Modulation of protein /cell functions by heparin/heparan

Child Health And Human Development

Investigators

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Abstract

This project elucidates the fundamental molecular design of the heparin/ heparan sulfate (H/HS) class of biological regulatory polysaccharides and studies how the high degree of diversity of its sulfated oligosaccharide (oligoS) structures relates to its parallel, multifunctional capacity to modulate the functional activity of numerous diverse protein partners in normal and disease processes (e.g., cell growth, cancer, secretion, multi-cell reactions in development, blood coagulation, physiological stability, and in infections by virus and other classes of human pathogens). Within this basic research map, the project pursues the development of H/HS-mimetic therapeutic agents against various disorders and pathogens (in particular HIV-1), based on the known glycobiology of the multifunctional family of H/HS and an H/HS-mimetic pharmaceutical which is comprised of a mixture of sulfated xylan oligocaccharides (S-oligoS) and closely mimics most of the biological actions of heparin. Newly devised and/or established biological, biochemical, and physical methods are utilized. Due to the complex diversity of sequences within H/HS chains, libraries of various unique H/HS oligoS have not been available for extended research. WE PREVIOUSLY devised an approach to overcome this roadblock. A family of H/HS-mimetic S-oligoS (a 24-Component library) was produced by fractionation of the pharmaceutical, thus generating a macro combinatorial strategy. This enabled us to examine whether a given heparin-mimetic function in vitro, like those of heparin, was governed by a degree of structural specificity within the family, and whether the active Component would likely lack bleeding toxicity as well. Both these characteristics are required indicators of usefulness for further drug development. We engaged several approaches to study the structure/function relations of the H/HS-mimetic inhibition of HIV-1. Early findings showed that Components of our H/HS-mimetic library displayed differential capacities to inhibit the cytotoxicity or the syncytium-forming toxicity of HIV-1, demonstrating that the inhibitions were governed by a degree of structural specificity. This supported our hypothesis that the anti-viral capacities of H/HS-mimetic S-oligoS follow rules of specificity, similar to those of H/HS and led us to the development of CpF-PkII, a highly active, anti-HIV-1 S-oligoS which was essentially free of anti-thrombin toxicity. Enlargement of preparative methods was devised within the limitations of the 400 mg/load imposed by the soft gel nature of the fractionating matrix, and a clinical preparation utilizing 40 grams of starting pharmaceutical has been underway to obtain sufficient agent for Phase I and other potential preliminary trials. THIS YEAR, further analysis of the structure/function relations of our S-oligoS library, including findings from studies on additional H/HS-modulating functions, showed that they are consistent with the known molecular glycobiology of H/HS, derived from the now classical model of antithrombin regulation of serine esterase enzymes in the clotting cascade, first discovered by Robert D. Rosenberg. 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 example, both the specific oligoS binding sequence plus an additional binding of another specific H/HS sequence, which is required to fully activate the protein against thrombin. Our CUMULATIVE FINDINGS have broad significance in laying to rest a long outdated bias that the heparin-like effects of sulfated but structurally unrelated polysaccharides must be non-specific and due to the high sulfate charge density. Below, we show also that the structure of the native polysaccharide dose not provide a valid clue about the structure of the active oligoS. THIS YEAR, we completed phase I fractionation of starting material and rechromatography to the CpF-PkII.stage is continuing; biological and physicochemical analyses are continuing. This putative drug would PROVIDE A SIGNIFICANT ADDITIONAL MECHANISM OF INHIBITION OF HIV-1 since its mode of action in vitro prevents the binding and entry of virus into target cells and the cell-cell spreading of the infection. Peak II also displays a moderate inhibitory capacity against the merozoite invasion of RBC by malarial parasites in vitro. Additional library Components are underway for studies on other potential therapeutic uses, e.g., the various anti-malarial capacities of the S-oligoS. Elucidation of the S-OLIGOS STRUCTURE by biochemical, chemical, and spectroscopic (FTIR, NMR, and dye-coupling) analyses revealed previously for the first time that the H/HS-mimetic S-oligoS were similar and significantly different from the primary structure of the native polyxylan which has one monomeric GlcA branch per 10 Xyl on the average. The primary structure of the S-oligoS contained a tetraS motif, one GlcA branch per 3 Xyl on average. This could generate a sulfated GlcA-xyl Di-S spaced by two xylose moieties along the backbone, each GlcA assuming a helical rotation of 180 degrees. Furthermore, FTIR and heteronuclear two-dimensional NMR proton-13C-correlation (HMQC) spectroscopy revealed for the first time that CpF-PkII contained some sugar moieties in an alternative chair conformation instead of the expected normal chair form; such sugars would have a shortening effect on the chain backbone. Further FTIR data indicated that these alternate sugar conformations are present in other H/HS-mimetic Components but that the ratio of normal to alternative forms differs among the various S-oligo. That H/HS-mimetic S-oligoS contain a tetraS motif involving GlcA in their back bone repeat and that their sugar conformations may be a governing factor in structural specificity of the protein-binding sugar sequences has SIGNIFICANT IMPORTANCE, e.g., in strategies for synthesis of oligoS drugs and the design of synthetic oligoS-protective antigens. It is of note that, more recently, the anticoagulant capacity of the synthetic antithrombin-binding pentaS was shown to reside uniquely in the form containing an alternative skewed-ring conformation in the iduronic acid moiety. THIS YEAR preparation of library Components for further studies on these structure/function relations have continued. Manuscripts in preparation: 1. Structure-function relations of a library of heparin-mimetc sulfated xylan oligosaccharides: Preparation of a potent HIV-1 inhibitor and structural characterization by FTIR and NMR spectroscopy and glucoronic acid analysis, by Audrey Larack Stone, Alexis F. Henry, Derek J. Melton, James McMahon and Maria O. Longas (Bioploymers); 2. Structure-function relations of a library of heparin-mimetic sulfated xylan oligosaccharides: Study of alternative chair sugar conformation by heteronuclear two-dimensional NMR 1H-13C-correlation spectroscopy and HMQC analysis, Maria O. Longas and Audrey Larack Stone (J. Biomedicine and Biotechnology); 3. Structure-Function relations of heparins/heparan sulfates and heparin-mimetic sulfated xylan oligosaccharides: A role of secondary structure in functional specificity as viewed from asymmetric metachromatic coupling reactions, by Audrey Larack Stone (Macromolecular Chemie).