An improved beta-lactamase reporter assay: multiplexing with a cytotoxicity readout for enhanced accuracy of hit identification.

A problem inherent to the use of cellular assays for drug discovery is their sensitivity to cytotoxic compounds, which can result in false hits from certain compound screens. To alleviate the need to follow-up hits from a reporter assay with a separate cytotoxicity assay, the authors have developed a multiplexed assay that combines the readout of a beta-lactamase reporter with that of a homogeneous cytotoxicity indicator. Important aspects to the development of the multiplexed format are addressed, including results that demonstrate that the IC(50) values of 40 select compounds in a beta-lactamase reporter assay for nuclear factor kappa B and SIE pathway antagonists are not affected by the addition of the cytotoxicity indicator.

Highly miniaturized formats for in vitro drug metabolism assays using vivid fluorescent substrates and recombinant human cytochrome P450 enzymes.

Highly miniaturized P450 screening assays designed to enable facile analysis of P450 drug interactions in a 1536-well plate format with the principal human cytochrome P450 enzymes (CYP3A4, 2D6, 2C9, 2C19, and 1A2) and Vivid fluorogenic substrates were developed. The detailed characterization of the assays included stability, homogeneity, and reproducibility of the recombinant P450 enzymes and the kinetic parameters of their reactions with Vivid fluorogenic substrates, with a focus on the specific characteristics of each component that enable screening in a low-volume 1536-well plate assay format. The screening assays were applied for the assessment of individual cytochrome P450 inhibition profiles with a panel of selected assay modifiers, including isozyme-specific substrates and inhibitors.

Overcoming compound interference in fluorescence polarization-based kinase assays using far-red tracers.

Kinase-mediated phosphorylation of proteins is critical to the regulation of many biological processes, including cell growth, apoptosis, and differentiation. Because of the central role that kinases play in processes that can lead to disease states, the targeting of kinases with small-molecule inhibitors is a validated strategy for therapeutic intervention. Classic methods for assaying kinases include nonhomogenous enzyme-linked immunosorbent assays or scintillation-based formats using [gamma-(32)P]ATP.