Identification of an Atg8-Atg3 protein-protein interaction inhibitor from the medicines for malaria venture malaria box active in blood and liver stage plasmodium falciparum parasites

Article abstract of DOI:10.1021/jm401675a

Atg8 is a ubiquitin-like autophagy protein in eukaryotes that is covalently attached (lipidated) to the elongating autophagosomal membrane. Autophagy is increasingly appreciated as a target in diverse diseases from cancer to eukaryotic parasitic infections. Some of the autophagy machinery is conserved in the malaria parasite, Plasmodium. Although Atg8's function in the parasite is not well understood, it is essential for Plasmodium growth and survival and partially localizes to the apicoplast, an indispensable organelle in apicomplexans. Here, we describe the identification of inhibitors from the Malaria Medicine Venture Malaria Box against the interaction of PfAtg8 with its E2-conjugating enzyme, PfAtg3, by surface plasmon resonance. Inhibition of this protein-protein interaction prevents PfAtg8 lipidation with phosphatidylethanolamine. These small molecule inhibitors share a common scaffold and have activity against both blood and liver stages of infection by Plasmodium falciparum. We have derivatized this scaffold into a functional platform for further optimization.

Full text of DOI:10.1021/jm401675a

Article
pubs.acs.org/jmc
Identification of an Atg8-Atg3 Protein−Protein Interaction Inhibitor
from the Medicines for Malaria Venture Malaria Box Active in Blood
and Liver Stage Plasmodium falciparum Parasites
Adelaide U.P. Hain,†,∥ David Bartee,‡ Natalie G. Sanders,§,∥ Alexia S. Miller,†,∥ David J. Sullivan,§,∥
Jelena Levitskaya,§,∥ Caren Freel Meyers,‡ and Jurgen Bosch*,†,§,∥
̈
†Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, 615 North Wolfe Street,
Baltimore, Maryland 21205 (United States)
‡Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street,
Baltimore, Maryland 21205 (United States)
§W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health,
Baltimore, Maryland 21205 (United States)
∥The Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, 615 North Wolfe Street,
Baltimore, Maryland 21205 (United States)
S
* Supporting Information
ABSTRACT: Atg8 is a ubiquitin-like autophagy protein in eukaryotes that is covalently
attached (lipidated) to the elongating autophagosomal membrane. Autophagy is increasingly
appreciated as a target in diverse diseases from cancer to eukaryotic parasitic infections. Some of
the autophagy machinery is conserved in the malaria parasite, Plasmodium. Although Atg8’s
function in the parasite is not well understood, it is essential for Plasmodium growth and survival
and partially localizes to the apicoplast, an indispensable organelle in apicomplexans. Here, we
describe the identification of inhibitors from the Malaria Medicine Venture Malaria Box against
the interaction of PfAtg8 with its E2-conjugating enzyme, PfAtg3, by surface plasmon resonance.
Inhibition of this protein−protein interaction prevents PfAtg8 lipidation with phosphatidyle-
thanolamine. These small molecule inhibitors share a common scaffold and have activity against
both blood and liver stages of infection by Plasmodium falciparum. We have derivatized this
scaffold into a functional platform for further optimization.
In most eukaryotes, lipidation of Atg8 to phosphatidylethanol-
amine (PE) in membranes normally requires proteolytic
processing of the C-terminus of Atg8 by Atg4 and activation
via adenosine 5′-triphosphate (ATP) followed by intermediate
thioester bond formation with the E1-activating enzyme Atg7.
Atg8 is then transferred to its E2-like conjugating enzyme Atg3,
forming a second thioester intermediate before being conjugated
to the nitrogen of PE (Figure 1).10 This process also requires
noncovalent interaction between Atg8 and Atg3 through a well-
characterized Atg8-interacting motif (AIM) in Atg3 and two
hydrophobic pockets, termed the W and L-site, in Atg8.11
Notably, in Plasmodium, Atg8 is synthesized with a C-terminal
glycine and therefore does not require activation by Atg4.
Recently published studies showed a drastic growth defect in
Plasmodium falciparum when levels of PfAtg7 were reduced.12
This along with the essentiality of Plasmodium Atg8 suggests that
targeting PfAtg8 lipidation is a good strategy for drug
intervention.9
INTRODUCTION
■
The malaria parasite, Plasmodium, is a major public health burden
in the developing world, and despite the existence of antimalarial
treatment active in blood stages of infection, there is a continual
need for novel drug design as the parasite develops resistance to
current treatments.1 Additionally, treatment for the hypnozoite-
causing species, Plasmodium vivax, requires primaquine, which
has severe side effects and causes hemolysis in patients with
glucose-6-phosphate dehydrogenase deficiency.2 Discovery of
novel compounds in antimalarial drug development is essential
for future intervention strategies.
Atg8 is the ubiquitin-like (Ubl) protein necessary for
formation and maturation of autophagosomes in autophagy in
Eukarya. In yeast and mammals, Atg8 is lipidated to the
autophagosome membrane, but in Plasmodium, Atg8 is partially
conjugated to the membrane of the apicoplast under non-
starvation conditions.3−6 The apicoplast is a nonphotosynthetic
chloroplast-like organelle present in apicomplexans and is
essential for isoprenoid synthesis.7 Under starvation conditions,
Atg8 relocates to acidic vesicles with Rab7 in and near the food
vacuole.8 Atg8 is essential to the Plasmodium parasite and has
been proposed as a target for antimalarial drug design.9
Received: October 28, 2013
Published: May 1, 2014
© 2014 American Chemical Society
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Figure 1. Overview of Atg8 Conjugation pathway in autophagy. (A) Visual representation of the localization of Atg8 in autophagy. Atg8, represented by
the rectangle, is essential to the elongation of the autophagosomal membrane. (B) Generic conjugation pathway shown for yeast system. In Plasmodium,
Atg8 is synthesized with a C-terminal glycine that does not require proteolytic processing for activation. The red line indicates step of pathway targeted
by our inhibitors.
