Linkers activity-based probes

Activity-based probes (ABPs) contain (1) a warhead— a class-selective electrophile that reacts primarily with a key amino acid in the active site of the proteins of a particular class and covalently attaches to it, plus a recognition element that directs the warhead more specifically to the active site (2) a reporter tag that allows detection and isolation and (3) a linker, which connects the elements with a proper distance and flexibility (Figure 1.7). ABPs have the potential to ... 

In their most basic form, activity-based probes consist of three distinct functional elements (Figure 2.1) a reactive group for covalent attachment to the enzyme, a linker region that can modulate reactivity and specificity of the reactive group, and a tag for identification and purification of modified... 

Combined with photocrossing linkers, ABPP has also been utilized to profile enzymes that do not form covalent bond with activity-based probes, including... 

Figure 13 ABPP probes for metalloproteases. (a) First generation of hydroxamate-based probes with rhodamine (Rh) and Gy3 as fluorescent tags and photocross-linkers for covalent modification, (b) Active site-directed labeling mechanism for a clickable second-generation probe. (c)The potent SAHA inhibitor for HDAC classes I and II and its derived ABPP probe.

Another approach to study kinase activity was introduced by Hagenstein et at 2 who designed an affinity-based probe equipped with a benzophenone photocross-linker for covalent modification of plant kinases. 

Figure 16.9 Structures of chemical components of activity/affinity probes The activity/affinity-based probe consists of reactive group and tag connected by a linker (L indicates the points of connection to the linker). Specific examples of each of the chemical components are shown. Two types of tags are commonly employed. The affinity tag for isolation/purification and the flnorescent or radiolabel tags for identification. Taken from Jeffery and Bogyo (2003)...

Another important class of substrate-based probes for proteases uses two or more fluorophores, that are self-quenched when in close proximity. " Multiple fluorophores can be linked to graft polymers containing peptide substrate sequences. When these linkers are cleaved by the protease, free fluorescent monomer can be detected. This class of probes has been widely used to study the activity of the cysteine cathepsin family of proteases aeross many diverse disease models. 

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