TOPOLOGY OF THE LOXORIBINE BINDING-SITE - STUDIES WITH INACTIVE LOXORIBINE ANALOGS

We have previously described a class of immunostimulatory ribonucleosi de, exemplified by loxoribine (7-allyl-8-oxoguanosine) and 8-bromoguan osine, that act-on cycling B cells to promote nonspecific proliferatio n and differentiation, and that synergize with Ag to recruit quiescent Ag-reactive B cells to undergo differentiation and high level Ab prod uction. In murine B cells, two distinct binding activities have been c haracterized that have dissociation constants that parallel their dist inctive dose-response profiles. The SJL mouse strain, which is hypores ponsive to the B cell proliferative properties of these molecules, exh ibits a dissociation constant of 10- to 20-fold lower affinity than th at of normal murine strains; its K-d for the higher affinity-binding i nteraction, however, is essentially normal. These observations suggest ed the existence of two distinct binding activities. Recently, immunos elective subgroups of loxoribine have been described, with an ability to stimulate individual components of the immune response that correla tes with specific changes in discrete domains of the nucleoside molecu le. By using a panel of nonstimulatory loxoribine analogues as inhibit ors, we now report further evidence for the existence of unique sites on loxoribine binding proteins that recognize distinct domains on nucl eoside analogues; changes in these regions confer immunologic selectiv ity. These data confirm the conclusions of previous studies, extend ou r understanding of the topology of subsites that are otherwise silent, and provide insight into the binding site mediating adjuvanticity. Th ese results allow more profound appreciation of the nature of structur al changes that will permit a tighter fit between ligand and binding s ite, ultimately promoting the design of more potent immunoselective ag ents.