Utilization of Meso Scale Discovery ELISA technology to quantitate the in vivo pharmacodynamic effects of Rapamycin treatment on the AKT pathway

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The integral role of multiple members of the PI3K/AKT pathway in cell survival has made this pathway a fertile hunting ground for the development of targeted cancer therapeutics. Generation of an optimal therapeutic agent to a specific target requires thorough characterization. This characterization often involves repetitive assessment of the effects of hundreds of related structures not only on the direct downstream effectors of the target, but on other members of the pathway of interest as well. The collection of in vitro cell culture data is valuable for the initial assessment of compound related target effects. Ultimately however, it is essential to evaluate the effects of treatment on members of the pathway and to directly demonstrate that the target is inhibited in vivo. Quantitating the phosphorylation status of multiple signaling pathway members and downstream effectors in treated tumor samples via western blot analysis is laborious and time-consuming, particularly in a medium throughput mode. Using the Meso Scale Discovery (MSD) ELISA technology, we were able to rapidly and efficiently assess the in vivo effects of treatment with the mTOR inhibitor Rapamycin on multiple phosphorylation sites for AKT, p70S6 Kinase, S6RP and GSK3β in HCT 116 xenograft tumors. Utilization of the MSD technology not only facilitated the thorough in vivo characterization of Rapamycin treatment on several members of the PI3K/AKT pathway, but allows for enhanced compound throughput without diminution of data collection during the course of an SAR effort for the development of a targeted cancer therapeutic agent.