Riaz Rouhani

A High-Throughput, Nonisotopic, Competitive Binding Assay for Kinases Using Nonselective Inhibitor Probes (ED-NSIP™)

A novel competitive binding assay for protein kinase inhibitors has been developed for high-throughput screening (HTS). Unlike functional kinase assays, which are based on detection of substrate phosphorylation by the enzyme, this novel method directly measures the binding potency of compounds to the kinase ATP binding site through competition with a conjugated binding probe. The binding interaction is coupled to a signal amplification system based on complementation of β-galactosidase enzyme fragments, a homogeneous, nonisotopic assay technology platform developed by DiscoveRx Corp. In the present study, staurosporine, a potent, nonselective kinase inhibitor, was chemically conjugated to a small fragment of β-galactosidase (termed ED-SS ). This was used as the binding probe to the kinase ATP binding pocket. The binding potencies of several inhibitors with diverse structures were assessed by displacement of ED-SS from the kinase. The assay format was specifically evaluated with GSK3α, an enzyme previously screened in a radio-active kinase assay (i.e., measurement of [33P]-γ-ATP incorporation into the kinase peptide substrate). Under optimized assay conditions, nonconjugated staurosporine inhibited ED-SS binding in a concentration-dependent manner with an apparent potency (IC50) of 11 nM, which was similar to the IC50 value determined in a radioactive assay. Furthermore, 9 kinase inhibitors with diverse structures, previously identified from chemical compound library screening, were screened using the competitive binding assay. The potencies in the binding assay were in very good agreement with those obtained previously in the isotopic functional activity assay. The binding assay was adapted for automated HTS using selected compound libraries in a 384-well microtiter plate format. The HTS assay was observed to be highly robust and reproducible (Z factors > 0.7) with high interassay precision ( R 2 > 0.96). Interference of compounds with the β-galactosidase signal readout was negligible. In conclusion, the DiscoveRx competitive kinase binding assay, termed ED-NSIP ™, provides a novel method for screening kinase inhibitors. The format is homogeneous, robust, and amenable to automation. Because there is no requirement for substrate-specific antibodies, the assay is particularly applicable to Ser/Thr kinase assay, in which difficulties in identifying a suitable substrate and antibody preclude development of nonisotopic assays. Although the nonselective kinase inhibitor, staurosporine, was used here, chemically conjugating the ED fragment to other small molecule enzyme inhibitors is also feasible, suggesting that the format is generally applicable to other enzyme systems.

Galactosidase Enzyme Fragment Complementation as a High-Throughput Screening Protease Technology

The authors describe a homogeneous, high-throughput screening (HTS) assay for measuring protease activity and detection of inhibitors. The assay comprises a cyclic β-galactosidase (β-gal) enzyme donor peptide (ED) containing a protease-selective cleavage sequence. Alone, the cyclic peptide is inactive, but when linearized following protease cleavage, ED complements with β-gal enzyme acceptor forming active β-gal enzyme. This then catalyzes the formation of either fluorescent or chemiluminescent products, with β-gal turnover providing a highly amplified signal, and thus an assay technology of high sensitivity. To demonstrate the utility of the technology, an EFC assay was developed to measure the activity of 2, caspase 3 and β-secretase. Using a cyclic ED containing the caspase 3 substrate sequence, DEVD, the EFC assay signal was linear with respect to caspase 3 concentration. The assay was very sensitive, being able to detect activity at low picogram amounts of caspase 3. For the β-secretase (BACE) EFC assay, a cyclic ED containing the Swedish mutant cleavage site of amyloid precursor protein (APP), SEVNLDAEFK, was used. In a similar fashion to the caspase 3 assay, the signal induced by BACE activity was linear with respect to enzyme concentration and was highly sensitive, being able to detect nanogram quantities of BACE. The assay was also more sensitive than a commercially available FRET-based assay of BACE activity. It is concluded that the EFC protease assay is a simple, flexible, and sensitive technology for HTS of proteases.