This project elucidates heparin- and heparan sulfate (H/HS)-mimetic actions of sulfated xylan oligosaccharides (S-oligoS) in malarial invasion and pathogenicity. H/HS and S-oligoS appear to modulate the parasites at various infectious stages and in their sequelae: 1) As an hepatocyte receptor for malarial sporozoite invasion through binding to the circumsporozoite membrane protein; 2) as a possible erythrocyte (RBC) receptor in the invasion of RBC by newly released merozoites, as well as involved in the complex rosetting reactions between parasitized-RBC (pRBC) and normal RBC; and 3) by sequestration of p-RBC to the microvasculature in cerebral and severe malaria. The molecular basis underlying these functional reactions of H/HS remain to be clarified. Exogenous heparin was shown by others to inhibit the parasite invasion of hepatocytes and RBC and to dissolve rosettes in vitro. It also rapidly dissolved rosettes in vivo. This clinical treatment for cerebral malaria, however, had to be abandoned because of bleeding toxicity. Utilizing our library of H/HS-mimetic S-oligoS and a macro combinatorial strategy to examine the degree of specificity in the putative H/HS hepatocyte receptor function in vitro , we had demonstrated that the potencies of the inhibition of liver stage sporozoite development were differentially displayed among eight S-oligoS Components (Cp). [Two Cp (av. mass region 7200 and 3600) exhibited concentration dependent, high inhibitory capacity (ID50 est. 1 uM), while four other Cp (broad av. mass range 2300 - 6600 lacked anti-sporozoite capacity]. These data indicated that this inhibition was governed by a degree of molecular specificity. In contrast, in the in vitro assay of invasion of RBC, high potency (IC50=0.14-0.27uM) H/HS-mimetic S-oligoS against malaria parasites appeared to be limited to the high mass region (av. 8600 and 13,400) of the library; these would have relatively high anti-thrombin capacity and aPTT values. Moderate inhibitory potency (IC50=0.54-0.69uM), however, was exhibited by S-oligoS in the intermediate av mass range of 5800-4700. [details in HD01315-07,08; HD008733-02] These studies revealed that a small S-oligoS (approx. a dodecamer, av. mass 4700), which would be devoid of anti-thrombin capacity and close to a low level of 1.5 in the aPTT test, might be an approach to treatment of malaria in adults and children. It is noteworthy that our high potency anti-HIV-1 agent, CpF-Pk II, which was devoid of capacity to inhibit sporozoite invasion, had moderate potency against malaria parasite invasion of RBC. Such capacities may be found to provide a dual usefulness in treating populations subject to both diseases. Research in this project was largely inactive this year, but will resume next year with renewed lab staff. Studies this year focused on development of enlarged and further devoluted preparations of the two anti-malaria sporozoite S-oligoS and the S-oligoS region moderately inhibitory against RBC invasion (av. mass = 5800). We now have 200-500 mg of first stage preparations ready for second stage purification. Our basic research will resume, continuing in three approaches. The first is to repeat and extend through collaboration the anti-sporozoite and anti-RBC invasion assays of the putative receptor-mimetic S-oligoS. The second is to identify and elucidate the S-oligoS ligands, using a modified gel-shift analysis for heparin oligoS-protein binding. This would reveal possible specific binding of S-oligoS to, and isolation of, their protein partners in the given membranes, e.g., fluorescent receptor (monodansylated S-oligoS) would mark possible shifts in gel migration upon its binding to specific protein sequences identified in the data banks on malarial proteins of the sporozoite membrane. The third approach in the basic research on our H/HS-mimetic library is to examine the potential specificity and usefulness of the S-oligoS against cerebral malaria in children and adults by means of in vitro assays on their capacity to rapidly dissolve the pathogenic rosettes of pRB.
