Development of Toxoplasma gondii calcium-dependent protein kinase 1 (Tg CDPK1) inhibitors with potent anti- toxoplasma activity

Article abstract of DOI:10.1021/jm201713h

Toxoplasmosis is a disease of prominent health concern that is caused by the protozoan parasite Toxoplasma gondii. Proliferation of T. gondii is dependent on its ability to invade host cells, which is mediated in part by calcium-dependent protein kinase 1 (CDPK1). We have developed ATP competitive inhibitors of TgCDPK1 that block invasion of parasites into host cells, preventing their proliferation. The presence of a unique glycine gatekeeper residue in TgCDPK1 permits selective inhibition of the parasite enzyme over human kinases. These potent TgCDPK1 inhibitors do not inhibit the growth of human cell lines and represent promising candidates as toxoplasmosis therapeutics.

Full text of DOI:10.1021/jm201713h

Article
pubs.acs.org/jmc
Development of Toxoplasma gondii Calcium-Dependent Protein
Kinase 1 (TgCDPK1) Inhibitors with Potent Anti-Toxoplasma Activity†
Steven M. Johnson,‡ Ryan C. Murphy,‡ Jennifer A. Geiger,⊥ Amy E. DeRocher,⊥ Zhongsheng Zhang,∥
Kayode K. Ojo,§ Eric T. Larson,∥ B. Gayani K. Perera,‡ Edward J. Dale,‡ Panqing He,§ Molly C. Reid,§
Anna M. W. Fox,§ Natascha R. Mueller,§ Ethan A. Merritt,∥ Erkang Fan,∥ Marilyn Parsons,⊥,#
Wesley C. Van Voorhis,*,§,# and Dustin J. Maly*,‡
‡Department of Chemistry, University of Washington, Seattle, Washington, United States
§Department of Medicine, Division of Allergy and Infectious Diseases, University of Washington, Seattle, Washington, United States
∥Department of Biochemistry, University of Washington, Seattle, Washington, United States
⊥Seattle Biomedical Research Institute, Seattle, Washington, United States
#Department of Global Health, University of Washington, Seattle, Washington, United States
S
* Supporting Information
ABSTRACT: Toxoplasmosis is a disease of prominent health concern that is caused by the
protozoan parasite Toxoplasma gondii. Proliferation of T. gondii is dependent on its ability to
invade host cells, which is mediated in part by calcium-dependent protein kinase 1
(CDPK1). We have developed ATP competitive inhibitors of TgCDPK1 that block
invasion of parasites into host cells, preventing their proliferation. The presence of a unique
glycine gatekeeper residue in TgCDPK1 permits selective inhibition of the parasite enzyme
over human kinases. These potent TgCDPK1 inhibitors do not inhibit the growth of human cell lines and represent promising
candidates as toxoplasmosis therapeutics.
age.6 Approximately 11% of the U.S. population is seropositive
INTRODUCTION
Toxoplasma gondii is a food and waterborne pathogen that can
■
for T. gondii, with most studies of European populations
reporting 20−35% seropositivity.1,7 In less developed countries,
these rates can reach 50−75%.6 Thus, many individuals who
become immunocompromised are at risk for developing acute
toxoplasmosis.
infect humans and all warm-blooded animals.1,2 It is acquired
by consumption of undercooked meat bearing tissue cysts or
by ingesting foods or water contaminated with oocysts shed
by infected felines. The oocysts are highly infectious and
environmentally stable, making infection by this route a
serious concern in areas where the water supply is not safe
and secure. During the initial infection, the parasites proliferate
rapidly as tachyzoites until controlled by the immune system.
At that point, the parasite transforms into the bradyzoite, a slow
growing stage, establishing a reservoir of tissue cysts in the
brain and other tissues. Periodically, the tissue cysts rupture,
releasing tachyzoites that again replicate rapidly. If not brought
under control by the immune system, this can cause re-
emergence of the disease. The result in immunocompromised
individuals is toxoplasmic encephalitis. In some regions of the
world, T. gondii infections even appear to be problematic in
immunocompetent individuals, such as foci in Brazil where up
to 17% of individuals suffer from ocular toxoplasmosis3 and in
French Guiana where severely life-threatening manifestations of
infection have been seen in immunocompetent patients.4 A
recent study suggests that a large fraction of individuals with
ocular toxoplasmosis also have tachyzoites in the blood.5 When
initial infection with T. gondii occurs during pregnancy, it can
be vertically transmitted, often leading to birth defects or
miscarriage. A recent review of the literature illuminates the
high prevalence of T. gondii infection in women of childbearing
In immunocompetent individuals, toxoplasmosis is usually
asymptomatic but can appear as mild flulike symptoms in some
instances. For these individuals, recovery usually occurs without
antimicrobial treatment. However, the fact that toxoplasma
infection is epidemiologically associated with schizophrenia
suggests that some immunocompetent individuals may also
suffer from other adverse health effects.8 For immunocompro-
mised individuals, intensive treatment is often required for the
infection, with additional suppressive therapy necessary for the
duration of the immunosuppression. This treatment can be for
life in the case of patients with AIDS, though with immune
reconstitution after highly active antiretroviral therapy,
suppressive treatment can be stopped. Before the era of highly
effective antiretrovirals, toxoplasmic encephalitis was the initial
AIDS-defining illness in up to 33% of cases.9 First line therapy
for toxoplasmosis typically involves a combination regimen
of pyrimethamine with a sulfa (sulfonamide) drug, such as
sulfadiazine.2 For patients with sensitivity to sulfa drugs,
clindamycin can be administered in lieu of sulfadiazine.
Received: December 20, 2011
Published: February 9, 2012
© 2012 American Chemical Society
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Figure 1. R1 and R2 substructures of compounds evaluated in this study.
