Modulation Of Protein And Cell Functions By Heparin/hepa

Child Health And Human Development

Investigators

Linked publications, trials & patents

Abstract

Our studies elucidate the molecular design of the heparin/ heparan sulfate class (H/HS) of modulatory polysaccharides, and how the diversity of its sulfated oligosaccharides relates to its capacity to modulate the activity of diverse proteins in normal and disease processes (e.g., cell growth, cancer, secretion, multi-cell reactions in development, blood coagulation, physiological stability, and infections by virus and other human pathogens). We have developed libraries of H/HS-mimetic sulfated oligosaccharide inhibitors of human disorders and pathogens (in particular HIV-1) isolated from a heparin-mimetic pharmaceutical which is comprised of a mixture of sulfated oligoxylans and closely mimics biological actions of the heparin family. Newly devised and/or established biological, biochemical, and physical methods are utilized. [HD 01315-01-08-10]. A MACRO COMBINATIONAL STRATEGY USING LIBRARIES OF H/HS-MIMETIC SULFATED GLYCURONYL-XYLAN OLIGOSACCHARIDES: Inhibition of HIV-1. Due to the diversity of sequences within H/HS chains, libraries of unique H/HS oligoS have not been available for research. Our libraries and macro combinatorial strategy overcame this barrier and enabled us to study whether a given in vitro heparin-mimetic function, like those of heparin, was governed by structure and whether the active component would likely to be free of coagulation blocking activity, two essential indicators of usefulness for further drug development. The inhibition of HIV-1 cytotoxicity and syncytium-formation by S-oligoS is governed by structural specificity; library component 9/10 (Pk II) exhibited high anti-viral and negligible anti-thrombin capacity. An enlarged preparation of this component is underway for Phase I testing as a new potential anti-AIDS agent [See HD001315-10]. SOLIS, A NEW HIV-1 INHIBITOR: The number of AIDS cases in the United States is reaching one million and the morbidity, and mortality of AIDS world wide are increasing. We completed most of the Stage 2 chromatography to obtain product from ~40 grams of pharmaceutical, and will continue next year with biochemical, biological, and physicochemical characterization of the product to refine the method for clinical preparation of Solis. We requested additional pharmaceutical source material to produce more Solis for the Phase I trial, which is being planned. Staffing difficulties, and the labor intensive nature of the scaled up methods for Solis production, such as is the need for multiple columns, have limited production. Solis would be an adjunct drug blocking binding and entry of virus into target cells, adding to the blockade of replication by current therapeutics. Our findings on structure-function relations of the S-oligoS libraries, including those on additional H/HS-modulating functions, were consistent with the molecular glycobiology of H/HS in the modulation of the clotting cascade by antithrombin. We completed study of an enlarged S-oligoS library suited for the identification and isolation of S-oligoS able to inhibit the rosetting of normal and PfRBC in cerebral malaria (for further study in our malaria project [1 Z01 HD008733-05 LDMI]. HUNT FOR LIGANDS: We propose to identify and isolate putative H/HS ligand(s) under conditions of tissue culture infection. Experiments to study binding of a bifunctional Solis probe to its ligand(s) and achieve chemical bonding by flash illumination will be conducted, time and staff permitting. Cell membranes will be purified, membrane proteins purified and analyzed for the fluorescent probe by gel electrophoresis. Our method of synthesis of a S-oligoS bifunctional probe using a bifunctional hydrazine derivative in reaction with the reducing aldehyde will be used for Solis. Such ligand(s) might be protective antigens and/or a means of obtaining endogenous H/HS receptors. Our studies revealed that the structure of the native xylan did not provide valid indication of the structure of the active S-oligoS. Experiments utilizing FTIR, HMQC heteronuclear two-dimensional NMR proton-13C correlation spectra and preliminary capillary HPLC/mass spectroscopy are planned for differentially functional Components.