9 research outputs found
WEHI-916 is lethal to <i>P. falciparum</i> 3D7.
<p>(A) Dose-response curves of <i>P. falciparum</i> 3D7 in the presence of 916, 024, or 025. EC<sub>50</sub> values are shown. (B) Parasitemia measured at 72 h (<i>y</i>-axis) following drug treatment at rings (30 min postinvasion) and replacement of the medium with inhibitor-free medium (wash-out) at the time intervals shown (<i>x</i>-axis). (C) Parasitemia at 72 h (<i>y</i>-axis) after replacement of inhibitor-free medium with media containing compounds at the intervals shown (<i>x</i>-axis). Parasitemia was determined by FACS in (AâC) and is relative to DMSO treatment in (B) and (C). Concentrations are as follows: 916, 024, 025 (15 ”M); CQ, chloroquine (150 ng/ml); ART, artemisinin (100 ng/ml). Error bars in (AâC) are mean ±SEM from duplicate experiments. (D) Light micrographs of Giemsa-stained parasites 16 and 32 h after drug treatment at early rings (15 ”M). 916-treated parasites failed to develop into trophozoites and did not recover. Ring parasites treated with E-64 (10 ”M) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001897#pbio.1001897-terKuile1" target="_blank">[22]</a> contained swollen food vacuoles (arrow) due to inhibition of proteases involved in hemoglobin degradation; however, treatment with DMSO, 916, 024, or 025 did not cause swelling. Swelling was quantified using 500 infected cells per condition in duplicate. Scale bar is 6 ”m.</p
PMV conservation and expression.
<p>(A) Structure and size of PMVHA proteins used in this study. Catalytic dyad residues DTG/DSG are shown including Asp to Ala mutations* in red. TM, transmembrane domain. (B) Immunoblot of infected erythrocytes with α-HA antibodies shows expression of PMVHA proteins in <i>P. falciparum</i>. Sizes indicate that the signal peptides were removed (PfPMVHA, 69.1 kDa; PvPMVHA, 60.9 kDa). (C) Immunoblotting of infected erythrocytes with rabbit α-PfPMV antibodies (Rα-PfPMV) validates they are specific for PfPMV. Endogenous PfPMV is the lower band (lanes 1, 3, 4, 5), and the larger band corresponds to 3à HA-tagged PfPMV (lanes 2, 4). Aldolase is a loading control in (B) and (C) and shows slight overloading of some lanes compared to others. (D, Top) Immunofluorescence micrographs show rabbit α-PfPMV antibodies (Rα-PfPMV, green) label PfPMV in the ER. Colocalizations were performed with mouse α-PfPMV antibodies (Mα-PfPMV, red), shown previously to label PMV in the ER <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001897#pbio.1001897-Klemba1" target="_blank">[16]</a>. (Middle) α-HA antibodies (red) label PfPMVHA (Top) and PvPMVHA (Bottom) in the parasite ER. (Bottom) α-HA antibodies (red) label PvPMVHA in the ER, as shown by clocalization with ERC (green). (E) Immunopurified PfPMVHA and PvPMVHA cleave KAHRP peptides containing the PEXEL sequence RTLAQ but not peptides containing point mutations R>K, L>I, or RL>A. Pf and Pv PMVmutHA proteins with catalytic D>A mutations did not cleave the KAHRP RTLAQ peptide. (F) Overexpression of PfPMVmutHA from episomes in <i>P. falciparum</i> 3D7 impairs growth relative to expression of a similar episomal construct encoding a mini PfEMP1HA reporter (miniVarHA). Parasites expressing episomes were selected on 5 nM WR99210 (WR). Two starting inocula were used in triplicate wells, and parasitaemia was determined at 72 h. *<i>p</i><.0001 (<i>t</i> test). Data are mean ± SEM from duplicate experiments.</p
PMV inhibition impairs protein export, PfEMP1 display, and cytoadherence.
