RapaLink-1 is a bitopic inhibitor that links an ATP-competitive inhibitor to an allosteric inhibitor via a tether to inhibit signaling by mammalian target of rapamycin complex 1 (mTORC1). Despite its large molecular weight, RapaLink-1 can enter cells and can cross the blood–brain barrier. To determine the basis of RapaLink-1’s cellular entry, Lou et al. performed a genome-wide CRISPR screen in K562 human lymphoblast cells and identified chemical–genetic interactions between RapaLink-1 and members of the interferon-induced transmembrane (IFITM) protein family, which are localized to plasma and endolysosomal membranes and whose primary function is to restrict viral entry. High expression of IFITM proteins was associated with increased sensitivity to RapaLink-1 across a large panel of cell lines. Loss of all three IFITM members increased resistance to RapaLink-1 and reduced RapaLink-1-mediated inhibition of mTORC1. The development of a fluorescent analog of RapaLink-1 revealed that IFITM proteins mediate uptake of RapaLink-1 through endocytic vesicles and into the intracellular space. The effects of IFITM proteins were specific for RapaLink-1, as no effects were observed with the individual components of RapaLink-1 or other mTOR inhibitors; this suggests that IFITM proteins may regulate the cellular entry of linked compounds specifically. To test this possibility, Lou et al. designed a bitopic inhibitor, DasatiLink-1, that targets the fusion oncoprotein BCR–ABL1; it links the ATP-competitive inhibitor dasatinib to an allosteric inhibitor that targets the myristoyl pocket. Loss of all three IFITM proteins reduced the potency of DasatiLink-1, in support of the proposal that IFITM proteins may recognize linked compounds. In addition, IFITM proteins also altered the sensitivity of proteolysis-targeting chimeras and linked molecular glue inhibitors, whereby longer linker lengths correlated with IFITM-mediated uptake. Although the mechanism of IFITM-mediated uptake of linked compounds remains to be determined, the findings from Lou et al. offer potential physiochemical requirements to inform linked compound design.
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