Cysteine Sulfhydryl Groups Research Articles

Nebulized Glutathione as a Key Antioxidant for the Treatment of Oxidative Stress in Neurodegenerative Conditions.

Glutathione (GSH), a tripeptide synthesized intracellularly, serves as a pivotal antioxidant, neutralizing reactive oxygen species (ROS) and reactive nitrogen species (RNS) while maintaining redox homeostasis and detoxifying xenobiotics. Its potent antioxidant properties, particularly attributed to the sulfhydryl group (-SH) in cysteine, are crucial for cellular health across various organelles. The glutathione-glutathione disulfide (GSH-GSSG) cycle is facilitated by enzymes like glutathione peroxidase (GPx) and glutathione reductase (GR), thus aiding in detoxification processes and mitigating oxidative damage and inflammation. Mitochondria, being primary sources of reactive oxygen species, benefit significantly from GSH, which regulates metal homeostasis and supports autophagy, apoptosis, and ferroptosis, playing a fundamental role in neuroprotection. The vulnerability of the brain to oxidative stress underscores the importance of GSH in neurological disorders and regenerative medicine. Nebulization of glutathione presents a novel and promising approach to delivering this antioxidant directly to the central nervous system (CNS), potentially enhancing its bioavailability and therapeutic efficacy. This method may offer significant advantages in mitigating neurodegeneration by enhancing nuclear factor erythroid 2-related factor 2 (NRF2) pathway signaling and mitochondrial function, thereby providing direct neuroprotection. By addressing oxidative stress and its detrimental effects on neuronal health, nebulized GSH could play a crucial role in managing and potentially ameliorating conditions such as Parkinson's Disease (PD) and Alzheimer's Disease (AD). Further clinical research is warranted to elucidate the therapeutic potential of nebulized GSH in preserving mitochondrial health, enhancing CNS function, and combating neurodegenerative conditions, aiming to improve outcomes for individuals affected by brain diseases characterized by oxidative stress and neuroinflammation.

Virus-like particle vaccine displaying an external, membrane adjacent MUC16 epitope elicits ovarian cancer-reactive antibodies

BackgroundMUC16 is a heavily glycosylated cell surface mucin cleaved in the tumor microenvironment to shed CA125. CA125 is a serum biomarker expressed by > 95% of non-mucinous advanced stage epithelial ovarian cancers. MUC16/CA125 contributes to the evasion of anti-tumor immunity, peritoneal spread and promotes carcinogenesis; consequently, it has been targeted with antibody-based passive and active immunotherapy. However, vaccination against this self-antigen likely requires breaking B cell tolerance and may trigger autoimmune disease. Display of self-antigens on virus-like particles (VLPs), including those produced with human papillomavirus (HPV) L1, can efficiently break B cell tolerance.ResultsA 20 aa juxta-membrane peptide of the murine MUC16 (mMUC16) or human MUC16 (hMUC16) ectodomain was displayed either via genetic insertion into an immunodominant loop of HPV16 L1-VLPs between residues 136/137, or by chemical coupling using malemide to cysteine sulfhydryl groups on their surface. Female mice were vaccinated intramuscularly three times with either DNA expressing L1-MUC16 fusions via electroporation, or with alum-formulated VLP chemically-coupled to MUC16 peptides. Both regimens were well tolerated, and elicited MUC16-specific serum IgG, although titers were higher in mice vaccinated with MUC16-coupled VLP on alum as compared to L1-MUC16 DNA vaccination. Antibody responses to mMUC16-targeted vaccination cross-reacted with hMUC16 peptide, and vice versa; both were reactive with the surface of CA125+ OVCAR3 cells, but not SKOV3 that lack detectable CA125 expression. Interestingly, vaccination of mice with mMUC16 peptide mixed with VLP and alum elicited mMUC16-specific IgG, implying VLPs provide robust T help and that coupling may not be required to break tolerance to this epitope.ConclusionVaccination with VLP displaying the 20 aa juxta-membrane MUC16 ectodomain, which includes the membrane proximal cleavage site, is likely to be well tolerated and induce IgG targeting ovarian cancer cells, even after CA125 is shed.