<p>(A) (Top) Immunofluorescent micrographs show Hyp8-GFP is exported and localizes within puncta in the infected erythrocyte. (Middle) Hyp8-HA localizes within SBP1-containing MCs. (Right) Immunoelectron microscopy shows Hyp8-HA localization at MCs. Scale bar is 100 nm. (B) Maximum intensity projection micrographs showing export of Hyp8-GFP to MCs and secretion of EXP2 to the parasitophorous vacuole membrane following treatment with DMSO or 916 (50 ”M). Puncta of nonexported GFP within the parasite and vacuole is shown (arrows). (C) GFP intensity in MCs (outside EXP2 signal) and within the parasite and parasitophorous vacuole (inside EXP2 signal) was quantified following drug treatment (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001897#s4" target="_blank">Materials and Methods</a>) and is presented as a ratio: exportedâ=âratio outside/inside EXP2. The number of infected cells counted (<i>n</i>) is shown. (D) GFP within the parasite and parasitophorous vacuole (inside EXP2 signal) was quantified across treatments and is presented as a ratio: nonexportedâ=âratio inside/outside EXP2. (E) EXP2 intensity in the parasitophorous vacuole membrane (red) was quantified across treatments and is presented as average (threshold) signal. (F) The number of GFP-positive MCs per infected erythrocyte was quantified across treatments. Error bars represent the mean ±SEM, and <i>p</i> values were determined by ANOVA in (CâF). (G) Surface-exposed PfEMP1 (VAR2CSA) on infected erythrocytes was measured following inhibitor treatment by FACS using monoclonal human PAM1.4 serum <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001897#pbio.1001897-Barfod1" target="_blank">[30]</a>. Geometric mean fluorescence of >100,000 cells per condition are shown relative to no treatment. Parasites received inhibitor at 15 ”M for 23 h (15 ”M) or 50 ”M for 12 h followed by a reduction to 15 ”M for 11 h (50>15 ”M). Shown is a single representative of duplicate experiments. Raw FACS data are presented in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001897#pbio.1001897.s007" target="_blank">Figure S7</a>. (H) Adhesion of infected red blood cells (iRBCs) to chondroitin sulfate A (CSA) under static conditions. Adherent iRBCs were counted in ten 0.28 mm<sup>2</sup> fields of view per sample from triplicate samples and are shown as number of iRBCs per mm<sup>2</sup>. Shown is a single representative of duplicate experiments. Data represent mean ±SEM. 95% confidence intervals are shown (grey dashed lines). 916 significantly reduced adhesion to CSA <i>p</i><.0001.</p
PMV knockdown or overexpression modulates sensitivity to WEHI-916.
<p>(A) Immunoblot with Rα-PfPMV antibodies shows successful integration of the PMVHA-<i>glmS</i> or -M9 plasmid (M9 is an inactive <i>glmS</i> riboswitch control). The upper band (α-PfPMV blot) in lane 1 (denoted by *) is nonspecific. The same blot is shown below, probed with α-HA antibodies. HSP70 is a loading control. (B) Knockdown of PMV in <i>P. falciparum</i> NF54 following 5 mM GlcN treatment. (Left) 0 h GlcN treatment of trophozoites causes no knockdown. (Center) The 24 h GlcN treatment of trophozoites causes âŒ80% knockdown of PMV in subsequent rings compared to ââGlcN.â (Right) 48 h GlcN treatment of trophozoites causes âŒ90% knockdown of PMV in subsequent trophozoites compared to ââGlcN.â A small degree of knockdown is seen for M9, indicating GlcN has a minor effect. (C) PMV knockdown by GlcN has no significant effect on parasite growth rate (<i>p</i>â=â.6250). Trophozoites were treated with 0 mM or 5 mM GlcN and parasitaemia determined 48 h later by flow cytometry. Data are % growth â+GlcNâ relative to ââGlcN,â and data are mean ±SEM of a representative of duplicate experiments. (D) PEXEL processing of PfEMP3-GFP in <i>P. falciparum</i> parasites expressing PMVHA-<i>glmS</i> is reduced more by 916 treatment when PMV is knocked down [+GlcN (5 mM for 48 h prior to addition of 916)]. Densitometry shows the ratio of uncleaved to PEXEL-cleaved protein in each lane beneath the blot. Note that PfEMP3-GFP expression is lower in â+GlcNâ parasites despite relatively similar HSP70 levels. (E) Dose-response curves of <i>P. falciparum</i> expressing PMVHA-<i>glmS</i> shows parasites have enhanced sensitivity to 916 following PMV knockdown (3.3-fold decrease in EC<sub>50</sub>). Parasitemia was determined 72 h after addition of 916 to ring parasites with or without PMV knockdown (knockdown ring parasites were obtained by adding 6 mM GlcN to trophozoites for 24 h). GlcN and 916 were maintained in the culture medium throughout. (F) Dose-response curves of <i>P. falciparum</i> overexpressing PvPMVHA or a mini PfEMP1HA reporter (miniVarHA) in the presence of 5 nM WR99210 show parasites have increased resistance to 916 when PMV is overexpressed (1.9-fold increase in EC<sub>50</sub>).</p
WEHI-916 blocks PEXEL cleavage in <i>P. falciparum</i>.
