9 research outputs found
Maturation dynamics of cultured <i>P. falciparum</i> and <i>P. cynomolgi</i> hepatic forms.
<p>The proportion of PE forms at different stages of maturation (as assessed by the extent of nuclear division) is presented for cultures of increasing age. Uninucleate parasite forms correspond to the small forms (PE-uni) shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018162#pone-0018162-g001" target="_blank">Figure 1</a>, while all others correspond to the mat-PE forms. The data was representative of three independent experiments.</p
Selection of <i>P. cynomolgi</i> PE-uni forms prior to screening for hypnozoiticidal activity.
<p>Cultures were treated with atovaquone (ATQ) at 67 ng/ml (182 nM) for three days starting on Day 5 post-inoculation in order to eliminate the mat-PE parasites. Subsequently the cultures were either left untreated, treated for a further two days with ATQ (as above), or primaquine (PQ) at 10 µg/ml (22 nM). The cultures were then stopped by methanol fixation on Day 10 and the hepatic forms present enumerated. The data was representative of three independent experiments. *, <i>p</i><0.05.</p
Slow growing and normally developing pre-erythrocytic (PE) forms.
<p>Two types of hepatic parasites can be clearly distinguished from days 5 post-infection onwards, in <i>in vitro</i> cultured human or <i>M. fascicularis</i> primary hepatocytes infected with <i>P. falciparum</i> or <i>P. cynomolgi</i> sporozoites, respectively. The representative photomicrographs were made on cultures fixed on Day 5 and Day 11 post-infection. The parasite and host nuclei are stained with DAPI (blue), while the parasites are labelled by an antibody specific to the HSP70 of the two parasite species (green). Mature PE forms (Mat-PE) and uninucleate small PE forms (PE-uni) are clearly distinguishable.</p
Construction and analysis of fluorescent <i>P. cynomolgi</i> using a novel centromere construct.
<p>(A) Dot matrix analysis of a <i>P. cynomolgi</i> and <i>P. vivax</i> putative centromere (PCEN). Graphical representation of a matrix analysis of a <i>P. cynomolgi</i> PCEN aligned against itself (<i>left</i>), <i>P. cynomolgi</i> PCEN against the <i>P. vivax</i> PCEN (<i>middle</i>) and <i>P. vivax</i> PCEN aligned against itself (<i>right</i>). The analysis was performed using Dotlet <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054888#pone.0054888-Junier1" target="_blank">[46]</a> as described before <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054888#pone.0054888-Iwanaga1" target="_blank">[22]</a>. The diagonal line within each analysis represents sequence identity, and the diagonal line indicates repetitive regions within each PCEN. Note the absence of the diagonal in the repetitive regions of the <i>P. cynomolgi</i> and <i>P. vivax</i> alignment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054888#pone-0054888-g001" target="_blank">Figure 1</a>, <i>middle</i> panel). (B) Schematic representation of the pPcyC-PAC-GFP<sub>hsp70</sub>-mCherry<sub>ef1α</sub> plasmid. The plasmid contains the <i>Tgdhfr-ts</i> selectable marker that confers resistance against pyrimethamine and two expression cassettes for constitutive expression of GFP and mCherry. Additionally, to maintain the plasmid throughout the life cycle, a putative <i>P. cynomolgi</i> centromere (PcyCEN) is included. (C) Schematic representation of the procedure used for transfection and analysis of <i>P. cynomolgi</i>. (D) PCR amplification of <i>gfp</i> and <i>mCherry</i> in PcyC-PAC-GFP<sub>hsp70</sub>-mCherry<sub>ef1α</sub> (PcyC-PAC) blood stage parasites. Wild type gDNA of <i>P. cynomolgi</i> M served as negative control. For a control PCR primers for the <i>circumsporozoite protein (csp)</i> were used. For primers used, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054888#pone.0054888.s001" target="_blank">Table S1</a>. (E) GFP and mCherry expression throughout the life cycle of <i>P. cynomolgi.</i> GFP and mCherry expression in pPcyC-PAC-GFP<sub>hsp70</sub>-mCherry<sub>ef1α</sub> transfected <i>P. cynomolgi</i> blood stage parasites (a ring and a trophozoite or gametocyte), in oocysts 5 days post mosquito feeding and in salivary gland sporozoites 12 days post feeding. In the Brightfield panel two salivary gland lobes can be distinguished; only one lobe contains sporozoites. In the panel on the right GFP and mCherry expression is shown in Hoechst 33342 stained day 6 liver stages. Note the autofluorescence of hepatocytes in the GFP channel in contrast to the mCherry channel. A small uninucleate (arrow) and a large multinucleate liver stage are visible, confirmed by staining of fixed parasites with anti-HSP70 antibodies (<i>lower right panel</i>). White bars correspond to 10 µm (blood and mosquito stages) and 50 µm (liver stages).</p
Dose-response curves for primaquine, atovaquone and pyrimethamine against the distinct hepatic forms observed for <i>P. falciparum</i> and <i>P. cynomolgi</i>.
