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 HD001315-03-05). 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. CURRENT STUCTURAL CONSIDERATIONS indicate the S-OligoS structure contains a tetrasaccharide motif of three xyloses and a glucuronic acid as a branch on the xylan chain i.e., -D-glucuronyl-alpha 1,2 beta 1,4 D-(xylyl)3 with up to one thirdof the GlcA O-methylated. The sugars are within 90 percent sulfated yielding a high negative charge density. Such motif could accommodate the subtle variations in geometries involving sulfates which would be components to provide for mutifunctional mimicry of the heparins. The mass of Cp 6 and 3 wouod accommodate the presence of 6 and 2 such motifs, resp. 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 differ. Enlagement of our H/HS-mimetic S-OligoS library to generate specific anti-malaria Components of mass class Cp 4 and 5 (8-9000) 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 (John Sacci, Jr.). This now promises to provide amounts of sporozoites to allow completion of full dose-response measurements of groups of S-oligoS in bioassays by comparison. 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 to the limit of our resources. Experiments to elucidate putative protein ligands using a modified gel-shift analysis and/or to identify protein ligands using fluorescent receptors (library of S-oligoS and/or heparin-oligoS) have been identified.
