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TAR RNA represents an attractive target for the intervention of human immunodeficiency virus type 1 (HIV-1) replication by small molecules. We now describe three small molecule inhibitors of the HIV-1 Tat-TAR interaction that target the RNA, not the protein. The chemical structures and RNA binding characteristics of these inhibitors are unique for each molecule. Results from various biochemical and spectroscopic methods reveal that each of the three Tat-TAR inhibitors recognizes a different structural feature at the bulge, lower stem, or loop region of TAR. Furthermore, one of these Tat-TAR inhibitors has been demonstrated, in cellular environments, to inhibit (a) a TAR-dependent, Tat-activated transcription and (b) the replication of HIV-1 in a latently infectious model. Drug discovery has traditionally involved a search for inhibitors of protein complexation to small molecules (e.g., substrates or ligands) or macromolecules (e.g., other proteins, nucleic acids, or polysaccharides). While agonists or antagonists of macromolecular receptors that become useful drugs must display a wide range of other attributes, including the appropriate physicochemical properties, pharmacokinetic and pharmacodynamic properties, stability, etc., they must first be protein ligands. The vast majority of available drugs act by binding noncovalently to a protein, preventing or (less often) stimulating that protein's complexation to a complement (1).Complexes of nucleic acid and proteins are key intermediates in all transcriptional and translational processes. Some nucleic acids (e.g., ribozymes) even form functional complexes with small molecules (2). Nonetheless, nucleic acids are widely viewed as ineffective targets for the discovery of low-molecular weight inhibitors. One reason is that the linear motif in single-stranded DNA and the repetitive motif in double-stranded DNA provide attractive targets for large, linear binding molecules (3), but unattractive targets for the small molecules that lead to orally available medications. Another reason is that the lack of tertiary structure in DNA does not afford the diverse topology associated with folded proteins.However, single-stranded RNA often folds into welldefined tertiary structures (4). Furthermore, such structures serve as docking sites for transcriptional activators (5) and substrates for self-splicing reactions (6, 7). Can such structured nucleic acids display sufficient shape diversity to permit the complexation of a small molecule at one site in a virtual sea of nucleic acid material? This is the essential question whose presumed negative answer hinders drug discovery at the nucleic acid level.Certain cis-acting RNA elements are essential for the gene expression of human immunodeficiency virus type 1 (HIV-1) (8). The functions and sequences of these RNA molecules have been well characterized (9-11). A segment of HIV-1 mRNA (residues 1-59), identified as the transactivation responsive element (TAR), adopts a stem-loop secondary structure consisting of a highly cons...

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A-Tris (3,4,7,8-tetramethyl-1,10-phenanthroline)ruthenium(ll) [A-Ru(TMP)2+] was found to be a distinctive molecular tool to examine the local variations in conformation along the strand. The metal complex binds cooperatively to A-form helices of various base sequences under conditions where little or no binding was found to analogous B-form DNAs. Photoactivated DNA cleavage may be coupled to this conformation-specific binding by taking advantage of the photophysical properties of ruthenium(ll) complexes. ARu(TMP)2+ cleaves preferentially 3H-labeled A-form polynucleotides upon irradiation with visible light. The photoinduced DNA strand scission is likely to be mediated by singlet oxygen, which leads to a preferential cleavage of guanine residues. Comparative mapping of cleavage sites on a linear pBR322 fragment for tris(phenanthroline)ruthenium(II), which binds to B-DNA and cleaves also by sensitization of singlet oxygen, and for Ru(TMP)2 I shows the selective binding of ARu(TMP)2+ to conformationally distinct sites along the fragment. These sites correspond to 5-to 13-base-pair homopyrimidine stretches.It has become increasingly clear that a remarkable conformational heterogeneity may be present along the DNA strand. Local variations in DNA structure include bends, kinks, cruciform loops, and even left-handed Z-DNA (1-5). Segments of altered conformation may serve as recognition sites for the binding of regulatory proteins, and indeed the correlation of conformationally distinct sites with the borders of gene coding regions has been observed (6-9).Small molecules that recognize and react at distinct sites along the DNA strand provide sensitive probes for the local variations in DNA structure. We have focused on chiral metal complexes in developing tools to map local DNA secondary structure (10). A-tris(4,7-diphenyl-1,10-phenanthroline)cobalt(III), for example, has been shown to cleave conformationally distinct sites, such as Z-DNA, and has been used to examine altered conformations along the simian virus 40 genome (6, 7, 11). We have determined that tris(3,4, 7,8-tetramethylphenanthroline)ruthenium(II) [Ru(TMP)"+] binds preferentially to A-form polynucleotides, displays chiral discrimination in its binding, favoring the A-isomer in binding to right-handed helices, and upon photoactivation promotes cleavage of the bound polymer (12). The complex associates with the polymer in a surface or groove-bound mode rather than through intercalation, the primary mode of binding for the parent chiral complex tris(phenanthroline)ruthenium(II) [Ru(phen)2+] (13). We report here the application of Ru(TMP)2+ to synthetic polynucleotides and to probe local A-form sites through photoactivated cleavage. Abbreviations: Ru(TMP)3 +, tris(3,4,7,8-tetramethylphenanthroline)ruthenium(II); Ru(phen)3+, tris(phenanthroline)ruthenium(II).

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