title: Identification of biologically active, HIV TAR RNA-binding small molecules using small molecule microarrays.
abstract: 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' untranslated region (UTR) of the HIV-1 genome to determine global structural changes in the presence of the molecule. Importantly, the interaction of compound 4 can be mapped to the TAR hairpin without broadly disrupting any other structured elements of the 5' UTR. Cell-based anti-HIV assays indicated that 4 inhibits HIV-induced cytopathicity in T lymphocytes with an EC50 of 28 μM, while cytotoxicity was not observed at concentrations approaching 1 mM.
authors.affiliation: HIV Drug Resistance Program, National Cancer Institute , Frederick, Maryland, United States .
authors.forename: Joanna
authors.initials: J
authors.lastname: Sztuba-Solinska
authors.forename: Shilpa R
authors.initials: SR
authors.lastname: Shenoy
authors.forename: Peter
authors.initials: P
authors.lastname: Gareiss
authors.forename: Lauren R H
authors.initials: LR
authors.lastname: Krumpe
authors.forename: Stuart F J
authors.initials: SF
authors.lastname: Le Grice
authors.forename: Barry R
authors.initials: BR
authors.lastname: O'Keefe
authors.forename: John S
authors.initials: JS
authors.lastname: Schneekloth
mesh_terms: D019380:Anti-HIV Agents / Q000737:chemistry* / Q000494:pharmacology; D045744:Cell Line, Tumor; D003588:Cytopathogenic Effect, Viral; D055808:Drug Discovery; D005470:Fluorometry; D016325:HIV Long Terminal Repeat / Q000187:drug effects* / Q000235:genetics; D015497:HIV-1 / Q000187:drug effects / Q000472:pathogenicity; D006801:Humans; D015394:Molecular Structure; D059372:Nucleotide Motifs / Q000235:genetics; D012367:RNA, Viral / Q000737:chemistry*; D054852:Small Molecule Libraries / Q000737:chemistry* / Q000494:pharmacology; D013601:T-Lymphocytes / Q000821:virology
chemical_list: D019380:Anti-HIV Agents; D012367:RNA, Viral; D054852:Small Molecule Libraries