Figure 2. Identification of a common scaffold that inhibits Atg8-Atg3 from the MMV malaria box screen. (A) Primary screen of MMV Malaria Box14
with SPR competition assay. The gray box denotes compounds meeting the threshold of greater than 25% inhibition. (B) Bar graph showing inhibition
of hits in primary screen, denoted by compound number. (C) Dose-dependent inhibition of PfAtg8-PfAtg3 interaction by MMV compounds. Inhibition
was measured with increasing amount of compound in SPR competition screen. Mean and standard deviation (SD) of three injections are shown. (D)
Thermal stability assay of PfAtg8CM with the three MMV hits. Error bars show SD of three measurements.
We previously elucidated the protein crystal structure of P.
falciparum Atg8 bound to a peptide corresponding to PfAtg3’s
AIM (PDB code 4EOY).13 Regions of diversity exist between the
human and Plasmodium system that may be exploitable through
small molecule inhibition. Our mutational and interaction
studies suggest that the Plasmodium Atg8-Atg3 interaction
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a
Table 1. Structures and Inhibition Data for PTA Compounds
a
PTA-containing compounds used in studies listed with PubChem Compound Identification (CID), MMV ID, chemical structure, molecular weight
(g/mol), IC50 in SPR and blood stage assays, and LEAN score, calculated as −log (IC50)/number of heavy atoms. Asterisks denote IC50 values
derived from the literature.
requires Atg8’s W/L site as well as the apicomplexan loop on
Atg8 (residues 67−76), termed the A-loop.13 Here, we report the
identification of a class of compounds that inhibit the
Plasmodium Atg8-Atg3 interaction and that inhibit in vitro
growth of P. falciparum in blood- and liver-stage assays,
presumably through prevention of PfAtg8 lipidation.
(4-methylphenyl)-4-pyridin-2-yl-1,3-thiazol-2-amine) (1), (2-
methylsulfanyl-N-(4-pyridin-2-yl-1,3-thiazol-2-yl)benzamide)
(2), (2-bromo-N-(4-pyridin-2-yl-1,3-thiazol-2-yl)benzamide)
(3), N-[2-chloro-5-(trifluoromethyl)phenyl]-2-[2-(4-
methylphenyl)pyrazolo[1,5-a]pyrazin-4-yl]sulfanylacetamide
(4), 1-[4-(dimethylamino)phenyl]-6,6-dimethyl-1,3,5-triazine-
2,4-diamine (5), and 2-N,3-N-bis(4-bromophenyl)quinoxaline-
2,3-diamine (6) (Figure 2A,2B, Table 1). In subsequent dose-
dependent studies, 4−6 demonstrated a constant level of
inhibition independent of concentration of the small molecule
and were not further investigated. Compounds 1−3 led to dose-
dependent inhibition with an SPR inhibitory concentration
(IC50 SPR) ranging from 6 to 18 μM (Figure 2C). Interestingly,
these compounds shared a common scaffold: 4-pyridin-2-yl-1,3-
thiazol-2-amine (PTA) incorporated as the N-substituent in
various anilines or benzamides. 1−3 were tested for their effect
on the stability of PfAtg8CM13 using fluorescence-based thermal
shift assays (TSAs). None of the compounds significantly
RESULTS
■
Screening of the MMV Malaria Box Library. Previously,
we developed a surface plasmon resonance (SPR)-based
competition assay to identify compounds that disrupt the
PfAtg8-PfAtg3 noncovalent interaction.13 PfAtg3 is immobilized
onto an SPR chip, and PfAtg8 is injected in the presence of
dimethyl sulfoxide (DMSO, control) or a compound (dissolved
in DMSO), and binding is measured by the SPR response. The
Medicines for Malaria Venture (MMV) Malaria Box of 200
druglike and 200 probelike molecules was screened at 5 μM in a
primary SPR screen (Figure 2A).14 Six compounds met the cutoff
for at least 25% inhibition of the PfAtg8-PfAtg3 interaction: (N-
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affected the melting temperature (Tm) indicating that they most
likely did not disturb the tertiary structure of PfAtg8 (Figure 2D).
Determination of the PTA Binding Site on PfAtg8. We
tested whether these compounds bound directly to PfAtg8 using
SPR. PfAtg8CM was immobilized onto an SPR Ni-nitrilotriacetic
acid (NTA) chip via a 12 histidine N-terminal tag. 1, chosen for
its better solubility, was injected over the chip, and binding was
measured. Compound 1 led to a dose-dependent increase in SPR
response, indicating binding to PfAtg8CM (Figure 3A). We next
sought to determine the binding site for the PTA compounds
with in silico docking.
group bound to the L-site of PfAtg8. Docking was repeated using
a version of the receptor for which the carbonyl of Lys47 was
input as a hydrogen bond acceptor constraint (Figure 3B inset). 3
had the same docking pose in both studies. 1 had a similar
binding pose to the highest ranked pose from the unconstrained
docking, while 2 was slightly different with the pyridine ring
rotated 90° in the W-site and the benzene ring positioned just
below and to the left of the L-site pocket with the methylsulfanyl
group reaching into the mostly hydrophobic L-site.
Compound 1 Shows Activity against Plasmodium Liver
Stages. The half maximal inhibitory concentrations (IC50) for
these compounds in P. falciparum 3D7 blood stages were
previously reported and are located on the NCBI PubChem
database (http://pubchem.ncbi.nlm.nih.gov). 1 has a reported
IC50 of 350−400 nM (PubChem bioassay ID (AID): 660866 and
449703).16 The reported IC50 for 2 ranged from 0.20 to 6.8 μM,
while 3 ranged from 1.36 to 4.52 μM (PubChem AID: 660866
and 449707).17 We focused on compound 1 for further studies
because the reported cytoxicity in human cell lines is much lower
than that of compounds 2 or 3 (PubChem AID: 660872, 685525,
and 449705).
