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, multi-functional capacity. This capacity is exerted through specific binding to, and thus modulating 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 viral and other infections). Established and/or newly devised biological, biochemical, and physical methods are utilized. [HD 01315-01-07] Previously: To reveal and study putative unique structures required for specific H/HS functions, and focusing on its anti-viral capacities against HIV-1, we devised a structurally simpler, model H/HS system because despite intensive biochemical research to prepare libraries of such H/HS structures, these are difficult to generate (especially in sufficient quantities for extended research needs) due to the complex diversity of oligoS sequences within H/HS. A model H/HS library of functionally different Components was prepared by low pressure liquid chromatography of a multifunctional heparin-mimetic pharmaceutical which is comprised of a mixture of chemically sulfated xylan oligosaccharides (S-oligoS). [HD 01315-01-06]. Briefly, Components were tested against five in vitro anticoagulant and antiviral target assays. This study showed that each functional capacity was governed by a degree of structural specificity as would be expected for H/HS. This established the potential usefulness of our model system and a potent anti-HIV-1 agent, CpF-PkII, devoid of anti-thrombin activity was isolated and characterized. An enlarged method suitable for producing sufficient CpF-PkII for potential Phase I testing was developed and is underway. About 30 percent is prepared. In addition, the above macro combinatorial strategy using the S-oligoS library was successfully extended to elucidate the anti-malaria parasite capacities attributed to H/HS [See HD 008733-01]. Structural features of the S-oligoS are studied by analytical, chemical, sugar and spectroscopic-FTIR, NMR, and dye-coupling analysis, and by titration. First we discovered that the GlcA to Xyl ratio in the S-oligoS was closer to 1:3 (sugar and proton NMR analyses), indicating that there is a tetrasaccharide motif (beta-1,4-linked trixyloside with one alpha 1,2-linked branch) in the H/HS mimetic structure, rather than that of a beta xylan chain with sparse (1:10) GlcA branches as previously assumed and accepted. [HD 01315-01-05] In addition, from FTIR, proton NMR and 13C heteronuclear correlation spectroscopy data on CpF-PkII and other Components we discovered that functional structures in our S-oligoS library contained sugar moieties in an alternative conformation as well as those in the expected normal chair form. Moreover, the relative proportions of axial sulfate groups that would be associated with the alternate ring form differed among Components with different functional specificities. [HD 01313-05] Since H/HS also contains axial sulfates on sugar moieties in an alternate chair form, this finding indicated a structural basis for the heparin-mimetic capacity of S-oligoS for the first time and highlighted that such sugar conformations may be important in function and specificity of H/HS. [HD 01315-03-04] Current: Some experiments from above were completed. We continue to characterize and expand the library of S-oligoS structures and are establishing it among colleagues studying diverse normal/pathogenic systems as a valid mimic of the H/HS structures which have the various functions ascribed to H/HS. Progress was significant in: 1) We reported this research in four presentations on the expanded library and our applications of the library to examine H/HS function. Results from a series of 12 highly characterized S-oligoS (of 24 studied) against six targets demonstrated that this library contained discrete structures which exhibited differential specificities against the targets and that the potent inhibitors could be identified. Moreover, Components are well characterized as to functional, physical and chemical properties and could now be used to elucidate the properties of the structure of the functional molecules of H/HS in the inhibition of other diseases or disorders (e.g., whether an inhibitor could be prepared devoid of anti-thrombin toxicity). From NMR, FTIR and mass studies, we also proposed that the structure of CpF-PkII is a (4-O Me D- glucuuronyl-alpha 1,2 beta 1,4 D-ylyltrisaccharide) dodecaglucuronyl-oligoxylose containiing multiple xylyl moieties in an alternative chair conformation. Such sugars have a shortening effect on the chain backbone which resembles that in heparin. The positions and nature of the alternative moieties remain to be elucidated. [Stone AL Differential structure-function relations in a family of heparin-mimetic sulfated glucuronyloligoxyloses in vitro: Malaria parasite, AIDS virus and blood coagulation inhibitors Gordon Research Conference on Proteoglycans, July 2002] 2) The first extended library samples were prepared and sent to Dr. Lubor Borsig (U. of Zurich) for his studies on heparin inhibition of tumor metastasis. 3) The potential importance of Cp11 as an agent against malaria sporozoites is a significant new finding in our studies. This H/HS mimetic has ideal characteristics for safe clinical application (See HD 008733-02) and could be produced by methods analogous to those developed for isolation of CpF-PkII. Cp 3 (highly active against parasite invasion of erythrocytes) and Cp 11 were prepared and analyses are on going for further structural characterization. 4) Heteronuclear two-dimensional NMR proton-13C-correlation spectroscopy using an inverted probe in a 600 MHz spectrometer at 60 degrees and analysis by the heteronuclear multi-dimensional quantum coherences (HMQC) method conclusively demonstrated the presence of alternative chair conformations. A hydrogen atom bonded to a sugar carbon that occurs in two distinct conformations would exhibit two complex interactions with that carbon. The NMR correlation spectrum of CpF-PkII displayed two pairs of 13C-satellite signals about 71 ppm on the carbon spectrum, showing unambiguously that at least one type of sugar moiety existed in two conformations, which confirmed the finding from FTIR. Detailed analysis of the correlation spectrum of Pk-II and FTIR spectroscopy of numerous Components now indicate that axial-like sulfates were present on sugar rings with alternative chair conformations in most S-oligoS and that in the case of PkII the axial sulfate appears to occur on carbon 3. The ratio of these forms among additional Components studied also varied. Samples of an anti-thrombin and the antimalaria S-oligoS have been prepared for correlation NMR spectroscopy and analyses are ongoing. 5) This finding, that sugar conformations may be a governing parameter in structural specificity of the protein-binding sugar sequences of H/HS, may also have far reaching important implications in the areas of development of strategies for synthesis of oligoS drugs and in the design of synthetic oligoS-protective antigens. The above studies will be continued and include initiation of testing of putative proteins as partners for CpF-PkII, Cp11 and Cp3 by modification of the gel shift analysis for heparin oligoS-protein binding. Preparation of CpF-PkII will be resumed when required staff arrives. [See HD 008733-02 for other references.]
| 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 |
