Selective, rapid and optically switchable regulation of protein function in live mammalian cells

Abstract

The rapid and selective regulation of a target protein within living cells that contain closely related family members is an outstanding challenge. Here we introduce genetically directed bioorthogonal ligand tethering (BOLT) and demonstrate selective inhibition (iBOLT) of protein function. In iBOLT, inhibitor-conjugate/target protein pairs are created where the target protein contains a genetically encoded unnatural amino acid with bioorthogonal reactivity and the inhibitor conjugate contains a complementary bioorthogonal group. iBOLT enables the first rapid and specific inhibition of MEK isozymes, and introducing photoisomerizable linkers in the inhibitor conjugate enables reversible, optical regulation of protein activity (photo-BOLT) in live mammalian cells. We demonstrate that a pan kinase inhibitor conjugate allows selective and rapid inhibition of the lymphocyte specific kinase, indicating the modularity and scalability of BOLT. We anticipate that BOLT will enable the rapid and selective regulation of diverse proteins for which no selective small-molecule ligands exist.

The use of unnatural amino acids to study and engineer protein function

“…Tethering of inhibitors to UAAs with bioorthogonal reactivity (iBOLT) installed close to the active site of kinases facilitates their potent, specific inactivation in mammalian cells (Figure 3b) [51 ]. Introducing a photo-isomerizable azo-benzene group into the linker connecting the inhibitor and the reactive group allows controlling the activity of the kinase by light (photo-BOLT).…”

“…These bispecific antibodies mediate the encounter between these cell types to induce neutralisation of the cancer cell. (b) Selective inhibition of kinases has been achieved by sitespecific coupling of a low affinity inhibitor close to the active site [51 ]. (c) Identification of cell type specific proteins in fruit flies by incorporation of reactive UAAs in response to sense codons using cell type specific promoters for the expression of the synthetase [52 ].…”

Playing with the Molecules of Life

“…This ncAA has been used to photoregulate enzyme activity by reversibly blocking the active site 247 or by moving a tethered inhibitor in and out of the active site by cis-trans photoisomerization of the azobenzene moiety. 259 …”

“…Light irradiation of different wavelengths results in cis/trans isomerization, blocking or exposing the active site. 259 E) Genetically-encoded decaging of a strained cycloalkene via reaction with a tetrazine, restoring a lysine residue and protein function. Proof-of-concept experiments were performed via caging luciferase, quenching luminescence until the tetrazine reagent is added.…”

Self-assembling peptide-based building blocks in medical applications

“…A recent approach called “π-clamping” tailored biomolecules with the help of a natural small peptide sequence, and is providing to be a very exciting strategy that expands the ability of bio-conjugation chemistry [63,64]. Another approach is bioorthogonal ligand tethering (BOLT), in which a genetically encoded, unnatural amino acid on a protein is bioorthogonally reactive with an inhibitor conjugate to enable reversible regulation of protein activity in mammalian cells [65]. The unnatural amino acids are commonly used probes for selective conjugation, and their use can be extended to several pairs of fluorophores with Förster resonance energy transfer in physiological conditions [66].…”