Leveraging an enzyme/artificial substrate system to enhance cellular persulfides and mitigate neuroinflammation

Abstract

Persulfides and polysulfides, collectively known as the sulfane sulfur pool along with hydrogen sulfide (H₂S), play a central role in cellular physiology and disease. Exogenously enhancing these species in cells is an emerging therapeutic paradigm for mitigating oxidative stress and inflammation that are associated with several diseases. In this study, we present a unique approach of using the cell's own enzyme machinery coupled with an array of artificial substrates to enhance the cellular sulfane sulfur pool. We report the synthesis and validation of artificial/unnatural substrates specific for 3-mercaptopyruvate sulfurtransferase (3-MST), an important enzyme that contributes to sulfur trafficking in cells. We demonstrate that these artificial substrates generate persulfides in vitro as well as mediate sulfur transfer to low molecular weight thiols and to cysteine-containing proteins. A nearly 100-fold difference in the rates of H₂S production for the various substrates is observed supporting the tunability of persulfide generation by the 3-MST enzyme/artificial substrate system. Next, we show that the substrate 1a permeates cells and is selectively turned over by 3-MST to generate 3-MST-persulfide, which protects against reactive oxygen species-induced lethality. Lastly, in a mouse model, 1a is found to significantly mitigate neuroinflammation in the brain tissue. Together, the approach that we have developed allows for the on-demand generation of persulfides in vitro and in vivo using a range of shelf-stable, artificial substrates of 3-MST, while opening up possibilities of harnessing these molecules for therapeutic applications.

Fig. 1. (A) Catalytic cycle of 3-MST: the sulfur of 3-MP is transferred to 3-MST to produce 3-MST-SS⁻ and pyruvate. (B) 3-MST-SS⁻ reacts with reducing agents containing two cysteine residues such as thioredoxin (Trx) to generate H₂S. 3-MST-SS⁻ can also generate a protein persulfide through protein–protein interaction or transfer sulfur to low molecular weight thiols such as GSH to produce glutathione persulfide (GSSH). (C) Thioacetate 1 is expected to be cleaved by esterase (ES) to produce the designed 3-MST substrate 2. This thiol is positioned to undergo a sulfur transfer reaction to produce 3-MST-SS⁻ and a ketone 3 as the byproduct.

Fig. 2. (A) Docking analysis of the active site of h3-MST with 3-MP shows a favorable conformation with the S–S bond distance as 4.4 Å. The R188 residue is 6.1 Å from the carboxyl group whereas the R197 residue is at a distance of 6.3 Å from the carbonyl group. (B) Docking analysis of the designed substrate 2a (R¹ = Ph, R² = H in Fig. 1C) reveals a similar anchoring of the substrate by the two arginine residues and the S–S bond distance was found as 5.2 Å. (C) HPLC analysis of 1a + ES in the presence of h3-MST shows the formation of the thiol 2a which is then subsequently converted to acetophenone 3a during 10 h. (D) Intrinsic fluorescence assay on 3-MST with dimer of ethyl 3-mercaptopyruvate (E3-MP) 4 shows a decrease in fluorescence intensity which is consistent with the generation of 3-MST-SS⁻. A similar result was observed with the unnatural substrate 2a. Ctrl refers to 3-MST alone. (E) Detection of 3-MST-SS⁻ using the modified tag-switch technique (Fig. S7a†), conducted with 4 and 1a + ES; The C238A 3-MST mutant treated under similar conditions showed a diminished band corresponding to the formation of 3-MST-SS⁻: (i) detection of 3-MST persulfide (ii) loading control. (F) Effect of persulfidation on the activity of GAPDH: GAPDH upon treatment with 1a + ES + 3-MST enhances its activity compared to GAPDH alone presumably due to the formation of the persulfide of GAPDH. Ctrl refers GAPDH + ES + 3-MST; 1a refers to co-incubation of 1a + ES + 3-MST followed by addition of GAPDH (absorbance 340 nm). (G) Structures of compounds 5 and 6. (H) Persulfide/polysulfide detection using SSP-2: Ctrl refers to 3-MST alone and 1a ctrl refers to 1a + ES only (100 μM); 1a refers to co-incubation of varying concentrations of 1a, ES and 3-MST; +DTT: addition of DTT; +IAM: addition of iodoacetamide, an electrophile that reacts with thiols; 4 refers to incubation of the compound with 3-MST; 5 and 6 refers to the incubation of the compounds with ES followed by treatment with 3-MST. (I) Extracted ion chromatogram from a mass spectrometry-based analysis of reaction products formed upon incubation of 1a and 3-MST in the presence of ES followed by addition of GSH as the thiol acceptor. Reaction of the reactive sulfur species (GSSH) with an electrophile monobromobimane (mBBr) was employed. LC/MS analysis revealed the formation of the GSS-bimane adduct (expected m/z = 530.1379; observed m/z = 530.1357) when 1a was incubated with ES and 3-MST. (J) Area under the curve (AUC) for the peak corresponding to GSS-bimane (Fig. 2I); Ctrl refers to 1a alone while 3-MST refers to 3-MST alone and 1a refers to 1a + ES + 3-MST.

