Site-Specific Labeling of Endogenous Proteins Using CoLDR Chemistry

Nir London,Adi Rogel,Neta Gurwicz,Ronen Gabizon,Rambabu N. Reddi,Pragati Kishore Prasad,Ziv Shulman,Haim Barr,Efrat Resnick

doi:10.1021/jacs.1c06167

Nir London, Adi Rogel + Show 7 more

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https://doi.org/10.1021/jacs.1c06167

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Abstract

Chemical modifications of native proteins can affect their stability, activity, interactions, localization, and more. However, there are few nongenetic methods for the installation of chemical modifications at a specific protein site in cells. Here we report a covalent ligand directed release (CoLDR) site-specific labeling strategy, which enables the installation of a variety of functional tags on a target protein while releasing the directing ligand. Using this approach, we were able to label various proteins such as BTK, K-RasG12C, and SARS-CoV-2 PLpro with different tags. For BTK we have shown selective labeling in cells of both alkyne and fluorophores tags. Protein labeling by traditional affinity methods often inhibits protein activity since the directing ligand permanently occupies the target binding pocket. We have shown that using CoLDR chemistry, modification of BTK by these probes in cells preserves its activity. We demonstrated several applications for this approach including determining the half-life of BTK in its native environment with minimal perturbation, as well as quantification of BTK degradation by a noncovalent proteolysis targeting chimera (PROTAC) by in-gel fluorescence. Using an environment-sensitive “turn-on” fluorescent probe, we were able to monitor ligand binding to the active site of BTK. Finally, we have demonstrated efficient CoLDR-based BTK PROTACs (DC50 < 100 nM), which installed a CRBN binder onto BTK. This approach joins very few available labeling strategies that maintain the target protein activity and thus makes an important addition to the toolbox of chemical biology.

Highlights

Selective modifications of native proteins in cells with chemical probes are a powerful tool to tune and investigate protein function, conformation, structure, cellular signaling, localization, and more
Ibrutinib, which is a highly potent covalent inhibitor of Bruton’s tyrosine kinase (BTK) that binds at its ATP-binding pocket, was used as the ligand to guide the selective labeling of BTK’s noncatalytic cysteine 481.38 The amine precursor for ibrutinib (Ibr-H; Figure 2) contains a piperidine moiety, which can be installed as a heterosubstituent on an α-methacrylamide and serve as a leaving group.[37]
While ibrutinib alone completely inhibited BTK’s activity, we show that all covalent ligand directed release (CoLDR) probes can rescue this inhibition

Summary

Introduction

Selective modifications of native proteins in cells with chemical probes are a powerful tool to tune and investigate protein function, conformation, structure, cellular signaling, localization, and more. These approaches, typically rely on overexpressed proteins, and the newly introduced domains can be large and perturb the very same process they aim to investigate.[13−15] Genetic code expansion enables site-specific incorporation of unnatural amino acids bearing bioorthogonal reactive handles.[16,17] The subsequent bio-orthogonal reaction with a suitable complementary reactive functionality allows effective and selective bioconjugation This circumvents the introduction of a large domain, but these methods are laborious and require engineered cells,[16] limiting their scope

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