HIV-infection in Americans is one million plus and rising, 50 times that worldwide. With no cure nor fully effective vaccine, and the successful long term control of viral load and deadly effects in developed countries threatened by toxicity, multi-drug resistant strains, and reliance on mostly protease and reverse transcriptase inhibitors (drugs approved by FDA plus 1 virus entry inhibitor), broad consensus counsels that inhibitors against viral components having other functions in HIV-1 attack are critically needed. Heparin was shown by earliest clinical investigators to inhibit binding and fusion of AIDS virus to its target CD4 cell in vitro, but heparin is an antithrombin-activator anticoagulant and as such may not be otherwise used clinically because of possible hemorrhagic toxicity. Early on at NIH, we discovered that heparin, thought to be a random polymer lacking secondary structure/ information content, assumed a helical structure in solution. We classified the various sulfated glycosaminoglycan biopolymers (GAG, mucopolysaccharides) based on specificity of their optically active structures, and demonstrated (AL Stone 1963/4 Biopoly- mers 2/3) that H/HS (and keratosulfate) comprise a Class of GAGs, separate from other GAG types. H/HS are highly sulfated, found in cells of the various tissues and comprise modulator receptor systems throughout the body where they not only govern the level of activity/function of numerous normal proteins and cell systems, but serve as receptors for various human pathogens in unclear mechanism(s). H/HS have a large diversity in sugar modifications and sulfate density (anionic density) along their polysaccharide chain. This structural diversity provides H/HS with a sufficiently large number of unique sequences to account for its known ability to modulate precisely the functions of many diverse normal and disease processes. e.g., development, growth, inflamation, and attack by viruses and other human pathogens. Due to the high degree of similarity of physicochemical properties of H/HS chains carrying these specific sequences, however, and absent the given specific ligands, libraries of the unique H/HS have not been obtainable for research. We developed a macro combinatorial type of heparin-mimetic family (library) based on the structure-function model for the antithrombin-dependent anticoagulant heparin (R.D. Rosenberg, et al). For this we selected a German pharmaceutical, chemically sulfated xylan, comprised of S-oligoS that mimicked unfractionated heparin in almost all its known biological actions, at similar uM concentrations, to obtain a pilot heparin-mimetic family of S-OligoS HD 01315-01-08-10. With this we demonstrated for the first time that in vitro inhibitions of HIV-1 cytotpathology and syncytium-formation were 1) each governed by a degree of structural specificity and 2)each separable from the anticoagulant S-oligoS of the pharmaceutical and from each other, which are two essential indicators of their usefulness for further drug development AL Stone, et al 1998 Glycoconjugate Journal 15:697-71. We devised enlarged procedures to enable clinical preparation of a highly active HIV-1 virus fusion inhibitor free of anti-thrombin toxicity (PK II, SOLIS) AL Stone and James McMahon, et al;SUMMARY OF PROPERTIES OF PK II;*IC 50 vs cell-killing (formazan assay) -- 0.20-0.3 ug/ml;*IC 50 vs virus-cell fusion -- 0.05-0.1 ug/ml;inhib. adherence CD4 cell to gp120 (concentration-dependent);anticoagulation vs thrombin -- less than 0.05-0.3 HU/mg;endotoxin content FDA LAL Assay -- less than 0.06 eu. *N.B.: IC 50 may be 7-10 x greater in a commercial lab;SOLIS: A POTENTIAL HIV-1 VIRAL ENTRY INHIBITOR: We are working on completing a clinical preparation of SOLIS for formulation in a small Phase I i.v.-administration safety trial. SOLIS would be an adjunct drug selected from the family library as an inhibitor of virus entry into CD4 cells ((See Table above). Mechanism of Action: Heparin has long been known to affect conformation of proteins as a means of modulating their biological function (e.g., AL Stone RD Rosenberg et al 1982 PNAS 79:7190, AL Stone P Epstein 1977 BBA 497:298). Thus, inhibition of conformational changes believed to be associated with formation of the immediate fusigenic structure of gp41 required for fusion progression, is a possible mechanism of action of SOLIS. It has long been established that orally administered S-oligoS are very poorly absorbed, necessitating an i.v. route of administration and consequently a relatively large amount of drug, and clinical studies can be flawed by use of indirect means to assess blood levels of the drug during i.v. trials. Currently: We completed the Stage II product, SOLIS, from first lot of pharmaceutical starting material;appx.1.6 g conjoined and stored at minus 75 degrees C. Importantly, prolonged negotiations to renew our Confidentiality Agreement and a materials transfer with the pharmaceutical company, to facilitate scientific progress and rapidly obtain starting pharmaceutical to prepare additional SOLIS, were completed this year, and a donation of 40 grams transferred. STUDIES ON COMBINATION THERAPY: Currently, Cp11 samples on hand were combined for studies on whether the addition of increasing amounts of Cp11 (relatively inactive vs HIV-1) to the standard Pk II doses in formazan cytotoxicity protection assay would effect capacity of Pk II to protect cells;for report see Z01 HD008733- . ENLARGEMENT OF LIBRARY: Further enlargement of our library in the preparation of Cp11 and of CpC (in Cp7,8A region) and 8B is in progress. CpC (smallest mass component retaining high activity against HIV-1 cyopathology in vitro) will be used to prepare derivatives (conjugates) using selected polypeptides for various immnological approaches, and for S-F studies (See below) on SOLIS;1) to obtain a potent immunogen that would produce a specific Ab reagent for direct measurement of blood SOLIS concentration after i.v. administration;2) to obtain Ab against putative viral fusion-specific conformations. VIRUS LIGANDS AND BIFUNCTIONAL SOLIS PROBE: Our proposal, See previous HD 00 1315xx, to identify and isolate putative H/HS ligand(s) under conditions of HIV-1 tissue culture infection in the presence of a bifunctional SOLIS probe, and thereby to achieve chemical bonding to endogenous viral/cell protein structures during virus attack and subsequently isolate the putative endogenous protein-probe will continue with further urified CpC. Such ligand(s) could be used to obtain endogenous receptors and/or might be protective antigens. DERIVATIZATION OF S-OLIGOS: S-oligoS of interest are derivatized following our procedures for momo-derivatizing library S-oligoS at the reducing end, at pH close to 3.0 (See earlier HD 00-1315xx). Our proposal to select particular synthetic polypeptides for linkage to produce specific immunologic reagents and immunogens is on going and based on our speculation of the mode of action of SOLIS. STRUCTURE-FUNCTION RELATIONS: Early on we discovered that S-oligoS remaining after chemical sulfation of the native xylan comprised a family of oligomers that range about 20 to less than 2 K in av mass. Compositional and structural analysis of Cps led us to propose a tetraS motif within the S-OligoS chains: D-glucuronyl-alpha 1,2 beta 1,4 D-(xylyl)3 motifs possibly increasing with mass. We also revealed presence of varying axial sulfates (alternative chair conformations) in these S-OligoS which are displayed in decreasing proportions with decreasing mass (See previous HD 00 1315xx). Currently we are preparing Cp II and Cp 8b for advanced FTIR spectral analysis and HMQC correlation spectra to detdetermine the relation of alternative chair structure in the S-OligoS and the inhibition of HIV-1 gp120 and gp41, and in anticoagulation.
