Biosynthesis and Reactivity of Cysteine Persulfides in Signaling

Hydrogen sulfide (H₂S) elicits pleiotropic physiological effects ranging from modulation of cardiovascular to CNS functions. A dominant method for transmission of sulfide-based signals is via posttranslational modification of reactive cysteine thiols to persulfides. However, the source of the persulfide donor and whether its relationship to H₂S is as a product or precursor is controversial. The transsulfuration pathway enzymes can synthesize cysteine persulfide (Cys–SSH) from cystine and H₂S from cysteine and/or homocysteine. Recently, Cys–SSH was proposed as the primary product of the transsulfuration pathway with H₂S representing a decomposition product of Cys–SSH. Our detailed kinetic analyses demonstrate a robust capacity for Cys–SSH production by the human transsulfuration pathway enzymes, cystathionine beta-synthase and γ-cystathionase (CSE) and for homocysteine persulfide synthesis from homocystine by CSE only. However, in the reducing cytoplasmic milieu where the concentration of reduced thiols is significantly higher than of disulfides, substrate level regulation favors the synthesis of H₂S over persulfides. Mathematical modeling at physiologically relevant hepatic substrate concentrations predicts that H₂S rather than Cys–SSH is the primary product of the transsulfuration enzymes with CSE being the dominant producer. The half-life of the metastable Cys–SSH product is short and decomposition leads to a mixture of polysulfides (Cys–S–(S)_<i>n</i>–S–Cys). These in vitro data, together with the intrinsic reactivity of Cys–SSH for cysteinyl versus sulfur transfer, are consistent with the absence of an observable increase in protein persulfidation in cells in response to exogenous cystine and evidence for the formation of polysulfides under these conditions

Does Perthionitrite (SSNO^–) Account for Sustained Bioactivity of NO? A (Bio)chemical Characterization

Hydrogen sulfide (H₂S) and nitric oxide (NO) are important signaling molecules that regulate several physiological functions. Understanding the chemistry behind their interplay is important for explaining these functions. The reaction of H₂S with <i>S</i>-nitrosothiols to form the smallest <i>S</i>-nitrosothiol, thionitrous acid (HSNO), is one example of physiologically relevant cross-talk between H₂S and nitrogen species. Perthionitrite (SSNO^–) has recently been considered as an important biological source of NO that is far more stable and longer living than HSNO. In order to experimentally address this issue here, we prepared SSNO^– by two different approaches, which lead to two distinct species: SSNO^– and dithionitric acid [HON­(S)­S/HSN­(O)­S]. (H)­S₂NO species and their reactivity were studied by ¹⁵N NMR, IR, electron paramagnetic resonance and high-resolution electrospray ionization time-of-flight mass spectrometry, as well as by X-ray structure analysis and cyclic voltammetry. The obtained results pointed toward the inherent instability of SSNO^– in water solutions. SSNO^– decomposed readily in the presence of light, water, or acid, with concomitant formation of elemental sulfur and HNO. Furthermore, SSNO⁻ reacted with H₂S to generate HSNO. Computational studies on (H)­SSNO provided additional explanations for its instability. Thus, on the basis of our data, it seems to be less probable that SSNO^– can serve as a signaling molecule and biological source of NO. SSNO^– salts could, however, be used as fast generators of HNO in water solutions

Synthesis and Pharmacological Evaluation of Novel Adenine–Hydrogen Sulfide Slow Release Hybrids Designed as Multitarget Cardioprotective Agents

This work deals with the design, synthesis, and evaluation of the cardioprotective properties of a number of novel hybrid compounds combining the adenine nucleus with a suitable H₂S slow-releasing moiety, coupled via a stable ether bond. The H₂S release rate of the hybrids and their ability to increase cGMP were estimated in vitro. The most promising derivatives <b>4</b> and <b>11</b>, both containing 4-hydroxythiobenzamide moiety as H₂S donor, were selected for further in vivo evaluation. Their ability to release H₂S in vivo was recorded using a new fully validated UPLC-DAD method. Both compounds reduced significantly the infarct size when administered at the end of sustained ischemia. Mechanistic studies showed that they conferred enhanced cardioprotection compared to adenine or 4-hydroxythiobenzamide. They activate the PKG/PLN pathway in the ischemic myocardium, suggesting that the combination of both pharmacophores results in synergistic cardioprotective activity through the combination of both molecular pathways that trigger cardioprotection