Activity-based probes: discovering new biology and new drug targets

Heal, William P.; Dang, Tam; Tate, Edward W.

doi:10.1039/c0cs00004c

Cited by 162 publications

(93 citation statements)

References 74 publications

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“…Protease activity profiling (also called activity-based protein profiling of proteases) is an easy and powerful method to monitor the active state of proteases in crude extracts or living organisms (Heal et al, 2011;Serim et al, 2012;Haedke et al, 2013;Willems et al, 2014). Protease activity profiling is based on the use of chemical probes that react covalently with the active site of proteases in an activitydependent manner.…”

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confidence: 99%

Subfamily-Specific Fluorescent Probes for Cysteine Proteases Display Dynamic Protease Activities during Seed Germination

Chandrasekar

Oeljeklaus

et al. 2015

Plant Physiology

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Cysteine proteases are an important class of enzymes implicated in both developmental and defense-related programmed cell death and other biological processes in plants. Because there are dozens of cysteine proteases that are posttranslationally regulated by processing, environmental conditions, and inhibitors, new methodologies are required to study these pivotal enzymes individually. Here, we introduce fluorescence activity-based probes that specifically target three distinct cysteine protease subfamilies: aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes. We applied protease activity profiling with these new probes on Arabidopsis (Arabidopsis thaliana) protease knockout lines and agroinfiltrated leaves to identify the probe targets and on other plant species to demonstrate their broad applicability. These probes revealed that most commercially available protease inhibitors target unexpected proteases in plants. When applied on germinating seeds, these probes reveal dynamic activities of aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes, coinciding with the remobilization of seed storage proteins.

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confidence: 99%

Subfamily-Specific Fluorescent Probes for Cysteine Proteases Display Dynamic Protease Activities during Seed Germination

Chandrasekar

Oeljeklaus

et al. 2015

Plant Physiology

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“…16 ABPP hinges on the use of activity-based probes (ABPs) to report on enzyme activity in cells, tissue or animals. 17 An ABP normally consists of a covalent, irreversible enzyme inhibitor featuring a reporter entity (fluorophore, biotin, bioorthogonal tag) to label the active site of the enzyme or enzyme family at hand. ABPP is unique in its ability to rapidly identify inhibitor activity and selectivity within large enzyme families in complex proteome samples.…”

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confidence: 99%

Highly Selective, Reversible Inhibitor Identified by Comparative Chemoproteomics Modulates Diacylglycerol Lipase Activity in Neurons

et al. 2015

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Diacylglycerol lipase (DAGL)-α and -β are enzymes responsible for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). Selective and reversible inhibitors are required to study the function of DAGLs in neuronal cells in an acute and temporal fashion, but they are currently lacking. Here, we describe the identification of a highly selective DAGL inhibitor using structure-guided and a chemoproteomics strategy to characterize the selectivity of the inhibitor in complex proteomes. Key to the success of this approach is the use of comparative and competitive activity-based proteome profiling (ABPP), in which broad-spectrum and tailor-made activity-based probes are combined to report on the inhibition of a protein family in its native environment. Competitive ABPP with broad-spectrum fluorophosphonate-based probes and specific β-lactone-based probes led to the discovery of α-ketoheterocycle LEI105 as a potent, highly selective and reversible dual DAGL-α/DAGL-β inhibitor. LEI105 did not affect other enzymes involved in endocannabinoid metabolism including abhydrolase domain-containing protein 6, abhydrolase domain-containing protein 12, monoacylglycerol lipase and fatty acid amide hydrolase and did not display affinity for the cannabinoid CB1 receptor. Targeted lipidomics revealed that LEI105 concentration-dependently reduced 2-AG levels, but not anandamide levels, in Neuro2A cells. We show that cannabinoid CB1-receptor-mediated short-term synaptic plasticity in a mouse hippocampal slice model can be reduced by LEI105. Thus, we have developed a highly selective DAGL inhibitor and provide new pharmacological evidence to support the hypothesis that ‘on demand biosynthesis’ of 2-AG is responsible for retrograde signaling.

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“…during infection of mammalian cells. Further probe modification may permit activity-based protein profiling (ABPP), [24,25] a complementary technique that could correlate AMPylated proteins with the activities of known AMP transferases, and provide new insights into the activity of as yet undiscovered enzymes in this class.…”

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confidence: 99%