥ Ryan Choi,‡ Molly C. Reid,‡ Katelyn R. Keyloun,‡ Kasey Rivas,‡
Latha Kallur Siddaramaiah,† Kenneth M. Comess,⊥ Kenneth P. Robinson,⊥ Philip J. Merta,⊥
Lemma Kifle,⊥ Wim G. J. Hol,† Marilyn Parsons,∥ Ethan A. Merritt,† Dustin J. Maly,§
Christophe L. M. J. Verlinde,† Wesley C. Van Voorhis,*,‡ and Erkang Fan*,†
†Department of Biochemistry, University of Washington, Seattle, Washington 98195, United States
‡Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, Seattle, Washington 98195, United
States
§Department of Chemistry, University of Washington, Seattle, Washington 98195, United States
∥Seattle Biomedical Research Institute, Seattle, Washington 98109, United States
⊥High Throughput Biology, Global Pharmaceutical R&D, AbbVie, North Chicago, Illinois 60064, United States
S
* Supporting Information
ABSTRACT: 5-Aminopyrazole-4-carboxamide was used as an alter-
native scaffold to substitute for the pyrazolopyrimidine of a known
“bumped kinase inhibitor” to create selective inhibitors of calcium-
dependent protein kinase-1 from both Toxoplasma gondii and
Cryptosporidium parvum. Compounds with low nanomolar inhibitory
potencies against the target enzymes were obtained. The most selective inhibitors also exhibited submicromolar activities in T.
gondii cell proliferation assays and were shown to be nontoxic to mammalian cells.
KEYWORDS: Toxoplasma gondii, Cryptosporidium parvum, calcium-dependent protein kinase-1, enzyme inhibitor, selectivity
likely of importance to the lifecycle of C. parvum for similar
reasons.
Toxoplasma gondii and Cryptosporidium parvum are apicomplex-
an parasites that cause serious diseases in humans (toxoplas-
mosis1,2 and cryptosporidiosis3) with inadequate treatment
options. C. parvum infection has been implicated in 15−20% of
childhood diarrhea cases in developing countries4,5 and can lead
to life-threatening illness in immunocompromised persons. The
only approved medicine for C. parvum infection, nitazoxanide,
is expensive and not very effective for treating immunocom-
promised patients.3 Toxoplasmosis also leads to life-threatening
situations in immunocompromised patients. T. gondii infection
of pregnant women can result in severe birth defects or
miscarriage. Current options are limited to sulfadiazine and
pyrimethamine, which can have toxic side effects and require
lifelong treatment for immunocompromised persons.1,2 Clearly,
new and effective therapy for treating T. gondii or C. parvum
infection is needed.
The calcium-dependent protein kinase-1 orthologues of both
T. gondii (TgCDPK1) and C. parvum (CpCDPK1) have
attracted interest as potential drug targets for these para-
sites.6−12 CDPK1 belongs to a family of serine/threonine
protein kinases found in plants and Apicomplexa but not in
humans or other animals. Recent genetic and chemical evidence
suggests that TgCDPK1 plays a critical role in the lifecycle of T.
gondii parasites by controlling the exocytosis of micronemes,
which are specialized organelles that contain a number of
proteins involved in parasite invasion and egress.6 CpCDPK1 is
From a drug discovery perspective, CDPK1 contains unique
structural features that can be targeted for parasite-specific
inhibition over human kinases. Crystal structures of TgCDPK1
and CpCDPK1 revealed an enlarged ATP-binding pocket due
to the presence of the smallest amino acid, glycine, at the
“gatekeeper” position adjacent to the adenine recognition
site.7,8 In most kinases, the amino acid at the gatekeeper
position is large although threonine or valine can be found at
this position. It is extremely rare for alanine or glycine to be
present at this position.13 Consequently, we and others have
explored the use of so-called “bumped kinase inhibitors”14
(BKIs), which allow selective inhibition of Tg/CpCDPK1s and
parasite proliferation.7,10,12 Our previous efforts have focused
on generating BKIs based on a pyrazolopyrimidine (PP)
scaffold (Figure 1); several of which are very potent and
selective inhibitors of Tg/CpCDPK1 that show good efficacies
against parasites and minimal toxicities to mammalian
cells.9,11,15 We also attempted to generate selective inhibitors
using an alternative acylbenzimidazole scaffold (Figure 1).
