Fluorescent Affinity Probe for mTOR Protein Synthesized via Click Chemistry and Its Applications

The mammalian target of rapamycin (mTOR) is a crucial threonine/serine kinase and plays a pivotal role in regulating cell growth and proliferation, making it a significant drug target for various diseases. Understanding the function of mTOR in living cells is essential for the diagnosis of certain diseases. However, so far, the in situ study of endogenous mTOR activity and its interaction with drugs in living cells remains a significant challenge. In this study, we synthesized a novel fluorescent probe utilizing click chemistry to in situ label the endogenous mTOR protein in living cells. Rapamycin, known for its high affinity to mTOR, was used as the probe's backbone. Initially, a modified connecting arm was introduced into the rapamycin structure, followed by the incorporation of <i>trans</i>-cyclooctene, resulting in the intermediate compound (RPT). Another intermediate compound (TA680) was synthesized by attaching the fluorescent dye (ATTO680) to a tetrazine. The fluorescence labeling of the mTOR protein in living cells was achieved through the inverse electron demand Diels-Alder (IEDDA) click reaction between cyclooctene in RPT and tetrazine in TA680. Fluorescence correlation spectroscopy (FCS) and fluorescence imaging confirmed that the probe specifically labeled endogenous mTOR in living cells. The TA680 demonstrated fluorescence quenching behavior, and the probe's fluorescence intensity increased 8-fold upon the click reaction with <i>trans</i>-cyclooctene-modified rapamycin. The interaction between endogenous mTOR and drugs in living cells was investigated. Compared to a traditional fluorescent probe based on rapamycin-modified fluorescent dyes, this new fluorescent probe exhibits turn-on fluorescence properties, making it highly suitable for mTOR studies in living cells.