title: NMR-based quality control approach for the identification of false positives and false negatives in high throughput screening.
abstract: The quality of the data generated in a high throughput screening (HTS) run is fundamental for selecting bona fide inhibitors and for ensuring the capture of the full richness of inhibitors present in a chemical library. For this purpose a quality control filter based on three one dimensional (1D) proton NMR experiments is proposed. The approach called SPAM (Solubility, Purity and Aggregation of the Molecule) Filter requires the acquisition of a 1D reference spectrum, a WaterLOGSY spectrum and/or a selective longitudinal relaxation filter spectrum for the identified hits dissolved in aqueous solution and in the presence of a water soluble reference molecule. This palette of experiments permits the rapid characterization of the identity, purity, solubility and aggregation state of the active compound. This knowledge is crucial for deriving accurate IC(50) and K(1) values of the inhibitors, for identifying false negatives and for detecting promiscuous inhibitors. Only compounds that pass through the SPAM Filter can be considered as starting points for medicinal chemistry efforts directed toward lead optimization. Examples of this approach in the identification of false positives in a screening run against the enzyme thymidine phosphorylase (TP) and the rescue of a false negative in a screening run against the Ser/Thr kinase AKT1 are presented.
authors.affiliation: Chemistry Department Nerviano Medical Sciences, Viale Pasteur 10, 20014 Nerviano, Milan, Italy. claudio.dalvit@nervianoms.com
authors.forename: Claudio
authors.initials: C
authors.lastname: Dalvit
authors.forename: Dannica
authors.initials: D
authors.lastname: Caronni
authors.forename: Nicola
authors.initials: N
authors.lastname: Mongelli
authors.forename: Marina
authors.initials: M
authors.lastname: Veronesi
authors.forename: Anna
authors.initials: A
authors.lastname: Vulpetti
mesh_terms: D000465:Algorithms; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology / Q000592:standards; D006801:Humans; D020128:Inhibitory Concentration 50; D007546:Isoquinolines / Q000737:chemistry / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D015394:Molecular Structure; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism; D011786:Quality Control; D011794:Quercetin / Q000737:chemistry / Q000494:pharmacology; D012995:Solubility; D013329:Structure-Activity Relationship; D013449:Sulfonamides / Q000737:chemistry / Q000494:pharmacology; D013678:Technology, Pharmaceutical / Q000379:methods* / Q000639:trends; D013939:Thymidine Phosphorylase / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D007546:Isoquinolines; D013449:Sulfonamides; D011794:Quercetin; D013939:Thymidine Phosphorylase; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide