Abstract
Over the past two decades, activity-based probes have enabled a range of discoveries, including the characterization of new enzymes and drug targets. However, their suitability in some labeling experiments can be limited by nonspecific reactivity, poor membrane permeability, or high toxicity. One method for overcoming these issues is through the development of “inducible” activity-based probes. These probes are added to samples in an unreactive state and require in situ transformation to their active form before labeling can occur. In this Review, we discuss a variety of approaches to inducible activity-based probe design, different means of probe activation, and the advancements that have resulted from these applications. Additionally, we highlight recent developments which may provide opportunities for future inducible activity-based probe innovations.
Highlights
Activity-based probes have been developed as effective tools for identifying active enzymes in biological samples.[1]
They enable the detection and characterization of target proteins through the formation of a covalent bond between the probe and an amino acid residue at the protein active site.[2]. This labeling reaction occurs via the “warhead” unit of the probe, commonly an electrophile, which is tethered through a linker to a detectable reporter group (Figure 1a)
The reporter group may be present during labeling or can be added in a subsequent step using a bioorthogonal reaction such as copper-catalyzed azide−alkyne cycloaddition, strain-promoted azide−alkyne cycloaddition, inverse electron demand Diels−Alder tetrazine ligation, and Staudinger−Bertozzi ligation.[4−7] Applications of probes have enabled extensive activity-based protein profiling of many cell types, leading to the characterization of novel drug targets,[8] enzyme inhibitors,[9] and even the discovery of new enzymatic activity.[10]
Summary
Activity-based probes have been developed as effective tools for identifying active enzymes in biological samples.[1]. Irradiation at 365 nm resulted in uncaging to form a nitrilimine, which acted as an effective electrophilic trap for the active site cysteine residues of the target DUBs.[61] Due to the inclusion of both labile cell penetrating peptides and a photocaged electrophilic warhead, the probe could undergo light induced labeling of DUBs in HeLa whole cells (Figure 2f), demonstrating the benefits of a combinatorial approach to inducible activity-based probe design. Examination of other commonly metabolized functional groups may help identify yet more classes of masked electrophilic warheads for application in inducible activity-based probes In each of these examples, the in situ activation of activitybased probes allowed whole cell protein profiling.[100] These publications demonstrated different approaches to probe design, masking either the recognition element or the warhead. The simplicity and elegance of this mode of activation ensures highly specific labeling and mimics the tuned enzyme−substrate reactivity native to biological systems
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