Researchers publishing in Physical Review X have discovered compounds that can double the efficiency of the sirtuin SIRT3 in processing NAD+.
Looking for a new way to boost enzymes
The researchers begin their paper by noting that most drugs administered to people are geared towards inhibition of particular enzymes in order to treat a disease. In this case, however, the goal is the opposite: to boost the function of an enzyme, thereby boosting a healthy phenotype rather than battling back a diseased one.
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Sirtuins are enzymes that have been heavily investigated in the context of aging. They rely on NAD+ to function, and these researchers describe them as being critical regulators of cellular pathways relating to aging [1]. Upregulating sirtuins has been found in considerable previous work to extend lifespan in mammals [2]. However, most methods of using drugs to boost sirtuins has relied on allosteric activation, a chemical process that relies on an existing substrate that might be limited in quantity [3].
Of course, as sirtuins rely on NAD+, there has been much work on directly influencing that instead. These researchers note two problems with that approach: as it is a common aspect of metabolism, boosting NAD+ across the board may result in broad side effects [4] and converting it into NADH relies on delivering it into cells that have functioning internal machinery [5], which, in the context of aging, is far from guaranteed.
Therefore, these researchers seek to allow sirtuins to do more with less: to continue to function adequately even when NAD+ is diminished. This, the researchers describe, is a trickier thing to do; while allosteric activators fundamentally rely on existing, evolved mechanisms, attempting to modulate these enzymes is similar to designing new enzymes outright.
Also, they needed a compound that works all the time: a steady-state activator. Previous work has created compounds that inhibit, rather than activate, sirtuins most of the time [6], only performing their desired function under specific conditions.
SIRT3 was chosen as the target for two reasons. The first is that it is known to have beneficial effects on mitochondria [7], and previous work has found that the benefits of NAD+ against mitochondrial dysfunction are due to SIRT3 [8]. The second is that natural mutations in the SIRT3 gene are connected to longevity [9].
Needle in a haystack
Using an advanced algorithm, the researchers searched a library of 1.2 million compounds by beginning with Honokiol, a compound that only activates SIRT3 under certain conditions. The researchers were able to find compounds that do steady state and non-steady state activation, with which they refined their experiments further with a close and detailed examination of the specific biochemistry involved, looking for compounds that have strong bonds to certain amino acids on the SIRT3 protein.
This initial work, however, was all done on computers. To verify their findings in the real world, the authors administered their compounds to real SIRT3 in a substrate. While a lot of this type of work uses fluorescent labeling, the authors eschewed that approach as it may have affected the results. One particularly strong compound, number 5689785, was identified as being a plausible drug after this screening process.
The researchers tested their new candidate against a control group, honokiol, and the well-known NAD+ precursor NMN. In nearly all cases, 5689785 performed favorably against these alternatives. Administering nicotinamide (NAM) to cells inhibits NAD+ enzymatic activity, but 5689785 was able to restore it in a way that honokiol could not.
Next steps
This is not a drug yet; it has not been formulated in a way that is consumable by living organisms, and so there were no animal studies done. What the researchers have is an initial compound with which to continue the process of drug development. Their goal was to prove that it is indeed possible to directly enhance the activity of sirtuins without relying on substrate-based methods. If this approach sees success in animal models, it could pave the way for drugs that, due to SIRT3’s mitochondrial effects, fight multiple aspects of aging.
Literature
[1] Kaeberlein, M., McVey, M., & Guarente, L. (1999). The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms. Genes & development, 13(19), 2570-2580.
[2] Roichman, A., Elhanati, S., Aon, M. A., Abramovich, I., Di Francesco, A., Shahar, Y., … & Cohen, H. Y. (2021). Restoration of energy homeostasis by SIRT6 extends healthy lifespan. Nature communications, 12(1), 3208.
[3] Sinclair, D. A., & Guarente, L. (2014). Small-molecule allosteric activators of sirtuins. Annual review of pharmacology and toxicology, 54(1), 363-380.
[4] Yang, T., & Sauve, A. A. (2006). NAD metabolism and sirtuins: metabolic regulation of protein deacetylation in stress and toxicity. The AAPS journal, 8, E632-E643.
[5] Hu, Q., Wu, D., Walker, M., Wang, P., Tian, R., & Wang, W. (2021). Genetically encoded biosensors for evaluating NAD+/NADH ratio in cytosolic and mitochondrial compartments. Cell reports methods, 1(7).
[6] Reverdy, C., Gitton, G., Guan, X., Adhya, I., Dumpati, R. K., Roy, S., … & Chakrabarti, R. (2022). Discovery of novel compounds as potent activators of Sirt3. Bioorganic & medicinal chemistry, 73, 116999.
[7] Van de Ven, R. A., Santos, D., & Haigis, M. C. (2017). Mitochondrial sirtuins and molecular mechanisms of aging. Trends in molecular medicine, 23(4), 320-331.
[8] Cantó, C., Houtkooper, R. H., Pirinen, E., Youn, D. Y., Oosterveer, M. H., Cen, Y., … & Auwerx, J. (2012). The NAD+ precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet-induced obesity. Cell metabolism, 15(6), 838-847.
[9] Bellizzi, D., Rose, G., Cavalcante, P., Covello, G., Dato, S., De Rango, F., … & De Benedictis, G. (2005). A novel VNTR enhancer within the SIRT3 gene, a human homologue of SIR2, is associated with survival at oldest ages. Genomics, 85(2), 258-263.