<p>(A) Immunoblotting with α-GFP antibodies shows dose-dependent inhibition of PEXEL cleavage of PfEMP3-GFP in parasites after 5 h 916 treatment at the indicated concentrations. Uncleaved protein (black arrow), PEXEL-cleaved protein (blue arrow), and degraded chimera in food vacuole (GFP only) are labeled. PfEMP3-GFP R>A PEXEL mutant is shown as a size control. (B) Immunoblot shows time-dependent inhibition of PEXEL cleavage in parasites. HSP70 is a loading control in (A) and (B) and densitometry of the uncleaved band in each lane is shown below the blots in (A) and (B). (C) Immunoblot shows 916 treatment (20 and 50 ”M for 5 h) causes accumulation of uncleaved PfEMP3-GFP and KAHRP-GFP (black arrow), but 024 and 025 have no effect. R>A PEXEL mutant size controls are shown. Signal peptide-cleaved species of KAHRP-GFP, but not PfEMP3-GFP, can be seen (red arrow). (D) Immunoblotting shows no defect (black arrow indicates the predicted size of uncleaved protein; âŒ30 kDa) in signal peptide cleavage (red arrow; 27 kDa) of SERA5s-GFP, which lacks a PEXEL, following treatment with 916, 024, or 025 (20 ”M for 5 h). Aldolase was a loading control in (C) and (D). (E) <sup>35</sup>S-Methionine/Cysteine labeling of PfEMP3-GFP in parasites reveals the rapid rate of translation, ER import, and N-terminal processing in <i>P. falciparum</i>. Uncleaved (black arrow), signal peptide-cleaved (red arrow), PEXEL-cleaved (blue arrow), and GFP-only (food vacuole) species are shown. Densitometry of each band per lane (colored traces) is shown. Uncleaved (Un, black), signal peptide-cleaved (SP, red), and PEXEL-cleaved (PEX, blue) bands are shown as percentage of total intensity per lane. Signal peptide-cleaved PfEMP3-GFP can be seen (red arrow, trace). (F) The experiment in (E) was performed after 916 treatment (20 ”M for 5 h). Uncleaved protein was most abundant. Note low intensity of bands indicated by red and blue arrows and delay in their appearance compared to (E). (G) Densitometry showing the ratio of PEXEL-cleaved to -uncleaved PfEMP3-GFP with or without 916 treatment (determined using 15 min lanes in (E) and (F)).</p
A PEXEL-mimetic inhibitor of PMV.