PfAtg8 is expressed and lipidated during the liver stage where it
partially localizes to the apicoplast.4 Treatment of early liver stage
parasites with the autophagy inhibitor 3-methyladenine is
reported to delay conversion of the parasite into its trophozoite
form.9 We therefore hypothesized that 1 would also have activity
against the liver stage. 1 was previously tested in Plasmodium
yoelii liver stage cultures and did not display >50% inhibition at
the screening concentration of 10 μM; an IC50 was not reported
(PubChem AID: 602118 and 602156).18,19 P. yoelii and
Plasmodium berghei are often used to test drugs for liver stage
inhibition as they are easier to culture. However, these are rodent
malaria models and may not be indicative of activity in P.
falciparum. Analysis of the W/L-site in these three species
revealed differences in the amino acid composition that could
affect drugs predicted to bind in that region (Figure 4). We
Figure 3. Identification of 1 binding site on PfAtg8CM. (A) Binding of 1
to PfAtg8. His12-PfAtg8CM was immobilized onto a nickel-charged NTA
SPR chip. 1 was injected over immobilized PfAtg8-variants in two
separate runs at four concentrations. Graph shows mean
SD of
binding from 1. (B) In silico docking of PTA compounds to PfAtg8CM
(PDB code 4EOY). Overall structure of PfAtg8 with W- and L-site and
A-loop demarcated. Inset shows best poses for each compound. The
predicted pose for unconstrained docking is shown in cyan. Docking was
also performed with a hydrogen bond constraint to the carbonyl of
Lys47, located between the W- and L-site. Predicted pose for
constrained docking is shown in green. For 1, an alternate pose, it is
highly ranked in the unconstrained docking and is enriched in the top 10
poses as shown in magenta.
The OpenEye software package (www.eyesopen.com)15 was
used to dock conformers of the compounds against the X-ray
structure of PfAtg8 (PDB code 4EOY).13 All three compounds
docked to the W-site of PfAtg8 (Figure 3B). 2 and 3 bound with
the pyridine ring in the W-site, whereas 1 was predicted to bind
with the pyridine ring in the L-site, the thiazole ring positioned
between the pockets, and the methylbenzene group in the W-
site. An alternate pose was enriched within the top 10 poses
output by the docking study in which 1 binds solely within the
W-site in a more compact conformation. 1 is missing a donor
oxygen compared to 2 and 3 and, therefore, may adopt a different
mode of binding to the W- and L-sites of PfAtg8. 2 docked with
the pyridine ring in the W-site, and the methylsulfanylbenzene
Figure 4. P. falciparum and P. yoelii Atg8 structural differences. PfAtg8
(PDB code 4EOY) is shown with surface and cartoon representation.
Amino acid changes between the species are shown in red with P.
falciparum letter and numbering followed by P. yoelii. W-site, L-site, and
A-loop pockets are shown in mesh in cyan, purple, and green,
respectively. P. falciparum Atg8 pocket sizes were calculated with
OpenEye VIDA visualization software (www.eyesopen.com).
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Figure 5. Effect of 1 treatment on the development of P. falciparum 3D7 GFP parasite in HC-04 cells in vitro. (A) Flow cytometry based detection of
Annexin-V positive and PI-positive cells in hepatocyte cultures treated with 1 (as described in Experimental section). Dot plot graphs demonstrate
representative pattern of staining, and bar graphs show summary (mean SD) of viable cell detection obtained in three independent hepatocyte
cultures. (B) Viable infected hepatocytes (GFP+/PI−) were detected by flow cytometry in HC-04 cultures 72 h post infection with P. falciparum. Dot
plot graphs demonstrate representative pattern of staining, and numbers reflect percentages of GFP positive cells in total PI negative cell populations.
(C) Summary (mean SD) of viable infected cell detection obtained in three independent hepatocyte cultures. (D) GFP-specific fluorescence was
assessed in infected cultures exposed to 1 or DMSO for 72 h. Histograms demonstrate one representative staining pattern, and numbers reflect mean
fluorescence intensity (MFI) in GFP-positive populations. Bar graphs reflect GFP-specific MFI (mean SD) in viable cell population detected in three
independent hepatocyte cultures.
utilized a recently established P. falciparum liver stage in vitro
model in which sporozoites isolated from infected mosquitos’
salivary glands invade HC-04 hepatocytes.20 HC-04 is a unique
immortalized cell line that exhibits the expression of biochemical
markers characteristic for normal hepatocytes and allows for the
full development of the human malaria parasite, P. falcipa-
rum.20−22 Using this system, we assessed the effect of 1 on the
development of P. falciparum 3D7-green fluorescent protein
(GFP) parasites23 in human hepatocytes in vitro. No change in
the viability of HC-04 cells was detected in response to treatment
with 3 μM or 30 μM of 1 for 96 h (Figure 5A). Using flow
cytometry, we observed about a 50% decrease in the proportion
of hepatocytes infected with P. falciparum 3D7-GFP sporozoites
(GFP+/propidium iodide (PI)- cells) in response to treatment
with 30 μM, but not with 3 μM of 1 (Figure 5B, C). Additionally,
there was a dose-dependent reduction in the intensity of GFP
fluorescence at both concentrations of 1, indicating inhibition of
parasite development within hepatocytes, at least in vitro (Figure
5D). Because 1 did not affect cell survival or cell growth of HC-04
cells (Figure 5A), the compound’s effect on the parasite is
unlikely to result from host cell cytotoxicity.
Validation of Drug Effect on PfAtg8 in Parasite
Cultures. We next sought to determine whether 1 had an effect
on PfAtg8 in P. falciparum blood stage cultures. In immunoblot
assays, very low levels of endogenous PfAtg8 were detected in
DMSO-treated control cells. Incubation of cells in minimal
media lacking human serum for 5 h led to a very slight increase in
PfAtg8. In contrast, treatment with 50 μM cytocidal levels of
compound 1 led to a drastic increase in PfAtg8 protein levels as
well as to a shift in mobility, likely corresponding to the
unlipidated form of PfAtg8. Overall protein levels were
unchanged, indicating an up-regulation or accumulation of
PfAtg8 in the presence of 1 under conditions that are likely
leading to cell death (Figure S1 of the Supporting Information).
