Development of a Split Esterase for Protein–Protein Interaction-Dependent Small-Molecule Activation

Abstract

Split reporters based on fluorescent proteins and luciferases have emerged as valuable tools for measuring interactions in biological systems. Relatedly, biosensors that transduce measured input signals into outputs that influence the host system are key components of engineered gene circuits for synthetic biology applications. While small-molecule-based imaging agents are widely used in biological studies, and small-molecule-based drugs and chemical probes can target a range of biological processes, a general method for generating a target small molecule in a biological system based on a measured input signal is lacking. Here, we develop a proximity-dependent split esterase that selectively unmasks ester-protected small molecules in an interaction-dependent manner. Exploiting the versatility of an ester-protected small-molecule output, we demonstrate fluorescent, chemiluminescent, and pharmacological probe generation, each created by masking key alcohol functional groups on a target small molecule. We show that the split esterase system can be used in combination with ester-masked fluorescent or luminescent probes to measure protein–protein interactions and protein–protein interaction inhibitor engagement. We demonstrate that the esterase-based reporter system is compatible with other commonly used split reporter imaging systems for the simultaneous detection of multiple protein–protein interactions. Finally, we develop a system for selective small-molecule-dependent cell killing by unmasking a cytotoxic molecule using an inducible split esterase. Presaging utility in future synthetic biology-based therapeutic applications, we also show that the system can be used for intercellular cell killing via a bystander effect, where one activated cell unmasks a cytotoxic molecule and kills cells physically adjacent to the activated cells. Collectively, this work illustrates that the split esterase system is a valuable new addition to the split protein toolbox, with particularly exciting potential in synthetic biology applications.

Genetically Encoded Activators of Small Molecules for Imaging and Drug Delivery

“…Recently, Dickinson and co-workers used asplit BS2 esterase that only becomes active upon protein-protein interaction. [81] BS2 can hydrolyze the model substrate p-nitrophenyl acetate with ac atalytic efficiency of about k cat /K m = 10 m À1 s À1 . [82] This system was used to release ester-protected fluorophores as well as bioactive compounds.Aremarkable novel feature of this GEA is its dependence on aprotein-protein interaction, which can be used for on-demand activation of substrates.A different bioorthogonal deprotection strategy of hydroxy groups was developed by Meggers,R eetz and co-workers, [83] who reported an engineered bacterial cytochrome p450 fatty acid hydrolase that selectively cleaves off apropargylic ether coumarin-derived substrate with k cat /K m = 76 m À1 s À1 .…”

Versatile labeling and detection of endogenous proteins using tag-assisted split enzyme complementation

“…Other existing split enzymes that could be tested as TASEC systems in tandem tagged cell lines include split-TEV protease 16 , split-Cre recombinase 17 , split-Firefly luciferase 18 , split-DamID 19 and split-esterase 20 . Though we have optimized the system in human cell lines, the TA-splitHalo systems we describe should be applicable in model systems across all three kingdoms (eukaryotes, prokaryotes, and archaea).…”

Genetically Encoded Activators of Small Molecules for Imaging and Drug Delivery

“…This work demonstrates that the cleavage of bioorthogonal esters can be used for the delivery of bioactive or fluorescent compounds with hydroxy groups (Figure A). Recently, Dickinson and co‐workers used a split BS2 esterase that only becomes active upon protein–protein interaction . BS2 can hydrolyze the model substrate p ‐nitrophenyl acetate with a catalytic efficiency of about k cat /K m =10 m −1 s −1 .…”