A high throughput assay for mammalian target of rapamycin (mtor) kinase activity

1537 Background : The goal of this study was to develop an automation-friendly, homogenous, time-resolved fluorescence resonance energy transfer (HTR-FRET) assay to screen for mTOR kinase inhibitors. The assay was adapted from an ELISA assay, which utilized a partially purified rat brain extract as the source of mTOR kinase activity and an 81-amino acid GST-p70S6 kinase fragment as the substrate.
 Methods : Protocols for enrichment of mTOR kinase activity from rat brain extract as well as an ELISA-based assay to measure mTOR kinase activity were transferred from the Burnham Institute. The extract was shown to contain activity which phosphorylated both mTOR-raptor subtrates and mTOR-rictor substrates. Using the GST-p70S6K fragment as the substrate, the assay was shown to be specific for mTOR by immunodepletion studies. The mTOR kinase activity was profiled against thirty-five known kinase inhibitors with only Wortmannin (>90% Inhibition) and LY294002 (27% Inhibition) identified as inhibitors. Rapamycin, a well-characterized inhibitor of mTOR-raptor was active as an inhibitor when pre-incubated with recombinant FKBP12. FKBP12 alone was not inhibitory. The FKBP12-Rapamycin complex at 1 μM resulted in 50% inhibition of mTOR activity suggesting the existence of a Rapamycin-resistant component of mTOR kinase activity. Using the HTR-FRET assay the mTOR activity was shown to be dependent on Mn2+ similar to other lipid kinases, and unlike protein kinases that typically prefer Mg2+.
 Results: The HTR-FRET assay was needed to screen a collection of 170,000 compounds. The screen resulted in finding compounds that were both ATP competitive and reversible. Recently recombinant mTOR has become available from Invitrogen. A number of compounds were tested with both enzyme sources and comparable IC50 values were obtained.
 Conclusion: We have successfully developed a homogenous HTR-FRET assay for mTOR kinase using purified rat brain extract as a source for mTOR enzyme and identified ATP competitive inhibitors of mTOR kinase.