Identificationof Biologically Active HIV TAR RNA-BindingSmall Molecules Using Small Molecule Microarrays

摘要

Identifying small molecules that selectively bind to structured RNA motifs remains an important challenge in developing potent and specific therapeutics. Most strategies to find RNA-binding molecules have identified highly charged compounds or aminoglycosides that commonly have modest selectivity. Here we demonstrate a strategy to screen a large unbiased library of druglike small molecules in a microarray format against an RNA target. This approach has enabled the identification of a novel chemotype that selectively targets the HIV transactivation response (TAR) RNA hairpin in a manner not dependent on cationic charge. Thienopyridine >4 binds to and stabilizes the TAR hairpin with a Kd of 2.4 μM. Structure–activity relationships demonstrate that this compound achieves activity through hydrophobic and aromatic substituents on a heterocyclic core, rather than cationic groups typically required. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) analysis was performed on a 365-nucleotide sequence derived from the 5′ untranslatedregion (UTR) of the HIV-1 genome to determine global structural changesin the presence of the molecule. Importantly, the interaction of compound >4 can be mapped to the TAR hairpin without broadly disruptingany other structured elements of the 5′ UTR. Cell-based anti-HIVassays indicated that >4 inhibits HIV-induced cytopathicityin T lymphocytes with an EC50 of 28 μM, while cytotoxicitywas not observed at concentrations approaching 1 mM.