Functionalised Cofactor Mimics for Interactome Discovery and Beyond

Abstract

Cofactors are required for almost half of all enzyme reactions, but their functions and binding partners are not fully understood even after decades of research. Functionalised cofactor mimics that bind in place of the unmodified cofactor can provide answers, as well as expand the scope of cofactor activity. Through chemical proteomics approaches such as activity-based protein profiling, the interactome and localisation of the native cofactor in its physiological environment can be deciphered and previously uncharacterised proteins annotated. Furthermore, cofactors that supply functional groups to substrate biomolecules can be hijacked by mimics to site-specifically label targets and unravel the complex biology of post-translational protein modification. The diverse activity of cofactors has inspired the design of mimics for use as inhibitors, antibiotic therapeutics, and chemo- and biosensors, and cofactor conjugates have enabled the generation of novel enzymes and artificial DNAzymes.

Keywords: Chemical Probes; Cofactors; Photoaffinity Labelling; Protein Modifications; Proteomics.

Figure 1

Examples of activity‐based cofactor probes designed to bind covalently to binding proteins to enable identification. A) Suite of clickable pyridoxal probes and schematic for profiling the PLP interactome. PL probes are taken up by bacterial cells, phosphorylated, and incorporated into PLP‐dependent enzymes. Following cell lysis, sodium borohydride‐mediated reduction of the imine bond and click ligation to enrichment tags enables identification of labelled enzymes by mass spectrometry (adapted from Hoegl et al. ^[21] ); B) mechanism of transfer of the biotin tag from an ATP mimic to a kinase active site lysine residue; C) vitamin A probe and D) electrophile trap pyridoxal mimics; e) AIZin‐1, a zinc‐responsive protein labelling reagent; PL: pyridoxal; PLP: pyridoxal phosphate; LC‐MS/MS: liquid chromatography tandem mass spectrometry.

Figure 2

Examples of photoaffinity‐functionalised cofactor mimics designed to identify substrate or binding proteins.

Figure 3

Examples of functionalised cofactor mimics designed to transfer labels onto substrate proteins. A) Alkyne‐ and photoaffinity‐labelled SAM mimics; B) alkyne‐ and photoaffinity‐labelled NAD⁺ mimics. Schematic for profiling the interaction network of PARylated proteins; C) probes based on the structure of coenzyme A; NAD⁺: nicotinamide adenine dinucleotide; PARPs: poly‐ADP‐ribose polymerases; MS: mass spectrometry; PARylation: polyADP‐ribosylation.

Figure 4

Examples of fluorophore‐functionalised cofactor mimics and cofactor‐based sensors.

Figure 5

Examples of cofactor mimics with artificial activity.