Blue Light Activated Rapamycin for Optical Control of Protein Dimerization in Cells and Zebrafish Embryos

Abstract

Rapamycin-induced dimerization of FKBP and FRB is the most commonly utilized chemically induced protein dimerization system. It has been extensively used to conditionally control protein localization, split-enzyme activity, and protein−protein interactions in general by simply fusing FKBP and FRB to proteins of interest. We have developed a new aminonitrobiphenylethyl caging group and applied it to the generation of a caged rapamycin analog that can be photoactivated using blue light. Importantly, the caged rapamycin analog shows minimal background activity with regard to protein dimerization and can be directly interfaced with a wide range of established (and often commercially available) FKBP/FRB systems. We have successfully demonstrated its applicability to the optical control of enzymatic function, protein stability, and protein subcellular localization. Further, we also showcased its applicability toward optical regulation of cell signaling, specifically mTOR signaling, in cells and aquatic embryos.

Efficient Activation of 6‐Nitropiperonyloxymethylene (NPOM)‐Caged Nucleosides with Visible Light

“…The comparatively reduced decaging efficiency for product formation of highly conjugated nitrobenzyl chromophores with extended π‐conjugation and with electron donating substituents resulting in donor‐π‐acceptor systems are not uncommon, as previous attempts to generate such groups with bathochromic absorption, such as 7‐dimethylamino nitrodibenzofuryl and 4‐methoxystyryl nitrobenzyl, have suffered from poor one‐photon quantum yields [29] . Such an observation was also reported for ANBE‐caged rapamycin where, despite full consumption of starting materials, release of the small molecule rapamycin was incomplete [22] . In our study, we observe additional small peaks in the chromatogram of the decaging reaction of 3 (Figure S5).…”

“…While we observe complete release of T 4 upon photolysis of 1 for both 2 and 5 min exposures (Table 1, color coded green), appearance of 4 from 3 lagged behind even after 10 min of illumination, thus exhibiting a modest 54 % release (Table 1). Contrary to 1 , it was surprising that we did not observe complete release of T from 3 even though the starting material shows complete disappearance (color coded green), leading us to hypothesize that the material is getting trapped at an intermediate stage during the photolysis process, thereby preventing complete release of T. Similar findings has been reported for this particular caging group [22] and are not uncommon with other caging groups [4] . A similar decaging trend was observed for 1 and 3 under 415 nm illumination as well.…”

Molecular Approaches to Protein Dimerization: Opportunities for Supramolecular Chemistry

“…Molecule glue approaches to induce protein dimerization have been demonstrated into two groups: 1) Asymmetric molecules such as Cyclosporin A ( Liu et al, 1991 ), FK506 ( Liu et al, 1991 ; Ho et al, 1996 ), FKCsA ( Belshaw et al, 1996a ), rapamycin ( Rivera et al, 1996 ), gibberellin ( Miyamoto et al, 2012 ), abscisic acid ( Liang et al, 2011 ), HaXS ( Erhart et al, 2013 ), TMP-HTag ( Ballister et al, 2014 ) and ATB-737 ( Hill et al, 2018 ) induce hetero-dimerization of proteins; 2) Symmetric molecules such as FK1012 ( Spencer et al, 1993 ), coumermycin ( Farrar et al, 1996 ) and (cyclosporin A) 2 ( Belshaw et al, 1996b ) induce homo-dimerization of proteins ( Table 1 ). For example, the natural product rapamycin has emerged as the biofunctional dimerizer to induce heterodimerization of proteins ( Choi et al, 1996 ; Liang et al, 1999 ; Bayle et al, 2006 ; Brown et al, 2015 ; Mangal et al, 2018 ; Courtney et al, 2021 ). The most prominent molecular feature of rapamycin is its two chemically distinct protein binding domains: one part of the molecule binds with high nanomolar affinity to the FK506-binding protein (FKBP12), the other molecular part to the FRB domain of mTOR, FRAP (FKBP-rapamycin associated protein), overall resulting in dimerization of the proteins involved.…”

Engineering Small Molecule Switches of Protein Function in Zebrafish Embryos

“…Conditional control of protein activity is an important tool for studying embryo development. − Very few general approaches exist that utilize small molecules as triggers for protein function in zebrafish. Rapamycin-induced protein dimerization and tamoxifen-induced nuclear translocation , have enabled conditional control of protein activity in embryos (Figure A,B). However, these methodologies are limited to split proteins and functional control through protein mislocalization, and they require protein domain fusions that may not always be functionally equivalent to the native protein.…”