Photo-activatable derivatives of small molecules are typically generated through the installation of a light-removable protecting group, a so called “caging group”, at a site crucial for biological activity of the small molecule.⁹ This renders the molecule inactive, until the caging group is removed through light-irradiation, typically with UV light of 365–405 nm.¹⁰ The feasibility of this approach has been demonstrated through successful photochemical regulation of numerous small molecules in cellular environments and multicellular organisms.^{9, 11} Here, we report the synthesis of a photocaged rapamycin analog (pRap, Figure 1a) which, together with an engineered FKBP (iFKBP), enables successful photo-control of the FKBP-FRB interaction (Figure 1b). The caged rapamycin is applied to regulate both protein dimerization and kinase activity in live cells. Interestingly, caging is not effective with unaltered FKBP, but requires the FKBP mutations described here. An analysis of the chemically accessible sites of rapamycin (Rap) revealed that the methoxy group on C-16 can undergo nucleophilic substitution^13–17 and β-elimination.¹² The hydroxyl groups at C-28 and C-40 can be protected with silyl groups,^13,14 and a trifluoromethylsulfonyl group.¹⁵ The lactone at C-34 can be hydrolyzed and eliminated^22–24 and, importantly, the hydroxyl group at C-40 can be converted into a carbonate group¹⁵ and be esterified.¹⁵, ^{20, 21} Thus, C-40 represents the most suitable site for chemical modification with a carbonate-linked caging group that can provide facile installation and quick photolysis. Importantly, based on the crystal structure of the ternary complex of rapamycin, FRB, and FKBP12, the hydroxyl group at C-40 undergoes hydrogen bond formation with the glutamine 53 of FKBP12 (Figure 1c).¹⁶ Thus, we hypothesized that disruption of that hydrogen bond through installation of a sterically demanding α-methyl-6-nitropiperonyloxycarbonyl (MeNPOC) group would prevent protein dimerization.

The caged rapamycin pRap was synthesized in one step from rapamycin (Rap) via chemoselective acylation with the mixed carbonate of 6-nitro-piperonyl alcohol and N-hydroxysuccinimide (NPOC-NHS, Figure 1a). The identity of purified pRap was confirmed by ¹H NMR and HRMS analysis (see Supporting Information).

We first tested whether pRap could induce dimerization of FKBP12 and FRB. For this we created a GFP-FRB protein fusion and wild-type FKBP12 fused to the N-terminus of focal adhesion kinase (FAK). FAK localizes prominently to focal adhesions in living cells,¹⁷ allowing us to test dimerization in vivo by observing rapamycin-mediated translocation of GFP-FRB into focal adhesions. Prior to live cell co-localization studies, the constructs were tested in pull-down assays, comparing the ability of pRap and Rap to mediate the intracellular dimerization of FKBP-FAK and FRB. Cells expressing both FKBP-FAK and FRB were treated with rapamycin or pRap for one hour, with or without irradiation. Complex formation was assayed by pulling down myc-FKBP-FAK from cell lysates and blotting for GFP-FRB (Supporting Figure 1a, b). Surprisingly, both small molecules generated dimerization with similar effectiveness, with or without UV irradiation (Supporting Figure 1a, b). These results were further confirmed by an mTOR activity assay (Supporting Figure 2). This data showed that the FKBP12-rapamycin-FRB complex was not sufficiently sensitive to rapamycin modification for a successful light-activation approach through photocaging.

We hypothesized that alteration of FKBP12 could be used to render the ternary rapamycin-FKBP-FRB interaction more sensitive to the functional groups of pRap affected by photolysis. We tested a recently developed modified FKBP, named iFKBP, that is proposed to have increased structural mobility of the Lys52-Glu54 loop positioned next to the C-40 hydroxyl group of rapamycin (Figure 1c).⁸ Using both an N-terminal iFKBP-FAK fusion (as tested above for FKBP) and a fusion of iFKBP internally, at position 413 of FAK (Figure 2a), we examined whether pRap could mediate heterodimerization of iFKBP and FRB in a light dependent manner. Indeed, pRap (at concentrations of up to 20 µM) failed to mediate interaction between iFKBP-FAK and GFP-FRB, while irradiation of pRap-treated cells with 365 nm UV light successfully removed the caging group and induced iFKBP-FRB dimerization (Figure 2b–d). Uncaging kinetics were dependent on both light dosage and pRap concentration. Importantly, in the presence of pRap, translocation of FRB into focal adhesions was observed only upon decaging, indicating successful protein dimerization between FAK-iFKBP and FRB in live cells (Figure 2e–f). These studies demonstrated that pRap can effectively mediate light-dependent protein heterodimerization when used with iFKBP rather than FKBP12.

