The mechanism of action of N-acetylcysteine (NAC)

[madman]

ABSTRACT

Initially adopted as a mucolytic about 60 years ago, the cysteine prodrug N-acetylcysteine (NAC) is the standard of care to treat paracetamol intoxication and is included on the World Health Organization’s list of essential medicines. Additionally, NAC increasingly became the epitome of an “antioxidant”. Arguably, it is the most widely used “antioxidant” in experimental cell and animal biology, as well as clinical studies. Most investigators use and test NAC with the idea that it prevents or attenuates oxidative stress. Conventionally, it is assumed that NAC acts as (i) a reductant of disulfide bonds, (ii) a scavenger of reactive oxygen species, and/or (iii) a precursor for glutathione biosynthesis. While these mechanisms may apply under specific circumstances, they cannot be generalized to explain the effects of NAC in a majority of settings and situations. In most cases, the mechanism of action has remained unclear and untested. In this review, we discuss the validity of conventional assumptions and the scope of a newly discovered mechanism of action, namely the conversion of NAC into hydrogen sulfide and sulfane sulfur species. The antioxidative and cytoprotective activities of per- and polysulfides may explain many of the effects that have previously been ascribed to NAC or NAC-derived glutathione.




1. Introduction: the popularity of N-acetylcysteine

2. Three classical narratives
2.1. The disulfide reductant narrative
2.2. The oxidant scavenger narrative
2.3. The glutathione replenishment narrative

3. Why use NAC rather than cysteine?

4. Hydrogen sulfide: the missing link in cysteine toxicity?

5. How does NAC circumvent the problem of cysteine toxicity?

6. Are the cytoprotective effects of NAC caused by low-level H2S production?

7. Are the cytoprotective effects of NAC caused by sulfane sulfur species?

8. The sulfane sulfur perspective: a new angle to look at the classical NAC narratives




9. Conclusion

In summary, previously favored explanations for the mechanism of action of NAC are either poorly supported (direct oxidant scavenging), restricted to very specific situations (disulfide reduction in lung mucins), or fail to explain all observations (GSH biosynthesis). A new conceptual framework for NAC’s mechanism of action is emerging, namely, as a Cys pro-drug that leads to modest elevations of H2S and sulfane sulfur species inside cells. The slow release of Cys from NAC allows for sustained sulfane sulfur production, providing protective effects -independently of GSH replenishment (Fig. 9). The sulfane sulfur branch of NAC metabolism opens new perspectives on its therapeutic use.

 

[madman]

Fig. 2. The three classical narratives of how NAC exerts its biological effects. Three narratives have been cultivated in the literature to explain the observed biological effects of NAC. The disulfide reductant narrative postulates that the beneficial effects of NAC are due to its capacity to reduce extra- and/or intracellular disulfide bonds. The oxidant scavenger narrative argues that the NAC sulfhydryl group (highlighted in red) is effective in removing one- and two-electron oxidants, such as hydrogen peroxide (H2O2), hypochlorous acid (HOCl), or hydroxyl radicals (•OH). The glutathione replenishment narrative proposes that NAC delivers Cys for GSH synthesis, hence boosting GSH levels

 

[madman]

Fig. 3. The GSH biosynthesis pathway and its regulation Two enzymatic steps facilitate the synthesis of GSH. The first step is catalyzed by γ-glutamylcysteine ligase (γ-GCL) which conjugates L-Cys and L-glutamate (L-Glu) into γglutamylcysteine (γ-GC). The second step is catalyzed by GSH synthase (GS), which incorporates L-glycine (L-Gly) to form the final GSH tripeptide. As L-Cys typically is the limiting substrate, supplementation with L-Cys (directly or through NAC) can restore GSH levels when depleted. However, in the absence of GSH depletion, feedback inhibition of γ-GCL activity will prevent GSH biosynthesis, even when extra L-Cys are available. At high concentrations, GSH competitively binds to the γ-GCL active site, preventing further GSH formation. A pharmacological inhibitor of GSH biosynthesis is L-buthionine sulfoximine (BSO), which irreversibly binds to the γ-GCL active site.

 

[madman]

Table 1 Chemical properties of Cys, NAC, and their oxidation products.

 

[madman]

Fig. 4. Three enzymatic systems generate H2S from Cys (A) Cystathionine-β-synthase (CBS) condenses Cys and homocysteine to form cystathionine and H2S. (B) Cystathionine-γ-lyase (CSE) eliminates H2S from Cys, forming pyruvate and NH3. (C) Cysteine aminotransferase (CAT) deaminates Cys by transamination to generate 3-mercaptopyruvate. 3-mercaptopyruvate sulfurtransferase (MPST) desulfurates 3-mercaptopyruvate to generate pyruvate and an enzyme-bound persulfide. MPST then transfers the outer sulfur of the persulfide to a dithiol acceptor molecule (R), in particular thioredoxin, which forms an intramolecular disulfide bond to release H2S.