Native and nonnative reactions in ethanolamine ammonia-lyase are actuated by different dynamics

We address the contribution of select classes of solvent-coupled configurational fluctuations to the complex choreography involved in configurational and chemical steps in an enzyme by comparing native and nonnative reactions conducted at different protein internal sites. The low temperature, first-order kinetics of covalent bond rearrangement of the cryotrapped substrate radical in coenzyme B12-dependent ethanolamine ammonia-lyase (EAL) from Salmonella enterica display a kink, or increase in slope, of the Arrhenius plot with decreasing temperature. The event is associated with quenching of a select class of reaction-actuating collective fluctuations in the protein hydration layer. For comparison, a nonnative, radical reaction of the protein interior cysteine sulfhydryl group with hydrogen peroxide (H2O2) is introduced by cryotrapping EAL in an aqueous H2O2 eutectic system. The low-temperature aqueous H2O2 protein hydration and mesodomain solvent phases surrounding cryotrapped EAL are characterized by using TEMPOL spin probe electron paramagnetic resonance spectroscopy, including a freezing transition of the eutectic phase that orders the protein hydration layer. Kinetics of the cysteine-H2O2 reaction in the EAL protein interior are monitored by DEPMPO spin trapping of hydroxyl radical product. In contrast to the native reaction, the linear Arrhenius relation for the nonnative cysteine-H2O2 reaction is maintained through the solvent-protein ordering transition. The nonnative reaction is coupled to the generic local, incremental fluctuations that are intrinsic to globular proteins. The comparative approach supports the proposal that select coupled solvent-protein configurational fluctuations actuate the native reaction, and suggests that select dynamical coupling contributes to the degree of catalysis in enzymes.

Graphene oxide/Fe3O4@β-lactoglobulin nanocomposite for the enrichment of trace Pb(II) from real samples

A new GO/Fe3O4@β-lactoglobulin sorbent was prepared for selective extraction of trace Pb(II). The GO/Fe3O4@β-lactoglobulin packed column advantageously permeates samples faster than nascent GO column ca. 5 fold. We experimentally demonstrated the developed SPE method for the enrichment of Pb(II) from real samples (tap, river and packaged drinking water) before ICP-OES determination. The sulfhydryl groups of the vicinal cysteine and the carboxylic groups were led to successful chelate formation with Pb(II) ions. The method shows a detection limit of 0.30 ng mL−1 for Pb(II) ion. The prepared sorbent has a surface area and pore volume of 1460.37 m2g−1 and 1.36 ccg−1, respectively. The preconcentration limit and preconcentration factor for Pb(II) was found to be 0.37 μg L−1 and 900, respectively using 3 mL of eluent. At 95% confidence level, the Student's t-test was less than the critical Student's t value (4.303). A coefficients of variation for five replicate measurements is 3.45%, indicates good precision of the method.

Covalent bonding between polyphenols and proteins: Synthesis of caffeic acid-cysteine and chlorogenic acid-cysteine adducts and their quantification in dairy beverages

Protein-polyphenol interactions affect the structure, stability, and functional properties of proteins and polyphenols. Oxidized polyphenols (o-quinones) react rapidly with the sulfhydryl group of cysteine (Cys) residues, inducing covalent bonding between proteins and polyphenols. However, quantitative data on such reactions remain elusive, despite the importance of depicting the significance of such interactions on food structure and function. This work reports the synthesis, purification, and characterization of caffeic acid-cysteine (CA-Cys) and chlorogenic acid-cysteine (CGA-Cys) adducts and their stable isotope analogs, CA-[13C3,15N]Cys and CGA-[13C3,15N]Cys. A sensitive LC-MS/MS isotope dilution method was developed to simultaneously quantify these adducts in foods and beverages. Protein-bound CA-Cys and CGA-Cys were detected in the micro-molar range in milk samples with added CA and CGA, confirming covalent bonding between milk proteins and CA/CGA. These adducts were detected in commercial coffee-containing beverages but not in cocoa-containing drinks. Furthermore, the adducts were found to be partially stable during enzymatic protein hydrolysis.

