SIMLIN: a bioinformatics tool for prediction of S-sulphenylation in the human proteome based on multi-stage ensemble-learning models.

BACKGROUND: S-sulphenylation is a ubiquitous protein post-translational modification (PTM) where an S-hydroxyl (-SOH) bond is formed via the reversible oxidation on the Sulfhydryl group of cysteine (C). Recent experimental studies have revealed that S-sulphenylation plays critical roles in many biological functions, such as protein regulation and cell signaling. State-of-the-art bioinformatic advances have facilitated high-throughput in silico screening of protein S-sulphenylation sites, thereby significantly reducing the time and labour costs traditionally required for the experimental investigation of S-sulphenylation. RESULTS: In this study, we have proposed a novel hybrid computational framework, termed SIMLIN, for accurate prediction of protein S-sulphenylation sites using a multi-stage neural-network based ensemble-learning model integrating both protein sequence derived and protein structural features. Benchmarking experiments against the current state-of-the-art predictors for S-sulphenylation demonstrated that SIMLIN delivered competitive prediction performance. The empirical studies on the independent testing dataset demonstrated that SIMLIN achieved 88.0% prediction accuracy and an AUC score of 0.82, which outperforms currently existing methods. CONCLUSIONS: In summary, SIMLIN predicts human S-sulphenylation sites with high accuracy thereby facilitating biological hypothesis generation and experimental validation. The web server, datasets, and online instructions are freely available at http://simlin.erc.monash.edu/ for academic purposes.

Dynamics of oxytetracycline, sulfamerazine, and ciprofloxacin and related antibiotic resistance genes during swine manure composting.

Understanding the dynamics of veterinary antibiotic and related antibiotic resistance genes (ARGs) during swine manure composting is crucial in assessing the environmental risk of antibiotics, which could effectively reduce their impact in natural environments. This study investigated the dissipation of oxytetracycline (OTC), sulfamerazine (SM1) and ciprofloxacin (CIP) as well as the behaviour of their corresponding ARGs during swine manure composting. These antibiotics were added at two concentration levels and two different methods of addition (single/mixture). The results indicated that the removal efficiency of antibiotics by composting were ≥85%, except for the single-SM1 treatment. The tetracycline resistance genes (TRGs) encoding ribosomal protection proteins (RPP) and efflux pump (EFP) and fluoroquinolone resistance genes (FRGs) could be effectively removed after 42 days. On the contrary, the TRGs encoding enzymatic inactivation (EI) and sulfonamide resistance genes (SRGs) were enriched up to 31-fold (sul 2 in single-low-SM1). Statistical analyses indicated that the behaviour of these class antibiotics and ARGs were controlled by microbial activity and significantly influenced by environmental factors (mainly C/N, moisture and pH) throughout the composting process.

Detecting Protein Sulfenylation in Cells Exposed to a Toxicant.

Protein sulfenylation is a post-translational modification that is linked to many cell signaling networks and specific protein functions, thus the detection of any sulfenylated protein after a toxicological exposure is of importance. Specifically, the detection of protein sulfenylation can provide multiple levels of mechanistic insight towards understanding the impact of a toxicological exposure. For instance, sulfenylation is caused by only a handful of reactive chemical species. Any altered sulfenylation suggests a change in cellular health, and the elucidation of the specific protein target that undergoes sulfenylation can help ascertain downstream targets and associated adverse outcomes. This document describes straightforward approaches to detect protein sulfenylation of total protein as well as individual proteins of interest with a focus on immunoblotting approaches. © 2017 by John Wiley & Sons, Inc.

[Enhancement of Sulfamerazine Degradation Under H2O2/KI System by Ultrasound and UVA Irradiation].