Leucovorin (folinic acid) is coadministered to mitigate the toxic
effects that pyrimethamine has on bone marrow. Additionally,
pyrimethamine is teratogenic and is thus contraindicated for
use in women during their first trimester of pregnancy. While
not as effective as pyrimethamine and sulfonamides, spiramycin
is recommended in these circumstances and has proven
moderately effective at reducing congenital transmission.2,10,11
Unfortunately, spiramycin has yet to gain FDA approval in the
United States. While other T. gondii antiparasitic drugs are
available, these agents also have significant drawbacks. Because
of the toxicity associated with current toxoplasmosis
therapeutics, complicated dosing regimens, and decreased
effectiveness of second-line treatments when pyrimethamine
and sulfonamides are contraindicated, there is the need to
develop new T. gondii antiparasitic drugs that are nontoxic to
humans and possess simpler dosing profiles.
In developing new toxoplasmosis therapeutics, we are
exploring enzyme targets that are involved in calcium-regulated
biological processes, such as host cell invasion, gliding motility,
and exocytosis.12,13 A key component of the signaling pathways
that regulate these events is the calcium-dependent protein
kinase CDPK1. As calcium levels increase, CDPK1 is activated,
leading to increased gliding and motility, which is important for
both parasite invasion and egress.14 Because T. gondii is an
obligate intracellular parasite that requires invasion of
mammalian host cells to proliferate, TgCDPK1 represents a
promising drug target for the development of antiparasitic
agents. We previously developed several ATP-competitive
inhibitors of TgCDPK1 enzymatic activity and confirmed that
TgCDPK1 inhibition prevents invasion of T. gondii into host
cells, blocking parasite proliferation.15,16 A critical consideration
of this antiparasitic strategy is to minimize perturbation of off-
target mammalian signaling pathways by selectively targeting
TgCDPK1 over the 518 kinases present in humans. We were
able to accomplish this goal by exploiting a unique sequence
and structural variation in the ATP-binding cleft of TgCDPK1,
where the presence of a small glycine gatekeeper residue
permits large hydrophobic substituents displayed from the C-3
position of the pyrazolopyrimidine scaffold to occupy an
adjacent hydrophobic pocket (Figures 1 and 2). Human kinases
contain gatekeeper residues with larger side chains that
sterically occlude access to this pocket. On the basis of
structure−activity relationships from our previous studies,15,16
we have developed an optimized panel of TgCDPK1 inhibitors.
Numerous compounds from this panel are extremely potent
inhibitors of TgCDPK1 activity in vitro and block T. gondii host
cell invasion and proliferation. Several lead candidates were
further shown to be highly selective for TgCDPK1 over a panel
of human kinases and additionally do not inhibit the growth of
human cell lines, suggesting that this antiparasitic strategy could
prove to be nontoxic to mammalian systems.
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activity.16 In that study, two distinct molecular series were
developed to optimize compounds for inhibition of TgCDPK1
enzymatic activity. The first series explored variation of the R2
substructure in the context of a naphthylmethylene R1-bearing
pyrazolopyrimidine core scaffold (substituent 10 in Figure 1).
From that series, several piperidine-containing R2 substructures
were found that confer potent inhibition of TgCDPK1
enzymatic activity, the best being the 4-piperidinemethyl R2
substructure of analogue 10n (TgCDPK1 IC50 = 15 nM). X-ray
crystallographic analysis showed that the 4-piperidinemethyl
group is oriented toward the αD-helix and makes a solvent
exposed salt bridge with the Glu135 side chain carboxylate (as
exemplified in the cocrystal structure of compound 15n bound
to TgCDPK1, presented in Figure 2B). The second series of
inhibitors evaluated variation at the R1 position and identified
several groups that were superior to the naphthylmethylene
substructure for conferring potent inhibition of TgCDPK1
activity. These contain R1 aryl groups directly linked to the
pyrazolopyrimidine core (i.e., through a Caryl−Caryl linkage),
which orients the R1 substructures toward the Gly128
gatekeeper residue and into an adjacent hydrophobic pocket
(Figure 2). This increases selectivity for TgCDPK1 over
potential off-target human kinases, which primarily contain
threonine and larger gatekeeper residues that hinder access to
this pocket.
On the basis of the structure−activity relationships generated
in our previous study, we have designed, synthesized, and
evaluated a subsequent panel of lead TgCDPK1 enzyme
inhibitors for their ability to prevent the invasion of T. gondii
parasites into host cells. In the first part of this study, we have
investigated a panel of R1 groups in the context of iPr, tBu, and
4-piperidinemethyl substituents at the R2 position (series a, b,
and n in Figure 1 and Table 1). To impart selective inhibition
for TgCDPK1 over human kinases, our efforts focused on R1
substructures that occupy the enlarged hydrophobic pocket
next to the Gly128 gatekeeper residue, such as substituted
phenyls, indoles, indazoles, naphthyls, and quinolines (Figure 1).
From previous structural studies,15 it appeared that increased
binding affinity could be gained by extending the R1 substructure
more deeply into the adjacent hydrophobic pocket (Figure 2C).
To explore this possibility, analogues with extended R1 groups
(series 14−25) were synthesized. Analysis of various R2 sub-
structures in the context of multiple R1 groups allows the inter-
dependence of these two positions to be explored.
Syntheses of pyrazolopyrimidine compounds with Pr or Bu
groups at the R2 position (series a and b) are outlined in
Scheme 1. Detailed procedures and characterization data for all
compounds are presented in the Supporting Information.
