Chemical Proteomics Approach for Profiling the NAD Interactome

Nicotinamide adenine dinucleotide (NAD⁺) is a multifunctional molecule. Beyond redox metabolism, NAD⁺ has an equally important function as a substrate for post-translational modification enzymes, the largest family being the poly-ADP-ribose polymerases (PARPs, 17 family members in humans). The recent surprising discoveries of noncanonical NAD (NAD⁺/NADH)-binding proteins suggests that the NAD interactome is likely larger than previously thought; yet, broadly useful chemical tools for profiling and discovering NAD-binding proteins do not exist. Here, we describe the design, synthesis, and validation of clickable, photoaffinity labeling (PAL) probes, 2- and 6-ad-BAD, for interrogating the NAD interactome. We found that 2-ad-BAD efficiently labels PARPs in a UV-dependent manner. Chemical proteomics experiments with 2- and 6-ad-BAD identified known and unknown NAD⁺/NADH-binding proteins. Together, our study shows the utility of 2- and 6-ad-BAD as clickable PAL NAD probes.