Dioxin-elicited decrease in cobalamin redirects propionyl-CoA metabolism to the β–oxidation-like pathway resulting in acrylyl-CoA conjugate buildup

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant that induces diverse biological and toxic effects, including reprogramming intermediate metabolism, mediated by the aryl hydrocarbon receptor. However, the specific reprogramming effects of TCDD are unclear. Here, we performed targeted LC-MS analysis of hepatic extracts from mice gavaged with TCDD. We detected an increase in S-(2-carboxyethyl)-L-cysteine, a conjugate from the spontaneous reaction between the cysteine sulfhydryl group and highly reactive acrylyl-CoA, an intermediate in the cobalamin (Cbl)-independent β–oxidation-like metabolism of propionyl-CoA. TCDD repressed genes in both the canonical Cbl-dependent carboxylase and the alternate Cbl-independent β–oxidation-like pathways as well as inhibited methylmalonyl-CoA mutase (MUT) at lower doses. Moreover, TCDD decreased serum Cbl levels and hepatic cobalt levels while eliciting negligible effects on gene expression associated with Cbl absorption, transport, trafficking, or derivatization to 5′-deoxy-adenosylcobalamin (AdoCbl), the required MUT cofactor. Additionally, TCDD induced the gene encoding aconitate decarboxylase 1 (Acod1), the enzyme responsible for decarboxylation of cis-aconitate to itaconate, and dose-dependently increased itaconate levels in hepatic extracts. Our results indicate MUT inhibition is consistent with itaconate activation to itaconyl-CoA, a MUT suicide inactivator that forms an adduct with adenosylcobalamin. This adduct in turn inhibits MUT activity and reduces Cbl levels. Collectively, these results suggest the decrease in MUT activity is due to Cbl depletion following TCDD treatment, which redirects propionyl-CoA metabolism to the alternate Cbl-independent β–oxidation-like pathway. The resulting hepatic accumulation of acrylyl-CoA likely contributes to TCDD-elicited hepatotoxicity and the multihit progression of steatosis to steatohepatitis with fibrosis.

Evidence of endogenously produced hydrogen sulfide (H2S) and persulfidation in male reproduction

Persulfidation contributes to a group of redox post-translational modifications (PTMs), which arise exclusively on the sulfhydryl group of cysteine as a result of hydrogen sulfide (H2S) action. Redox-active molecules, including H2S, contribute to sperm development; therefore, redox PTMs represent an extremely important signalling pathway in sperm life. In this path, persulfidation prevents protein damage caused by irreversible cysteine hyperoxidation and thus maintains this signalling pathway. In our study, we detected both H2S and its production by all H2S-releasing enzymes (cystathionine γ-lyase (CTH), cystathionine β-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (MPST)) in male reproduction, including spermatozoa. We provided evidence that sperm H2S leads to persulfidation of proteins, such as glyceraldehyde-3-phosphate dehydrogenase, tubulin, and anchor protein A-kinase. Overall, this study suggests that persulfidation, as a part of the redox signalling pathway, is tightly regulated by enzymatic H2S production and is required for sperm viability.

A novel ratiometric fluorescent probe for the detection and imaging of cysteine in living cells

Cysteine is an essential amino acid for humans and can be generated from the conversion of methionine or the interconversions with cystine. The detection of cysteine in the living system is of great importance for the diagnosis of related diseases. Based on the classical cysteine response mechanism, the sulfhydryl group of cysteine undergoes Michael addition with the acrylate to rapidly respond to form a seven-membered ring that specifically recognizes cysteine. In the present work, we designed a novel ratiometric fluorescent probe N-1, which contained the benzo[cd]indol fluorophore and the response group acrylate. As the concentration of cysteine increased, the fluorescence of I485 was significantly enhanced while the fluorescence intensity at 635 nm was weakened. Moreover, probe N-1 showed excellent sensitivity and selectivity to Cys with the calculated LOD of 4.0 × 10-9 M. Probe N-1 was successfully used for imaging cysteine in the living Hela cells with low cytotoxicity, indicating that this ratiometric fluorescent probe would be a novel and useful tool to monitor cysteine in the physiological or pathological processes.