The degradation of Sulfamerazine(SMR) enhanced by molecular iodine under ultrasound/H2O2/KI and UVA/H2O2/KI was investigated. The main affecting parameters, iodine generation, active species and degradation products in the two systems were discussed as well. The experimental results showed that sulfamerazine degradation was effectively enhanced in both systems, and the enhancement of ultrasound was much better. The initial pH had an obvious effect on sulfamerazine removal in the range of 2.6-5.6, and the SMR removal efficiency decreased with initial pH value. Iodine radicals (I2-·, I·) were determined as the main species in ultrasound/H2O2/KI and UVA/H2O2/KI systems. HPLC/MS/MS analysis indicated that iodo-benzene was detected in both system.

Revealing the Mechanistic Pathway of Acid Activation of Proton Pump Inhibitors To Inhibit the Gastric Proton Pump: A DFT Study.

Acid-related gastric diseases are associated with disorder of digestive tract acidification due to the acid secretion by gastric proton pump, H+,K+-ATPase. Omeprazole is one of the persuasive irreversible inhibitor of the proton pump H+,K+-ATPase. However, the reports on the mechanistic pathway of irreversible proton pump inhibitors (PPIs) on the acid activation and formation of disulfide complex are scarce in the literature. We have examined the acid activation PPIs, i.e., timoprazole, S-omeprazole and R-omeprazole using M062X/6-31++G(d,p) in aqueous phase with SMD solvation model. The proton pump inhibitor is a prodrug and activated in the acidic canaliculi of the gastric pump H+,K+-ATPase to sulfenic acid which can either form another acid activate intermediate sulfenamide or a disulfide complex with cysteine amino acid of H+,K+-ATPase. The quantum chemical calculations suggest that the transition state (TS5) for the disulfide complex formation is the rate-determining step of the multistep acid inhibition process by PPIs. The free energy barrier of TS5 is 5.5 kcal/mol higher for timoprazole compared to the S-omeprazole. The stability of the transition state for the formation of disulfide bond between S-omeprazole and cysteine amino acid of H+,K+-ATPase is governed by inter- and intramolecular hydrogen bonding. The disulfide complex for S-omeprazole is thermodynamically more stable by 4.5 kcal/mol in aqueous phase compared to disulfide complex of timoprazole, which corroborates the less efficacy of timoprazole as irreversible PPI for acid inhibition process. It has been speculated that sulfenic acid can either form sulfenamide or a stable disulfide complex with cysteine amino acid residue of H+,K+-ATPase. The M062X/6-31++G(d,p) level of theory calculated results reveal that the formation of tetra cyclic sulfenamide is unfavored by ∼17 kcal/mol for S-omeprazole and 11.5 kcal/mol for timoprazole compared to the disulfide complex formation in each case. The DFT calculations have further shed light on the acid activation process of R- and S-isomers of omeprazole. The calculated results suggest that the efficacy of these isomers lie on their metabolic pathway and excretion from human body.

Does the Transcription Factor NemR Use a Regulatory Sulfenamide Bond to Sense Bleach?

Reactive chlorine species (RCS), such as hypochlorous acid (i.e., bleach), are antimicrobial oxidants produced by the innate immune system. Like many redox-regulated transcription factors, the Escherichia coli repressor NemR responds to RCS by using the reversible oxidation of highly conserved cysteines to alter its DNA-binding affinity. However, earlier work showed that RCS response in NemR does not depend on any commonly known oxidative cysteine modifications. We have now determined the crystal structure of NemR, showing that the regulatory cysteine, Cys106, is in close proximity to a highly conserved lysine (Lys175). We used crystallographic, biochemical, and mass spectrometric analyses to analyze the role of this lysine residue in RCS sensing. Based on our results, we hypothesize that RCS treatment of NemR results in the formation of a reversible Cys106-Lys175 sulfenamide bond. This is, to our knowledge, the first description of a protein whose function is regulated by a cysteine-lysine sulfenamide thiol switch, constituting a novel addition to the biological repertoire of functional redox switches.

Convenient microwave-assisted synthesis of lipophilic sulfenamide prodrugs of metformin.

