Fluorescently labeled analogues of dofetilide as high-affinity fluorescence polarization ligands for the human ether-a-go-go-related gene (hERG) channel.

Novel fluorescent derivatives of dofetilide (1) have been synthesized. Analogues that feature a fluorescent probe attached through an aliphatic spacer to the central tertiary nitrogen of 1 have high affinity for the hERG channel, and affinity is dependent on both linker length and pendent dye. These variables have been optimized to generate Cy3B derivative 10e, which has hERG channel affinity equivalent to that of dofetilide. When bound to cell membranes expressing the hERG channel, 10e shows a robust increase in fluorescence polarization (FP) signal. In a FP binding assay using 10e as tracer ligand, Ki values for several known hERG channel blockers were measured and excellent agreement with the literature Ki values was observed over an affinity range of 2 nM to 3 muM. 10e blocks hERG channel current in electrophysiological patch clamp experiments, and computational docking experiments predict that the dofetilide core of 10e binds hERG channel in a conformation similar to that previously predicted for 1. These analogues enable high-throughput hERG channel binding assays that are rapid, economical, and predictive of test compounds' potential for prolonged QT liabilities.

Involvement of beta-site APP cleaving enzyme 1 (BACE1) in amyloid precursor protein-mediated enhancement of memory and activity-dependent synaptic plasticity.

The amyloid precursor protein (APP) undergoes sequential cleavages to generate various polypeptides, including the amyloid-beta protein (Abeta), which forms amyloid plaques in Alzheimer's disease (AD), secreted APPalpha (sAPPalpha) which enhances memory, and the APP intracellular domain (AICD), which has been implicated in the regulation of gene transcription and calcium signaling. The beta-site APP cleaving enzyme 1 (BACE1) cleaves APP in an activity-dependent manner to form Abeta, AICD, and secreted APPbeta. Because this neural activity was shown to diminish synaptic transmission in vitro [Kamenetz F, Tomita T, Hsieh H, Seabrook G, Borchelt D, Iwatsubo T, Sisodia S, Malinow R (2003) Neuron 37:925-937], the prevailing notion has been that this pathway diminishes synaptic function. Here we investigated the role of this pathway in vivo. We studied transgenic mice overproducing APP that do not develop AD pathology or memory deficits but instead exhibit enhanced spatial memory. We showed enhanced synaptic plasticity in the hippocampus that depends on prior synaptic activity. We found that the enhanced memory and synaptic plasticity are abolished by the ablation of one or both copies of the BACE1 gene, leading to a significant decrease in AICD but not of any other APP cleavage products. In contrast to the previously described negative effect of BACE1-mediated cleavage of APP on synaptic function in vitro, our in vivo work indicates that BACE1-mediated cleavage of APP can facilitate learning, memory, and synaptic plasticity.

Early evaluation of compound QT prolongation effects: a predictive 384-well fluorescence polarization binding assay for measuring hERG blockade.

INTRODUCTION: A large number of drugs from a variety of pharmacological classes have been demonstrated to cause adverse effects on cardiac rhythm, including the life-threatening arrhythmia Torsades de Pointes. These side effects are often associated with prolongation of the QT interval and are mediated via blockade of the human ether-a-go-go related gene (hERG) encoded potassium channel. In order to manage this risk in the pharmaceutical industry it is desirable to evaluate QT prolongation as early as possible in the drug discovery process. METHODS: Here we describe the development of a 384-well fluorescence polarization (FP) binding assay compatible with high-throughput assessment of compound blockade of the hERG channel during the lead optimisation process. To characterise the fluorescent ligand that was developed, competition binding studies, kinetic studies and electrophysiology studies were performed. Furthermore, to validate the assay as a key screening method a series of competition binding studies were performed and correlated with functional data obtained via patch-clamp. RESULTS: Evaluation of the assay indicates that high quality data is obtained (Z'>0.6), that the K(i) values determined are equivalent to more traditional radiometric methods and that it is predictive for functional hERG blockade as assessed by patch clamp. DISCUSSION: Whilst FP assays, utilizing a variety of fluors, have become well established for the evaluation of G-protein-coupled receptor (GPCRs) and kinase ligand interactions, this technique has not been applied widely to the study of ion channels. Therefore, this represents a novel assay format that is amenable to the evaluation of thousands of compounds per day. Whilst other assay formats have proven predictive or high throughput, this assay represents one of few that combines both attributes, moreover it represents the most cost effective assay, making it truly amenable to early assessment of hERG blockade.