Guided by compound-bound TgCDPK1 structures, we
Received: August 13, 2013
Accepted: November 13, 2013
Published: November 13, 2013
© 2013 American Chemical Society
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ACS Medicinal Chemistry Letters
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Table 1. Initial Assessment of CDPK1 Inhibitors Based on a
5-Aminopyrazole-4-carboxamide Scaffold
Figure 1. Different chemical scaffolds for CDPK1 inhibitors.
obtained potent inhibitors based on this scaffold that
unfortunately lacked cellular activity.16 In our continued efforts
to explore alternative scaffolds in the hope of finding
compounds with different physicochemical and pharmacolog-
ical profiles for drug discovery, we report here the design of Tg/
CpCDPK1 inhibitors based on a 5-aminopyrazole-4-carbox-
amide (AP) scaffold (Figure 1).
Inhibitor design was based on the crystal structures of
TgCDPK1 in complex with several PP and acylbenzimidazole
derivatives.7,15 The 4-amino group of the PP scaffold and the
adjacent N5 nitrogen atom make hydrogen bonds to the hinge
region of TgCDPK1, projecting the Ar1 group at the C3
position into the hydrophobic pocket adjacent to the
gatekeeper residue and the R2 group at the N1 position into
the ribose-binding pocket. The acylbenzimidazole scaffold
allows Ar1 and R2 substituents to be oriented in the same
directions and pockets as PP-based inhibitors. It is the
combined effect of Ar1 and R2 groups that enhances selectivity
for CDPK1 even over human kinases that contain a relatively
small gatekeeper residue, such as the threonine gatekeeper of
the tyrosine kinase Src.15 Using pyrazole-4-carboxamide as an
alternative core scaffold, we expected the 4-carboxamide group
to replace the pyrimidine to form specific hydrogen bonds to
the hinge region of CDPK1 and maintain the relative
orientations of Ar1 and R2. Incorporation of the 5-amino
group allows the formation of an intramolecular hydrogen bond
to the amide oxygen atom in order to preorganize the
compound for binding to CDPK1 and reduce loss of binding
entropy. The pyrazole moiety has been utilized before as a
scaffold for the discovery of kinase inhibitors such as those
targeting p38α MAP kinase at an allosteric binding site outside
the ATP binding pocket.17,18 However, our design explores a
different functional group display and targets an alternative site
within the kinase domain.
The synthesis of AP derivatives with N1 and C3 substitutions
has been reported in the literature,19,20 and some of the
derivatives were used as intermediates for other classes of
kinase inhibitors.19 We used similar approaches to synthesize a
few derivatives containing selective combinations of Ar1 and R2
groups to compare directly with matching analogues based on
the PP scaffold, as well as one compound with the pyrazole
scaffold without the 5-amino group. The results are shown in
Table 1. It was gratifying to observe that even the first
compound (1) with 2-naphthyl at C3 and t-butyl at N1 is a very
potent inhibitor of both parasite CDPK1s. Compound 1 is
slightly more potent than the corresponding PP scaffold
analogue (compound 2). The same trend exists for the pair of
compounds 3 and 4, which contain 6-ethoxy-2-naphthyl groups
at their C3 positions. Compound 5, which lacks the 5-amino
group of compound 3, is an important control in this series.
Compound 5 possesses 30-fold lower potency against the target
enzymes relative to 3, indicating that keeping the 5-amino
a
Reported in ref 11. Assay was performed in a pH 7.5 buffer
containing 1 mM EGTA, 2 mM CaCl2, 10 μM ATP, and 40 μM
syntide-2 peptide substrate (PLARTLSVAGLPGKK−OH). Enzyme
concentration: 2.0 nM of TgCDPK1 or 0.80 nM of CpCDPK1.
group on the pyrazole scaffold to restrict the conformation and
orientation of the 4-carboxamide group through an anticipated
intramolecular hydrogen bond is very desirable. These
examples provided confidence that our design approach for
CDPK1 inhibitors is working as expected.
