title: High throughput screening of library compounds against an oligonucleotide substructure of an RNA target.
abstract: Approximately 300,000 compounds from selected libraries were screened against a subdomain of a hepatitis C viral (HCV) RNA using a high throughput flow injection mass spectrometry (FIA-MS) method with automated data storage and analysis. Samples contained 2 microM RNA target and 10 microM of each of up to ten ligands. Preliminary studies to optimize operational parameters used the binding of aminoglycosides to the A44 subdomain of bacterial RNA. Binding (confirmed by titration) and sensitivity were maximized within the constraints of the library and throughput. The mobile phase of 5 mM ammonium acetate in 50% isopropanol maintained the noncovalent complexes and provided good detection by electrospray mass spectrometry. Additionally, this composition maximized general solubility of the various classes of compounds including the oligonucleotide and organic library molecules. Cation adduction was insignificant in this screen although some solute and target dependent acetate adduction was observed. The ion trap mass spectrometer provided sufficient mass resolution to identify complexes of RNA with known components of the library. Converted mass spectral data (netCDF) were subjected to two types of statistical evaluation based on binding. The first algorithm identified noncovalent complexes that correlated with the molecular weights of the injected compounds. The second yielded the largest peak in the noncovalent complex region of the spectrum; this spectrum may or may not correlate with expected well components. Sixty-three compounds were confirmed to bind by more stringent secondary testing. Titrations, which were carried out with selected binding compounds, yielded a range of dissociation constants. Biological activity was observed for eleven confirmed binders.
authors.affiliation: Eli Lilly and Company, Indianapolis, Indiana 46285, USA. gooding_karen@lilly.com
authors.forename: Karen B
authors.initials: KB
authors.lastname: Gooding
authors.forename: Richard
authors.initials: R
authors.lastname: Higgs
authors.forename: Barry
authors.initials: B
authors.lastname: Hodge
authors.forename: Eric
authors.initials: E
authors.lastname: Stauffer
authors.forename: Beverly
authors.initials: B
authors.lastname: Heinz
authors.forename: Kevin
authors.initials: K
authors.lastname: McKnight
authors.forename: Krista
authors.initials: K
authors.lastname: Phipps
authors.forename: Michael
authors.initials: M
authors.lastname: Shapiro
authors.forename: Malcolm
authors.initials: M
authors.lastname: Winkler
authors.forename: Wai-Leung
authors.initials: WL
authors.lastname: Ng
authors.forename: Randall K
authors.initials: RK
authors.lastname: Julian
mesh_terms: D008024:Ligands; D013058:Mass Spectrometry; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009930:Organic Chemicals / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D008024:Ligands; D009843:Oligoribonucleotides; D009930:Organic Chemicals; D012313:RNA