After treating the parasites for 6 hours, a dose-dependent
increase in delipidation of PfAtg8 is already observed at 12.5 μM
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of 1, and at 25 μM, unlipidated PfAtg8 is the predominant species
(Figure 6A, Figures S2−S4 of the Supporting Information).
amide coupling. Compound 7 can be tethered through a
dialkoxyamine linker to a library of aldehydes and can be
screened using our primary SPR competition assay against the
PfAtg8-PfAtg3 interaction.24 In docking studies, 7 bound the W-
and L-site of PfAtg8 in a fashion similar to compound 2 with the
functional handle positioned toward the A-loop pocket (Figure
7A). A gain in parasite selectivity for such bifunctional analogues
is expected as the A-loop is missing in the human Atg8
homologues.13 To confirm binding to PfAtg8, we tethered 7 to
(+)-biotinamidohexanoic acid hydrazide (BACH) through its
reactive aldehyde group and tested binding with SPR. The
biotinylated PTA compound 8 was immobilized onto a
neutravidin coupled SPR chip. His12-PfAtg8CM injected at
various concentrations led to a dose-dependent increase in
SPR response indicating PfAtg8 directly binds 8 with a KD of 540
nM. In contrast, the human Atg8 homologue, microtubule-
associated protein light chain 3 (hLC3) showed much lower
affinity for 8 with a KD of 18 μM, indicating specificity of the PTA
scaffold for P. falciparum (Figure 7B). Additionally, recombinant
PfAtg3 did not bind immobilized 8 (data not shown) in
agreement with our docking studies suggesting binding to the W-
site of PfAtg8.
Compound 7 was converted to the hydrochloride salt and was
subjected to acid-catalyzed acetal formation with methanol and
trimethyl orthoformate under microwave irradiation to provide
the dimethyl acetal compound 9, an unreactive derivative, to
confirm the inhibitory activity of the starting platform (Table 1,
Scheme 1). 4-(Dimethoxymethyl)-N-(4-pyridin-2-yl-1,3-thiazol-
2-yl)benzamide 9 has a similar shape and distribution of the
acceptor and donor pairs as the original PTA compounds (Figure
7C) and docked onto PfAtg8 in a fashion similar to 7 (Figure
7A).
SPR studies confirmed that 9 inhibited the PfAtg8-PfAtg3
interaction with an IC50 of 2.86 μM in SPR (Figure 8A). We next
measured growth inhibition of P. falciparum 3D7 by 1 using the
SYBR green I assay.25 This assay exploits the absence of nuclei in
erythrocytes with a fluorescent dye that is unquenched upon
binding to nucleic acids, preferentially double-stranded DNA. In
two of three independent experiments, the IC50 of 1 was 768 nM,
similar to previously published results, while in a third
experiment, the IC50 was 3.3 μM, resulting in an average IC50
of 1.61 1.47 μM.16 Using this assay, 9 had a potency similar to
that of compound 1 against the blood stage of P. falciparum with
an average IC50 of 1.48 0.6 μM (Figure 8B).
Figure 6. Effect of 1 on PfAtg8 protein levels. (A) Dose-dependent
immunoblot analysis of P. falciparum treated with DMSO or 3.375, 6.75,
12.5, or 25 μM 1 for 6 h. Chloroquine (CQ) at 50 nM was used as a
positive control of autophagy inhibition. Atg8-PE has a faster migration
than unlipidated Atg8 with SDS-PAGE. Arrows indicate the migration of
lipidated and unlipidated PfAtg8. The blot was probed with antibody
against TgAtg8, demonstrated to be cross reactive against PfAtg8.40 (B)
Blood smears of P. falciparum after treatment with DMSO or 50 μM 1
for 5 h, observed at 100× magnification. Representative images for
different stages are shown, progressing from ring stage on the left to late
schizont on the right.
When investigating the soluble versus insoluble membrane
fraction, PfAtg8 could only be detected in the soluble fraction
with the number of parasites used per lane. We attribute this to
the low amount of PfAtg8 present in the untreated control and to
reaching the detection limit of our assay (data not shown). At the
treatment concentration and duration used in the study, parasite
morphology appeared normal (Figure 6B).
Synthesis of a Novel PTA Derivative with a Functional
Handle. Our studies indicated that the PTA scaffold is a good
platform for hit-optimization. We synthesized a PTA-benzalde-
hyde derivative, 4-formyl-N-(4-pyridin-2-yl-1,3-thiazol-2-yl)-
benzamide (7), with a functional handle extending off the
common hydrophobic ring system (Table 1, Scheme 1). 7 was
prepared from commercially available PTA and 4-formyl benzoic
acid through a dicyclohexylcarbodiimide (DCC)-promoted
DISCUSSION
■
We identified compound 1 through a screen for inhibitors against
the Plasmodium Atg8-Atg3 protein−protein interaction (PPI).
Targeting protein−protein interactions has long been over-
looked in the drug development field. It was thought to be
difficult because of the shallower nature of many pockets and the
more extensive network of residues involved in the interaction
a
Scheme 1
a
Reagents and conditions. (a) DCC, ACN, 50 °C; (b) 1, 1 M HCl; 2, CH(OMe)3, p-toluene sulfonic acid-H2O, MeOH, microwave irradiation 130
°C.
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Figure 7. Analysis of PTA-derivatives. (A) In silico docking of PTA-derivatives on PfAtg8. 7 (yellow) and 9 (green) were docked onto the constrained
receptor of PfAtg8CM (PDB code 4EOY) with OpenEye docking suite.15 (B) Direct binding of the PTA scaffold to PfAtg8. PfAtg8CM and hLC3 were
injected over immobilized 8, and binding was measured with SPR. (C) Superposition of small molecule hits derived from MMV Malaria Box with 9. The
individual molecular surfaces with their corresponding electrostatic potential are depicted in side and top view. All four molecules share the PTA moiety
and were superimposed using ROCS via shape complementarity and Tanimoto color scoring function.46 The figure was prepared with Vida and was
rendered in PovRay (www.povray.org).
compared to an enzymatic site. However, PPIs also offer the
opportunity for more selectivity, an important concept when
targeting a eukaryotic parasitic protein within a eukaryotic host.
