Increasing resistance to current HIV-1 therapy underlines the need for novel antiretroviral agents.The protein Tat is critical for viral replication and therefore is an attractive potential target for the discovery of antiretroviral agents [1].Our approach to Tat inhibitors involves the use of conformationally constrained peptidomimetics.A library of peptidomimetics was derived, by grafting Tat-related sequences onto a-hairpin-inducing D-Pro-L-Pro template [2].The peptidomimetics were tested in vitro for their activity against HIV and BIV TAR.One mimetic (BIV2) was identified that binds tightly to both HIV and BIV TAR, The solution structure of the BIV2-BIV TAR complex revealed all the important intermolecular contacts that account for the sequence specificity and affinity of complex formation and facilitated the development of more potent ligands.New libraries of mimetics were designed based on these structural data and the molecules were tested for their activity against BIV and HIV-1 TAR.Single amino acid substitution within BIV2 severely affected the affinity providing a foundation of structure-based design in the Tat-TAR system.Moreover,the total overall charge of our compounds was reduced to enhance their pharmacological potential.Several mimetics from these new libraries proved to be HIV-1 TAR ligands of improved affinity.The peptidomimetics with highest affmity to HIV-1 TAR were tested in reconstituted transcription of the HIV-1 promoter in HeLa cell extracts.Some of our ligands were found to inhibit Tat-specific transactivation of transcriptional elongation.These new ligands represent leads for the development of more potent and specific Tat inhibitors.