Development of novel chemical inducers of dimerization to regulate proteins with high spatial and temporal precision

The function of proteins is often regulated by the formation of multi-protein complexes or translo- cation events. Over the last few decades, several methods have been developed to force proteins to specific locations or induce protein-protein interactions. One of these approaches is based on small bi-functional molecules, so-called chemical inducers of dimerisation (CIDs), which bind to two protein tags. Therefore, two proteins of interest fused to the tag protein, are brought into close proximity in the presence of the CID. Since the pioneering report by Schreiber and Crabtree in 1996, a variety of novel CIDs were designed and used to regulate protein dimerisation and translocation. However, most of these systems exhibit relatively low temporal resolution, deter- mined by the cell permeability of the small molecule. One of the few exceptions is rapamycin, a naturally occurring CID, which rapidly induces dimerisation between FKBP12 and FRB. Due to its immunosuppressant properties, several researchers used the ”bump and whole” approach to design C16 rapamycin derivatives, which do not bind to wild-type FRB, but to a geneti- cally engineered mutant. Although, various nucleophiles were introduced at the C16 position, no general method to introduce carbamates was published. Here we describe an efficient Lewis acid- mediated method to substitute rapamycin at the C16 position with various nucleophiles under mild conditions. Furthermore, we performed NMR experiments to elucidate the exact mech- anism of the mentioned method. We observed that treatment of rapamycin with the Lewis acid BF3-Et2O in CH2Cl2, leads to the heterolytic cleavage of the C16 methoxy group and the formation of a BF3-carbocation complex, which does not react with carbamates. The addition of THF further stabilises the carbocation, which results in a fast reaction with the nucleophile. To demonstrate the utility of this novel method, we introduced a series of representative carbamates at the C16 position of rapamycin. One of the major challenges, when synthesising rapalogs, is that small impurities of rapamycin or rapamycin by-products still inhibit mTOR. Therefore, we extensively purified the diastereomers of C16 phenyl carbamate (pcRap) with preparative HPLC. In a proof of concept, (R)-pcRap successfully induced dimerisation of FKBP and FRB fusion proteins without interfering with the mTOR pathway.
The application of light controllable tools is an alternative approach to regulate the localisation and the activity of proteins with high spatiotemporal precision. Here we report the synthe- sis of a series of photoactivatable HaXS dimerisers, which contain a photocleavable linker be- tween the HaloTag-reactive chloroalkane ligand and the SNAP-tag-reactive O6-benzylguanine. Chemical modification of the photocleavable linker afforded three powerful photocleavable and cell-permeable CIDs, which could be cleaved independently and orthogonally. Furthermore, in a proof of concept, one of these photocleavable HaXS molecules was successfully used to translocate proteins of interest to the plasma membrane, late endosomes, lysosomes, Golgi, mitochondria and the actin cytoskeleton. Subsequent irradiation of a specific location with either a FRAP-laser or a mercury lamp with a DAPI filter set, readily liberated the anchored proteins. Furthermore, we demonstrated the utility of photocleavable CIDs and explicitly MeNV-HaXS in kinetic studies of protein dynamics and the manipulation of subcellular enzyme activities. Therefore, MeNV- HaXS was used to anchor a nuclear probe to the Golgi. Subsequent irradiation of cells triggered the release and the relocalisation of the nuclear probe into the nucleus.