In Vitro and In Vivo Metabolic Activation and Hepatotoxicity of Environmental Pollutant 2,6-Dimethylphenol.

2,6-Dimethylphenol (2,6-DMP) is an environmental pollutant found in industrial wastewater. Exposure to 2,6-DMP is of increasing concern as it endangered reportedly some aquatic animals. In this study, we investigated the metabolic activation and hepatotoxicity of 2,6-DMP. 2,6-DMP was metabolized to an o-quinone methide intermediate in vitro and in vivo. The electrophilic metabolite was reactive to the sulfhydryl groups of glutathione, N-acetyl cysteine, and cysteine. NADPH was required for the formation of the reactive metabolite. The quinone methide intermediate reacted with cysteine residues to form hepatic protein adduction. A single dose of 2,6-DMP induced marked elevation of serum ALT and AST in mice. Both the protein adduction and hepatotoxicity of 2,6-DMP showed dose dependency.

Fluorescence Studies: A9 Peptide, Functionalized with a Fluorogenic Probe, Interacts with Its Receptor Model HER2-DIVMP.

A recently developed synthetic protocol allowed for the functionalization of the active peptide A9 with a fluorogenic probe, which is useful for studying biomolecular interactions. Essentially, a nucleophilic attack on a halo-substituted benzofurazan is selectively performed by a cysteine sulfhydryl group. The process is assisted by the basic catalysis of activated zeolites (4 Å molecular sieves) and promoted by microwave irradiation. Fluorescence studies revealed that a donor-acceptor pair within the peptide sequence was introduced, thus allowing a deeper investigation on the interaction process between the peptide ligand and its receptor fragment. The obtained results allowed us to come full circle for all the currently understood structural determinants that were found to be involved in the binding process.

Phytohormonal Regulation Through Protein S-Nitrosylation Under Stress.

The liaison between Nitric oxide (NO) and phytohormones regulates a myriad of physiological processes at the cellular level. The interaction between NO and phytohormones is mainly influenced by NO-mediated post-translational modifications (PTMs) under basal as well as induced conditions. Protein S-nitrosylation is the most prominent and widely studied PTM among others. It is the selective but reversible redox-based covalent addition of a NO moiety to the sulfhydryl group of cysteine (Cys) molecule(s) on a target protein to form S-nitrosothiols. This process may involve either direct S-nitrosylation or indirect S-nitrosylation followed by transfer of NO group from one thiol to another (transnitrosylation). During S-nitrosylation, NO can directly target Cys residue (s) of key genes involved in hormone signaling thereby regulating their function. The phytohormones regulated by NO in this manner includes abscisic acid, auxin, gibberellic acid, cytokinin, ethylene, salicylic acid, jasmonic acid, brassinosteroid, and strigolactone during various metabolic and physiological conditions and environmental stress responses. S-nitrosylation of key proteins involved in the phytohormonal network occurs during their synthesis, degradation, or signaling roles depending upon the response required to maintain cellular homeostasis. This review presents the interaction between NO and phytohormones and the role of the canonical NO-mediated post-translational modification particularly, S-nitrosylation of key proteins involved in the phytohormonal networks under biotic and abiotic stresses.

Zeolites employed as basic catalyst for nucleophilic substitution reactions: An analysis of the adopted approach and hypothesized new perspectives

The present article reports on an excursus of substitution reactions performed on different halo-compounds and several cyclic sulfamidates that are readily accessible to nucleophilic attack. Essentially, a cysteine sulfhydryl group is employed as nucleophile and the process is promoted by the basic sites of activated Zeolites A (4 Å molecular sieves). The catalysis occurs on the external surface of the zeolite, as also assessed by the FT-IR analysis executed on molecules adsorbed on the catalyst. The moderate basicity of the zeolite lattice surface is the prerequisite for conducting chemoselective and in some case stereoselective peptide modifications. The developed methodology allows an efficient one-pot introduction of exogenous moieties into peptides, useful for developing peptidomimetic and/or peptide-based probes.

