Identification of Heparin Modifications and Polysaccharide Inhibitors of Plasmodium falciparum Merozoite Invasion That Have Potential for Novel Drug Development

Abstract

Despite recent successful control efforts, malaria remains a leading global health burden. Alarmingly, resistance to current antimalarials is increasing and the development of new drug families is needed to maintain malaria control. Current antimalarials target the intraerythrocytic developmental stage of the Plasmodium falciparum life cycle. However, the invasive extracellular parasite form, the merozoite, is also an attractive target for drug development. We have previously demonstrated that heparin-like molecules, including those with low molecular weights and low anticoagulant activities, are potent and specific inhibitors of merozoite invasion and blood-stage replication. Here we tested a large panel of heparin-like molecules and sulfated polysaccharides together with various modified chemical forms for their inhibitory activity against P. falciparum merozoite invasion. We identified chemical modifications that improve inhibitory activity and identified several additional sulfated polysaccharides with strong inhibitory activity. These studies have important implications for the further development of heparin-like molecules as antimalarial drugs and for understanding merozoite invasion.

Keywords: Plasmodium falciparum; heparin; invasion; merozoite; polysaccharide.

FIG 1

Growth-inhibitory activity of fractionated heparin and heparan sulfate compounds. Fractionated heparin and heparan sulfate tetra- and hexasaccharides were tested for growth-inhibitory activity in two-cycle assays. Data are means ± SEMs from two assays performed in duplicate. Abbreviations: HS, heparan sulfate; hep, heparin; 4-mer, tetrasaccharide; 6-mer, hexasaccharide; Ac1 and Ac2, N-sulfation at positions 1 and 2, respectively; Ac0, a lack of N-sulfation; S0 to S9, O-sulfation at the indicated position; *, different preparations of the same fraction.

FIG 2

Identification of chondroitin sulfates with inhibitory activity against P. falciparum merozoite invasion. (A) CSD, CSE, and highly sulfated CSB polysaccharides were tested in growth inhibition assays at concentrations of 0 to 100 μg/ml. Data are means ± SEMs from three assays performed in duplicate. (B) Fractionated CSE hexasaccharides were tested in invasion inhibition assays. The degree of sulfation is 5, 6, or 7 sulfate groups per molecule. Data are means ± ranges from one assay performed in duplicate.

FIG 3

CSE disrupts initial contact of the merozoite with the RBC but not heparin binding to MSP1-42. (A, B) Flow cytometry of late-stage parasite cultures, in which parasite stages were differentiated on the basis of ethidium bromide staining, was used to track parasite rupture (in which the results are given as percent schizonts) (A) and merozoite invasion (in which the results are given as percent ring forms) (B) in cultures treated with CSE at 100 μg/ml and uninhibited cultures treated with PBS. After 3 h of incubation, there were increased frequencies of schizonts and decreased ring forms in cultures incubated with CSE. Data are the means ± SEMs from two assays performed in duplicate. *, P < 0.05. (C) Live video microscopy of merozoite invasion in the presence of CSE. Merozoites were able to make initial contact with the RBC, but the contact was not sustained and the merozoites disassociated from the RBC surface. Times (in seconds) are indicated in the lower right corner, and the white arrows highlight a single merozoite that attached to and then disassociated from the RBC. (D) Heparin bead binding assays with P. falciparum protein extract. The protein extract was incubated with heparin beads along with soluble inhibitors, as indicated. Unbound and bead-bound fractions were probed for MSP1-42 binding via Western blotting. MSP1-42 was found in the unbound fraction when it was incubated with heparin as a soluble inhibitor, indicating that soluble heparin was able to outcompete MSP1-42 for binding. However, MSP1-42 was found in the bound fraction when it was incubated with soluble de-6-OS-heparin, CSE, or CSC, indicating that these compounds were not able to compete with heparin binding. (E) MSP1-42 coated on ELISA plates was incubated with heparin-BSA along with soluble heparin, CSC, and CSE at increasing concentrations. The binding of heparin-BSA was detected with anti-BSA antibodies. Soluble heparin, but not CSE or CSC, inhibited the binding of heparin-BSA to MSP1-42.

FIG 4

Invasion-inhibitory activity of sulfated polysaccharides. Highly active HLMs and sulfated carbohydrates were tested in invasion inhibition assays to confirm their activity against merozoite invasion. All compounds were tested at 10 μg/ml. Data are expressed as the percent inhibition from one assay performed in duplicate relative to the inhibition caused by PBS as a reference control. Three CSC-negative controls were included in the assay, and all were noninhibitory (data not shown). †, prepared using pyridine sulfur trioxide complex; $, prepared using piperidine-N-sulfonic acid.