A convenient microwave-assisted synthesis of lipophilic sulfenamide prodrugs of antidiabetic agent, metformin, is reported in this study. These acyclic prodrugs were synthesized directly from selected disulfides with basic metformin and silver nitrate by a one-pot reaction under microwave irradiation. The prepared prodrugs had significantly increased lipophilicity, which resulted in excellent permeability of the octylthio prodrug of metformin across a Caco-2 cell monolayer. According to our preliminary in vivo studies, the octylthio prodrug was also absorbed mostly intact after oral administration in rats. In conclusion, this study shows that these types of more lipophilic sulfenamide prodrugs can be promising candidates to improve permeability and passive absorption of highly water-soluble metformin.

Determination of the permeability characteristics of two sulfenamide prodrugs of linezolid across Caco-2 cells.

The purpose of this work was to study the permeability of two relatively lipophilic sulfenamide prodrugs of linezolid (clogP 0.85), N-(phenylthio)linezolid (1, clogP 2.77) and N-[(2-ethoxycarbonyl)ethylthio]linezolid (2, clogP 1.43), across Caco-2 cell monolayers. Both prodrugs were found to convert to linezolid in the donor compartment presumably from the reaction with free thiol groups on proteins on the surface of the Caco-2 cells, as no conversion was seen in the donor compartment media per se. Neither of the prodrugs could be detected in the receptor phase from either apical (AP) to basolateral (BL) or BL to AP studies. However, the appearance of linezolid in the receptor phase was biphasic with an initial rapid phase suggesting that the prodrugs were indeed more permeable, and for a short period, some prodrug was able to permeate in competition with conversion to linezolid on the donor phase surface. It appears that the prodrug was able to permeate was rapidly converted to linezolid prior to acceptor phase appearance. The second slower phase was due to the permeability of the donor-phase-formed linezolid, with the slopes similar to those from control experiments with linezolid. The limitations and possible utility of oral sulfenamide prodrugs are discussed.

The thermodynamics of thiol sulfenylation.

Protein sulfenic acids are essential cysteine oxidations in cellular signaling pathways. The thermodynamics that drive protein sulfenylation are not entirely clear. Experimentally, sulfenic acid reduction potentials are hard to measure, because of their highly reactive nature. We designed a calculation method, the reduction potentials from electronic energies (REE) method, to give for the first time insight into the thermodynamic aspects of protein sulfenylation. The REE method is based on the correlation between reaction path-independent reaction energies and free energies of a series of analogous reactions. For human peroxiredoxin (Tpx-B), an antioxidant enzyme that forms a sulfenic acid on one of its active-site cysteines during reactive oxygen scavenging, we found that the reduction potential depends on the composition of the active site and on the protonation state of the cysteine. Interaction with polar residues directs the RSO(-)/RS(-) reduction to a lower potential than the RSOH/RSH reduction. A conserved arginine that thermodynamically favors the sulfenylation reaction might be a good candidate to favor the reaction kinetics. The REE method is not limited to thiol sulfenylation, but can be broadly applied to understand protein redox biology in general.

[Coordinative compounds of zinc with N-substituted thiocarbamoyl-N'-pentamethylensulfenamides--activity modifiers of enzymes of proteolytic and glycolytic action].