Extra-platelet low-molecular-mass thiols mediate the inhibitory action of S-nitrosoalbumin on human platelet aggregation via S-transnitrosylation of the platelet surface

Nitrosylation of sulfhydryl (SH) groups of cysteine (Cys) moieties is an important post-translational modification (PTM), often on a par with phosphorylation. S-Nitrosoalbumin (ALB-Cys34SNO; SNALB) in plasma and S-nitrosohemoglobin (Hb-Cysβ93SNO; HbSNO) in red blood cells are considered the most abundant high-molecular-mass pools of nitric oxide (NO) bioactivity in the human circulation. SNALB per se is not an NO donor. Yet, it acts as a vasodilator and an inhibitor of platelet aggregation. SNALB can be formed by nitrosation of the sole reduced Cys group of albumin (Cys34) by nitrosating species such as nitrous acid (HONO) and nitrous anhydride (N2O3), two unstable intermediates of NO autoxidation. SNALB can also be formed by the transfer (S-transnitrosylation) of the nitrosyl group (NO+) of a low-molecular-mass (LMM) S-nitrosothiol (RSNO) to ALB-Cys34SH. In the present study, the effects of LMM thiols on the inhibitory potential of ALB-Cys34SNO on human washed platelets were investigated. ALB-Cys34SNO was prepared by reacting n-butylnitrite with albumin after selective extraction from plasma of a healthy donor on HiTrapBlue Sepharose cartridges. ALB-Cys34SNO was used in platelet aggregation measurements after extended purification on HiTrapBlue Sepharose and enrichment by ultrafiltration (cutoff, 20 kDa). All tested LMM cysteinyl thiols (R-CysSH) including l-cysteine and L-homocysteine (at 10 µM) were found to mediate the collagen-induced (1 µg/mL) aggregation of human washed platelets by SNALB (range, 0–10 µM) by cGMP-dependent and cGMP-independent mechanisms. The LMM thiols themselves did not affect platelet aggregation. It is assumed that the underlying mechanism involves S-transnitrosylation of SH groups of the platelet surface by LMM RSNO formed through the reaction of SNALB with the thiols: ALB-Cys34SNO + R-CysSH ↔ ALB-Cys34SH + R-CysSNO. Such S-transnitrosylation reactions may be accompanied by release of NO finally resulting in cGMP-dependent and cGMP-independent mechanisms.

Visible‐Light‐Driven Fluoroalkylation of Tryptophan Residues in Peptides

AbstractTrifluoromethylated and fluoroalkylated cyclic λ3‐iodanes and their acyclic salts were used for visible light‐driven fluoroalkylation of tryptophan and tryptophan‐containing peptides in aqueous media. In comparison to previously reported fluoroalkylation using similar reagents and sodium ascorbate as reductant, the photochemical process did not require any additive or catalyst and was more selective for Trp versus other aromatic amino acids due to the gradual production of fluoroalkyl radicals over the whole irradiation period. However, in the presence of Cys residues, both methods were not selective and cysteine sulfhydryl groups were fluoroalkylated in side reactions. Spectroscopic and photochemical investigations as well as quantum chemical calculations provided insight into the reaction mechanism. The process was found to be photoinduced involving the formation of fluoroalkyl radical from the excited state of λ3‐iodane.

