Affinity-based protein profiling-driven discovery of myricanol as a Nampt activator.

[Display omitted] • A photo-affinity probe pMY was designed to identify the targets of myricanol (MY). • Nampt was identified as a direct target of MY via activity-based protein profiling. • MY sensitizes insulin action in C2C12 myotubes by activating Nampt. • pMY is the first probe to label Nampt. Herein, we synthesized an affinity-based probe of myricanol (pMY) with a photo-affinity cross-linker to initiate a bioconjugation reaction, which was applied for target identification in live C2C12 myotubes. Pull-down of biotinylated pMY coupled with mass spectroscopy and Western blotting revealed that pMY can bind with nicotinamide phosphoribosyltransferase (Nampt), a rate-limiting enzyme in the nicotinamide adenine dinucleotide salvage pathway. Cellular thermal shift assay, drug affinity responsive target stability assay and recombinant protein labeling further validated the direct interaction between myricanol and Nampt. Myricanol did not affect the protein expression of Nampt, but enhanced its activity. Knock-down of Nampt totally abolished the promoting effect of myricanol on insulin-stimulated glucose uptake in C2C12 myotubes. Taken together, myricanol sensitizes insulin action in myotubes through binding with and activating Nampt. [ABSTRACT FROM AUTHOR]