The influence of a number of coordinative compounds of zinc with N-substituted thiocarbamoil-N'-pentamethylensulfenamides on activity of elastase, alpha-L-rhamnosidase and alpha-galactosidases evidence for a possibility of their usage as stimulators or inhibitors of enzymes tested have been studied. It was shown that all the compounds in concentration of 0.1 and 0.01% inhibited by 90-100% Bacillus thuringiensis 27-88Els+ elastase activity. [Zn(L2)Br2], [Zn(L1)(NCS)2] and [Zn(L3)(NCS)2] at 20 h exposition activated Cryptococcus albidus 1001 alpha-L-rhamnosidase activity. The rest of compounds influenced it on the control level or inhibited it by 7-23%. The obtained results testify that essential role is not played by separate fragments (L-ligand and anions), but by molecules of zinc complexes as a whole. All the studied complexes, exept for [Zn(L3)(NCS)2], induced alpha-L-rhamnosidase activity of Eupenicillium erubescens 248 (7 to 60%). All zinc compounds (concentration 0.01%, exposition time - 60 min) influenced at the control level Aspergillus niger and Cladosporium cladosporioides alpha-galactosidases activity, however inhibited (up to 20%) activity of Penicillium canescens alpha-galactosidase. The increasing of exposition time of the compounds tested with enzymes up to 20 h testify to selective action of separate compounds on enzymes tested. The data obtained prove, that the character of interaction of zinc complexes is changed depending on the enzyme tested and its strain-producer.

MDCK cell permeability characteristics of a sulfenamide prodrug: strategic implications in considering sulfenamide prodrugs for oral delivery of NH-acids.

The objective of this Letter is both to report the permeability results of a linezolid-based sulfenamide prodrug in an MDCK cell model (enterocyte surrogate system) and to discuss the strategic implications of these results for considering sulfenamide prodrugs to enhance the oral delivery of weakly acidic NH-acids (e.g., amides, ureas, etc.). The two main findings from this study are that the sulfenamide prodrug does not appear to survive intracellular transport due to conversion to linezolid and that there appears to be an apically-oriented surface conversion pathway that can additionally serve to convert the sulfenamide prodrug to linezolid upon approach of the apical membrane. It is hoped that these findings, along with the discussion of the strategic implications, will facilitate a greater awareness of the potential strengths and weaknesses inherent in the sulfenamide prodrug approach for enhancing the oral delivery of weakly acidic NH-acid drugs.

Preparation, characterization and in vivo conversion of new water-soluble sulfenamide prodrugs of carbamazepine.

Improved synthetic methods are reported for the preparation of sulfenamide derivatives of carbamazepine (CBZ) for evaluation as prodrugs. These sulfenamide prodrugs were designed to rapidly release CBZ in vivo by cleavage of the sulfenamide bond by chemical reaction with glutathione and other sulfhydryl compounds. Physicochemical characterization and in vivo conversion of a new prodrug of CBZ was evaluated to further establish the proof of concept of the sulfenamide prodrug approach.

A complex thiolate switch regulates the Bacillus subtilis organic peroxide sensor OhrR.

Oxidation of protein thiolates is central to numerous redox-regulated processes. Bacillus subtilis OhrR is an organic peroxide sensor that represses expression of an inducible peroxiredoxin, OhrA. Here, we present evidence that oxidation of the sole cysteine residue in OhrR leads to a sulfenic acid-containing intermediate that retains DNA-binding activity: further reaction to generate either a mixed disulfide (S-thiolation) or a protein sulfenamide (sulfenyl-amide) derivative is essential for derepression. Protein S-thiolation protects OhrR from overoxidation and provides for a facile regeneration of active OhrR by thiol-disulfide exchange reactions. The sulfenamide can also be reduced by thiol-disulfide exchange reactions, although this process is much slower than for mixed disulfides. Recovery of oxidized OhrR from B. subtilis identifies three distinct S-thiolated species, including mixed disulfides with a novel 398-Da thiol, cysteine, and CoASH. Evidence for in vivo formation of the sulfenamide derivative is also presented.

Comparison of the determination of four sulphonamides and their N4-acetyl metabolites in swine muscle tissue using liquid chromatography with ultraviolet and mass spectral detection.

