A fluorescence polarization based assay for the identification and characterization of polymerase inhibitors.

A homogenous fluorescence polarization (FP) assay was developed to monitor the enzymatic activity of polymerases. Under the optimized conditions established in this study, the assay provides highly robust and reproducible data. Miniaturization of the assay for high-throughput screening and compound testing was also performed. The sensitivity of the newly developed assay was confirmed using 2',3'-dideoxyadenosine-5'-triphosphate (ddATP), a chain-elongating inhibitor of the polymerase reaction. Side-by-side comparison of the presented fluorescence polarization assay with already well established PicoGreen® fluorescence intensity assay revealed that the performance of both formats is comparable with good assay sensitivity. However, the direct ratiometric readout of the presented FP assay makes it superior over existing colorimetric and fluorescence intensity based assays in terms of susceptibility to false positives. Moreover, due to its generic nature the presented FP assay can be applied to other polymerases and is compatible with identification of inhibitors and requirements of hit-to-lead programs.

Identification of 12/15-lipoxygenase as a regulator of axon degeneration through high-content screening.

Axon degeneration is a programed process that takes place during development, in response to neuronal injury, and as a component of neurodegenerative disease pathology, yet the molecular mechanisms that drive this process remain poorly defined. In this study, we have developed a semi-automated, 384-well format axon degeneration assay in rat dorsal root ganglion (DRG) neurons using a trophic factor withdrawal paradigm. Using this setup, we have screened a library of known drugs and bioactives to identify several previously unappreciated regulators of axon degeneration, including lipoxygenases. Multiple structurally distinct lipoxygenase inhibitors as well as mouse DRG neurons lacking expression of 12/15-lipoxygenase display protection of axons in this context. Retinal ganglion cell axons from 12/15-lipoxygenase-null mice were similarly protected from degeneration following nerve crush injury. Through additional mechanistic studies, we demonstrate that lipoxygenases act cell autonomously within neurons to regulate degeneration, and are required for mitochondrial permeabilization and caspase activation in the axon. These findings suggest that these enzymes may represent an attractive target for treatment of neuropathies and provide a potential mechanism for the neuroprotection observed in various settings following lipoxygenase inhibitor treatment.

Fragment-based discovery of BACE1 inhibitors using functional assays.

Novel nonpeptidic inhibitors of beta-secretase (BACE1) have been discovered by employing a fragment-based biochemical screening approach. A diverse library of 20000 low-molecular weight compounds were screened and yielded 26 novel hits that were confirmed by biochemical and surface plasmon resonance secondary assays. We describe here fragment inhibitors cocrystallized with BACE1 in a flap open and flap closed conformation as determined by X-ray crystallography.

A multiplexed approach to hit finding.

Assay formats and screening technologies can deliver more than one readout per measurement and therefore can be considered to be information-rich. This holds true for biophysical and selected biochemical assays, and in particular for cellular assay formats, where the term 'high-content assay' describes the most complex and advanced paradigm of small-molecule screening available to date. Given the multifactorial nature of the lead generation and optimization process in drug discovery, the enhanced information-density offered by multiplex screening technologies is considered to be beneficial to an informed hit selection for optimization. While hard evidence for an enhancement in hit and lead selection is only now emerging, multiplexed screening technologies will fulfill their promise in drug discovery if our understanding of target pharmacology and the complexity of biological systems can be advanced in parallel.