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Fluorescence spectroscopy is one of the most widely used tools in drug discovery; however, specific criteria for compound fluorescence are not well defined. While a highly sensitive method of detection, fluorescence-based sensing is susceptible to assay interference due to intrinsically fluorescent compounds. Current methods of fluorescence prediction do not consider high-throughput compound screening (HTCS) requirements and therefore are not applicable for filtering large chemical databases. The studies described herein aim to establish structural and electronic criteria for fluorescent emission in HTS, identify structure-fluorescence relationships (SFR) for novel sets of fluorophores, and develop fluorescence-based assays using an explicit empirical definition of fluorescence. Our working definition of fluorescence caters to the unique environment of HTS and is described as BGA: bright (?5.0% fluorescence intensity of fluorescein at the same concentration), green (emission at 535 nm ± 25 nm upon excitation at 485 nm ± 20 nm), and aqueous (fluorescence observed in a buffered solution at a specific concentration). Implementing a decision tree learning-based algorithm based on calculable molecular properties, we were able to predict BGA fluorescence with 93% accuracy for a sample library of compounds (N = 2,231).
From this sample library, two novel fluorophores were identified. Spectral evaluation of these fluorophores resulted in the validation of a dual fluorophore with red and green emissions and a bright green fluorophore with chemical and fluorescent stability in varying solvents and bioavailability in zebrafish. Finally, we have developed a high-throughput fluorescence polarization (FP) assay, utilizing a novel fluorescein-labeled compound (Kd = 0.378 ?M) and a PAN construct, to identify small molecules that bind to the PAN endonuclease active site of influenza virus. This FP assay provides direct target validation information for active compounds, is suitable for HTS and fragment-based compound screening studies, and has resulted in the identification of novel endonuclease inhibitors. Our work suggests that with a basic understanding of the fundamental requirements for fluorescence in an HTS environment, SFR can be predicted, and interference due to intrinsically fluorescent compounds can be controlled within assay settings.