RecA, a protein pivotal in bacteria’s ability to mutate and resist antibiotic treatment, is an attractive therapeutic target, however efforts to find small molecule drugs that block its function have been limited by the sensitivity of the assays used to detect its ATP hydrolysis (ATPase) activity. Recently researchers at The University of North Carolina at Chapel Hill overcame this roadblock by using BellBrook Labs’ Transcreener ADP Assay as an exquisitely sensitive ATPase Assay, allowing them to identify novel RecA inhibitors that were active in bioassays with live bacteria (Assay Drug Dev Technol 2011 Dec 22).
Madison, WI (PRWEB) May 04, 2012 —The continued emergence of antibiotic resistant bacteria is a growing public health concern that threatens both human and animal health and the lack of appropriate therapies remains an urgent unmet need. RecA is an attractive antibacterial drug target because it is ubiquitous and highly conserved in bacteria, with only distant homologs in humans, and is a key player in bacteria’s ability to mutate and resist antibiotic treatment. RecA uses the energy of ATP hydrolysis to drive DNA strand exchange, so ATPase activity provides a convenient way to monitor a very complicated reaction. Previous efforts to discover inhibitors of RecA fell short because the ATPase assays required high enzyme and substrate concentrations, resulting in deleterious effects on RecA conformation. Recently, The University of North Carolina at Chapel Hill’s Scott Singleton and his team successfully used BellBrook Labs’ Transcreener ADP2 Assay as a robust RecA ATPase assay (Z’ = 0.92) using ATP and enzyme concentrations more than 10-fold lower than previous efforts, thus eliminating the RecA conformational constraint. Under optimized assay conditions for a high throughput screen of greater than 110,000 small molecules, they identified 79 inhibitors with IC50 less than 10 µM and 35 that were active in bioassays with live bacteria (Assay Drug Dev Technol 2011 Dec 22).
Over the last century, the use of antibacterial therapies has significantly impacted both human and animal health. However, the selection pressures inherent with the widespread use of antibacterials have contributed to the increased prevalence of antibacterial resistant bacteria globally. The discovery of effective RecA inhibitors should address the mutative properties of antibiotic resistant bacteria allowing complementary antibacterial therapy to function as intended.
About Transcreener: 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, 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. Visit BellBrook’s website for more information.