This project pursues the development of therapeutic agents against malaria and the invasion and pathogenicity of the malaria parasite, based on our knowledge of the glycobiology of the multifunctional family of heparin/heparan sulfate (H/HS) oligo- and polysaccharides and their mimetic sulfated xylan oligosaccharides (S-oligoS). Severe malaria kills 1-2 million globally per year; 90% are young children. Currently, there is no preventive vaccine, and malaria parasites are increasingly resistant to the long-standing anti-malarial drugs. H/HS appears to be involved as an hepatocyte receptor for malarial sporozoite invasion through binding to the circumsporozoite membrane protein (Nussenzweig & coworkers, 1993)and possibly other proteins; as a possible erythrocyte (RBC) receptor for merozoite invasion of RBC: in rosetting reactions between parasitized-RBC (pRBC) and normal RBC; as well as in sequestration of p-RBC to the microvasculature in cerebral and severe malaria. Exogenous heparin was shown by others to inhibit the parasite invasion of hepatocytes and RBC and to dissolve rosettes in vitro. In clinical studies, heparin was shown to rapidly inhibit severe malaria in children by M. Munir, et. al., in 1980, but some incidence of bleeding toxicity precluded further use. We had prepared a library of H/HS-mimetic oligoS and developed a macro combinatorial strategy (first applied to examine heparin's anti-HIV-1 capacity [HD0135-01]) which enables us to examine whether a given heparin-mimetic function, like those of heparin, was governed by a degree of structural specificity within the family, and whether the active Component would likely lack anticoagulant capacity as well. [HD01315-03-07]. Both these characteristics would be required indicators of usefulness in further drug development. This approach enabled us to examine the putative H/HS hepatocyte receptor function with John Sacci and show a degree of structural specificity in the heparin-mimetic inhibition of Plasamodium yoeli invasion of hepatocytes using his Liver Stage Development Inhibition Assay ; two Components of mass av region 7200 and 3600 exhibited concentration dependent, high inhibitory capacity (ID50 est. 1 uM), the latter expected to lack anticoagulant toxicity [HD008733-03 LDMI]. Differential capacities against Pl. falciparum invasion of RBC, studied with Milhous and Kyle (WRAIR), were also displayed among our library Components. The specific structural requirements for high potency in this inhibition (IC50=0.14-0.27uM), however, were different from those seen in the hepatocyte invasion assays [HD008733-03]. High potency resided in Components larger than mass av 8000 which would be associated with relatively high anti-thrombin capacity and aPTT values. OligoS of mass av 5800 down to 4700 which would lack anti-thrombin toxicity, displayed moderate inhibitory potency (IC50=0.54-0.69uM). The distinction between putative Pl. falciparum proteins as ligands in the invasion of hepatocyte and RBC, however, has blurred as additional parasite proteins are studied, e.g., the apical membrane antigen-1 (AMA-1) is located on the surface of the sporozoite as well as on the erythrocyte during parasite invasion (Silvie O, et al.December 2003). AMA-1 and TRAP are processed during sporozoite invasion of hepatocytes by putative serine esterases. This year, further analysis of our anti-malarial data showed that it is consistent with the established molecular glycobiology of H/HS in the antithrombin regulation of the clotting cascade. Here, activation of a single inhibitory protein is multifunctional and two types of binding may occur between H/HS and antithrombin. One involves the oligoS sequence which provides most of the specific binding strength and effectuates an activating conformational change in antithrombin against the given enzyme. The second is nuanced, involving in the case of thrombin for exampleee, both the specific oliigoS binding sequence plus an additional binding of another specific H/HS sequence to antithrombin, which is required to fully activate the protein against thrombin. We therefore expanded preparation of our H/HS-mimetic library to generate appropriate Components for continuing the above antimalarial studies. Further experiments were planned for continuation of the studies with collaborators at WRAIR on the in vitro inhibition of RBC and hepatocyte invasion. Application of our expanded library in studies of the capacity of Components to inhibit Pfl RBC resetting in vitro is planned to examine the potential for therapeutic use of H/HS-mimetics against cerebral malaria in children and adults. This year again, research on this project was limited by delays in staffing. We have planed experiments, which will start when research staffing is completed, to elucidate the H/HS-mimetic ligands, by modification of a gel-shift analysis of heparin oligoS-protein binding and testing the possible protein partners using fluorescent receptor Components (monodansylated) which would mark possible shifts in gel migration upon binding. A manuscript entitled, Structure-function relations of heparin-mimetic sulfated xylan oligosaccharides the in vitro inhibition of hepatocyte invasion by Plasmodium Yoelii and of Plasmodium falciparum development/invasion of erythrocytes, by Audrey L. Stone, John Sacci and Will K Milhous, remains in preparation.