Microwave-assisted Suzuki−Miyaura reactions were employed
for the coupling of R1 boronic acids or boronate pinacol
esters to the respective pyrazolopyrimidine halide intermedi-
ates 26 and 27.16,17 For compounds 17a−25a, containing
extended R1 substructures, Suzuki−Miyaura coupling of the
TBDMS-protected boronic acid conveniently afforded the
deprotected naphthol intermediate directly, which was sub-
sequently alkylated with the respective R′ halide. Synthesis of
the 4-piperidinemethylpyrazolopyrimidine compounds (series n)
is outlined in Scheme 2. The pyrazolopyrimidine core scaffold
intermediate 32 was synthesized according to previously reported
procedures.16,17 Pyrazolopyrimidine 32 was then alkylated with
mesylate 30 to afford the key intermediate 34. Suzuki−Miyaura
coupling of the respective R1 boronic acids or boronate pinacol
esters and subsequent naphthol alkylations were performed as
Figure 2. X-ray crystallographic structure of compound 15n bound to
wild type T. gondii CDPK1 (PDB accession code: 3SX9).18 (A)
Complete view of the 15n·TgCDPK1 cocrystal structure. (B)
Expanded view of the ATP binding site from part A (protein
backbone ribbons have been removed for clarity). The backbone
carbonyl of Glu129 and NH of Tyr131 hydrogen-bond with the
exocyclic amine and adjacent endocyclic nitrogen of the pyrazolopyr-
imidine core, respectively (green dashed lines). The Glu135 side chain
carboxylate makes a solvent exposed salt bridge with the piperidine R2
ring, which is protonated under physiological conditions (red dashed
line). The R1 aryl substructure is oriented toward the Gly128
gatekeeper residue and into the adjacent hydrophobic pocket. The
mutant Met128 residue (yellow) has been overlaid to show the steric
clash that would occur between a larger gatekeeper side chain with the
inhibitor R1 substructures. The catalytic Lys80 and Asp195 side chains
are also shown for context. (C) Rotated side/bottom view of the ATP
binding site from part B.
i
t
RESULTS AND DISCUSSION
■
Molecular Design and Synthesis. We have previously
shown that pyrazolopyrimidine-based molecules, variably
substituted at the R1 and R2 positions of the core scaffold
(Figure 1), are potent inhibitors of TgCDPK1 enzymatic
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Table 1. Enzymatic Assay (IC50) and T. gondii Proliferation
Scheme 1. General Synthetic Procedures for Compound
a
(EC50) Results for Compounds with Variable R1
Series a and b
a
Substructures (1−25) across the R2 Series a, b, and n
kinase enzymatic IC50 (μM)
TgCDPK1
(G128M)
T. gondii proliferation
EC50 (μM)
compd
TgCDPK1
SRC
1
2
3
b
>5
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
0.94
>3
>3
>3
1.3
>3
1.1
>10
>10
0.13
>10
1.3
>6.25
>6.25
0.23
n
b
n
a
>5
0.0045
0.058
0.019
0.51
b
a
0.0073
0.040
0.58
5.1
0.041
4
5
a
0.0050
0.0041
0.0022
0.012
0.65
0.18
>10
1.5
0.080
0.0030
1.7
b
n
a
6
0.15
n
a
0.081
>10
1.2
7
0.0091
0.018
0.27
n
a
>10
0.29
0.52
2.1
>6.25
0.12
a
(i) Na2CO3, Pd(PPh3)4, boronic acid or boronate pinacol ester, H2O/
DME, 80 °C; (ii) TFA/CH2Cl2 (for Boc-protected R1 substructures
6 and 7); (iii) R′ halide, K2CO3, DMF, room temperature or 80 °C.
8
0.0036
0.013
b
b
n
a
0.044
0.12
9
0.0025
0.0037
0.13
>10
8.8
>6.25
described above for the series a and b compounds. Boc-containing
compounds were deprotected using 50% TFA in CH2Cl2 and
converted to their HCl salts for enzymatic and cell-based
evaluation (results are discussed below and presented in Tables
1 and 2).
10
b
n
a
0.031
2.2
0.015
>10
0.065
0.12
>10
0.20
>10
4.1
2.2
11
0.0050
0.0079
0.0025
0.024
0.072
0.35
b
n
a
From the compounds described in Table 1, the 6-
ethoxynaphthyl R1 group (15) were identified as the best
substructure for conferring potent inhibition of TgCDPK1
enzymatic activity and T. gondii cell proliferation (vide infra).
Therefore, a second series of compounds containing a 6-
ethoxynaphthyl R1 and variable R2 substructures was generated
(Figure 1, substructures a−w). Syntheses of these compounds
are presented in Scheme 3. For the piperidinylamide and
sulfonamide compounds (15l, 15m, 15q, and 15r), the
pyrazolopyrimidine intermediate core 32 was alkylated with
mesylates 36, 37, 40, and 41, respectively, followed by
microwave-assisted Suzuki−Miyaura coupling with 6-ethox-
ynaphthyl-2-boronic acid as described above (Scheme 3A).
Compounds 15o and 15p were synthesized by reductive
alkylation of 15n with formaldehyde and acetaldehyde,
respectively, using sodium cyanoborohydride (Scheme 3B).
The remaining series 15 compounds were synthesized by first
appending the 6-ethoxynaphthyl R1 substructure to the
pyrazolopyrimidine core scaffold 32 through microwave-
assisted Suzuki−Miyaura couplings, affording intermediate 44.
Mitsunobu alkylation of the remaining R2 groups using resin-
bound PPh3, followed by Boc-deprotection with TFA/CH2Cl2,
afforded compounds 15c, 15e, 15g, and 15s. Reductive
alkylation of the deprotected intermediates, with respective
R2 aldehydes, provided final compounds 15d, 15f, 15h−k, and
15t−w (compounds 15k and 15w required additional Boc-
deprotection). All compounds were purified by preparatory RP-
HPLC, and amine-containing compounds were converted to
their HCl salts for enzymatic and cell-based evaluation (results
are discussed below).
0.63
12
13
14
0.52
n
b
n
a
0.018
>6.25
0.11
0.0031
0.0054
0.0060
0.020
>10
0.67
2.0
>6.25
0.021
0.010
0.41
b
n
a
0.0049
0.0050
0.033
>10
0.38
2.2
15
0.018
b
n
n
a
0.0025
0.0034
0.010
>10
>10
0.55
>10
0.20
>10
0.30
>10
0.26
1.1
0.052
0.058
0.33
16
17
n
a
0.0038
0.0006
0.0030
0.0032
0.0026
0.0054
0.20
0.50
18
19
20
0.010
0.12
n
a
>6.25
0.16
n
a
0.31
b
n
a
0.0037
0.0009
0.0037
0.0008
0.0023
0.011
>10
0.78
>10
0.041
3.1
0.21
21
22
23
24
25
0.090
0.060
0.0033
0.13
n
a
n
a
0.28
1.8
0.14
n
a
0.0013
0.0055
0.0007
0.0040
0.0007
1.1
0.28
0.38
0.27
1.6
0.012
0.10
n
a
0.013
0.46
Pyrazolopyrimidines Are Potent Inhibitors of
TgCDPK1 Enzymatic Activity. Compounds were first
evaluated for their ability to inhibit the in vitro enzymatic activity
of wild type T. gondii CDPK1. Inhibition was determined
n
a
All results are the averages of at least three assays. Heat map
representation of IC50 and EC50 results are presented in Table 2.
