This project elucidates the actions of heparin and heparan sulfate (H/HS) and heparin-mimetic sulfated xylan oligosaccharides (S-oligoS) in malarial invasion and pathogenicity. H/HS and HS-proteoglycan (PG) appear to exert multifunctional modulation of the malaria parasites at various infectious stages and in their sequelae: 1) HS-PG was shown to be an hepatocyte receptor for malarial sporozoite invasion through its binding to the circumsporozoite membrane protein (Nussenzweig & coworkers, 1993); 2) The erythrocyte (RBC) membrane H/HS might be a receptor for invasion of normal RBC by newly released merozoites; it is involved as a receptor in the complex reactions between parasitized-RBC and normal RBC in the rosetting reaction and 3) in 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 is known to inhibit the parasite invasion of hepatocytes and RBC in vitro, and it also rapidly dissolves rosettes in vitro and in vivo. Heparin was tried as a treatment for cerebral malaria but, although was abandoned because of bleeding toxicity. Last year we used our S-oligoS library of H/HS-mimetic structures to examine whether there was a degree of specificity in the H/HS hepatocyte receptor functions.[Methods and details in [HD008733-02] Briefly: following our macro combinatorial strategy [HD 01315-07], the capacity of eight S-oligoS Components to inhibit hepatocyte invasion by malaria sporozoites in vitro was tested in a two point, 10-fold dose range against the Inhibition of Liver Stage Development Assay (ILSDA) using Plasmodium yoelii (J. Sacci, 2001). [These inhibition studies using our S-oligoS library are ongoing with the collaboration of John Sacci at The Walter Reed Army Institute of Research (WRAIR), Silver Spring, MD.] Freshly prepared sporozoites from heads and thorax of infected mosquitoes were used to infect 24h murine hepatocyte monolayer cultures for 3 hours, after which cultures were washed and incubated. At 48 h schizonts are visualized and counted to measure the percent inhibition. The eight Components displayed differential potencies, indicating that this inhibition was governed by a degree of molecular specificity. Four were inactive, notably including CpF-PkII. Only two of the 8 S-oligoS exhibited significantly high and concentration-dependent inhibitory capacity, Cp11, av 3500 in mass (appx a dodecamer which would be devoid of anti-thrombin capacity and close to a low level of 1.5 in the aPTT test). The other Component was ~ 7000 in mass (~22 saccharides). The estimated IC50 of these S-oligoS was ~1 uM. A study on the capacity of these S-oligoS to inhibit the invasion of erythrocytes was insufficient due to the choice of a ng high dose range. One S-oligoS, Cp3 of mass av 13400, had sufficient potency to yield an IC50 of 0.26 nM vs 0.41 nM for chloroquine. Current: Bioassays were repeated and/or further analyzed with regard to structure-function relations of the S-oligoS inhibition of the malaria parasites. Inhibition studies of P. falciparum development/invasion of erythrocytes using our S-oligoS library are ongoing with the collaboration of the laboratory of WK Milhouse, at WRAIR. Briefly, pRBC (parasitemia 0.4% with a 1% hematocrit) are incubated 72 h. in RPMI-1640. Incorporation of radiolabled hypoxanthine by parasites is measured in the absence or presence of inhibitor to measure the degree of inhibition. Two clones are tested and two standard anti- malarial drugs are positive controls. In a series of eight Components, Cp3 and Cp4B (of relatively high mass 13,400 and 8600, resp.) were identified as the most potent inhibitors of the parasites in this assay (IC50 = 0.14 and 0.26 uM with clone D6 and 0.17 and 0,23 uM with clone W2 ). These Components would have unacceptable anti-thrombin potency (range appx 73 and 45 H units/mg) and about 4 aPTT units vs our goal of less than 2. Other S-oligoS in the series, however, exhibited a moderately high potency level (IC50 = 0.54 - 0.69 with both clones), two of which (Cp8B and CpF-PkII) would be low or devoid of anti-thrombin toxicity. S-oligoS with mass less than about 4600 were inactive in this assay. [AL Stone (2001) Structure-Function Relations of heparin-mimetic sulfated oligoxylans in the inhibition of the invasion of hepatocytes by malaria parasites in vitro Glycobiology Vol. 11:901-902;] Differences in potencies and five structural characteristics among the library Components as measured in these inhibition of malaria parasite assays as well those in the inhibition of HIV-1 and thrombin assays were compared. [Stone AL Differential structure-function relations in a family of heparin-mimetic sulfated glucuronyloligo-xyloses in vitro: Malaria parasite, AIDS virus and blood coagulation inhibitors Gordon Research Conference on Proteoglycans, July 2002; Longas MO and Stone AL Structure-function relations of heparin-mimetic sulfated oligoxylans: Study of alternate chair conformation by NMR spectroscopy, Gordon Research Conference on Proteoglycans, July 2002.] The significant findings from these data are: 1) They indicate that the structural requirements for functional H/HS in the invasion of hepatocytes by malarial sporozoites are different from those involved in invasion of RBC. In the former, there appears to be a high degree of structural specificity which is relatively independent of mass (S-oligoS having wide differences in mass were inactive while within the same range (appx 3600 to appx 7200) two Cps have relatively high potency, with a small S-oligoS, Cp11, displaying the highest. In contrast, H/HS involvement in the invasion of RBC seems to require a relatively large molecule for maximum function, but moderate inhibitory potency of the S-oligoS was retained down to a mass of appx 4700 (such Cps have displayed axial-to-equatorial sulfate band intensity ratios of appx 0.3 or greater). This is similar to our findings in the case of the inhibition of HIV-1. Additional library Components should be tested. 2) The finding that Cp 11 is a relatively highly potent inhibitor of malaria parasite invasion of the heptocyte is an important lead to its possible clinical usefulness in children since it would be expected to be devoid of anti-thrombin toxicity and have tolerable elevation of the aPTT. 3) The finding that our HIV-1 inhibitor, CpF-PkII, was moderately active in inhibiting the invasion of RBC by malaria parasites suggests that this agent might be delivered in a manner that serves both purposes in populations that suffer from both diseases. These results help to establish that our semi-synthetic H/HS-mimetic library can substitute for as yet unavailable H/HS libraries to elucidate the molecular basis underlying the various modulator functions ascribed to H/HS. Our strategy in using this particular pharmaceutical heparin-mimetic is particularly useful because this basic research purpose of the library we have produced can be translated directly from bench- to-bedside due to the demonstrated pharmaceutical properties of the S-oligoS Compon-ents. The studies will be extended (e.g., on Cp11, 4B and 3) to better define the structural requirements and nature of H/HS functions and those of potential drugs. In addition we expect to start studies to identify putative protein partners for the H/HS modulators of malaria parasite invasion. Because the gel shift assay for measuring heparin-binding to a protein requires radioactive H oligoS for radiation detection, we must first try to adapt the assay to study the S-oligoS by a sensitive metachromatic measurement instead.
