Janus liquid handling platform

The screen was performed at the Case Western Reserve University School of Medicine Small Molecule Drug Development Core facility on 10K “pre-aged” (precultured for 45–50 PD) NHDF. The collection of biologically active molecules was compiled from LOPAC library (Sigma) and Bioactive Compound Library (Selleck Chemicals). A total of 2684 mechanistically annotated compounds were used for the screening. A set of redox catalysts were added to the screen, including: VA, dimethyl-nitrosoaniline, diethyl-nitrosoaniline, 1-nitroso-2-naphthol, 2-nitroso-1-naphthol, CYPMPO, DMPO, methylene blue and coniferron (Sigma-Aldrich). Stock solutions were prepared in DMSO at 10 mM. For screening, 384-well assay plates were prepared with final drug concentrations of 5 μM using a Janus liquid handling platform (Perkin Elmer) equipped with 50 nL pin transfer tool (V&P Scientific). For concentration–response studies, lead compounds were retested at eight concentrations in two-fold dilutions. A final DMSO concentration of 0.1% was not exceeded in the screening assay and in hit validation. The negative controls contained the same percentage of vehicle/DMSO. In each screening plate two vehicle columns (32 wells) served as controls. In each titration plate, four vehicle columns (64 wells) served as controls.

The Approved Oncology Drugs Set VIII, consisting of 133 FDA-approved chemotherapeutic drugs was kindly provided by the National Cancer Institute Division of Cancer Treatment and Diagnosis/ Developmental Therapeutics Program (http://dtp.cancer.gov) of the National Institutes of Health (Bethesda, MD, USA). Drugs were provided at concentrations of 10 mM in 100% dimethyl sulfoxide (DMSO). Stock plates were diluted in 96- or 384-well polypropylene V-bottom microplates using an automated JANUS liquid handling platform (Perkin Elmer).