Great methodological and technological advances have been
made recently using this approach with promising leads in cancer
drug development.26−28 In the case of our inhibitor, in addition
to its potential as a therapeutic drug, any 1-derived inhibitor
could serve as a tool to elucidate the function of PfAtg8 during
different stages of the malaria life cycle as its function is currently
unclear.29 Treatment of P. falciparum with high levels of 1 led to a
drastic increase in PfAtg8 protein levels, presumably the
unlipidated form as judged by its migration in sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This
increase could be due to an up-regulation of PfAtg8 synthesis to
compensate for inhibition or due to a buildup of existing protein
levels because of a blockade in autophagic degradation. In yeast,
nitrogen starvation leads to induction of Atg8 expression while
inhibition of later stages of autophagy leads to accumulation and
even greater protein levels of Atg8.30,31 Further studies are
necessary to determine if PfAtg8 is up-regulated at the
transcriptional, translational, or degradation level in response
to treatment with 1.
The IC50 in parasite cultures was much lower than against the
protein−protein interaction as measured via SPR. This could be
due to an accumulation of the drug inside the parasite or could be
because even slight inhibition of PfAtg8 lipidation has drastic
effects on parasite growth, similar to reaching the tipping point
on a balance. An alternative explanation is the result of off-target
effects; however, the observed delipidation of PfAtg8 could not
be attributed to an off-target effect. 1 was previously reported to
have low cytotoxicity with an LD50 (lethal dose) of 18.2 μM in
HepG2 cells and half maximal cytotoxicity concentration (CC50)
and IC50 of 32 μM in Huh7 cells (PubChem AID: 685525,
660872, 449705). In our study, we did not observe cell death in
HC-04 cells at 30 μM. Together, this indicates 1 may be a good
starting scaffold for antimalarial drug design.
Compound 1 had lower activity in the liver stage than in the
blood stage, which could either indicate that PfAtg8 is less
important in the liver stage or that less compound is delivered to
the parasite in liver cells. There is precedence for this variability in
the efficacy of antimalarials between the liver and blood stages.32
Compound 1 showed 50% inhibition of liver stage parasites at 30
μM, which is within the range for cytotoxicity in human cells. We
suggest the use of 1 and the PTA scaffold for further probe
development to increase specificity and potency in both the liver
and blood stages. All PTA-containing MMV compounds had a
ligand efficiency by atom number (LEAN) score greater than 0.3
in the blood stage assay, indicative of good ligand efficiency and
good potential for future drug optimization.33 This combined
with the higher affinity of 8 for PfAtg8 compared to human LC3
makes the PTA scaffold a promising starting point for expansion.
We are currently pursuing optimization through a combinatorial
oxime library approach using 7 with the goal to extend the
inhibitor into the A-loop pocket and to gain selectivity toward
PfAtg8.
Our SPR data confirm that 1 directly binds to PfAtg8, while
our docking studies suggest that the PTA scaffolds of compounds
1−3, 7, and 9 bind the W-site. The W-sites of Atg8 homologues
in mammals and yeast participate in numerous protein−protein
interactions through binding to the aromatic residue of an AIM,
including nonautophagic proteins.35−38 Therefore, our inhibitor
and its derivatives could be used to identify novel Plasmodium
Atg8 interactions as well as to confirm paralogous interactions
known to occur in yeast and mammalian cells.
P. yoelii as a Liver Stage Model. Taken together, our
bioinformatics analysis of the PTA-binding site and the SPR
binding studies strongly suggest that the amino acid differences
near the L- and W-site may have contributed to the failure of 1 in
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Surface Plasmon Resonance Assays. SPR runs were conducted
on a Biacore 3000 instrument (GE Healthcare) at 25 °C with a flow rate
of 50 μL/min, unless otherwise specified. Running buffer (RB)
consisted of 1× phosphate-buffered saline (PBS) (1 mM KH2PO4, 5.6
mM Na2HPO4, 154.5 mM NaCl, pH 7.4), 0.01% v/v P20, and varying
amounts of DMSO (Quality Biologicals). Binding and equilibrium
constants were determined with Scrubber (BioLogic). A double
referencing method was applied to correct for nonspecific binding to
the chip with interspersed blank injections correcting for baseline drifts.
Changes in refractive index because of DMSO were accounted for with a
DMSO calibration curve.
MMV SPR Competition Assay. MBP-PfAtg3 was immobilized
onto a CM5 chip (Biacore) as described previously with MBP
immobilized on a reference flowcell.13 Compounds were added to 300
nM His6-PfAtg8CM in RB at a final concentration of 5 μM, and 40 μL was
injected, followed by a 12.5 μL injection of 2 M MgCl2 for dissociation
and regeneration of the SPR chip surface. The final DMSO
concentration in SPR runs was 3%.
SPR Dose-Dependent Inhibition. Thirty microliters of PfAtg8CM
at 200 nM was injected in the presence of a 2-fold dilution series of
compound (highest concentration of 50 μM) or equivalent volume of
DMSO (final DMSO concentration was 1%). Each injection was
followed by a 10 μL injection of 2 M MgCl2. All measurements were
conducted in triplicate.
Direct Binding of 1 to PfAtg8CM. His12-PfAtg8CM was injected over
an NTA-chip (GE Healthcare) preconditioned with nickel, leading to
capture of 3000 response units (RUs). Running buffer contained 3%
DMSO. A 2-fold-dilution series of compounds, highest concentration of
75 μM, was injected over PfAtg8 variants at 40 μL/min.
Direct Binding of 8 to PfAtg8CM. 8 was injected over a neutravidin-
coated SPR chip (GE Healthcare) on one flowcell, with 130 RUs
immobilized, while BACH was injected over a reference flowcell (150
RUs immobilized). His6-PfAtg8CM, Human LC3, or His6-PfAtg3 was
injected in duplicate in running buffer containing 10 mM HEPES pH
7.5, 150 mM NaCl, 0.05% P20. Protein was dissociated from the chip
after each cycle with 10 μL injection of 20 mM HEPES pH 7.4, 1% w/v
SDS regeneration solution.
Synthesis of PTA Derivatives. General. All reagents were obtained
from commercial suppliers and were used without further purification.
Acetonitrile was distilled after drying on CaH2 and then was stored over
3 Å molecular sieves. Yields of all reactions refer to the purified products.
Dynamic Adsorbents 32−63 μm silica gel was used for flash column
chromatography, and 250 μm F254 plates were used for thin layer
Figure 8. Validation of 7 as starting point for optimization. (A)
Inhibition of PfAtg8-PfAtg3 interaction by 9. SPR response of PfAtg8
injected over immobilized PfAtg3 was measured in the presence of
increasing concentration of 9. IC50 was determined as 2.86 μM. Mean
SD of three injections are shown. (B) Inhibition of blood stage parasites
9. SYBR green I assays were used to measure inhibition by Chloroquine
(CQ), 1, and 9. Growth inhibition curves are shown for one experiment
with IC50 values from two to three experiments in table inset.
chromatography (TLC). Microwave-assisted reactions were carried out
1
using a Biotage Initiator Microwave Synthesizer (300 W). H and 13
C
NMR spectra were acquired on a Bruker Avance III 500 spectrometer
operating at 500 MHz for H and 125 MHz for 13C. Chemical shift
1
the P. yoelii liver stage drug-screening assay. Two residue
differences (I8V, P9S) adjacent to the W-site may lead to a
widening of the W-site in P. yoelli because of their shorter side
chains, and one residue (V62I) participating directly in the L-site
results in an extended side chain and therefore may decrease the
volume of the L-site pocket. Additionally, L115M just below the
L-site binding pocket may also contribute to a decreased volume
as a secondary shell residue. Care and emphasis on choice of
model system should be taken into account when studying
protein−ligand interactions, as single amino acid substitutions
may have drastic effects on binding.
values are reported as δ (ppm) relative to CHCl3 at δ 7.27 ppm and
1
DMSO at δ 2.50 ppm for H NMR and CHCl3 at δ 77.0 ppm and
DMSO at δ 39.51 ppm for 13C NMR. Mass spectrometry analysis was
carried out at University of Illinois at Urbana-Champagne, School of
Chemical Sciences, Mass Spectrometry Laboratory. The purity of
synthesized compounds was ≥95% as analyzed by high-performance
liquid chromatography (HPLC, Beckman Gold Nouveau System Gold)
on a C18 column (Grace Alltima 3 μm C18 analytical Rocket column, 53
mm × 7 mm) using triethylammonium acetate buffer (50 mM, pH 7)
and acetonitrile (ACN) as eluent, flow rate 3 mL/min, and detection at
300 nm.
4-Formyl-N-[4-(pyridin-2-yl)-1,3-thiazol-2-yl]benzamide (7).
To a solution of 4-(pyridin-2-yl)-1,3-thiazol-2-amine (0.059 g, 0.33
mmol) in acetonitrile (2.0 mL) was added sequentially dicyclohex-
ylcarbodiimide (0.076 g, 0.37 mmol), 4-formylbenzoic acid (0.050 g,
0.33 mmol), and N,N-dimethylamino pyridine (0.012 g, 0.10 mmol).
The mixture was heated at 50 °C for 17 h and then was allowed to cool to
ambient temperature. Solids were removed by vacuum filtration, and the
resulting filtrate was condensed under reduced pressure. The resulting
yellow solid was redissolved in CHCl3 (5 mL), and 1 M HCl (5 mL) was
added to give a yellow-tan emulsion at the liquid−liquid interface. This
solid was collected by centrifugation at 4000 rpm for 5 min followed by
manual collection of the resulting cake (this acid precipitation was
EXPERIMENTAL SECTION
Materials. All reagents, unless specified, were purchased from
Sigma−Aldrich.
■
Protein Expression and Purification. Proteins were cloned,
expressed, and purified as described elsewhere.13 His12-PfAtg8CM
variants were expressed and purified with Cobalt-NTA affinity columns
similar to His6-PfAtg8CM, as previously published with the exception that
proteins were eluted from cobalt-charged TALON resin (Clonetech) in
buffer containing 50 mM ethylenediaminetetraacetic acid (EDTA)
rather than imidazole.13
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Journal of Medicinal Chemistry
Article
necessary to remove closely eluting impurities). The solid was then
purified by silica flash column chromatography (dichloromethane-
(DCM):MeOH:triethylamine 94:5:1) Rf =0.32. The product was
obtained as a yellow powder (23 mg, 22% yield). 1H NMR (500
MHz, DMSO-d6) δ (ppm) = 12.95 (br. s., 1H), 10.13 (s, 1H), 8.63 (d, J
= 3.93 Hz, 1H), 8.31 (d, J = 8.17 Hz, 2H), 8.07 (d, J = 8.33 Hz, 2H), 8.03
(d, J = 7.86 Hz, 1H), 7.92 (s, 1H) 7.91 (td, J = 2.00 Hz, 8.75 Hz, 1H),
7.35 (ddd, J = 1.10, 4.79, 7.47 Hz, 1H) 13C NMR (500 MHz, DMSO-d6)
δ (ppm) = 192.90, 164.74, 158.91, 152.03, 149.54, 149.38, 138.50,
137.30, 137.15, 129.41, 128.94, 122.88, 120.06, 112.30. High-resolution
mass spectrometry (HRMS, electrospray ionization, ESI) m/z: calcd
310.0650 (M − H+); found 310.0651 (M − H+).
4-[[2-[6-[5-[(3aR, 4R, 6aS)-2-Oxo-1, 3, 3a, 4, 6, 6a-
hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]-
hexanoyl]hydrazinyl]methyl]-N-(4-pyridin-2-yl-1,3-thiazol-2-
yl)benzamide (8). Twenty-five microliters 50 mM 7 dissolved in
DMSO was incubated with 20 μL 50 mM BACH (5-[(3aS,4S,6aR)-2-
oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]-N-(6-hydrazin-
yl-6-oxohexyl)pentanamide) (Sigma-Aldrich) dissolved in DMSO and
10 μL sodium acetate (pH 4.5) with 0.02% sodium azide at 37 °C
overnight. Final concentration of 8 was 18 mM.
