Figure 7. The pure candidate compound specifically prevents FREP1 from binding iRBC, gametocytes, and ookinetes.

(a) The candidate compound specifically inhibited the binding of FREP1 protein to P. falciparum parasites as demonstrated by IFA. The first and second column detected FREP1 and parasite nuclei respectively. Merging column one and two generated the third column, which shows the co-localization of P. falciparum (nuclei) and FREP1 binding. The fourth column shows the bright views of the cells. No FREP1 signals were detected without adding FREP1 (1^st row, arrows points to gametocytes or ookinetes). Incubating FREP1 with P. falciparum gametocytes and ookinetes supplemented with 1% DMSO showed the co-localization of FREP1 and gametocytes or ookinetes (2^nd row). Addition of the candidate compound (40 μg/mL) reduced the interaction between FREP1 and gametocytes/ookinetes (3^rd row). The fourth row shows the bright field. Of note, many DAPI-positive dots that do not match with red spots are merazoites (free parasites), suggesting FREP1 does not bind merazoites well. (b) The intensity of red fluorescence indicated that the compound significantly prevented FREP1 from binding to gametocytes or ookinetes. (c) The candidate compound did not affect the ELISA reaction to detect FREP1, supporting the compound specifically interferes with FREP1-iRBC lysate interaction. Treatments: 1: FREP1 (7.5 μg/mL) plus DMSO (1%); 2: FREP1 (7.5 μg/mL) plus the candidate compound (40 μg/mL); 3: BSA (7.5 μg/mL) plus DMSO (1%).