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

Ratia, K. M.; Shen, Z.; Gordon-Blake, J.; Lee, H.; Laham, M. S.; Krider, I. S.; Christie, N.; Ackerman-Berrier, M. S.; Penton, C.; Knowles, N. G.; Musku, S. R.; Fu, J.; Musku, G. R.; Xiong, R.; Thatcher, G. R. J.

doi:10.1101/2022.10.21.513220

bioRxiv · 10.1101/2022.10.21.513220

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

Ratia, K. M.·Shen, Z.·Gordon-Blake, J.·Lee, H.·Laham, M. S.·Krider, I. S.·Christie, N.·Ackerman-Berrier, M. S.·Penton, C.·Knowles, N. G.·Musku, S. R.·Fu, J.·Musku, G. R.·Xiong, R.·Thatcher, G. R. J.

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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.

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Ratia, K. M., Shen, Z., Gordon-Blake, J., Lee, H., Laham, M. S., Krider, I. S., Christie, N., Ackerman-Berrier, M. S., Penton, C., Knowles, N. G., Musku, S. R., Fu, J., Musku, G. R., Xiong, R., Thatcher, G. R. J.. 2022-10-22. The mechanism of nicotinamide phosphoribosyltransferase whereby positive allosteric modulation elevates cellular NAD+. https://doi.org/10.1101/2022.10.21.513220

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