RESUMO
Förster resonance energy transfer (FRET) techniques have proven invaluable for probing the complex nature of protein-protein interactions, protein folding, and intracellular signaling events. These techniques have traditionally been implemented with the use of one or more fluorescence band-pass filters, either as fluorescence microscopy filter cubes, or as dichroic mirrors and band-pass filters in flow cytometry. In addition, new approaches for measuring FRET, such as fluorescence lifetime and acceptor photobleaching, have been developed. Hyperspectral techniques for imaging and flow cytometry have also shown to be promising for performing FRET measurements. In this study, we have compared traditional (filter-based) FRET approaches to three spectral-based approaches: the ratio of acceptor-to-donor peak emission, linear spectral unmixing, and linear spectral unmixing with a correction for direct acceptor excitation. All methods are estimates of FRET efficiency, except for one-filter set and three-filter set FRET indices, which are included for consistency with prior literature. In the first part of this study, spectrofluorimetric data were collected from a CFP-Epac-YFP FRET probe that has been used for intracellular cAMP measurements. All comparisons were performed using the same spectrofluorimetric datasets as input data, to provide a relevant comparison. Linear spectral unmixing resulted in measurements with the lowest coefficient of variation (0.10) as well as accurate fits using the Hill equation. FRET efficiency methods produced coefficients of variation of less than 0.20, while FRET indices produced coefficients of variation greater than 8.00. These results demonstrate that spectral FRET measurements provide improved response over standard, filter-based measurements. Using spectral approaches, single-cell measurements were conducted through hyperspectral confocal microscopy, linear unmixing, and cell segmentation with quantitative image analysis. Results from these studies confirmed that spectral imaging is effective for measuring subcellular, time-dependent FRET dynamics and that additional fluorescent signals can be readily separated from FRET signals, enabling multilabel studies of molecular interactions. © 2013 International Society for Advancement of Cytometry.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Processamento de Imagem Assistida por Computador/métodos , Células HEK293 , Humanos , Microscopia ConfocalRESUMO
In view of the emerging clinical indications for Phosphodiesterase 9 inhibitors e.g. treatment of Alzheimer, diabetes, cancer, and the limited number of its selective inhibitors which possess a single chemical scaffolds, a structure-based approach was undertaken to mine the ZINC database by virtual screening to identify novel PDE9 inhibitors. The database, which was never reported to have been used before for discovery of PDE9 inhibitors, was screened against the ligand binding pocket of the PDE9 complex (PDB:4GH6) using molecular docking programs, MOE and AutoDock Vina in PyRx. Three different scoring functions were used to evaluate the docking poses and scores of the compounds, and the compounds were selected through consensus selection, thus reducing the margin of error in docking. The highest scoring compounds were then selected and purchased for in vitro testing as PDE9 inhibitors and cancer growth inhibitory agents. This led to the discovery of three previously unreported potent PDE 9 inhibitory compounds with two unique chemical scaffolds. Consistent with the role of PDE9 in cancer cell growth, the compounds also inhibited the growth of breast tumor cell lines, MCF-7 and MDA-468 at concentrations comparable to those that inhibited PDE9.