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contrast to analogues that contain a naphthylmethylene group
at this position. For example, series 15 compounds (6-ethoxynaphthyl
R1) that are alkylated, acetylated, or sulfonylated on the
piperidine rings of their R2 substructures maintain low nano-
molar inhibition of TgCDPK1 enzymatic activity, whereas
series 10 analogues (naphthylmethylene R1) containing similar
modifications demonstrate IC50 values in the high nanomolar
range. Consistently potent inhibition across a wide range of R1
and R2 substructures suggests there is ample chemical space at
either position to exploit for optimizing the pharmacological
properties of these inhibitors.
Scheme 2. General Synthetic Procedures for Compound
Series n
a
To determine how a larger gatekeeper residue affects
compound binding, inhibitors were tested against a TgCDPK1
mutant enzyme that contains methionine at this position
(Gly128Met). Molecular modeling predicts that the increased
steric bulk of this residue should clash with large R1
substructures (Figure 2B). In addition, the Gly128Met mutant
was selected as a drug-resistant mutant because it maintains
enzymatic activity comparable to that of wild type TgCDPK1
and is able to complement for loss of endogenous enzyme
activity in T. gondii parasites. In nearly all cases, the presence of
the larger methionine side chain abolishes the inhibitory activity
of these molecules (IC50 values are generally >3 μM). Even for
compounds 22n, 24n, 25n, 15h, 15k, and 15s−w, which show
some activity against Gly128Met TgCDPK1, the differences in
IC50 values between the wild type and mutant enzymes are
>250-fold (with the exception of 15w, which displays a 68-fold
difference). Thus, the presence of a small gatekeeper residue
provides a distinct preference for binding to the wild type
enzyme. These results are promising for the development of
pyrazolopyrimidine inhibitors as potential antiparasitic drugs
because it suggests we should be able to obtain selectivity for
TgCDPK1 over human kinases, which do not contain
gatekeeper residues as small as Gly or Ala.
Lead Compounds Selectively Inhibit TgCDPK1 over
Human Kinases and Do Not Inhibit Growth of Human
Cell Lines. While compound evaluation in the Gly128Met
TgCDPK1 enzymatic assay was an important surrogate for
gauging potential inhibition of off-target kinases that contain
larger gatekeeper residues in an otherwise identical binding site,
further evaluation was performed against a panel of human
kinases. Compounds were first tested against SRC kinase, as
prior selectivity studies have demonstrated that similar
pyrazolopyrimidine-based inhibitors preferentially target this
enzyme.19−21 Since SRC contains a threonine gatekeeper
residue, which is one of the smallest amino acid side chains
present at this position in human kinases, we selected this
enzyme as a surrogate for probing potential off-target kinase
activity. Inhibition of this enzyme serves as a first-pass filter to
prioritize lead candidates for subsequent cell-based evaluation.
Inhibition values were determined using a previously reported
radioactive kinase assay.16
The activities of the first series of compounds against SRC
are shown in Table 1. While we had initially envisioned the R1
substructure providing the primary determinant for obtaining
selective inhibition of TgCDPK1 over human kinases (i.e., a
steric clash of the R1 substructures with the larger gatekeeper
residue side chains obstructs ligand binding), this appears to be
only partially true. For 93% of the compounds tested, IC50
values are at least 25-fold higher for SRC than for TgCDPK1
(Table 1). Thus, substitution at the R1 substructure is a
significant determinant of selective inhibition of TgCDPK1.
However, further examination of the results presented in Table 1,
a
(i) Boc2O, CH2Cl2; (ii) methanesulfonyl chloride, TEA, CH2Cl2,
0 °C to room temperature; (ii) Cs2CO3, DMF, room temperature or
80 °C; (iv) Na2CO3, Pd(PPh3)4, boronic acid or boronate pinacol
ester, H2O/DME, 80 °C; (v) R′ halide, K2CO3, DMF, room
temperature or 80 °C; (vi) TFA/CH2Cl2.
using a previously reported luminescence-based kinase assay.16
Although a large percentage of the compounds tested displayed
very potent inhibition of TgCDPK1, several notable trends
were observed. While the absence of a hydrophobic substituent
at the R1 position renders these pyrazolopyrimidine com-
pounds inactive against TgCDPK1 (the IC50 values for 1b and
1n are >5 μM, Table 1), incorporation of R1 substructures as
small as a phenyl group confers potent enzymatic inhibition.
Overall, 86% of the compounds tested have IC50 < 25 nM
against TgCDPK1 (Table 1). When compared with results
obtained in our previous study,16 it is evident that coupling of
an R1 substructure to the pyrazolopyrimidine core through a
direct Caryl−Caryl linkage provides analogues with superior
potency relative to those that contain a methylene spacer (for
example, series 10). As observed from crystallographic studies,
this direct linkage orients the R1 substructures toward the
adjacent pocket next to the Gly128 gatekeeper residue, allow-
ing large hydrophobic groups to make extensive contacts
(Figure 2).18 In the context of the 6-ethoxynaphthyl R1 sub-
structure (series 15), it appears that a wide variety of sub-
stitutions at the R2 position are well tolerated for maintaining
potent TgCDPK1 enzymatic inhibition (Table 3), which is in
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Table 2. Heat Map Representation of (A) Wild Type TgCDPK1 Enzymatic IC50 from Table 1, (B) SRC Enzymatic IC50 from
a
Table 1, and (C) Wild Type T. gondii Proliferation EC50 from Table 1
a
Blue represents compounds with desirable IC50 or EC50 values, transitioning to red for compounds with undesirable activities.