 

[madman]

Fig. 5. Two mechanisms by which NAC can increase intracellular Cys levels. In principle, NAC can deliver Cys in direct (grey) and indirect (orange) ways. How NAC enters cells is not fully understood. Anion exchanger 1 (AE1) may play a role in erythrocytes. Following import, NAC is deacetylated by aminoacylase 1 (ACY1), releasing Cys in the process. Alternatively, NAC can deliver Cys indirectly through disulfide exchange with oxidized Cys in the plasma (Cys-S-S-R), to release reduced Cys (Cys-SH), which is then rapidly imported by the neutral amino acid transporter ASCT1. Although direct evidence is lacking, it seems likely that NAC-S-S-Cys can be imported into cells by the system Xc - anion exchanger. Inside the cell, NAC-S-S-Cys is reduced by intracellular disulfide reducing systems, releasing Cys and NAC, the latter being deacetylated into Cys by ACY1.

 

[madman]

Fig. 6. Intracellular pathways leading from Cys to persulfides. Two main routes lead to the generation of persulfides. The first involves Cys-derived H2S as an intermediate (left side), the second involves the direct transfer of sulfur (right side). H2S-dependent persulfide generation is mainly catalyzed by sulfide: quinone oxidoreductase (SQR), with potential contributions from cytochrome c (CYT C), but may also to some extent result from the non-enzymatic reaction between H2S and sulfenic acids (R-SOH). H2S-independent persulfide generation is based on the transfer of sulfur from Cys-derived 3-mercaptopyruvate (3MP-SH) to thiols. Additionally, cysteinyl-tRNA synthetase (CARS) has recently been proposed to act as a cysteine persulfidase, although the exact mechanism remains unknown (the dashed line indicates uncertainty about the mechanism).

 

[madman]

Fig. 7. Three hypotheses potentially explaining persulfide-mediated cytoprotection. (A) The first hypothesis argues that persulfidation of Cys residues on proteins changes their activity and/or signaling properties in an adaptive manner, e.g. to alter metabolism or gene expression. (B) The second hypothesis argues that protein persulfidation protects thiol groups against irreversible oxidative damage. While sulfinic (SO2H) and sulfonic (SO3H) acid residues cannot be repaired, perthiosulfinic (SSO2H) and perthiosulfonic (SSO3H) acid residues are easily repaired by a disulfide reductase, such as thioredoxin (TRX). Reduction regenerates the original thiol and presumably releases the outer sulfur as sulfur dioxide (SO2) or sulfite (SO3 2-). (C) The third hypothesis argues for a scavenging role of (low-molecular-weight) persulfides, as they are more reactive towards one- and two-electron oxidants than thiols. This is explained by (i) the α-effect, which increases the nucleophilicity of the persulfide’s outer sulfur atom, and (ii) for two-electron oxidants, the higher availability of the reactive thiolate (i.e., lower pKa). In addition, persulfides are proposed to act as highly efficient radical scavengers. They form stable perthiyl radicals, unable to propagate radical chain reactions, and capable of recombining into tetrasulfides, thereby eliminating radicals from the system.

 

[madman]

Fig. 8. NAC-derived metabolites facilitate disulfide-reducing and oxidant scavenging activities. The conventional narratives assume that disulfide-reducing and antioxidative activities are inherent to NAC. However, evidence suggests that these activities are facilitated by NAC-derived metabolites, GSH, and H2S/sulfane sulfur species. On the one hand, the replenishment of previously depleted GSH levels supports enzymatic disulfide reduction by glutaredoxins (GRX), electrophile detoxification by glutathione S-transferases (GST), and (lipid) peroxide scavenging by glutathione peroxidases (GPX). On the other hand, sulfane sulfur species can act as direct radical scavengers, and protect proteins against irreversible oxidative damage. Additionally, persulfide modifications on proteins may change their activity and signaling properties to trigger stress-adaptive cellular responses.

 

[madman]

Fig. 9. The rate of Cys delivery may determine the difference between cytotoxic and cytoprotective effects. Direct Cys administration leads to a rapid and steep rise in intracellular Cys, which is metabolized into H2S and sulfane sulfur species. The sudden increase in the concentration of these molecules leads to cytotoxicity, associated with the blockage of the mitochondrial ETC and potentially other enzymatic metal centers. In contrast, NAC elevates intracellular Cys at a much slower and more steady rate, allowing for a low-level H2S and sulfane sulfur production, stimulating mitochondrial bioenergetics and protecting cells against oxidative damage.