A discharge-assisted LC-MS method has been developed and validated for the analysis of four sulphonamides (sulphathiazole, sulphadiazine, sulphamerazine and sulphadimidine) and their N4-acetyl metabolites in the muscle of swine treated with Polysulpha-Complex, which contains all four drugs. The clean-up procedure developed involved chloroform-acetone extraction followed by Sep-Pak silica solid-phase extraction. In parallel a LC-UV method was validated using the same clean-up procedure. Blank tissue was fortified at levels between 20 and 100 micrograms/kg. [13C]sulphadimidine was used as internal standard. The samples were analysed with thermospray LC-MS. The [M + H]+ ion was the major ion in all cases and was employed for single-ion monitoring. The limits of detection (LOD) were below 25 micrograms/kg and the limits of quantification (LOQ) for most sulphonamides were ca. 100 micrograms/kg. Incurred muscle tissues were measured by both LC methods and the concentrations of the sulphonamides were found to be similar. However, the LC-MS procedure is more suitable for confirmatory analysis due to its specificity.

Elucidation of the role of hydrophobic bonding in influencing intestinal absorption of model sulfonamides and revealing possible mechanism of drug absorption in rat model.

A recirculation technique was used to study the first-order kinetics of intestinal absorption of un-ionized sulfadiazine, sulfamerazine, and sulfamethazine in rats in situ at 32, 35, and 38 degrees C. The absorption rate constant (Kab) of each sulfonamide increased with increase in temperature and, at each temperature, Kab was the highest for sulfamethazine and the lowest for sulfadiazine. Applying the activated complex formation theory, the energy of activation (Ea), free energy of activation (delta F*), enthalpy of activation (delta H*), and entropy of activation (delta S*) of absorption were determined for the sulfonamides to gain some insight into the mechanism of their intestinal absorption. The high values of delta F* indicated that the barrier for sulfonamide absorption was great. For each drug, the value of delta H* was positive and that the delta S* negative. However, delta H* and delta S* were the highest for sulfamethazine and the lowest for sulfadiazine, thus revealing the influence of hydrophobic bonding in increasing Kab of the sulfonamides with the increase in methyl group content of their molecules. By considering the facts that (1) the microvillus membrane of the intestinal absorptive cells regulates the rate of passive absorption of drugs, (2) the microvillus membrane is rich in proteins, which are located external to the membrane and exposed to the intestinal fluid, and (3) hydrophobic bonding contributes to the activation parameters of absorption, it was postulated that the activated complex formed in the absorption process consisted of a transient association of the sulfonamide molecules with some protein component of the microvillus membrane.

Development of a screening method for five sulfonamides in salmon muscle tissue using thin-layer chromatography.

A thin-layer chromatographic (TLC) method was developed for the analysis of five sulfonamides [sulfadiazine (SDZ), sulfamerazine (SMRZ), sulfamethazine (SMTZ), sulfadimethoxine (SDMX) and sulfapyridine (SP)] in salmon muscle tissue. "Matrix solid-phase dispersion" was employed whereby the tissue sample was ground with C18-derivatized silica gel. This material was packed into a column and washed with 10% toluene in hexane (discarded) followed by dichloromethane which was evaporated. The residue was chromatographed on a high-performance TLC plate using ethyl acetate-n-butanol-methanol-aqueous ammonia (35:45:15:2, v/v). Sulfonamides were detected after spraying the plate with a solution of fluorescamine. Method parameters were determined by analyzing spiked salmon muscle tissue samples. The method detection limits at the 99% confidence level were 0.11, 0.44, 0.07, 0.13 and 0.13 ppm for SDZ, SMRZ, SMTZ, SDMX and SP, respectively. The lowest-detectable levels were approximately 0.04 ppm for SDZ, SMTZ, SDMX and SP, and 0.10 ppm for SMRZ. The average recoveries of analyses were 61, 63, 60, 63 and 57% for SDZ, SMRZ, SMTZ, SDMX and SP, respectively, and were found to be analyst-dependent. The method was found to give linear detector responses for all analytes over spiking levels ranging from 0 to 2 ppm.