where we have evaluated R1 substructures in the context of
group at the R1 position, was tested against SRC to further
probe this interesting phenomenon (Table 3). In this series, it
appears that compounds that contain a methylene spacer
adjacent to the pyrazolopyrimidine core display greater
selectivity for TgCDPK1 inhibition over SRC. 4-Piperidine-
methyl-containing compounds (n−r) are particularly selective
and display no inhibition of SRC activity at compound
concentrations upward of 10 μM. Thus, it is evident that the
degree of selective inhibition of TgCDPK1 over SRC results
t
iPr (a), Bu (b), and 4-piperidinemethyl (n) R2 substituents,
demonstrates a striking trend where the iPr- and tBu-containing
compounds are consistently less selective for TgCDPK1 over
SRC than the 4-piperidinemethyl analogues (differences are
readily observed in the inhibition heat maps in parts A and B of
Table 2 and Figure S1 in the Supporting Information). The
second compound series, where we have evaluated R2
substructures in the context of a 6-ethoxynaphthyl (15)
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Scheme 3. General Synthetic Procedures for Compound
Series 15
Table 3. Enzymatic Assay (IC50) and T. gondii Proliferation
(EC50) Results for Compounds with Variable R2 (a−w)
Substructures and a 6-Ethoxynaphthyl Group (Series 15) at
a
a
the R1 Position
kinase enzymatic IC50 (μM)
TgCDPK1
(G128M)
T. gondii proliferation
EC50(μM)
compd TgCDPK1
15-
SRC
a
b
c
d
e
f
0.0050
0.033
>3
>3
>3
>3
>3
>3
>3
2.6
>3
>3
0.98
>3
>3
>3
>3
>3
>3
>3
1.5
1.4
2.0
2.9
0.27
0.37
2.2
0.018
0.013
>10
>10
>10
>10
5.0
1.0
0.012
0.96
0.97
0.86
0.045
0.050
0.090
0.16
0.34
0.029
0.035
0.052
0.14
0.33
0.20
0.0091
0.0081
0.0026
0.0024
0.0034
0.0043
0.0043
0.0061
0.0028
0.0025
0.0029
0.0050
0.011
g
h
i
5.6
8.7
j
>10
0.73
1.6
k
I
m
n
o
P
q
r
0.81
>10
>10
>10
>10
>10
3.3
0.032
s
0.0019
0.0022
0.0027
0.0030
0.0043
0.14
0.049
0.10
0.18
1.2
t
5.2
u
v
w
6.3
6.7
8.2
a
All results are the averages of at least three assays.
from a synergy between the R1 and R2 substructures. A more
detailed discussion of the structural underpinnings for this
synergy will be presented in a forthcoming X-ray crystallog-
raphy manuscript.18
Several lead compounds (14a, 14n, 15a, 15h, 15n, 15o, and
16n) were further evaluated in an expanded panel of human
kinases that all contain threonine gatekeeper residues (ABL,
LCK, p38α, EPHA3, CSK, and EGFR). In general, similar
inhibition trends were observed for ABL, p38α, EPHA3, CSK,
and EGFR as described above (Table 4). In comparison to
SRC, compounds generally display increased inhibition of LCK,
equipotency against ABL and EGFR, and decreased inhibition
of p38α, EPHA3, and CSK, which is similar to trends that have
been previously reported.19 Importantly, we generally observe
>1000-fold differences between IC50 values for TgCDPK1 over
human kinases for our lead inhibitors (14n, 15h, 15n, 15o, and
16n; refer to Table S1 in the Supporting Information for
specific selectivity ratios between the human kinase IC50 values
compared to TgCDPK1). While these compounds have only
been tested against a small panel of enzymes that were
envisioned to be of primary concern for inhibition, these results
suggest that our lead candidate T. gondii therapeutics should
interact minimally with potential off-target human kinases.
As an indicator for potential host cell toxicity during toxo-
plasmosis therapy, we further evaluated our panel of TgCDPK1
inhibitors (14a, 14n, 15a, 15h, 15n, 15o, and 16n) for their
ability to inhibit the growth of human neutrophil (HL-60) and
lymphocyte (CRL-8155) cell lines. Assays were performed with
a similar procedure as previously reported.16 These compounds
a
(A) (i) Ac2O, TEA, CH2Cl2, then methanesulfonyl chloride; (ii)
methanesulfonyl chloride, TEA, CH2Cl2; (iii) Cs2CO3, DMF, 80 °C;
(iv) Na2CO3, Pd(PPh3)4, boronic acid or boronate pinacol ester,
H2O/DME, 80 °C in microwave. (B) (i) Na2CO3, Pd(PPh3)4,
6-ethoxynaphthalene-2-boronic acid, H2O/DME, 110 °C; (ii) Boc-R2-
OH, PS-PPh3, DIAD, THF, room temperature to 70 °C; (iii) TFA/
CH2Cl2; (iv) sodium methoxide in MeOH, then 2% AcOH,
R″-aldehyde, NaBH3CN.
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Table 4. Enzymatic Assay Results (IC50) for an Expanded Panel of Human Kinases and Growth Inhibition (GI50) of Human Cell
a
Lines
kinase enzymatic IC50 (μM)
human cells, GI50 (μM)
compd
TgCDPK1
SRC
ABL
LCK
p38α
EPHA3
CSK
EGFR
HL-60
CRL-8155
14a
14n
15a
15h
15n
15o
16n
0.0060
0.0049
0.0050
0.0024
0.0025
0.0029
0.0034
0.67
>10
0.38
5.6
0.82
>10
1.7
0.079
0.62
0.052
0.12
0.96
3.3
>10
>10
>10
>10
>10
>10
>10
1.6
2.6
>10
3.1
10
0.51
>10
0.70
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
3.7
>10
>10
4.6
>10
>10
>10
>10
>10
>10
>10
10
>10
>10
>10
>10
a
All results are the averages of at least three assays.
exhibited no inhibition of cell growth at concentrations up to
10 μM (Table 4). To better define potential therapeutic
windows for our toxoplasmosis drug candidates, we are
performing growth inhibition studies using higher compound
concentrations and identifying drug levels required to clear
parasitic infection in mammalian challenge models. Results
from these studies will be presented elsewhere.
these cellular assays are likely to be complicated and beyond the
scope of this manuscript.
