Article Figures & Data
Figures
- FIG 1
(A and B) Chemical structures of wALADin1 (A) and wALADin2 (B). (C) wALADin1 and derivatives were tested at 62.5 μM for 72 h in a P. falciparum erythrocyte culture. Parasitemia was determined by counting erythrocytes in blood smears. Chloroquine at 100 nM (CQ) was used as a positive control. Dashed vertical lines separate different experiments. (D) Dose-response curves for wALADin compounds 1, 4, and 7. Curves were fit to a sigmoidal (four-parameter) equation with GraphPad Prism 5.0 [log(inhibitor versus normalized response – variable slope); bottom = 0; top = shared by all data sets]. (E to G) Time course of parasitemia in P. falciparum culture in the presence of wALADin1 (E), compound 4 (F), and compound 7 (G) for 4 days. (H) Structure-activity profile of wALADin1 benzimidazoles in P. falciparum culture. The substituents R1, R2, and R3 are highlighted in gray boxes. Dashed arrows indicate positional changes of substituent groups; continuous arrows indicate replacement with a different chemical group. Arrow or font color indicates an enhancement in antiplasmodial activity (green) or a loss of activity (purple). (I) wALADin1 and compound 4 inhibited replication of T. gondii in LLC-MK2 cells, as determined by real-time PCR, with IC50s 1 order of magnitude (wALADin1) or several orders of magnitude (compound 4) higher than for P. falciparum, indicating specificity of the antiplasmodial effect (D). The graph shows means ± standard errors of the means of results for ≥2 experiments for pyrimethamine and wALADin1 and as means of results for a single experiment for compound 4.
- FIG 2
P. berghei ANKA sporozoites were extracted from the salivary glands of infected Anopheles stephensi mosquitoes on days 19 to 23 postinfection, taken up in RPMI medium supplemented with 3% BSA, and used on the same day. (A) Compound 7 potently inhibited the gliding motility of P. berghei ANKA sporozoites. A total of 10,000 sporozoites were added to glass slides precoated with 3% BSA–RPMI medium and allowed to glide for 30 min before fixation with 4% paraformaldehyde–phosphate-buffered saline. Gliding sporozoites were differentiated from adhesive but nongliding sporozoites by a trail of circumsporozoite protein (CSP) detected on the slide by immunofluorescence microsocopy after staining with monoclonal anti-CSP antibody and anti-mouse Alexa Fluor 488 (both at 1:300 in 10% fetal calf serum-PBS) (6). “X” indicates the absence of both sporozoites and CSP trails on the slide. (B) Compound 7 (ALPin1) inhibited the invasion of sporozoites into Huh7 cells. A total of 25,000 cells were seeded onto Lab-Tek 8-well chamber slides 1 day before infection with sporozoites for 90 min (MOI, 0.4). Parasite numbers were determined by fixation and staining using the two-color host cell invasion method. (C and D) For extraerythocytic stage development assays, Huh7 cells were infected with sporozoites for 90 min before free parasites were washed off and compounds were added. Cells were fixed after 24 h, 40 h, or 60 h with ice-cold methanol, followed by staining of liver stage parasites with monoclonal anti-P. berghei HSP70 antibody and goat anti-mouse Alexa Fluor 488 secondary antibody (13). None of the tested wALADins had any effect (up to 40 μM) on the number (C) or the size (D) of liver stage parasites. (E) A total of 6,000 Huh7 cells were seeded onto a 96-well plate before incubation with a 40 μM concentration of the indicated compound or with 0.5% DMSO for 72 h at 37°C and 5% CO2 in triplicate experiments. Neither compound 4 nor compound 7 affected Huh7 cell viability or replication as assessed with alamarBlue reagent. The inhibitor of actin polymerization cytochalasin D (CytD) served as a positive-control inhibitor for motility and invasion studies; primaquine (Pq) was used for intrahepatic stages. NC, negative control. (F) Chemical structure of compound 7 (ALPin1).
Tables
- TABLE 1
Antiplasmodial activity of wALADin1 benzimidazoles is not correlated with inhibitory activity against PfALAD
Compound R1 residue R2 residue Position of R3 residue P. falciparum erythrocyte culture LD50 (μM)a PfALAD IC50 (μM)b 1 (wALADin1) 3-CF3-benzyl 2-[(2-thienylcarbonyl)amino]ethyl C-5 39.3 ± 11.7 (n = 4) ∼568 2 H 2-[(2-thienylcarbonyl)amino]ethyl C-5 NA ND 3 3-CF3-benzyl H C-5 NA NI 4 3-CF3-benzyl 2-[(2-thienylcarbonyl)amino]ethyl C-6 7.7 ± 1.7 (n = 3) NI 5 3-CF3-benzyl 2-[(2-thienylcarbonyl)amino]ethyl C-4 NA ND 6 3-CF3-benzyl 2-[(2-thienylcarbonyl)amino]ethyl C-7 NA ∼625 7 (ALPin1) 4-CF3-benzyl 2-[(2-thienylcarbonyl)amino]ethyl C-5 12.8 ± 0.02 (n = 2) NI 8 4-CF3-benzyl H C-5 NA ND 9 CH3 H C-5 NA ND 10 H H C-5 NA ND 11 (wALADin2) Tricyclic quinoline derivative NA NI - TABLE 2
PCR conditions for T. gondii repeat DNA and M. mulatta β-actin PCRa
Parameter T. gondii repeat DNA PCR M. mulatta β-actin PCR Forward primer 5′-GATATCAGGACTGTAGATGAAGG-3′ (300 nM) 5′-GATGAGATTGGCTTTA-3′ (300 nM) Reverse primer 5′-GCGTCGTCTCGTCTAGATC-3′ (300 nM) 5′-AACCGACTGCTGTCACCTTC-3′ (300 nM) Hybridization probe 5′-6-FAM-AAGCGACGAGAGTCGGAGAGGGAG-3′-BHQ-1 (50 nM, TaqMan) 1× SYBR green dye Conditions 0.5 U HotStar Taq; 50 μM dNTPs; 4 mM MgCl2 0.5 U HotStar Taq; 50 μM dNTPs; 3 mM MgCl2 Cycling protocol 15 min at 95°C followed by 45 cycles of 10 s at 95°C and 30 s at 60°C (fluorescence acquisition) 15 min at 95°C followed by 45 cycles of 15 s at 94°C, 20 s at 58°C, and 20 s at 72°C (fluorescence acquisition) ↵a 6-FAM, 6-carboxyfluorescein; BHQ-1, black hole quencher 1; dNTPs, deoxynucleoside triphosphates.
