The Use of TrkA-PathHunter Assay in High-Throughput Screening to Identify Compounds That Affect Nerve Growth Factor Signaling

Abstract

The TrkA-PathHunter cell-based assay was used in high-throughput screening (HTS) to identify compounds that inhibit nerve growth factor (NGF)/TrkA signaling. The assay was conducted in a 384-well format, and typical Z' values during HTS ranged from 0.3 to 0.8. The reproducibility of IC50 values was good, and the use of cryopreserved cells was well tolerated, as judged by assay parameters such as Z' and S/B and by comparison of IC50 values obtained with cells in culture. During hit deconvolution, TrkA-kinase inhibitors were identified with ATP-competitive as well as non-ATP-competitive mechanisms of action. Furthermore, other mechanisms of action such as NGF and TrkA antagonists were also identified. Because of the different molecular mechanisms identified, it is possible that subsequent optimization work to increase affinity and selectivity might lead to compounds that could have a better chance to evoke clinical efficacy without the adverse effects observed for nonselective TrkA inhibitors.

Identification of amino acid residues of nerve growth factor important for neurite outgrowth in human dorsal root ganglion neurons

“…Activation of TrkA at tyrosine 490 (Y490) and the subsequent recruitment of the SHC1 adaptor protein were measured as described previously (Forsell et al., ) using the U2OS‐TrkA/p75‐SHC1 chemiluminescent cell assay from DiscoverX. NGF‐R100E was significantly more potent to enhance activation of TrkA and to induce SHC1 recruitment ( p = .0395) than wild‐type NGF (Figure a, Table ).…”

“…The enzyme fragment complementation (EFC) technique used for measuring activation of the TrkA receptor has been described previously (Forsell et al., ). Briefly, the assay is a proximity‐based assay from DiscoverX where the interaction between the phosphorylated TrkA receptor at tyrosine 490 (Y490) and the intracellular signalling protein SHC1 (Src homology 2 domain containing transforming protein 1) is accompanied by complementation between two parts of β‐galactosidase, leading to an active β‐galactosidase enzyme.…”

Design, Synthesis and Inhibitory Activity of Photoswitchable RET Kinase Inhibitors

“…Encouraged by the clear difference in inhibitory effect observed in the isolated enzyme, we turned to demonstrate the effect also in a living system. Thus, the inhibitory activity of E - 4 and Z - 4 was evaluated in a cell-based functional assay, which utilizes β-galactosidase-based enzyme fragment complementation technology to produce enzyme-activity correlated luminescence readout 27 28 29 . Compound 4 was added to live cells expressing the RET kinase (see Methods section for incubation details) in two identical preparations, followed by preincubation for 3 h at 37 °C.…”

Using the BioAssay Ontology for Analyzing High-Throughput Screening Data

“…More cost-effective methods and methods with higher throughput are constantly being developed and marketed. During assay development early in a discovery program, several methods are often compared and evaluated for each target, 11,12,37 which leads to the optimization and progression of the method that best meets the criteria of cost, automation, and robust detection of tool compounds. The assay method preference for a specific target or target class is not obvious and differs between screening sites and organizations, since choice of assay method is also influenced by retrospective analysis of success with a particular technology within the organization, the experience and bias of the researchers, and the availability of reagents and instruments.…”

“…Standardized annotation of in-house HTS assays using BAO is demonstrated by previously published in-house screening assay development experiments ( Table 1). [10][11][12] The assay design and detection methods of the in-house HTS assays have been analyzed together with 233 primary PubChem HTS assays. 7 Annotated PubChem assays have been provided by the BAO team and are mainly biochemical assays, G protein-coupled receptor (GPCR) assays, and various assays detected by luminescence.…”