5 research outputs found
Transition State Mimetics of the Plasmodium Export Element Are Potent Inhibitors of Plasmepsin V from P. falciparum and P. vivax
Following
erythrocyte invasion, malaria parasites export a catalogue
of remodeling proteins into the infected cell that enable parasite
development in the human host. Export is dependent on the activity
of the aspartyl protease, plasmepsin V (PMV), which cleaves proteins
within the Plasmodium export element
(PEXEL; RxLāxE/Q/D) in the parasiteās endoplasmic reticulum.
Here, we generated transition state mimetics of the native PEXEL substrate
that potently inhibit PMV isolated from Plasmodium
falciparum and Plasmodium vivax. Through optimization, we identified that the activity of the mimetics
was completely dependent on the presence of P<sub>1</sub> Leu and
P<sub>3</sub> Arg. Treatment of P. falciparum-infected erythrocytes with a set of optimized mimetics impaired
PEXEL processing and killed the parasites. The striking effect of
the compounds provides a clearer understanding of the accessibility
of the PMV active site and reaffirms the enzyme as an attractive target
for the design of future antimalarials
Pyridyl Benzamides as a Novel Class of Potent Inhibitors for the Kinetoplastid Trypanosoma brucei
A whole-organism screen of approximately
87000 compounds against Trypanosoma
brucei brucei identified a number of promising compounds
for medicinal chemistry optimization. One of these classes of compounds
we termed the pyridyl benzamides. While the initial hit had an IC<sub>50</sub> of 12Ā Ī¼M, it was small enough to be attractive
for further optimization, and we utilized three parallel approaches
to develop the structureāactivity relationships. We determined
that the physicochemical properties for this class are generally favorable
with particular positions identified that appear to block metabolism
when substituted and others that modulate solubility. Our most active
compound is <b>79</b>, which has an IC<sub>50</sub> of 0.045
Ī¼M against the human pathogenic strain Trypanosoma
brucei rhodesiense and is more than 4000 times less
active against the mammalian L6 cell line
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 (AāC) and is relative to DMSO treatment in (B) and (C). Concentrations are as follows: 916, 024, 025 (15 ĀµM); CQ, chloroquine (150 ng/ml); ART, artemisinin (100 ng/ml). Error bars in (AāC) are mean Ā±SEM from duplicate experiments. (D) Light micrographs of Giemsa-stained parasites 16 and 32 h after drug treatment at early rings (15 ĀµM). 916-treated parasites failed to develop into trophozoites and did not recover. Ring parasites treated with E-64 (10 ĀµM) <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 Āµm.</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 Ī±-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 Ī±-PfPMV antibodies (RĪ±-PfPMV) validates they are specific for PfPMV. Endogenous PfPMV is the lower band (lanes 1, 3, 4, 5), and the larger band corresponds to 3Ć 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 Ī±-PfPMV antibodies (RĪ±-PfPMV, green) label PfPMV in the ER. Colocalizations were performed with mouse Ī±-PfPMV antibodies (MĪ±-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) Ī±-HA antibodies (red) label PfPMVHA (Top) and PvPMVHA (Bottom) in the parasite ER. (Bottom) Ī±-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 Ā± SEM from duplicate experiments.</p
PMV knockdown or overexpression modulates sensitivity to WEHI-916.
<p>(A) Immunoblot with RĪ±-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 (Ī±-PfPMV blot) in lane 1 (denoted by *) is nonspecific. The same blot is shown below, probed with Ī±-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 ā¼80% knockdown of PMV in subsequent rings compared to āāGlcN.ā (Right) 48 h GlcN treatment of trophozoites causes ā¼90% knockdown of PMV in subsequent trophozoites compared to āāGlcN.ā 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>ā=ā.6250). Trophozoites were treated with 0 mM or 5 mM GlcN and parasitaemia determined 48 h later by flow cytometry. Data are % growth ā+GlcNā relative to āāGlcN,ā and data are mean Ā±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 ā+GlcNā 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