THE BASIC DOMAIN IN HIV-1 TAT PROTEIN AS A TARGET FOR POLYSULFONATED HEPARIN-MIMICKING EXTRACELLULAR TAT ANTAGONISTS

Heparin binds extracellular HIV-1 Tat protein and modulates its HIV lo ng terminal repeat (LTR)-transactivating activity (M, Rusnati, D. Colt rini, P. Oreste, G. Zoppetti, A Albini, D. Noonan, F. d'Adda di Fagagn a, M. Giacca, and M. Presta (1997) J. Biol. Chem. 272, 11313-11320). O n this basis, the glutathione S-transferase (GST)-Tat(R49/52/53/55/56/ 57A) mutant, in which six arginine residues within the basic domain of Tat were mutagenized to alanine residues, was compared with GST-Tat f or its capacity to bind immobilized heparin. Dissociation of the GST-T at(R49/52/53/55/56/57A). heparin complex occurred at ionic strength si gnificantly lower than that required to dissociate the GST-Tat heparin complex. Accordingly, heparin binds immobilized GST-Tat and GST-Tat(R 49/52/53/55/56/57A) with a dissociation constant equal to 0.3 and 1.0 mu M, respectively. Also, the synthetic basic domain Tat-(41-60) compe tes with GST-Tat for heparin binding. Suramin inhibits [H-3]heparin/Ta t interaction, I-125-GST-Tat internalization, and the LTR-transactivat ing activity of extracellular Tat in HL3T1 cells and prevents I-125-GS T-Tat binding and cell proliferation in Tat-overexpressing T53 cells. The suramin derivative C-14-PNU 145156E binds immobilized GST-Tat with a dissociation constant 5 times higher than heparin and is unable to bind GST-Tat(R49/52/53/55/56/57A). Although heparin was an antagonist more potent than suramin, modifications of the backbone structure in s elected suramin derivatives originated Tat antagonists whose potency w as close to that shown by heparin. In conclusion, suramin derivatives bind the basic domain of Tat, prevent Tat/heparin and Tat/cell surface interactions, and inhibit the biological activity of extracellular Ta t. Our data demonstrate that tailored polysulfonated compounds represe nt potent extracellular Tat inhibitors of possible therapeutic value.