<p>The infected primary hepatocyte cultures were exposed to the varying drug concentrations from Day 5 to Day 8 post-inoculation and fixed on D8. The curves obtained for the PE-uni forms are presented in red, while those for the Mat-PE forms are presented in black. The data was representative of two independent assays for pyrimethamine (for both parasite species) and for primaquine (<i>P. falciparum</i>); and of three independent experiments for atovaquone (both parasite species) and primaquine (<i>P. cynomolgi</i>). *, p<0.05.</p
Flow cytometry and cell sorting of <i>P. cynomolgi</i> liver stage parasites, including hypnozoite-forms.
<p>(A) Liver stage parasites used for flowcytometry as detected by anti-HSP70 antibodies 3 days and (B) 6 days post hepatocyte infection. White bars correspond to 50 µm. Note that day 3 cultures contain uniform small parasites while day 6 cultures contain both small and large liver stages (arrows). Flow cytometric plots of PcyC-PAC-GFP<sub>hsp70</sub>-mCherry<sub>ef1α</sub> (PcyC-PAC) <i>P. cynomolgi</i> liver stage parasites show a single GFP positive population compared to wild type parasites 3 days post hepatocyte infection (A, Gate 1) and two GFP positive populations 6 days post hepatocyte infection (B, Gates 2 and 3). The y-axis represents the PE-Texas Red Channel (for detection of autofluorescence), while the x-axis represents the GFP signal. (C) Post-sorting images of PcyC-PAC-GFP<sub>hsp70</sub>-mCherry<sub>ef1α </sub><i>P. cynomolgi</i> liver stage parasites ‘GFPlow’ (Gate 2) and ‘GFPhigh’ (Gate 3) parasites sorted at day 6 post hepatocyte infection. The upper panel shows a GFP/Brightfield overlay while the lower panel shows mCherry/Brightfield overlay. The panels below show close-ups of the sorted parasites revealing the size differences between the ‘GFPlow’ and ‘GFPhigh’ populations. White bars correspond to 50 µm.</p
High-Throughput Luciferase-Based Assay for the Discovery of Therapeutics That Prevent Malaria
In order to identify the most attractive
starting points for drugs that can be used to prevent malaria, a diverse
chemical space comprising tens of thousands to millions of small molecules
may need to be examined. Achieving this throughput necessitates the
development of efficient ultra-high-throughput screening methods.
Here, we report the development and evaluation of a luciferase-based
phenotypic screen of malaria exoerythrocytic-stage parasites optimized
for a 1536-well format. This assay uses the exoerythrocytic stage
of the rodent malaria parasite, Plasmodium berghei, and a human hepatoma cell line. We use this assay to evaluate several
biased and unbiased compound libraries, including two small sets of
molecules (400 and 89 compounds, respectively) with known activity
against malaria erythrocytic-stage parasites and a set of 9886 diversity-oriented
synthesis (DOS)-derived compounds. Of the compounds screened, we obtain
hit rates of 12–13 and 0.6% in preselected and naïve
libraries, respectively, and identify 52 compounds with exoerythrocytic-stage
activity less than 1 μM and having minimal host cell toxicity.
Our data demonstrate the ability of this method to identify compounds
known to have causal prophylactic activity in both human and animal
models of malaria, as well as novel compounds, including some exclusively
active against parasite exoerythrocytic stages
High-Throughput Luciferase-Based Assay for the Discovery of Therapeutics That Prevent Malaria
In order to identify the most attractive
starting points for drugs that can be used to prevent malaria, a diverse
chemical space comprising tens of thousands to millions of small molecules
may need to be examined. Achieving this throughput necessitates the
development of efficient ultra-high-throughput screening methods.
Here, we report the development and evaluation of a luciferase-based
phenotypic screen of malaria exoerythrocytic-stage parasites optimized
for a 1536-well format. This assay uses the exoerythrocytic stage
of the rodent malaria parasite, Plasmodium berghei, and a human hepatoma cell line. We use this assay to evaluate several
biased and unbiased compound libraries, including two small sets of
molecules (400 and 89 compounds, respectively) with known activity
against malaria erythrocytic-stage parasites and a set of 9886 diversity-oriented
synthesis (DOS)-derived compounds. Of the compounds screened, we obtain
hit rates of 12–13 and 0.6% in preselected and naïve
libraries, respectively, and identify 52 compounds with exoerythrocytic-stage
activity less than 1 μM and having minimal host cell toxicity.
Our data demonstrate the ability of this method to identify compounds
known to have causal prophylactic activity in both human and animal
models of malaria, as well as novel compounds, including some exclusively
active against parasite exoerythrocytic stages
Lead Optimization of Imidazopyrazines: A New Class of Antimalarial with Activity on <i>Plasmodium</i> Liver Stages
Imidazopyridine <b>1</b> was identified from a phenotypic
screen against <i>P. falciparum</i> (Pf) blood stages and
subsequently optimized for activity on liver-stage schizonts of the
rodent parasite <i>P. yoelii</i> (Py) as well as hypnozoites
of the simian parasite <i>P. cynomolgi</i> (Pc). We applied
these various assays to the cell-based lead optimization of the imidazopyrazines,
exemplified by <b>3</b> (KAI407), and show that optimized compounds
within the series with improved pharmacokinetic properties achieve
causal prophylactic activity <i>in vivo</i> and may have
the potential to target the dormant stages of <i>P. vivax</i> malaria