Three compounds (15n, 15o, and 16n) were evaluated in
cell-based experiments using T. gondii parasites expressing the
Gly128Met TgCDPK1 gatekeeper mutant, to further validate
that TgCDPK1 is the primary target for the observed
antiparasitic activity. The Gly128Met TgCDPK1 variant is
functionally active and complements the loss of endogenous
wild type enzyme activity, permitting parasite invasion and
proliferation in host cells. For all three compounds, a significant
increase in T. gondii proliferation EC50 (8- to 26-fold) was
observed for parasites expressing the Gly128Met TgCDPK1
mutant over those expressing wild type TgCDPK1 at similar levels
or nontransfected parasites (Figure 3B and Figure S2 in the
Supporting Information). The observed inhibitory effects at higher
compound concentrations (EC50 ≈ 1−3 μM for the Gly128Met
TgCDPK1 expressing parasites) could indicate off-target inhibition
of another parasite kinase. However, the dramatic EC50 shifts seen
for all three inhibitors demonstrate that TgCDPK1 is the primary
target for blocking parasite invasion and proliferation.
Potent TgCDPK1 Enzymatic Inhibitors Block the
Proliferation of T. gondii Parasites. Having developed
compounds that selectively inhibit TgCDPK1 over a panel of
human kinases and do not inhibit the growth of human cell
lines, we further investigated the most potent TgCDPK1
enzymatic inhibitors (IC50 < 25 nM) for their efficacy in
blocking the invasion of T. gondii parasites into human foreskin
fibroblast cells. Since T. gondii is an obligate intracellular
parasite, inhibition of host cell invasion blocks parasite
replication, which was measured as a surrogate according to a
slightly modified version of a previously reported procedure.15
In these cellular assays, several prominent trends were
observed. Notably, compounds 1b and 1n, which do not
contain an R1 substituent and are inactive against TgCDPK1
enzymatic activity, do not block T. gondii cell invasion/
proliferation. Of the compounds that do potently inhibit
TgCDPK1 enzymatic activity (IC50 < 25 nM), an impressive
84% also effectively block T. gondii cell invasion/proliferation
(EC50 < 1 μM). Importantly, no inhibitor toxicity was observed
against the human foreskin fibroblasts used in this assay. Thus,
it seems unlikely that the decreased parasite growth is an
artifact of host cell inhibition. Many of the 4-piperidinemethyl
compounds are potent inhibitors of T. gondii proliferation. In
particular, compounds bearing the 6-ethoxynaphthyl R1
substructure (series 15) are potent enzymatic and cell
proliferation inhibitors across nearly the entire R2 tsubstructure
CONCLUSIONS
■
In the present study, we have evaluated over 70 ATP-
competitive inhibitors of TgCDPK1 for their utility as potential
toxoplasmosis therapeutics. We found that inhibition of
TgCDPK1 enzymatic activity correlates strongly with inhibition
of T. gondii proliferation. Of the 64 compounds that are potent
inhibitors of TgCDPK1 enzymatic activity in vitro (IC50 < 25
nM), 83% exhibit EC50 < 1 μM in a parasite invasion/
proliferation assay. Furthermore, 38% exhibit EC50 < 100 nM.
In a rescue experiment, expression of the drug-resistant
Gly128Met TgCDPK1 enzyme in parasites verified that
inhibition of endogenous wild type CDPK1 is the primary
mechanism of action for these compounds. By virtue of the
small glycine gatekeeper residue in wild type TgCDPK1, these
“bumped” kinase inhibitors are able to selectively inhibit the
parasite enzyme over human kinases, which contain gatekeeper
residues larger than glycine that sterically hinder inhibitor
binding. This selectivity is mimicked in cellular assays where
compounds potently inhibit T. gondii proliferation but do not
affect the growth of human cell lines. Compounds exhibiting
large therapeutic windows between inhibition of T. gondii
proliferation and human cell growth (e.g., >100−1000×) are
undergoing additional preclinical drug development testing to
evaluate pharmacological properties such as solubility,
pharmacokinetics, pharmacodynamics, and metabolism. The
results obtained here poise us for future studies to evaluate lead
candidates possessing favorable properties in parasitic challenge
models in mice as a therapeutic proof of principle.
i
panel. However, compounds containing an Pr or Bu group at
the R2 position (series a and b, respectively) are generally more
potent inhibitors in the cell proliferation assay than their
4-piperidinemethyl analogues (series n). This is readily
observed in Figure 3A and in the inhibition heat map presented
in Table 2C. The a and b series of inhibitors are significantly
more hydrophobic than the 4-piperidinemethyl-containing
compounds (the piperidine amine would be protonated
under physiological conditions), which may increase their
membrane permeability. In addition, the greater potential of
i
t
pyrazolopyrimidine inhibitors with Pr and Bu substituents at
the R2 position to inhibit off-target mammalian and parasitic
kinases (as suggested by their more potent inhibition of SRC
and other human kinases) may lead to enhanced cellular
activity. While it is interesting to speculate on the biophysical
underpinnings of these differences, SAR rationalizations in
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Journal of Medicinal Chemistry
Article
without further purification. Reaction progress was monitored by
thin-layer chromatography on silica gel 60 F254 coated glass plates
(EM Sciences). Chromatography was performed using an IntelliFlash
280 automated flash chromatography system, eluting on prepacked
Varian SuperFlash silica gel columns with hexanes/EtOAc or CH2Cl2/
MeOH gradient solvent systems. For preparative HPLC purification,
samples were chromatographically separated using a Varian Dynamax
Microsorb 100-5 C18 column (250 mm × 21.4 mm), eluting with
H2O/CH3CN or H2O/MeOH gradient solvent systems (+0.05%
TFA). The purity of all final compounds was determined by two
analytical RP-HPLC methods, using an Agilent ZORBAX SB-C18 (2.1
mm × 150 mm) or Varian Microsorb-MV 100-5 C18 column (4.6 mm
× 150 mm) and eluting with either H2O/CH3CN or H2O/MeOH
gradient solvent systems (+0.05% TFA) run over 30 min. Products
were detected by UV at λ = 254 nm, with all final compounds
displaying >95% purity. NMR spectra were recorded on Bruker 300 or
500 MHz spectrometers at ambient temperature. Chemical shifts are
1
reported in parts per million (δ) and coupling constants in Hz. H
NMR spectra were referenced to the residual solvent peaks as internal
standards (7.26 ppm for CDCl3, 2.50 ppm for DMSO-d6, and 3.34
ppm for CD3OD). Mass spectra were recorded with a Bruker Esquire
liquid chromatograph−ion trap mass spectrometer. Inhibitors were
synthesized through several different routes, as represented in Schemes
1−3. Syntheses of compounds 1b, 3a, 4a, 5a, 10a, 10b, 10n, 11a, 11b,
12a, 14a, 15a, 26, 27, 31, and 32 have been previously reported.16,17
All other synthesis and compound characterization data are presented
in the Supporting Information.