Pharmacokinetics and renal clearance of sulphadimidine, sulphamerazine and sulphadiazine and their N4-acetyl and hydroxy metabolites in pigs.

The effect of molecular structure on the drug disposition and protein binding in plasma, the urinary recovery, and the renal clearance of sulphamerazine (SMR), sulphadiazine (SDZ), and sulphadimidine (SDM) and their N4-acetyl and hydroxy derivatives were studied in pigs. Following IV administration of SDM, SMR and SDZ, their mean elimination half-lives were 12.4 h, 4.3 h and 4.9 h respectively. The plasma concentrations of parent sulphonamide were higher than those of the metabolites, and ran parallel. The acetylated derivatives were the main metabolites; traces of 6-hydroxymethylsulphamerazine and 4-hydroxysulphadiazine were detected in plasma. The urine recovery data showed that in pigs acetylation is the major elimination pathway of SDM, SMR and SDZ; hydroxylation became more important in case of SMR (6-hydroxymethyl and 4-hydroxy derivatives) and SDZ (4-hydroxy derivatives) than in SDM. In pigs methyl substitution of the pyrimidine side chain decreased the renal clearance of the parent drug and made the parent compound less accessible for hydroxylation. Acetylation and hydroxylation speeded up drug elimination, because their renal clearance values were higher than those of the parent drug.

Pharmacokinetics, metabolism, and renal clearance of sulfadiazine, sulfamerazine, and sulfamethazine and of their N4-acetyl and hydroxy metabolites in calves and cows.

The effect of molecular structure on the drug disposition and protein binding in plasma and milk, the urinary recovery, and the renal clearance of sulfadiazine, sulfamerazine, and sulfamethazine and of their N4-acetyl and hydroxy derivatives were studied in calves and cows. Sulfadiazine was highly acetylated and was slightly hydroxylated. Sulfamerazine and sulfamethazine were hydroxylated predominantly at the methyl group of the pyrimidine side chain; hydroxylation of the pyrimidine ring itself was more extensive for sulfamethazine than for sulfamerazine. At dosages between 100 and 200 mg/kg of body weight, sulfamethazine had a capacity-limited elimination pattern, which was not observed for sulfadiazine or sulfamerazine. The concentrations of the parent sulfonamide and its metabolites in plasma and milk were parallel, the latter being lower. Metabolite concentrations in milk were at least 8 times lower than those of the parent drug. Metabolism speeds drug elimination, producing compounds with renal clearance values higher than those of the parent drug. The effect on the metabolism and renal clearance of methyl substitution in the pyrimidine side chain is discussed.

Pharmacokinetics and renal clearance of sulfamethazine, sulfamerazine, and sulfadiazine and their N4-acetyl and hydroxy metabolites in horses.

Plasma disposition, protein binding, urinary recovery, and renal clearance of sulfamethazine (SMZ), sulfamerazine (SMR), and sulfadiazine (SDZ) and their N4-acetyl and hydroxy derivatives were studied in 4 horses in a crossover trial. The plasma concentration-time curves of the metabolites paralleled those of the parent drug in the elimination phase. Sulfamethazine and SMR were extensively metabolized. In plasma and urine, the main metabolite of the 3 sulfonamides tested was the 5-hydroxypyrimidine derivative, which was highly glucuronidated. Difference in elimination half-life of SMZ, SMR, and SDZ could be related to difference in metabolism and renal clearance values. Metabolism speeds drug elimination, producing compounds with higher renal clearance values than those of the parent drug. Methyl substitution in the pyrimidine side chain increased hydroxylation of the parent drug, but prolonged the persistence of the sulfonamides studied in the body. The high concentration of N4-acetyl and hydroxy metabolites of SMZ and SMR in plasma and urine decreased the potential antibacterial activity of the parent drugs. Sulfadiazine was less metabolized, and microbiologically determined SDZ concentrations in plasma and urine were slightly lower than those measured by high-performance liquid chromatography.