This application is for the design of high affinity soluble analogs of the verotoxin (VT) receptor glycolipid, globotriaosylceramide(Gb3) which can efficiently compete with surface Gb3 of pediatric renal target cells and thereby prevent systemic VT from binding to initiate HUS following gastrointestinal infection with VT producing Escherichia coli (VTEC). We have used a combination of molecular modelling, based on the x-ray crystal structure of the B subunit pentamer, binding of the different VTs to deoxy-Gb3 analogs and analysis of site specific mutations which alter receptor binding, to define the Gb3 binding site within the verotoxin B subunit. This site lies in the cleft between adjacent subunits and comprises Aspl7, Glu28, Thr21, and Glu65. Using this model we have determined the hydrogen bonding network between the bound sugar and amino acids within the receptor site. The carboxyl groups of three amino acids are particularly heavily involved. The synthesis of galabiose analogs containing amino substitutions at the hydroxyl groups involved in these interactions should generate analogs of Gb3 with higher VT binding affinity. Although the VT/Gb3 binding is mediated by the galabiose moiety, the free oligosaccharide is an extremely poor inhibitor of VT/Gb3 binding, indicating the important role of the lipid moiety in binding. Our studies indicate that this is due to an effect on the relative lipid/carbohydrate conformation. We have designed a new synthetic strategy to make soluble glycolipid mimics in which the lipid moiety of the glycolipid has been truncated and derivatized with bulky, rigid hydrophobic substituents. Unlike the free oligosaccharide, such soluble Gb3 mimics are potent inhibitors of VT/Gb3 binding in vitro. We propose to optimize the coupling procedure and characterize the interaction of VT with these soluble receptor mimics. In addition, the combination of amino derivatized receptor carbohydrate analogs in a soluble glycolipid-mimetic format will likely provide extremely potent inhibitors of VT/membrane Gb3 binding.
The specific aims of this application are the development of potent soluble inhibitors capable of prevention of membrane Gb3 binding by VT in vitro assay, VT mediated cytotoxicity in sensitive cultured cells and a dog model of HUS. Our long-term goals are to establish a therapeutic modality for the administration of such soluble, Gb3-based, competitive receptor analogs, following diagnosis of acute VTEC-induced diarrhea in infants. Timely administration of such analogs should prevent the renal targeting of any systemic VT which might otherwise initiate the endothelial damage which can result in renal infarct and the severe sequelae of HUS.
