Heparin was known to inhibit the rosetting and cytoadhesion of parasitized Plasmodium falciparum erythrocytes (PfRBC) to normal RBC and the endothelium, and to clear blockage of the microcirculation and ameliorate life-threatening symptoms of cerebral malaria when administered in children; it also inhibited the initial malaria parasite invasion of hepatocytes (and possibly RBC). Malaria ranks in the top three deadliest diseases globally (approximately 300 million clinical cases per year). 1-3% of the Pf parasites are highly virulent, causing severe and cerebral malaria and the death of about 2 million people per year (90 % young children). There is no preventive vaccine, and malarial parasites are increasingly resistant to anti-malarial drugs. This project has previously applied a macro combinatorial srategy to study the heparin inhibitions in vitro, utilizing our library of sulfated oligoxylans. These were prepared from a Heparin/Heparan sulfate (H/HS)-mimetic pharmaceutical that mimics numerous discrete biological actions of the heparin family. (See [HD001315-03-07]). The capacity of P. yoelii sporozoites (freshly isolated from infected mosquitos) to invade hepatocytes was measured; differential potencies of inhibition of the parasite invasion by S-OligoS was demonstrated; highest potency and concentration- dependence resided in two S-OligoS in mass class about 7200 and 3700 with 43 and 56 percent inhibition at 3.5 and 5 micromolar, resp. The structure of the inhibitor sequence in not clear. Current structural considerations would accommodate the presence of a maximum of 6 and 3 putative -D-glucuronyl-alpha 1,2 beta 1,4 D-(xylyl)3 motifs with O-methyl groups on up to 35 per cent of the GlcA moieties, resp. Planned Capillary HPLC-mass spectoscopy of S-oligoS will analyze for the presence and importance of the putative motif. Studies on the Inhibition of the erythrocyte invasion stage of malarial parasites revealed that S-OligoS of mass class less than 4500 exhibited very low inhibitory capacity, while high potency was associated with S-OligoS of relatively high mass class equal to or greater than 10,000. Such data suggests that the molecular reactions underlying the inhibition of the two parasite stages were different. Enlagement of our H/HS-mimetic S-OligoS library to generate specific anti-malaria Components continues. Recent advances in malaria research include the first method for generation of sporozoites (and other stages of the malaria parasite) in a cell free culture. This now promises to provide larger amounts of sporozoites in a time short enough to allow completion of full dose- response measurements of groups of S-oligoS in bioassays by comparison. Although our project has had limited activity this year because of limitations in resources, we plan to resume our studies on inhibition of sporozoites, parasitized RBC, and rosetting as a path towards stable, inexpensive heparin-mimetic anti-malarials against initial infection, pathologies, and/or acute cerebral malaria. Experiments to elucidate putative protein ligands using a modified gel-shift analysis of heparin oligoS-protein binding and/or to identify protein ligands using fluorescent receptors (library of S-oligoS and/or heparin-oligoS) are also planned.
