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.
Subject(s)
High-Throughput Screening Assays/methods , Nerve Growth Factor/metabolism , Protein Kinase Inhibitors/pharmacology , Receptor, trkA/metabolism , Signal Transduction/drug effects , Adenosine Triphosphate/metabolism , Cell Line , Humans , Inhibitory Concentration 50 , Nerve Growth Factor/antagonists & inhibitors , Neurites/drug effects , Neurites/metabolism , Receptor, trkA/antagonists & inhibitorsABSTRACT
The pharmacology and regulation of Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel activity is intricate due to the physiological function as an integrator of multiple chemical, mechanical, and temperature stimuli as well as differences in species pharmacology. In this study, we describe and compare the current inhibition efficacy of human TRPA1 on three different TRPA1 antagonists. We used a homology model of TRPA1 based on Kv1.2 to select pore vestibule residues available for interaction with ligands entering the vestibule. Site-directed mutation constructs were expressed in Xenopus oocytes and their functionality and pharmacology assessed to support and improve our homology model. Based on the functional pharmacology results we propose an antagonist-binding site in the vestibule of the TRPA1 ion channel. We use the results to describe the proposed intravestibular ligand-binding site in TRPA1 in detail. Based on the single site substitutions, we designed a human TRPA1 receptor by substituting several residues in the vestibule and adjacent regions from the rat receptor to address and explain observed species pharmacology differences. In parallel, the lack of effect on HC-030031 inhibition by the vestibule substitutions suggests that this molecule interacts with TRPA1 via a binding site not situated in the vestibule.
