Advances in Activity-Based Sensing Probes for Isoform-Selective Imaging of Enzymatic Activity

Abstract

Until recently, there were no generalizable methods for assessing the effects of post-translational regulation on enzymatic activity. Activity-based sensing (ABS) has emerged as a powerful approach for monitoring small-molecule and enzyme activities within living systems. Initial examples of ABS were applied for measuring general enzymatic activity; however, a recent focus has been placed on increasing the selectivity to monitor a single enzyme or isoform. The highest degree of selectivity is required for differentiating between isoforms, where the targets display significant structural similarities as a result of a gene duplication or alternative splicing. This Minireview highlights key examples of small-molecule isoform-selective probes with a focus on the relevance of isoform differentiation, design strategies to achieve selectivity, and applications in basic biology or in the clinic.

Keywords: activity-based sensing; fluorescence; molecular imaging; probe development; rational design.

Figure 1.

General schematic depicting enzyme-catalyzed activation of an isoform-selective ABS probe to afford a fluorescent product.

Figure 2.

a) Chemical structure of AlDeSense. b) Normalized fluorescence turn-on of AlDeSense after incubation with ALDH isoforms. c) Confocal images of K562 cells stained with AlDeSense. Further permissions related to the material excerpted should be directed to the American Chemical Society. https://pubs.acs.org/doi/10.1021/acscentsci.8b00313. d) Chemical structure of red-AlDeSense.

Figure 3.

The chemical structures of a) TCFB; b) 4-ethylbenzyl substrate on a 3-hydroxylflavone backbone; c) DDAB; d) 4-MOMMP; and e) MMB. f) MMB docked into the active site of CE1. Reprinted (adapted) with permission from ref. . Copright 2019 American Chemical Society.

Figure 4.

a) The chemical structure of CoxFluor. b) CoxFluor docked into the active site of COX-2. c) Normalized fluorescent turn-on of CoxFluor with various enzymes. d) Normalized fluorescent turn-on of CoxFluor under 0.1, 1, or 21% oxygen concentrations. Reprinted (adapted) from ref. with permission from John Wiley and Sons, Inc. Copyright 2020.

Figure 5.

Chemical structures of a) NBCeN; b) NeiPN; and c) BnXPI. Docking of d) NBCeN in the CYP1A1 active site. Reprinted (adapted) from the Royal Society of Chemistry from ref. .; e) BnXPI bound in the CYP2J2 active site. Reprinted (adapted) with permission from ref. . Copyright 2019 American Chemical Society.

Figure 6.

a) The chemical structure of (S)-Sulfox-1. b) Images of E. coli cells at OD600 treated with (S)-Sulfox-1 (left) or treated with (S)-Sulfox-1 and inhibitor (right). Reprinted (adapted) with permission from ref. with permission from John Wiley and Sons, Inc. Copyright 2016.

Figure 7.

Chemical structures of a) U1 and b) Probe 1. c) Confocal images of HeLa and NIH-3T3 cells after incubation with Probe 1 for 1 hour. False coloring for signal from the initial probe (blue) and activated probe (red). d) Relative ratio of turned-over probe to initial fluorescence. Reprinted (adapted) with permission from ref. . Copyright 2018 American Chemical Society.

Figure 8.

a) Chemical structure of TG-mPC. b) TG-mPC fluorescence enhancement after incubation with various NNPs. Reprinted (adapted) with permission from ref. . Copyright 2011 American Chemical Society.

Figure 9.

a) Schematic of PSPtide sensing mechanism. b) The change in fluorescence as a result of PP2A, in lysates, can be resolved using calyculin A. c) The change in probe fluorescence decreases when the PP2ACα subunit is knocked down with siRNA. Reprinted (adapted) with permission from ref. . Copyright 2016 American Chemical Society.