The mechanism of nicotinamide phosphoribosyltransferase whereby positive allosteric modulation elevates cellular NAD⁺

Abstract

In aging and disease, cellular NAD+is depleted by catabolism to nicotinamide (NAM) and NAD+supple-mentation is being pursued to enhance human healthspan and lifespan. Activation of nicoti namide phosphoribosyl -transferase (NAMPT), the rate-limiting step in NAD+biosynthesis, has potential to increase salvage of NAM. Novel NAMPT positive allosteric modulators (N-PAMs) were discovered in addition to demonstration of NAMPT activati on by biogenic phenols. The mechanism of activation was revealed through synthesis of novel chemical probes, new NAMPT co-crystal structures, and enzyme kinetics. Binding to a rear channel in NAMPT regulates NAM binding and turnover, with biochemical observations being replicated by NAD+measurements in human cells. The mechanism of action of N-PAMs identifies, for the first time, the role of the rear channel in regulation of NAMPT turnover coupled to feedback inhibition by NAM. N-PAM inhibition of low affinity, non-productive NAM binding via the rear channel, causes a right-shif t in KI(NAM) that accompanies an increase in enzyme activity. Conversion of an N-PAM to a high-affinity l igand blocks both high and low affinity NAM binding, ablating enzyme activity. In the presence of an N-PAM, NAMPT boosts NAD+biosynthesis at higher NAM concentrations, in addition to relieving inhibition by NAD+. Since cellular stress often leads to enhanced catabolism of NAD+to NAM, this mechanism is relevant to supporting cellular N AD+levels in aging and disease. The tight regulation of cellular NAMPT is differentially regulated by N-PAMs and other activators, indicating that different classes of pharmacological activators may be engineered for cell and tissue selectivity.

Channeling Nicotinamide Phosphoribosyltransferase (NAMPT) to Address Life and Death

“…Small molecule N-PAMs were discovered by HTS using a coupled enzyme assay (NAMPT/NMNAT/NAD cycling) that identified hits that increased enzyme activity. 150 Cocrystal structures were obtained for early leads, NP-A1R, NP-A1S, and ZN-2-43S, and for ZN-2-29S obtained from further optimization (Figure 6C; Table S3). 150,151 These structures demonstrated that N-PAMs bind to the rear channel of NAMPT in the absence or presence of NAM (Figure 7D).…”

“…150 Cocrystal structures were obtained for early leads, NP-A1R, NP-A1S, and ZN-2-43S, and for ZN-2-29S obtained from further optimization (Figure 6C; Table S3). 150,151 These structures demonstrated that N-PAMs bind to the rear channel of NAMPT in the absence or presence of NAM (Figure 7D). The structure of an N-PAM from a different chemical series, NP-A3, was also obtained, again showing occupancy of the NAMPT rear channel (Figure 6C; Table S3).…”

“…The rear channel is a key feature for NAMPT inhibitors as discussed above and evidenced by multiple cocrystal structures. Small molecule N-PAMs were discovered by HTS using a coupled enzyme assay (NAMPT/NMNAT/NAD cycling) that identified hits that increased enzyme activity . Cocrystal structures were obtained for early leads, NP-A1R, NP-A1S, and ZN-2-43S, and for ZN-2-29S obtained from further optimization (Figure C; Table S3).…”

“…Therefore, as we increase the binding affinity of ligands to the rear channel, both productive and nonproductive pathways will be inhibited. Across the 70 NP-A1 series N-PAMs, increased affinity led to increased potency for activation but reduced maximal activation, with some high affinity ligands showing minimal or no activation . From a lead optimization perspective, it is frustrating that increasing affinity/potency does not translate to increased activity.…”

“…It is conceivable that SBI-797812 (and perhaps SBI-type activators) share a similar mechanism with N-PAMs in inhibiting nonproductive NAM binding. SBI-797812 displaces an FK866-like fluorescent probe from the NAMPT rear channel with affinity comparable to the potency for enzyme activation . Nonproductive binding occurs when the phosphoenzyme breaks down without turnover of NAM to NMN.…”