This project elucidates the fundamental molecular design of the heparin/heparan sulfate class of biological regulatory polysaccharides (H/HS) and studies how the high degree of diversity of its sulfated oligosaccharide (oligoS) structures relates to its parallel, multifunctional capacity. This capacity is exerted through specific binding to, and thus modulation of the functional activity of, many different protein partners in normal and disease processes (e.g., cell growth, secretion, multi-cell reactions in development, blood coagulation, physiological stability, and in infections by virus and other classes of human pathogens). Established and/or newly devised biological, biochemical, and physical methods are utilized. [HD 01315-01-08]. Due to the complex diversity of oligoS sequences within H/HS chains, libraries of unique H/HS structures have not been prepared in quantities needed for extended research. This project has engaged several approaches to study structural requirements for the functions ascribed to H/HS, in particular its capacity to inhibit HIV-1: 1) CHARACTERIZATION AND PREPARATION OF A NEW CLASS OF HIV-1 INHIBITOR: We previously developed a H/HS-mimetic library by low pressure liquid chromatography fractionation of a H/HS- mimetic sulfated xylan pharmaceutical which mimicks most of the known biological actions of H. 24 sulfated oligosaccharide (S-oligoS) Components (Cps) were obtained. Cps were tested against in vitro anticoagulant and anti-HIV-1 assays [HD 01315-01-06]. Findings showed that a degree of structural specificity governed both heparin-mimetic capacities of the pharmaceutical. This led to the preparation and characterization of a highly active anti-HIV-1 agent which was essentially free of anti-thrombin toxicity (CpF-PkII). An enlarged method for producing sufficient CpF-PkII for potential Phase I testing was developed [HD 01315-04-05] and a clinical preparation is underway utilizing 40 grams of starting pharmaceutical [HD 01315-05-08]. Currently, about 50% is processed to the second or first stage purification. Two modifications were developed for faster processing: An on-line detector monitors the chromatographic separation, eliminating post-column analyses; high reproducibility among the required series of columns now allows analyses of fewer fractions to identify product (sharper cut). We expect with staff to complete the preparation of 3-3.5 grams of CpF-PkII in FY 04, and then to prepare the requirements for Phase I testing. This putative adjunct drug would provide an additional mechanism of inhibition of HIV 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. Our basic research studies [HD 01315-03-06] with staff will resume and include: Protein or protein regions (from literature and data banks on HIV and CD4 membrane proteins) will be tested as putative ligands for CpF-PkII by modifying the gel shift analysis for heparin oligoS-protein binding (Rosenberg, R. D. and coworkers) and using a fluorescent derivative to mark the migration of S-oligoS in the gel. Further characterization of structure-function relation of CpF-PkII and other S-oligoS by capillary HPLC/mass spectrometry will be examined. 2) DEVELOPMENT OF H/HS-MIMETIC LIBRARY of S-OLIGOS: The heparin-mimetic S-oligoS are now characterized by physical,chemical,and various functional properties and may be used to identify and examine structural properties of the functionality of S-oligoS and H/HS in the inhibition of other diseases or disorders. We successfully utilized the library to examine the anti-malaria parasite capacities of H/HS [See HD008733-02-03]. Cps also were sent to colleagues for their comparison studies on the inhibition of tumor metastasis by H. Preliminary results were positive and additional Cps will be sent for further comparison. 3) ELUCIDATION OF THE S-OLIGOS STUCTURE. Structural features of the library have been studied by biochemical, chemical, and spectroscopic (FTIR, NMR, and dye-coupling) analyses and by titration. We discovered early that the heparin-mimetic structures differed markedly from the then generally accepted model(a sulfated polyxylose with sparse glucuronic acid (GlcA) branches, one to 10 xyloses (Xyl) on average). The S-oligoS Cps contained about I GlcA to 3 Xyl and thus were largely a tetrasaccharide motif, one alpha 1,2-linked GlcA-branch per beta-1,4-linked trixylyl sequence [HD 01315-01-05]. Such motifs would display sulfated GlcA-Xyl- groupings spaced along the chain by only two xylose rings and each GlcA could assume a helical rotation of 180 degrees. Subsequent studies further diminished apparent dissimilarities between the macrostructures of H and the S-oligoS. FTIR, proton NMR and heteronuclear two-dimensional NMR proton-13C-correlation spectroscopy (analyzed by the heteronuclear multi-dimensional quantum coherences (HMQC) method) on CpF-PkII revealed that some sugar moieties were in an alternative chair conformation instead of the expected normal chair form [HD 01313-05-08]. Only one of the two sugars was seen to assume this form, and the doublet signals appeared to arise from only one sulfated carbon position. This would be consistent with a mono- sulfated Xyl which contains a GlcA branch. As with H, such sugars would have a shortening effect on the chain backbone. This finding points out an additional parameter for possible diversity in the functional structures of these heparin-mimetics as well as that of H/HS. The data taken together led us to propose a structure for CpF-PkII, (4-O Me D- GlcA-alpha 1,2 beta 1,4 D-xylyltrisaccharide) dodecaglucuronyl-oligoxylose, having several moieties of either Xyl or less likely GlcA in the alternative chair conformation. Recent FTIR data demonstrated that alternative chair characteristics are also displayed by other S-oligoS. Furthermore, the relative ratio of such alternate ring forms to the normal chair differed among Cps with different functions and mass [HD 01313-05-08]. Current 1H-13C heteronuclear NMR spectroscopy of the low mass sporozoite inhibitor (Cp 11) did not reveal doublet peaks which signal these alternative forms. This data is consistent with the low ratio of alternate forms found by FTIR analysis of Cp 12 in the low mass region. The notion that sugar conformations may be a governing factor in structural specificity of the protein-binding sugar sequences of H/HS may have a general significance, e.g., in strategies for synthesis of oligoS drugs and the design of synthetic oligoS-protective antigens. Structure-function studies including FTIR and 1H-13C correlation NMR spectroscopy on further purified inhibitors will continue.
| Wright, T E; Milam, K A; Rougee, L et al. (2011) Agreement of umbilical cord drug and cotinine levels with maternal self-report of drug use and smoking during pregnancy. J Perinatol 31:324-9 |