Recently, we developed a new method for the regulation of protein kinases in live cells.⁸ Insertion of iFKBP at a structurally conserved position within the catalytic domain of several kinases, including FAK, rendered the kinases inactive. Our previous studies indicate that insertion of the iFKBP increases the mobility of the critical G-loop in the catalytic domain of FAK, resulting in the inhibition of catalytic activity.⁸ Formation of iFKBP-rapamycin-FRB complex through addition of rapamycin significantly restricts iFKBP dynamics, thus stabilizing the G-loop and rescuing the kinase activity.⁸ This was used to achieve specific control of kinases in living cells with high temporal resolution.⁸

Here we tested the light-mediated regulation of FAK activity with pRap using RapR-FAK (rapamycin-regulated FAK) as a model (Figure 3a). Myc-tagged RapR-FAK was co-expressed with GFP-FRB in HEK293T cells. As in the published validation of RapR-FAK, we examined rapamycin’s ability to induce RapR-FAK phosphorylation of the N-terminal fragment of paxillin,¹⁸ a signal transduction adaptor protein and natural FAK substrate. Unlike rapamycin, pRap was completely inactive at concentrations of up to 20 µM, producing no detectable paxillin Tyr31 phosphorylation. UV irradiation alone did not activate RapR-FAK in the absence of pRap, but irradiation in the presence of pRap (1–20 µM) induced activation of RapR-FAK through protein complex formation with FRB (Figure 3b), leading to robust phosphorylation of paxillin. Light-mediated interaction between RapR-FAK and GFP-FRB was further confirmed by co-immunoprecipitation of the two proteins (Figure 3b) and by translocation of FRB to focal adhesions (Figure 2f) upon pRap irradiation.

Finally, we examined whether light induced activation of pRap could be used to control cell behavior. FAK activation has been shown to produce large dorsal membrane ruffles⁸. We therefore examined effects of RapR-FAK activation on the membrane dynamics of HeLa cells. In the presence of pRap, but without UV irradiation, transfected HeLa cells displayed normal, small peripheral ruffles around the border of the cell (Figure 3c), consistent with inactive RapR-FAK. In contrast, UV irradiation (365 nm) produced very large and dynamic ruffles across the dorsal cell surface (Figure 3d and Supporting Movie 1). This UV-induced phenotype was displayed in 40% of analyzed cells (9 of 22 cells), in excellent agreement with effects of regular rapamycin on RapR-FAK (56% positive cells).⁸

To explore how modification of FKBP to iFKBP was able to render pRap inactive until irradiated with UV light, we performed discrete molecular dynamics (DMD) simulations.¹⁹ Within the sampled conformations we identified the dominant ensemble and compared the localization of residues that form the iFKBP-FRB and FKBP12-FRB interfaces. Surprisingly, our simulations demonstrated that binding of the caged rapamycin to iFKBP is at least as strong as that to FKBP12, but the interaction of the added piperonyloxycarbonyl moiety with iFKBP distorts the protein’s binding-competent conformations and prevents binding of FRB. The most notable difference between the iFKBP and FKBP complexes is a significant distortion of the FRB-binding interface formed by the segment Asp41-Leu49 (Figure 4a–b; Supporting Movies 2–5). The interface area formed between pRap and iFKBP is larger than that between pRap and FKBP12. Due to its higher structural plasticity, the iFKBP protein is able to deform and create additional contacts with pRap, which is unachievable by the more rigid and stable FKBP12. Based on these simulations, we suggest that strong binding between iFKBP and pRap distorts the FRB-binding interface in FKBP12, and thus prevents further binding to FRB.

In summary, we have developed a new photocaged analog of rapamycin, pRap, which can be used together with iFKBP, an engineered version of FKBP12, for the light-mediated regulation of protein dimerization. We demonstrated successful use of this new dimerization system by modulating protein interactions for two different FAK-iFKBP fusions in living cells. Furthermore, we achieved light-mediated activation of an engineered protein kinase, FAK, and demonstrated light-induced changes in cell behavior characteristic of this kinase. Rapamycin-mediated protein dimerization and regulation of kinases have lacked the precise spatial and temporal control of light-mediated processes. Our new caged rapamycin approach will significantly enhance the many existing methodologies that use rapamycin for the regulation of protein activity in cells and multicellular organisms. We are currently exploring the synthesis of caged, orthogonal rapalogs and two-photon activated caged rapamycin.

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