Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme for the nicotinamide adenine dinucleotide (NAD) salvage pathway in mammals. The activity of NAMPT fine-tunes intracellular NAD+ levels, thereby regulating cellular metabolism and controlling the activity of NAD-dependent enzymes, including sirtuins, PARPs, and NADases. NAD+ levels decline with aging, and NAD+ homeostatic imbalance can be found in virtually all age-related diseases, including neurodegeneration, diabetes, and cancer. Thus, a pharmacological boost of intracellular NAD+ levels through NAMPT activation has enormous therapeutic potential. Herein, we performed a 20,000-compound high throughput screen (HTS) in a coupled enzyme assay measuring the formation of NAD+. NAMPT activating hits were identified, and co-crystal structures were obtained that showed activators bind to an allosteric channel. Structural-guided optimization led to multiple NAMPT activators with low nanomolar potency, the binding of which was validated by fluorescence polarization assays, surface plasmon resonance assays, and X-ray co-crystal structures. Optimized NAMPT activators were shown to increase NAD+ levels in a THP-1 cell model. Overall, our approach represents a novel approach to raise intracellular NAD+ levels and to explore the therapeutic potential of NAD+ boosting.

Discovery and characterization of allosteric NAMPT activators

ACS Fall 2021

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