Role of glutathione on acrylamide inhibition: Transformation products and mechanism

Glutathione (GSH) is a potential inhibitor for acrylamide (AA) in heated food. In the present study, the inhibition pathways of GSH on AA were investigated in the asparagine(Asn)/glucose(Glc)/GSH model system. In comparison to the Asn/Glc model system, three unique molecular ions (m/z 470, 379, and 193) were identified in the Asn/Glc/GSH model system. Those molecular ions were confirmed as the Amadori rearrangement products which formed in the reaction between Glc and GSH, as well as the addition reaction products between AA and the sulfhydryl group (-SH) of GSH and cysteine (Cys). The competition between Asn and GSH for Glc in the competitive reactions was assumed to be the major pathway. Additionally, the elimination reaction between AA and GSH or between AA and Cys also played a minor role in the inhibition of AA. The variances of precursors, intermediates, and final products provided quantitative evidence for the above pathways.

Spectral and photochemical diversity of tandem cysteine cyanobacterial phytochromes

The atypical trichromatic cyanobacterial phytochrome NpTP1 from Nostoc punctiforme ATCC 29133 is a linear tetrapyrrole (bilin)-binding photoreceptor protein that possesses tandem-cysteine residues responsible for shifting its light-sensing maximum to the violet spectral region. Using bioinformatics and phylogenetic analyses, here we established that tandem-cysteine cyanobacterial phytochromes (TCCPs) compose a well-supported monophyletic phytochrome lineage distinct from prototypical red/far-red cyanobacterial phytochromes. To investigate the light-sensing diversity of this family, we compared the spectroscopic properties of NpTP1 (here renamed NpTCCP) with those of three phylogenetically diverged TCCPs identified in the draft genomes of Tolypothrix sp. PCC7910, Scytonema sp. PCC10023, and Gloeocapsa sp. PCC7513. Recombinant photosensory core modules of ToTCCP, ScTCCP, and GlTCCP exhibited violet-blue-absorbing dark-states consistent with dual thioether-linked phycocyanobilin (PCB) chromophores. Photoexcitation generated singly-linked photoproduct mixtures with variable ratios of yellow-orange and red-absorbing species. The photoproduct ratio was strongly influenced by pH and by mutagenesis of TCCP- and phytochrome-specific signature residues. Our experiments support the conclusion that both photoproduct species possess protonated 15E bilin chromophores, but differ in the ionization state of the noncanonical "second" cysteine sulfhydryl group. We found that the ionization state of this and other residues influences subsequent conformational change and downstream signal transmission. We also show that tandem-cysteine phytochromes present in eukaryotes possess similar amino acid substitutions within their chromophore-binding pocket, which tune their spectral properties in an analogous fashion. Taken together, our findings provide a roadmap for tailoring the wavelength specificity of plant phytochromes to optimize plant performance in diverse natural and artificial light environments.

SIMLIN: a bioinformatics tool for prediction of S-sulphenylation in the\xa0human proteome based on multi-stage ensemble-learning models

BackgroundS-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.ResultsIn 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.ConclusionsIn 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.

Some indices of the cats’ protein metabolism under the obesity

Obesity is one of the most widely-distributed metabolic disturbances in cats, herewith the animals’ number with a given pathology is constantly increasing. The excess mass and obesity lead to the extreme metabolic disturbances including all the chains of metabolism. The cats belong to wild animals and are characterized by a high level of protein metabolism, herewith they are incapable to keep up with the level of the particular amino acids under their constant introduction into the feeds. The intensity of glucogenesis for which the amino acids are viscous material and are involved into the process of dissemination under the obesity is constantly increasing. It results in increasing the amount of final product of nitrogen metabolism. The paper researches the total protein content as well as that of albumin, ammonia, urine, creatinine and creatine kinase activity of the cats’ blood which had the excess mass or obesity. In addition, it has become necessary to determine the content of amino acids cysteine sulfhydryl groups, which essentially contributes to the process of cats’ metabolism. To perform the research two groups of cats (female and male) aged 3–7 were formed, the control group consists of 7 clinically healthy animals that corresponds to the BCS 4–5 index according to the nine grading scale, the experimental group consists of 5 animals of the same age and they have BCS – 7–9 index, that testifies to the excess mass and obesity processes. The concentration of the total protein, albumin, ammonia, urine, creatinine as well as of creatine kinase activity of the total and free(nonprotein) sulfhydryl groups was found in the cats’ blood. The significant decreasing of the albumin part has been revealed in cats with the excess mass and obesity that causes the liver function disturbances and results in increasing of its protein decomposition. The increasing quality of the ammonia content by 31% and urine by 21% has been revealed in cats of the experimental group, however the individual ranges of given indexes within both control and experimental groups were considerable ones. The creatinine concentration and creatine kinase activity in cats with the obesity increases, that testifies to the destructive processes intensification in the muscular tissues. Sulfhydryl groups of the cysteine are of great importance for the metabolic processes in particular in the processes of detoxication, disease-resistance as well as of antioxidant processes. The total concentration of SH-blood groups made no essential difference in cats of the control and experimental groups, while the concentration of free SH-blood groups of experimental animals was by 31% less than in its control analogues. Thus, the obesity has an essential effects on the protein metabolism that causes the amino acids decomposition processes as well as the accumulation of the metabolic final products and free sulfhydryl groups reducing.

