and leukemia, and is also involved in protein aggregate formation in some neurodegenerative disorders.
Identification of inhibitors to clarify its role in disease and as potential drug scaffolds is therefore of high interest to investigators in a number of fields. However like many ATPases, p97 is a challenging target for high throughput screening because it has low activity and a high ATP requirement, a combination that demands an assay method with high sensitivity and outstanding signal to noise. A customer interested in finding p97 inhibitors for mechanistic studies recently asked BellBrook to develop an HTS assay for p97 using the company’s Transcreener ADP Assay, which has been used successfully in a number of similar ATPase screening projects (J Biomol Screen. 2010 15(3):279-86; Assay Drug Dev Technol. 2011 Dec 22). The customer had two different p97 protein preparations to evaluate and wanted to compare fluorescence polarization and TR-FRET readouts. BellBrook scientists delivered optimized FP (fluorescence polarization) and TR-FRET (time-resolved Förster resonance energy transfer) based p97 ATPase assays with outstanding signal windows in one week (Transcreener p97 Case Study).
Transcreener® is a universal, high throughput biochemical assay platform based on detection of nucleotides, including ADP, GDP, AMP, GMP and UDP which are formed by thousands of cellular enzymes, many of which catalyze the covalent regulatory reactions that are central to cell signaling and represent new opportunities for therapeutic intervention. It relies on highly specific antibodies that detect nucleotides coupled with homogenous fluorescent detection formats suitable for automated HTS applications. A single Transcreener assay can be used with any enzyme that produces a given nucleotide, allowing detection of hundreds of different drug targets. It is the only enzyme assay method that allows direct detection of nucleotide enzyme products without the use of additional coupling or reporter enzymes, which are a common source of interference by chemical library compounds. BellBrook currently holds five issued patents on the Transcreener technology.
The Transcreener ADP assay relies on selective immunodetection of ADP, and has been formatted in three different fluorescent detection modes: fluorescence polarization, fluorescence intensity and time-resolved Förster resonance energy transfer. With low nanomolar sensitivity, accommodation of ATP concentrations from 0.1 to 1,000 micromolar, and deck and signal stability of more than 24 hours, it has been broadly adopted for screening kinases and other ATP-dependent enzymes since its introduction in 2006. Recent enhancements include a more sensitive monoclonal antibody and the development of assay conditions that allow direct determination of IC50 values from raw fluorescence data; i.e., without using a standard curve.
About BellBrook Labs. BellBrook Labs, LLC develops detection reagents and microfluidic devices that accelerate the discovery of more effective therapies for cancer and other debilitating diseases. Transcreener® is a patented high throughput screening assay platform that was introduced in 2005 and is used to identify inhibitors for kinases and other types of protein drug targets. The iuvo™ Microconduit Array technology and assay screening service is a line of unique microscale devices for miniaturization and automation of advanced cell models that are more representative of human physiology. Visit BellBrook’s website for more information: http://www.bellbrooklabs.com.
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