T. gondii CDPK1 Enzymatic Assays. Expression, purification, and
enzymatic evaluation of wild type and Gly128Met gatekeeper mutant
TgCDPK1 were performed as described previously.15,16 Briefly,
enzymatic reactions were performed with 4 nM of either wild type
or Gly128Met TgCDPK1 in assay buffer containing 20 mM HEPES
(pH 7.5), 0.1% BSA, 10 mM MgCl2, 1 mM EGTA, 2 mM CaCl2,
10 μM ATP, and 40 μM syntide-2 peptide substrate (peptide sequence:
PLARTLSVAGLPGKK-OH). After incubation for 90 min at 30 °C, the
enzymatic reactions were terminated by adding EGTA to a final con-
centration of 5 mM. The amount of ATP remaining in solution was
evaluated using the Kinase Glo luciferase assay from Promega, with sample
luminescence read using a Microbeta 2 plate reader (Perkin-Elmer, Waltham,
MA). Results were converted to percent inhibition, and IC50 values were
calculated using nonlinear regression analysis in GraphPad Prism. Com-
pounds were evaluated in triplicate in eight-point dilutions (3-fold dilution
series) during the enzymatic reactions.
Human Kinase Enzymatic Assays. All compounds were
evaluated in a primary counterscreen against SRC kinase using either
the truncated catalytic kinase domain (SRCKD) or the three domain
enzyme (SRC3D). Results from compounds tested with both KD and
3D enzymes demonstrated no significant difference in IC50 values and
are thus reported together simply as inhibition of “SRC” kinase. Lead
compounds were further evaluated against a small panel of additional
human kinases: ABL, LCK, p38α, EPHA3, CSK, and EGFR.
Compounds were evaluated in 10-point, 3-fold dilution series ranging
from 10 μM to 0.5 nM during the enzymatic reactions, as per
previously reported procedures.16 Results were converted to percent
inhibition, and IC50 values were calculated using nonlinear regression
analysis in GraphPad Prism. Experiments were performed in triplicate
or quadruplicate. Assay buffers, enzyme concentrations, substrate
peptide sequences and concentrations, and enzymatic reaction times
are listed in the assay-specific details presented below. All assays were
performed using <5 μM ATP ([ATP] ≪ Km).
Figure 3. Enzymatic and cellular inhibition of TgCDPK1. (A)
Correlation between T. gondii cell proliferation EC50 and TgCDPK1
enzymatic IC50 results. The solid lines at EC50 > 6.25 μM and IC50 > 5
μM represent the upper detection limits for compounds tested in the
respective assays. Of the 62 compounds displaying TgCDPK1
enzymatic IC50 < 0.025 μM, 86% are also potent inhibitors of T.
gondii invasion/proliferation (EC50 < 1 μM, points in bottom left
quadrant). (B) Comparison of EC50 curves between wild type T. gondii
(open squares) and parasites expressing either wild type TgCDPK1
(open circles) or the drug resistant Gly128Met TgCDPK1 mutant
(closed circles). Expression of the drug resistant Gly128Met
TgCDPK1 mutant rescues cells from the potent antiproliferative
effects of inhibitor 15n.
(1) SRC. Kinase concentration during the enzymatic reaction:
1 nM for SRCKD or 2 nM for SRC3D. Assay buffer: 33.5 mM
HEPES, pH 7.5, 6.7 mM MgCl2, 1.7 mM EGTA, 67 mM NaCl,
2 mM Na3VO4, 0.08 mg/mL BSA, γ32P ATP (0.2 μCi/well).
Enzymatic reaction time: 30 min for SRCKD or 60 min for
SRC3D. Substrate peptide sequence and concentration: Ac-
EIYGEFKKK-OH (100 μM).
EXPERIMENTAL PROCEDURES
■
(2) ABL. Kinase concentration during the enzymatic reaction:
1 nM for ABLKD or 2 nM for ABL3D. Assay buffer: 33.5 mM
General Synthetic Methods. Unless otherwise stated, all
chemicals were purchased from commercial suppliers and used
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HEPES, pH 7.5, 6.7 mM MgCl2, 1.7 mM EGTA, 67 mM NaCl,
2 mM Na3VO4, 0.08 mg/mL BSA, γ32P ATP (0.2 μCi/well).
Enzymatic reaction time: 30 min for ABLKD and 60 min for
ABL3D. Substrate peptide sequence and concentration: Ac-
EAIYAAPFAKKK-OH (100 μM).
EC50 < 0.5 μM were repeated at least once. For assays to test the
role of the gatekeeper residue, the above procedure was followed
except that three T. gondii cell lines expressing β-galactosidase in a
“wild type” background (as above in the standard assay) or also
expressing HA-TgCDPK1 or HA-Gly128Met TgCDPK1 were assayed
in parallel.
(3) LCK. Kinase concentration during the enzymatic reaction:
10 nM. Assay buffer: 75 mM HEPES, pH 7.5, 15 mM MgCl2,
3.75 mM EGTA, 150 mM NaCl, 2 mM Na3VO4, 0.08 mg/mL
BSA, γ32P ATP (0.2 μCi/well). Enzymatic reaction time: 60 min.
Substrate peptide sequence and concentration: Ac-EIYGEFKKK-OH
(100 μM).
