MODULATION OF PROTEIN AND CELL FUNCTIONS BY HEPARIN/HEPARAN SULFATES

Child Health And Human Development

Investigators

Linked publications, trials & patents

Abstract

The project elucidates the fundamental molecular design of heparins and heparan sulfates (H/HS) and studies structure- function relations of their specific modulations of diverse normal and diseased protein and cell membrane systems, using biological, biochemical, and physical approaches. We examine H/HS as a modulator of viral infectivity, and isolate and study Components (S-oligoS) of a heparin-mimetic, sulfated xylan drug which, as does heparin, inhibits the infectivity of HIV-1 in vitro: 1) We had revealed differential functional potencies and physical properties of the S-oligoS Components, which led to our isolation and identification of a minimal-sized, potent anti-HIV-1 S-oligoS, CpF and CpF-PkII [HD 01315-01-03]. Moreover, CpF is separated from antithrombin activity which is associated with different structurally specific Components. Upscaled procedures were explored for use in obtaining a CpF-PkII drug against AIDS in humans [HD 01315-04-05]. Our optimized procedures are reliably reproducible and continue to be labor intensive, with ~ 8 % yield. Multiple preparations to obtain gram quantities of CpF-PkII for a Phase I trial remain a primary focus and continue in cooperation with the drug manufacturer with whom we have negotiated a confidential agreement (30% of goal are now completed through final step). 2) Structure-function relations are studied by FTIR, exciton-absorption and NMR spectroscopy. Proton NMR and sugar analysis support our proposal of a tetrasaccharide grouping in heparin-mimetic structures (a beta 1,4-linked trixyloside containing an alpha 1,2 D-GlcA branch) [HD01315-01-03,05]. FTIR of CpF-PkII revealed sugars in the alternate as well as normal chair forms (axial (ax) as well as expected equatorial (eq) sulfates) [HD01315-03-04], and these structures are displayed differentially among Components. Structures containing axial sulfates may be of significance in functional specificity: a high mass, antithrombin Component (4B) was associated with 1.7-fold greater proportion of such alternate chair conformations than PK-II [HD01313-05] and we find by FTIR that anticaoagulant, anti-HIV-1 Component 4A displays an ax-to-eq sulfate ratio of 3.1 compared with 0.11 for that of Component 12B which is inactive against thrombin and low in anti-HIV-1 capacity. From proton NMR spectra of PkII, signals at 4.83, 4.93, 4.6 and 5.18 ppm suggest alternate chair forms. A 13C-proton correlation study of PkII designed to elucidate the sugar(s) and carbon atom(s) associated with ax sufate groups is in progress. 3) The molecular mechanism underlying the inhibition of HIV-1 by S-oligoS and H/HS are investigated: Fluorescent photoactive-crosslinking CpF-PkI-probes were synthesized [HD 01315-05]. Studies to mprove % derivatization by direct nucleophilic addition to the aldehyde using aminonaptholsulfonic acid hydrazide continue. These unique probes will assist in studies to identify and isolate a putative cell membrane S-oligoS receptor involved in viral infectivity. 4) Heparin-mimetic inhibition of the malarial parasite by Components in vitro is examined: Components are prepared and currently assayed for specificity.