4-(Dimethoxymethyl)-N-[4-(pyridin-2-yl)-1,3-thiazol-2-yl]-
benzamide (9). 7 dissolved in CHCl3 was treated with 1 M HCl as
described above to form the hydrochloride salt. 7 HCl (0.040 g, 0.12
mmol) was suspended in MeOH (0.5 mL), and trimethylorthoformate
(0.010 mL, 0.91 mmol) was added followed by p-toulene sulfonic acid
monohydrate (0.003 g, 0.012 mmol). This solution was heated by
microwave irradiation at 130 °C in a sealed vial for 5 min and then was
stirred at ambient temperature for 36 h at which time a precipitate
formed. The solvent was removed under reduced pressure, and the
residue was dissolved in DCM (10 mL) and was washed with saturated
NaHCO3 (10 mL) and brine (10 mL) and was dried with Na2SO4.
Condensation under reduced pressure yielded the product as a yellow
powder (26 mg, 61% yield). 1H NMR (500 MHz, CDCl3) δ (ppm) =
9.81 (br s, 1H), 8.64 (d, J = 4.24 Hz, 1H), 7.96 (d, J = 8.17 Hz, 2H), 7.91
(d, J = 7.86 Hz, 1H), 7.74 (s, 1H), 7.74 (td, J = 1.73, 7.70 Hz, 1H), 7.62
(d, J = 8.17 Hz, 2H), 7.22 (dd, J = 4.95, 6.84 Hz, 1H), 5.47 (s, 1H), 3.35
(s, 6H) 13C NMR (500 MHz, CDCl3) δ (ppm) = 164.24, 158.12,
152.24, 149.85, 149.62, 143.32, 136.84, 131.80, 127.51, 127.29, 122.69,
120.50, 112.21, 102.08, 52.71. HRMS (ESI) m/z: calcd 356.1069 (M −
H+); found 356.1070 (M − H+).
well plate and were treated with 3 μM or 30 μM of 1 in complete
medium for 96 h, and a relevant amount of DMSO was used as a vehicle
control. Detection of Annexin-V positive and PI-positive cells in
hepatocyte cultures was done by flow cytometry according to the
manufacturer’s instruction (Invitrogen, Life Technologies, Grand
Island, NY, U.S.).
P. falciparum Blood Stage Culturing. P. falciparum 3D7 and
FCR3 cultures were maintained using modified, previously published
methods at 37 °C, 2% hematocrit of human red blood cells.39 Complete
culture media consisted of sterile RPMI 1640 media (Life Technologies)
supplemented with 10% human serum and 0.005% hypoxanthine and
buffered with final concentrations of 0.6% HEPES and 0.26% NaHCO3.
The FCR3 strain was maintained at 3% CO2 and 5% O2, 92% N2
atmosphere, while the 3D7 strain was maintained at 5% CO2, 5% O2,
and 90% N2 atmosphere.
Immunoblot Analysis of P. falciparum Blood Stage. P.
falciparum FCR3 (generously provided by Dr. J. Smith, Seattle BioMed)
asynchronous culture, 25% parasitemia, was washed in starvation media
lacking human serum and was resuspended in complete media with 50
μM compound 1 or equivalent DMSO or in starvation media with
equivalent DMSO for 5 h. RBCs were harvested with centrifugation and
were lysed with 0.2% saponin, and RBC lysate was removed through
three 1× PBS washes. Parasites were harvested by centrifugation and
were washed in 1× PBS with complete EDTA-free protease inhibitors
(Roche) and were lysed by repeated vortexing and boiling in SDS
reducing sample buffer. Lysates were separated with SDS-PAGE on a
4−20% polyacrylamide gel and were subjected to Western blotting with
1:400 α-TgAtg8, demonstrated to be cross-reactive with PfAtg840
(generously provided by Dr. P. Roepe, Georgetown University). HRP-
conjugated secondary antibodies (Southern Biotech) were detected by
SuperSignal West Femto (Thermo Scientific) or Amersham ECL Prime
(GE Healthcare) chemiluminescent substrate. AP-conjugated secon-
dary antibodies (EMD Millipore) were detected using NBT/BCIP
(Promega) colorimetric stain. Total protein levels were visualized with
ProAct Membrane Stain (Amresco) and were quantified with ImageJ.41
Parasite morphology at time of harvesting was visualized with light
microscopy at 100× magnification on Olympus BX53 system
microscope (Olympus America, Inc.).
SYBR Green I Growth Inhibition Assay. Ten microliters of 10×
compound diluted in RPMI 1640 media (Gibco) with a constant
concentration of 1% DMSO was added to a 96 well plate (Costar), 90
μL of 1.5% ring stage, synchronized with 5% w/v sorbitol P. falciparum
3D7 parasites, 1% hematocrit, in culture media with 10% v/v human
serum with 10 μg/mL gentamycin. Each compound concentration and
1% v/v DMSO controls were run in triplicate. Plates were incubated at
37 °C in 5% O2, 5% CO2, and 90% N2 for 72 h. Plates were frozen,
thawed, and incubated with 100 μL 2× SYBR green in lysis buffer (20
mM Tris pH 7.5, 5 mM EDTA, 0.008% Saponin, 0.08% TritonX-100) in
the dark for at least 1 h. Fluorescence was measured with a plate reader
(HTS 7000, PerkinElmer) at excitation/emission wavelengths of 485/
535 nm.
Thermal Shift Assays. Assays were conducted in 1× PBS with
1:1800 final dilution of SYPRO orange dye (Invitrogen). Measurements
were made in triplicate for each condition. The concentration of His12-
PfAtg8CM or His6-PfAtg8CM in assay was 65 μM. Fluorescence was
measured from 20 to 80 °C in a Biorad C1000 thermal cycler. One
hundred microliters of PTA compounds or equivalent volume of DMSO
was added to His6-PfAtg8CM
.
