Analysis of ubiquitin E3 ligase activity using selective polyubiquitin binding proteins.

The ubiquitin proteasome pathway controls the cellular degradation of ~80-90% of the proteome in a highly regulated manner. In this pathway, E3 ligases are responsible for the conjugation of ubiquitin to protein substrates which can lead to their destruction by the 26S proteasome. Aberrant E3 ligases have been implicated in several diseases and are widely recognized as attractive targets for drug discovery.

High-throughput screening for Kv1.3 channel blockers using an improved FLIPR-based membrane-potential assay.

Voltage-gated K(+) channels are potential drug targets for an increasing number of disease indications. Searching for compounds that modulate K(+) channel activities by high-throughput screening (HTS) is becoming a standard approach in the drug discovery effort. Here the authors report an improved fluorometric imaging plate reader (FLIPR) membrane potential assay for Kv1.

Old and new pharmacology: positive allosteric modulation of the alpha7 nicotinic acetylcholine receptor by the 5-hydroxytryptamine(2B/C) receptor antagonist SB-206553 (3,5-dihydro-5-methyl-N-3-pyridinylbenzo[1,2-b:4,5-b']di pyrrole-1(2H)-carboxamide).

The alpha7 nicotinic acetylcholine receptor (nAChR) has been implicated in Alzheimer's disease and schizophrenia, leading to efforts targeted toward discovering agonists and positive allosteric modulators (PAMs) of this receptor. In a Ca2+ flux fluorometric imaging plate reader assay, SB-206553 (3,5-dihydro-5-methyl -N-3-pyridinylbenzo [1, 2-b:4,5 -b']-di pyrrole-1(2H)-carboxamide), a compound known as a 5-hydroxytryptamine(2B/2C) receptor antagonist, produced an 8-fold potentiation of the evoked calcium signal in the presence of an EC(20) concentration of nicotine and a corresponding EC(50) of 1.5 muM for potentiation of EC(20) nicotine responses in GH4C1 cells expressing the alpha7 receptor.

Ion channel screening.

Ion channels are attractive targets for drug discovery with recent estimates indicating that voltage and ligand-gated channels account for the third and fourth largest gene families represented in company portfolios after the G protein coupled and nuclear hormone receptor families. A historical limitation on ion channel targeted drug discovery in the form of the extremely low throughput nature of the gold standard assay for assessing functional activity, patch clamp electrophysiology in mammalian cells, has been overcome by the implementation of multi-well plate format cell-based screening strategies for ion channels. These have taken advantage of various approaches to monitor ion flux or membrane potential using radioactive, non-radioactive, spectroscopic and fluorescence measurements and have significantly impacted both high-throughput screening and lead optimization efforts.

Orbital MALT lymphoma in a patient with Graves' ophthalmopathy: a unique observation.

A 57-year-old woman with a six-year history of Graves' Disease with ophthalmopathy developed unilateral periorbital swelling. CT scan suggested a lacrimal mass. Biopsy revealed MALT lymphoma.

Characterization of the cloned full-length and a truncated human target of rapamycin: activity, specificity, and enzyme inhibition as studied by a high capacity assay.

The mammalian target of rapamycin (mTOR/TOR) is implicated in cancer and other human disorders and thus an important target for therapeutic intervention. To study human TOR in vitro, we have produced in large scale both the full-length TOR (289 kDa) and a truncated TOR (132 kDa) from HEK293 cells. Both enzymes demonstrated a robust and specific catalytic activity towards the physiological substrate proteins, p70 S6 ribosomal protein kinase 1 (p70S6K1) and eIF4E binding protein 1 (4EBP1), as measured by phosphor-specific antibodies in Western blotting.

Development and comparison of two nonradioactive kinase assays for I kappa B kinase.

In response to diverse stimuli, the transcription factor NF-kappaB is activated by the IKK kinase complex containing two kinases (IKKalpha and IKKbeta) that phosphorylate IkappaB, an inhibitory protein of NF-kappaB. The phosphorylation of IkappaB results in ubiquitination and degradation of IkappaB, allowing NF-kappaB to translocate to the nucleus where it regulates its target genes. To elucidate the role of IKK in the NF-kappaB signaling pathway, we have developed and characterized two quantitative, sensitive, and nonradioactive assays for evaluating IKKbeta activity: a dissociation-enhanced lanthanide fluorescence immunoassay called DELFIA and a homogeneous time-resolved fluorescence resonance energy transfer assay called LANCE.