A Click Chemistry Mediated in Vivo Activity Probe for Dimethylarginine Dimethylaminohydrolase

Abstract

Asymmetric N(omega),N(omega)-dimethyl-l-arginine (ADMA) is an endogenously produced inhibitor of human nitric oxide synthase and an emerging biomarker for cardiovascular disease. Concentrations of ADMA are controlled by two isoforms of its catabolic enzyme dimethylarginine dimethylaminohydrolase (DDAH), the dysregulation of which has been studied as a mediating factor for endothelial dysfunction. A two-part, click-chemistry mediated activity-based probe, N-but-3-ynyl-2-chloroacetamidine, is shown to label myc-tagged DDAH-1 expressed in HEK 293T cells, but not an inactive mutant or inhibited enzyme. A two-color Western blotting technique is used to determine the in vivo IC(50) value for a reversible inhibitor of DDAH-1, N(5)-(1-iminopropyl)-l-ornithine, indicating this compound's bioavailability and its competition for binding to the active site. This probe provides a novel tool for the analysis of DDAH-1 activity in normal and pathophysiological states and should allow more meaningful studies of the etiology of endothelial dysfunction.

Activity‐Based Protein Profiling: Recent Advances in Probe Development and Applications

“…[43] DDAH-1 and DDAH-2, two isoforms with different tissue distributions, control the physiological concentration of asymmetric N w ,N w -dimethyl-l-arginine (ADMA), an endogenously produced inhibitor of human nitric oxide synthase. The design of probe 19 was based on 2-chloroacetamidine (CAA), which selectively modifies the active site cysteine of DDAH.…”

“…[79] To identify its cellular target, Taunton and co-workers incorporated photo-leucine, a diazirine-based photo-reactive analogue of leucine, into the scaffold of the cyclodepsipeptide (43). After incubation of this probe with an endoplasmic reticulum microsome fraction, followed by UV irradiation, a target of this HUN-7293-based probe was identified as Sec61a, the largest subunit of the Sec61 complex, which forms the translocation channel through which all proteins transit.…”

Applications for Activity-based Probes in Drug Discovery

“…This has included the development of probes that target non-enzymatic proteins such as GABA receptors 60 and acetylcholine receptors. 61 In addition, there have been recent reports of probes that target other diverse enzyme classes including sulfatases, 62 deaminases, 35 dimethylaminohydrolases, 63 lipases, 64,65 esterases, 65,66 nitrilases, 67 cytochrome P450s, 68 gylcosidases, 69 gylcanases 70 and beta galactosidase. 71 This ever-expanding list of enzyme families and receptors that can be targeted by ABPs suggests that the field continues to advance.…”

Design, synthesis, and in vitro evaluation of an activity-based protein profiling (ABPP) probe targeting agmatine deiminases

“…Similar studies have also demonstrated the ability of this halo-acetamidine 'warhead' to alkylate the active site cysteine of the related PAD and DDAH enzymes. [19][20][21][22] Growing interest in this family of enzymes, due in part to their potential as therapeutic targets, warrants the development of tools that will increase our understanding of the physiological roles that these enzymes play. The development of an activity-based protein profiling (ABPP) probe that selectively labels active AgD enzymes could aid in identifying novel AgDs, enable the monitoring of activity levels under various conditions, and facilitate the discovery of new inhibitors through screening methodologies (e.g., the FluoPol-ABPP methodology).…”

“…The development of an activity-based protein profiling (ABPP) probe that selectively labels active AgD enzymes could aid in identifying novel AgDs, enable the monitoring of activity levels under various conditions, and facilitate the discovery of new inhibitors through screening methodologies (e.g., the FluoPol-ABPP methodology). 22,23 The mechanism based inactivation of AgDs by ABCA make this inhibitor an ideal lead compound for the development of a probe that will selectively and covalently append a reporter tag to members of this family. Herein we describe the design, synthesis, and evaluation of such an ABPP probe.…”