In Vitro Infection of Human Hepatocytes with P. falciparum
3D7-GFP. The HC-04 cell line (ATCC, Manassas, VA, U.S.) was
maintained in complete medium (IMDM containing 2.5% FCS, 100
units/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine,
all from GIBCO, Life Technologies, Grand Island, NY). P. falciparum
3D7-GFP parasite strain23 was propagated in the Parasitology Core
facility, the Johns Hopkins Malaria Research Institute. In vitro infection
of human hepatocytes was done as described previously.20 Briefly,
salivary glands were sequestered from infected Anopheles gambiae
mosquitoes at day 17 after exposure to infective blood meal, and
homogenates were separated on an OptiPrep Density Gradient (Sigma-
Aldrich, St. Louis, MO) at 12 000g for 10 min. Sporozoites were
collected from the gradient interface, were washed in complete medium,
were counted using a hemocytometer, and were incubated with HC-04
cells at 3:1 sporozoite to hepatocyte ratio for 2 h at 37 °C. Infected
cultures were further propagated in complete medium alone or in
medium supplemented with 3 μM or 30 μM of 1. Flow cytometry based
detection of infected cells was done 72 h post infection using
FACSCalibur flow cytometer (BD Biosciences) and was analyzed
using FlowJo software (Tree Star, Inc., Ashland, OR).
In Silico Docking. Docking was conducted using the OpenEye
software package using standard parameters if not specified otherwise
(www.eyesopen.com).15 A receptor for PfAtg8 was made with
make_receptor from OEDocking toolkit without constraints covering
the whole molecule to detect potential binding pockets on the surface.
Three main pockets (W-site, L-site, A-site) were detected with 377, 271,
and 513 Å3 volumes, used in first docking studies. A second receptor was
generated with specific constraints to the carbonyl of Lys47 as hydrogen
bond donor. A maximum of 2000 conformers for each compound from
the MMV Malaria Box was prepared with Omega2.42,43 FRED was used
to dock these conformers onto both the constrained and unconstrained
receptor.15 Docking results were visualized using the OpenEye
visualization software, VIDA.
Generation of Homology Models for P. yoelii and P. berghei
Atg8. Using iTasser, models of P. yoelii and P. berghei Atg8 were
generated with PDB code 4EOY as the parent molecule.13,44 Default
values as suggested by the Web server were used to generate these
models. Sequences for P. yoelii (PYYM_0504500) and P. berghei
(PBANKA_050410) were obtained from PlasmoDB.45
Effect of 1 on the viability of in vitro propagated HC-04 cells was
monitored as follows: 0.3 × 106 cells per well were seeded into the 24-
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Journal of Medicinal Chemistry
Article
Human Subjects and Hazards. P. falciparum, a human pathogen, is
cultivated in human erythrocytes. The organisms are maintained in
licensed BSL2 facilities, and approvals are obtained annually for all
consortia laboratories. Human erythrocytes are obtained either
commercially or from healthy volunteers under Johns Hopkins IRB-
approved protocols. Because these cells are provided to the lab without
identifiers, their use does not constitute human investigation.
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ASSOCIATED CONTENT
* Supporting Information
■
S
A detailed analysis of the PfAtg8 Western blot with
quantification. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: 410-614-4742. Fax: 410-955-2926. E-mail: jbosch@jhu.
edu, jubosch@jhsph.edu.
■
(8) Tomlins, A. M.; Ben-Rached, F.; Willams, R. A. M.; Proto, W. R.;
Coppens, I.; Ruch, U.; Gilberger, T. W.; Coombs, G. H.; Mottram, J. C.;
Muller, S.; Langsley, G. Plasmodium falciparum Atg8 implicated in both
̈
Author Contributions
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Colombo, M. I.; Coombs, G. H.; Coppens, I.; Jayabalasingham, B.;
Langsley, G.; de Castro, S. L.; Menna-Barreto, R.; Mottram, J. C.;
Navarro, M.; Rigden, D. J.; Romano, P. S.; Stoka, V.; Turk, B.; Michels,
P. A. Autophagy in protists. Autophagy 2011, 7, 127−158.
The manuscript was written through contributions of all authors.
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manuscript. D.B. and C.F.M. synthesized compounds. A.U.H.,
J.L., N.S., A.S.M., D.S., and J.B. designed research. The funders
had no role in study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
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Ishihara, N.; Mizushima, N.; Tanida, I.; Kominami, E.; Ohsumi, M.;
Noda, T.; Ohsumi, Y. A ubiquitin-like system mediates protein
lipidation. Nature 2000, 408, 488−492.
Notes
The authors declare no competing financial interest.
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Ohsumi, Y.; Inagaki, F. Autophagy-related protein 8 (Atg8) family
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ACKNOWLEDGMENTS
■
We thank the five anonymous reviewers, who have helped us to
improve our manuscript. We thank Stephanie Trop, Peter
Dumoulin, and Jinxia Ma for dissection of P. falciparum-infected
mosquitoes for our assays. We thank Dr. Krista Matthews for
technical help. We thank the Johns Hopkins Malaria Research
Institute parasite and insectary facility for assistance with our
experiments. This work was partially funded through The
Bloomberg Family Foundation (J.B.), a grant (NIH AI099704
for C.F. M. and D.B.), and a Predoctoral fellowship from the
Johns Hopkins Malaria Institute for A.U.P.H. We thank Dr. P.
Roepe (Georgetown University), Dr. Anthony Sinai (University
of Kentucky), and Dr. N. Kumar (Tulane University) for
antibodies and Dr. J. Smith (Seattle BioMed) for the FCR3 strain
of P. falciparum. We thank The Medicines for Malaria Venture for
supply of the malaria box.
ABBREVIATIONS
■
Pf, P. falciparum; SPR, surface plasmon resonance; TSA, thermal
shift assay; CM, cysteine mutant; AIM, Atg8 family interacting
motif; RBC, red blood cell; RU, response units; PTA, 4-pyridin-
2-yl-1,3-thiazol-2-amine
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