(4) p38α. Kinase concentration during the enzymatic reaction:
2 nM. Assay buffer: 75 mM HEPES, pH 7.5, 15 mM MgCl2,
3.75 mM EGTA, 150 mM NaCl, 2 mM Na3VO4, 0.08 mg/mL
BSA, 1.9 mM BME, γ32P ATP (0.2 μCi/well). Enzymatic
reaction time: 180 min. Substrate peptide and concentration:
myelin basic protein (0.2 mg/mL).
ASSOCIATED CONTENT
* Supporting Information
■
S
Tabulation of IC50-fold differences between human kinases and
TgCDPK1; graphical comparison of SRC and TgCDPK1
enzymatic IC50 results; Tg cell proliferation EC50 shifts with
Gly128Met TgCDPK1 mutant for compounds 15o and 16n;
synthesis and characterization data for all compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(5) EPHA3. Kinase concentration during the enzymatic reaction:
10 nM. Assay buffer: 30 mM HEPES, pH 7.5, 38 mM MgCl2,
630 μM EGTA, 2 mM Na3VO4, 40 μg/mL BSA, γ32P ATP (0.2
μCi/well). Enzymatic reaction time: 120 min. Substrate peptide
and concentration: myelin basic protein (0.2 mg/mL).
(6) CSK. Kinase concentration during the enzymatic reaction:
5 nM. Assay buffer: 75 mM HEPES, pH 7.5, 15 mM MgCl2,
3.75 mM EGTA, 150 mM NaCl, 2 mM Na3VO4, 0.2 mg/mL
BSA, γ32P ATP (0.2 μCi/well). Enzymatic reaction time:
180 min. Substrate peptide sequence and concentration: Ac-
KKKKEEIYFFF-OH (130 μM).
Accession Codes
†The PDB code for the X-ray crystallographic structure of
compound 15n bound to wild type T. gondii CDPK1 is 3SX9.
AUTHOR INFORMATION
Corresponding Author
■
*For W.C.V.V.: phone, 206-543-2447; fax, 206-616-4898;
e-mail, wesley@uw.edu. For D.J.M.: phone, 206-543-1653;
fax, 206-685 7002; e-mail, maly@chem.washington.edu.
Notes
(7) EGFR. Kinase concentration during the enzymatic reaction:
1 nM. Assay buffer: 37.5 mM Tris, pH 7.5, 15 mM MgCl2,
0.75 mM EGTA, 0.75 mM Na3VO4, 0.015% Triton X-100, 3.75 mM
DTT, 0.08 mg/mL BSA, 2 mM ATP, γ32P ATP (0.2 μCi/well).
Enzymatic reaction time: 30 min. Substrate peptide and
concentration: poly Glu-Tyr substrate (0.2 mg/mL).
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful for the technical assistance of Suzanne Scheele
from the Parsons lab. This work was funded by the National
Institute of General Medical Sciences Grant R01GM086858
(D.J.M.) and the National Institute of Allergy and Infectious
Diseases Grants R01AI080625 (W.C.V.V.) and R01AI067921
(E.A.M. and W.C.V.V.). J.A.G. was supported by a training
grant from the National Institute of Allergy and Infectious
Diseases (Grant T32AI007509). The authors are solely
responsible for the content.
Human Cell Growth Inhibition Assays. Lead compounds were
evaluated for potential toxicity against two human cell lines: HL-60
(neutrophil) and CRL-8155 (lymphocytic) cells. Cells were grown in
either IMDM (HL-60) or RPMI-1640 (CRL-8155) growth medium
supplemented with 10% heat inactivated fetal calf serum and 2 mM
L-glutamine. HL-60 growth medium additionally contained 25 mM
HEPES and 1% penicillin/streptomycin. CRL-8155 growth medium
additionally contained 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/
L glucose, and 1.5 g/L sodium bicarbonate. Cells were grown in the
presence of 10 μM test compound for 48 or 72 h at 37 °C and 5%
CO2 in 96-well flat-bottom plates (Corning). Growth was quantified
using Alamar blue as a developing reagent and detecting sample
absorbance at λ = 570 nm (600 nm reference wavelength). Percent
growth inhibition by test compounds were calculated based on
cultures incubated with DMSO negative and tipifarnib (R115777)
positive controls (0% and 100% growth inhibition, respectively). All
assays were performed in triplicate.
T. gondii Cell Proliferation Assays. The invasion assay was
performed as previously described,15 with slight modifications to
improve assay sensitivity and reliability. Compounds were diluted in
DMEM maintaining 0.5% DMSO. T. gondii clonal parasites (103)
expressing β-galactosidase as a reporter (genotype RHΔhxgprt,
β-galactosidase, GFP15 for the standard protocol) were mixed with the
medium containing the compounds (200 μL) and incubated at 37 °C,
5% CO2, for ∼5 min. The parasite/compound mixture was added to
96-well plates containing confluent human fibroblast cell layers (from
which growth medium was aspirated) and incubated for 44 h at 37 °C
and 5% CO2. As a control, a dilution series of T. gondii (103−0)
parasites was grown in the same conditions described above but
without compound. Plates were visually inspected for evidence of
cytotoxic effects on fibroblasts. β-Galactosidase was then assayed using
chlorophenol red β-galactopyranose (Sigma) as a substrate.22 Plates
were developed for ∼1.5 h at 37 °C. Absorbance was measured at
595 nm on a SpectraMax M2 (Molecular Devices) microplate reader.
Each experiment was performed in triplicate, and experiments yielding
ABBREVIATIONS USED
■
ATP, adenosine triphosphate; Boc, tert-butyloxycarbonyl;
CDPK1, calcium-dependent protein kinase 1; CSK, C-terminal
SRC kinase; DIAD, diisopropyl azodicarboxylate; DME,
dimethoxyethane; EC50, half-maximal effective concentration;
EGFR, epidermal growth factor receptor kinase; EPHA3, EPH
receptor A3 kinase; IC50, half-maximal inhibitory concentration;
LCK, lymphocyte-specific protein tyrosine kinase; TBDMS,
tert-butyldimethylsilyl
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