<p>(A) Nomenclature for each residue in the PEXEL substrate (circles) and each pocket of the PMV active site (semicircles) with respect to the cleavage site (arrow). (B) Compound structures in this study include PMV inhibitor WEHI-916 and control analogs WEHI-024 and WEHI-025. (C) Structural model of PfPMV bound to WEHI-916. Residues forming the S<sub>3</sub> site, Try177, Glu179, and Glu215, form interactions with the guanidine side chain of WEHI-916. The Leu side chain of WEHI-916 packs tightly against the side-chain groups of Ile116, Tyr177, and Val227 in the S<sub>1</sub> site. The statine hydroxyl forms hydrogen bonds with the two catalytic aspartate residues Asp118 and Asp365. (D) Inhibition of PfPMV and PvPMV by WEHI-916 (blue) and weak activities of WEHI-024 (red) and WEHI-025 (brown). The grey box summarizes compound activity against BACE-1 and Cathepsin D (CathD) and lack of toxicity against human HEpG2 cells and Human Foreskin Fibroblasts (HFF).</p
Optimization of 2âAnilino 4âAmino Substituted Quinazolines into Potent Antimalarial Agents with Oral in Vivo Activity
Novel
antimalarial therapeutics that target multiple stages of
the parasite lifecycle are urgently required to tackle the emerging
problem of resistance with current drugs. Here, we describe the optimization
of the 2-anilino quinazoline class as antimalarial agents. The class,
identified from publicly available antimalarial screening data, was
optimized to generate lead compounds that possess potent antimalarial
activity against <i>P. falciparum</i> parasites comparable
to the known antimalarials, chloroquine and mefloquine. During the
optimization process, we defined the functionality necessary for activity
and improved <i>in vitro</i> metabolism and solubility.
The resultant lead compounds possess potent activity against a multidrug
resistant strain of <i>P. falciparum</i> and arrest parasites
at the ring phase of the asexual stage and also gametocytogensis.
Finally, we show that the lead compounds are orally efficacious in
a 4 day murine model of malaria disease burden
Optimization of 2âAnilino 4âAmino Substituted Quinazolines into Potent Antimalarial Agents with Oral in Vivo Activity
Novel
antimalarial therapeutics that target multiple stages of
the parasite lifecycle are urgently required to tackle the emerging
problem of resistance with current drugs. Here, we describe the optimization
of the 2-anilino quinazoline class as antimalarial agents. The class,
identified from publicly available antimalarial screening data, was
optimized to generate lead compounds that possess potent antimalarial
activity against <i>P. falciparum</i> parasites comparable
to the known antimalarials, chloroquine and mefloquine. During the
optimization process, we defined the functionality necessary for activity
and improved <i>in vitro</i> metabolism and solubility.
The resultant lead compounds possess potent activity against a multidrug
resistant strain of <i>P. falciparum</i> and arrest parasites
at the ring phase of the asexual stage and also gametocytogensis.
Finally, we show that the lead compounds are orally efficacious in
a 4 day murine model of malaria disease burden
Discovery of Potent and Selective Benzothiazole Hydrazone Inhibitors of BclâX<sub>L</sub>
Developing potent
molecules that inhibit Bcl-2 family mediated
apoptosis affords opportunities to treat cancers via reactivation
of the cell death machinery. We describe the hit-to-lead development
of selective Bcl-X<sub>L</sub> inhibitors originating from a high-throughput
screening campaign. Small structural changes to the hit compound increased
binding affinity more than 300-fold (to IC<sub>50</sub> < 20 nM).
This molecular series exhibits drug-like characteristics, low molecular
weights (<i>M</i><sub>w</sub> < 450), and unprecedented
selectivity for Bcl-X<sub>L</sub>. Surface plasmon resonance experiments
afford strong evidence of binding affinity within the hydrophobic
groove of Bcl-X<sub>L</sub>. Biological experiments using engineered
Mcl-1 deficient mouse embryonic fibroblasts (MEFs, reliant only on
Bcl-X<sub>L</sub> for survival) and Bax/Bak deficient MEFs (insensitive
to selective activation of Bcl-2-driven apoptosis) support a mechanism-based
induction of apoptosis. This manuscript describes the first series
of selective small-molecule inhibitors of Bcl-X<sub>L</sub> and provides
promising leads for the development of efficacious therapeutics against
solid tumors and chemoresistant cancer cell lines