High-Throughput Screening Platform Identifies Small Molecules That Prevent Sequestration ofPlasmodium falciparum–Infected Erythrocytes

Abstract

This 2-step screening approach should be efficient for identification of antiadhesion drug candidates for falciparum malaria.

Plasmodium falciparum infected erythrocytes can bind to host receptors integrins αVβ3 and αVβ6 through DBLδ1_D4 domain of PFL2665c PfEMP1 protein

“…Further, to confirm that IE expressing PFL2665c protein can bind αVβ3 integrin, we used the PFL2665c-expressing E9 parasite line, previously isolated from parent NF54 by limiting dilutions 23 . As PFL2665c also contains CD36-binding CIDRα2.3_D3 domain 24 (Fig.…”

“…Selection of the NF54 line E9 by limiting dilution, which expresses the single var gene, PFL2665, has been described earlier 23 . The aliquots of this line (expanded in 10–15 growth cycles and frozen) were thawed and grown in human O+ erythrocytes at 2% hematocrit in complete RPMI 1640 medium supplemented with 10% human serum and 40 μg/mL gentamicin sulfate for about 2–5 growth cycles (as described in 31 ) before binding experiments.…”

“…Each construct is fused with Green Fluorescence Protein at the C-terminus and the green fluorescence indicates expression of the full-length protein. Integrin receptors αVβ3 (R&D Systems, Cat #3050-AV) and αVβ6 (R&D Systems, Cat #3817-AV) were biotinylated and their binding to bead-bound PfEMP1 domains (1000 to 2000 beads in the reaction) was measured in duplicates on BioPlex 200 machine (BioRad) as described before 23 using concentrations of the biotinylated receptor indicated in the text and figure legends. As the negative control in these experiments, beads with immobilized HisAdEx construct 8,21 , which contains all the same parts as recombinant domain constructs but short irrelevant 37-amino acid long peptide instead of PfEMP1 domain, were used.…”

“…Binding of IE lines to various receptors (CSA from Sigma cat # C9819, and ICAM1, CD36, αVβ6, and αVβ3 – all from R&D Systems, cat ## 720-IC, 1955-CD, 3817-AV, 3050-AV, respectively) or control (BSA) immobilized on Petri dishes were performed using previously published methods and media supplemented with 1 mM CaCl 2 and 1 mM MgCl 2 23,36 . Before the adhesion assays, IE in trophozoite stage were enriched by magnetic LD columns (Milteni Biotec, cat#130-042-901) as described by manufacturer.…”

Rosette-Disrupting Effect of an Anti-Plasmodial Compound for the Potential Treatment of Plasmodium falciparum Malaria Complications

“…These 23 potential hits were cherry-picked and assayed again at 10 μM. In addition, five recently identified anti-sequestration compounds (from a biochemical screening of 10,000 ChemBridge DIVER-Set library compounds) were included 13 . The latter were assayed at 100 μM in accordance with the published study, with the following ChemBridge IDs were 5210644, 5210653, 5306995, 6917839 and 9121948.…”