Scheme 1. Compound 4 is the dimer of E3-MP and in pH 7.4 buffer, dissociates to produce the E3-MP.

Fig. 3. (A) A methylene blue assay was used to measure the rate of H₂S generation with 1a in the presence of h3-MST or b3-MST and DTT as the reducing agent. Nearly identical rates of H₂S generation were observed. (B) 3-MST KD refers to knock-down of expression of 3-MST in A549 cells while scrambled refers to A549 cells containing non-targeting scrambled shRNA. The H₂S donor used is an esterase-sensitive COS/H₂S donor that has been previously characterized. H₂S levels were assessed using a previously reported dye NBD-fluorescein (see ESI, Fig. S20†). Ctrl refers to untreated cells. Scale bar represents 200 μm.

Scheme 2. Proposed mechanism: thioacetate 1 is cleaved by esterase or DTT (HPLC in Fig. S23, ESI†) to produce the thiol 2, which is then turned over by 3-MST to produce 3-MST-SS⁻ and an enol(ate). The enol(ate) in aqueous buffer is rapidly converted to the ketone 3. 3-MST-SS⁻ in the presence of a reducing agent produces H₂S. 3-MST-SS⁻ can react with low molecular weight thiols such as GSH to produce GSSH. Under non-reducing conditions, 3-MST-SS⁻ can further turn over 2, generating the ketone 3; the likely byproduct of this reaction is polysulfide.

Fig. 4. (A) HPLC analysis of a reaction mixture containing 2a and 3-MST in the presence of DTT showed the gradual disappearance of 2a and concomitant formation of 3a. Curve fitting to first order kinetics gave rate constants of 0.99 h⁻¹ and 0.75 h⁻¹ for the disappearance of 2a and the formation of 3a, respectively. (B) Hammett analysis of rate constants of H₂S generation from unnatural substrates (see Table 1) with wt h3-MST. Linear regression analysis yielded a slope of +1.11 (R² = 0.8). (C) Docking of 2k in the active site of h3-MST shows S–S bond distance of 10.7 Å which is substantially larger when compared with the low energy conformation of 2a (5.2 Å). (D) Docking of 2k in the active site of h3-MST shows steric clashes of ortho-Me group of 2k with A185 and R188 residues in a higher energy conformation. (E) Cell viability assay conducted on N2a cells: Cells were pre-treated with 25 μM of compounds 1a, 1b, 1d, 1j and 1k for 12 h and then exposed to MGR-1 (25 μM) for 4 h. Ctrl refers to untreated cells. Cell viability was determined using a standard MTT assay. Results are expressed as mean ± SD (n = 3). ***p < 0.001 vs. MGR-1. (F) Cell viability assay conducted on N2a cells: cells that were pre-treated with 1a, 3a or 6 were then treated with a cell permeable ROS generator MGR-1. A dose-dependent protection of cells from MGR-1 induced cell death by 1a was observed. The byproduct ketone 3a or the negative control 6 did not show any protection against the cytotoxic effects of MGR-1. All data are presented as mean ± SD (n = 3 per group). ***p < 0.001 vs. MGR-1.

Fig. 5. (A) Effects of 1a on quenching hydrogen peroxide (H₂O₂) generated by MGR1:A549 cells treated with: (i and ii) dye control; (iii and iv) 25 μM MGR-1; (v and vi) 25 μM of 1a for 12 h followed by addition of the 25 μM MGR-1 for 1 h. Intracellular H₂O₂ was detected using a reported H₂O₂-sensor TCF-B (25 μM). Scale bar represents 200 μm. (B) Biomarkers for oxidative stress: Three cell lines (A549, mouse embryonic fibroblasts (MEF) and N2a) were pre-treated with vehicle, 1a or 6 and exposed to MGR-1 following which NAD⁺/NADH ratio and GSSG/GSH ratio were determined using an ELISA assay. All data are presented as mean ± SD (n = 5 per group). ***p < 0.001 vs. MGR-1. (C and D) Mouse endotoxin shock model: animals were treated with 1a (20 mg kg⁻¹) or NaSH (20 mg kg⁻¹) 4 h prior to treatment with lipopolysaccharide (LPS, 5 mg kg⁻¹). 30 min post-administration of LPS, another dose of 1a or NaSH was given. The brain tissue samples were harvested followed by measurement of: (C) Pro-inflammatory cytokines, TNF-α and IL-6 using a standard ELISA assay. All data are presented as mean ± SD (n = 6 per group). ***p < 0.001 vs. LPS; and (D) prostaglandins PGE2/D2 using LC/MS. All data are presented as mean ± SD (n = 10 per group). ***p < 0.001 vs. LPS.