Cellular electron transfer relies on nicotinamide adenine dinucleotide cofactors, specifically NAD⁺. NAD⁺ is a cofactor for hundreds of enzymes, including dehydrogenases and oxidoreductases, and is crucial for glycolysis and ATP synthesis. Lipid and fatty acid biosynthesis and processing rely on enzymes that are dependent on NAD⁺/NADP⁺. Consequently, low levels of cellular NAD⁺ can lead to cell death and is associated with aging and disease, spurring the study of dietary supplements to replenish NAD⁺. Three families of proteins with NADase activity, PARPs, SIRTs, and CD38, are responsible for the catabolism of NAD⁺, requiring the salvage of NAM to replenish cellular NAD⁺, which relies on the rate-limiting enzyme nicotinamide phosphoribosyltransferase (NAMPT). Pharmacological activation of NAMPT provides an alternative to dietary supplements. Novel NAMPT-positive allosteric modulators (N-PAMs) were discovered in addition to the demonstration of NAMPT activation by biogenic phenols. The mechanism of activation was revealed through synthesis of chemical probes, characterization of new NAMPT co-crystal structures, kinetic analysis, and biophysical measurements. Binding of N-PAMs to a rear channel in NAMPT regulates NAM binding and turnover, with biochemical observations being replicated by NAD⁺measurements in human cells. NAMPT activity is regulated by substrate (NAM, ATP) inhibition and inhibition by downstream products (NAD⁺ and NADH). All reported families of genuine NAMPT activators, including N-PAMs, bind to a rear channel that accesses the NAM binding pocket and active site. We propose that N-PAMs function by inhibiting non-productive NAM binding, by competing for the rear channel, preventing NAM access to the active site. This also prevents the dysregulated ATPase activity of NAMPT. The mechanism accounts for higher binding affinity leading to loss of efficacy. N-PAMs allow productive NAM binding, which is associated with high affinity binding of NAM. Perplexingly, the rear channel of NAMPT also accommodates classical NAMPT inhibitors. Furthermore, potent 3-pyridyl substituted inhibitors have regioisomeric congeners (4-pyridyl) that activate NAMPT with an ill-defined mechanism that is differentiated from N-PAMs. Future progress in developing N-PAMs and other NAMPT activator families as therapeutic agents will require a more detailed understanding of the allosteric mechanism and evidence that this mechanism is valid in animal and human tissues.

Martha Ackerman-Berrier

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