Additional interesting structure−activity relationship (SAR)
information was revealed through compounds 6−11 in Table 1
that explore the effect of substitutions at the N1 position
containing a piperidine moiety. In our previous investigations,
incorporation of a piperidine moiety at N1 on the PP scaffold
had several beneficial effects. It improved compound solubility,
inhibitory potency against the parasite enzyme, and selectivity
over mammalian enzymes (compounds 7 and 9 versus
compounds 2 and 4).11,15 However, a similar improvement in
potency is not preserved in the new AP series, as both
compounds 6 and 8 lose potency compared to the matched PP
analogues 7 and 9, or when compared to compounds 1 and 3
with the same core scaffold but containing a t-butyl group at the
N1 position. From compounds 10 and 11, it seems that the
contribution of a methyl piperidine moiety at the N1 position
to inhibitory potency is very limited. Possible reasons for the
different SAR of the two series of compounds may include
differences in electronic and conformational features of the two
cores. The PP core is a flat fused ring system, while the
exocyclic amide group of 5-amino-pyrazole-4-caboxamide may
adopt conformations that deviate from perfect planarity.
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Nevertheless, it is clear from data presented in Table 1 that the
SAR we obtained with PP scaffold is not directly transferrable
to the AP series, and new SAR needs to be derived.
Table 3. SAR Study of C3 Substitution
We divide the SAR work into two major parts, as listed in
Tables 2 and 3. For derivatives shown in Table 2, the Ar1 is
Table 2. SAR Study of N1 Substitution
fixed as 2-naphthyl, and the N1 substitution R2 is varied with
small aliphatic chains and rings, some of which contain nitrogen
or oxygen atoms to decrease lipophilicity. Overall, these small
substitutions do not improve potency over the t-butyl analogue
1, but variants such as 18, with a trifluoroethyl substitution,
have potency very close to compound 1. Similar substituents
may serve as an alternative to the t-butyl group in the future to
optimize pharmacokinetic properties. The relatively small range
of potency changes for the R2 variations may be partly due to
the fact that the R2 group is expected to occupy the ribose-
binding pocket, a region that is exposed to solvent and can
accommodate various functional groups.
Because the t-butyl group seems to be optimal for the N1
position, it was fixed for the analogues in Table 3, which
contain a range of substituted and fused double ring Ar1
systems at the C3 position. Compounds 24−27 are naphthyl-
based substituents, with various alkoxy groups at the 6-position.
These compounds possess good inhibitory potency, similar to
the PP series.11 Compounds 27−33 explore fused ring systems
that contain 5-membered rings and hydrophilic substitutions.
Unfortunately, all the modifications led to a loss of potency,
indicating that these fused rings do not fit the binding pocket
well. Coming back to two 6-membered ring fusions, while
chromenone and quinolin-2-yl rings led to poor inhibitors
(34−36), quinolin-3-yl or quinolin-6-yl were tolerated (37−
39). Overall, 6-alkoxy-naphth-2-yl and 7-alkoxy-quinolin-3-yl
groups are the best aromatic C3 substituents investigated
(compounds 25, 26, 38, and 39).
We were able to obtain a crystal structure of the TgCDPK1-
35 complex at 2.0 Å. Superposition of the structures of
TgCDPK1 in complex with 35 and with a PP analogue 27
shows clearly that the new AP core can preserve the projections
of N1 and C3 substitutions as well as hydrogen bond
interactions with TgCDPK1 seen for the PP core (Figure 2).
This structure confirms our original design strategy. The amide
group of the AP core is essentially on top of the amino-
pyrimidine moiety of the PP core. Further away, on the
naphthyl/quinolinyl and t-butyl substitutions, the matching
carbons are 0.5−0.6 Å apart. It is not clear if the deviations are
caused by the difference in the aromatic rings or by the overall
electronic configurations of the two inhibitors. It is certainly not
sufficient to explain why the two series of inhibitors have
different tolerance of methyl piperidine substituents on the N1
position (Table 1) since these substitutions project distally into
the ribose binding pocket and the solvent.
A select group of compounds with low nanomolar IC50s for
CDPK1s were tested for inhibition of a mammalian kinase with
a small gatekeeper residue (Src), for inhibition of T. gondii cell
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ACS Medicinal Chemistry Letters
Letter
carboxamide scaffold can effectively replace the pyrazolopyr-
imidine core of the BKI series, enabling design of novel CDPK1
inhibitors that offer additional opportunities for tuning their
pharmacokinetic properties in our drug discovery efforts.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic procedures, compound characterization data, crys-
tallography table, and kinase selectivity table. This material is
available free of charge via the Internet at http://pubs.acs.org.
Accession Codes
The PDB accession code for the X-ray crystal structure of
TgCDPK1-35 complex is 4M84.
AUTHOR INFORMATION
■
Corresponding Authors
Figure 2. Close-up view of inhibitors in an overlay of TgCDPK1-
bound 35 (2.0 Å, PDB 4M84, green structure; nitrogen in cyan,
oxygen in red, and sulfur in yellow) and 2 (PDB 3I7C, ref 7; carbon in
orange and water in pink). The key hydrogen bonds to the backbone
oxygen of Glu129 and NH of Tyr131 are the same in both scaffolds.
*(W.C.V.V.) E-mail: wesley@uw.edu.
*(E.F.) E-mail: erkang@uw.edu.
Funding
K.R.K. was supported by a training scholarship from the
University of Washington Plein Endowment for Geriatric
Pharmacy Research. J.A.G. was supported by a training grant
from the National Institute of Allergy and Infectious Diseases
(Grant T32AI007509). Research reported in this publication
was supported by the National Institute of Allergy and
Infectious Diseases and the National Institute of General
Medical Sciences of the National Institutes of Health under
award numbers R01AI089441, R01GM086858, and
T32AI007509. The content is solely the responsibility of the
authors and does not necessarily represent the official views of
the National Institutes of Health.
proliferation and for cytotoxicity against a mammalian cell line
(CRL8155) using reported procedures.11 The results are
summarized in Table 4. Some of the compounds were several
Table 4. Further Characterization of Select Inhibitors
cytotoxicity
Src IC50
(μM)
selectivity index
(Src/TgCDPK1)
T. gondii
EC50 (μM)
(CRL8155) EC50
compd
(μM)
1
1.3
650
0.26
0.39
0.26
0.32
0.072
1.1
>40
>30
>30
>40
>40
>40
>30
3
7.3
1825
245
Notes
18
19
25
38
39
0.98
2.7
The authors declare no competing financial interest.
225
6.9
>3500
>1600
>15000
>10
>30
ABBREVIATIONS
■
0.22
AP, 5-aminopyrazole-4-carboxamide; BKI, bumped kinase
inhibitor; CDPK1, calcium-dependent protein kinase-1; PP,
pyrazolopyrimidine; SAR, structure−activity relationship
thousand-fold selective for TgCDPK1 over Src. Most inhibited
parasite proliferation at submicromolar concentrations, and all
of them demonstrated low toxicity to mammalian cells. These
compounds are good candidates for further investigation of
pharmacological properties and efficacies in animal models by
oral administration. For example, 39 was given orally to mice at
10 mg/kg and showed an average Cmax of 10 μM at 60 min after
dosing and an area under the curve (AUC) of 2500 μM·min. It
was further profiled for kinome-wide selectivity using a panel of
80 human kinases representing different subfamilies of the
kinome tree.21 Compound 39 did not show significant
inhibition at 10 μM for 78 of the 80 kinases using a
fluorescence based competition assay.22 The remaining two
kinases, Kdr and Prkcn, were inhibited >1000-fold weaker by
39 than CpCDPK1 (Supporting Information). Follow-up
optimization to improve compound solubility and extend
half-life by oral administration is currently underway.
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