Inhibitors of Glyceraldehyde 3-Phosphate Dehydrogenase and Unexpected Effects of Its Reduced Activity.

The review describes the use of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) inhibitors to study the enzyme and to suppress its activity in various cell types. The main problem of selective GAPDH inhibition is a highly conserved nature of the enzyme active site and, especially, Cys150 environment important for the catalytic action of cysteine sulfhydryl group. Numerous attempts to find specific inhibitors of sperm GAPDH and enzymes from Trypanosoma sp. and Mycobacterium tuberculosis that would not inhibit GAPDH of somatic mammalian cells have failed, which has pushed researchers to search for new ways to solve this problem. The sections of the review are devoted to the studies of GAPDH inactivation by reactive oxygen species, glutathione, and glycating agents. The final section discusses possible effects of GAPDH inhibition and inactivation on glycolysis and related metabolic pathways (pentose phosphate pathway, uncoupling of the glycolytic oxidation and phosphorylation, etc.).

1892P - Enzalutamide (ENZA) and apalutamide (APA) In vitro chemical reactivity studies and activity in a mouse drug allergy model (MDAM)

BackgroundENZA and APA are androgen receptor inhibitors approved for treatment of castration-resistant prostate cancer (CRPC) and nonmetastatic CRPC, respectively. Their chemical structures differ with ENZA having 2-cyanophenyl and dimethyl moieties and APA 2-cyanopyridyl and cyclobutyl moieties. In the SPARTAN clinical trial, APA treatment was reported to be associated with an increased incidence of skin rash compared with the placebo group, which was not observed with ENZA in the PROSPER trial in a similar patient population; therefore, we examined their in vitro chemical reactivity and hypersensitivity potential in an MDAM. MethodsTo assess possible reactivity of cyanopyridine, in vitro studies examining the chemical stability in buffer (+/- glutathione) and covalent binding to proteins (eg, bovine serum albumin [BSA]) were evaluated. For the MDAM, C57BL/6 mice were injected subcutaneously with APA, ENZA, or RD162 (Analogue 1), daily for 3 days at 25, 50, or 100mg/kg/day. Brachial lymph nodes were harvested on day 6 and lymph node cellularity was analyzed by flow cytometry. ResultsThe 2-cyanopyridine moiety of APA was found to be chemically reactive with the thiol nucleophile glutathione, resulting in rearranged thiazoline products. Radiolabeled APA, but not ENZA, was shown to react with mouse and human plasma proteins. Thiol nucleophiles decreased the extent of covalent binding to the model protein BSA while amine and alcohol nucleophiles had no effect, suggesting a reaction with cysteine sulfhydryl groups of proteins. In the MDAM, dose-dependent higher lymph node cellularity was observed for the APA groups (compared with the vehicle control), suggesting immune activation. ENZA and Analogue 1 demonstrated substantially less covalent binding activity and no change in lymph node cellularity (compared with vehicle) in the MDAM. ConclusionsThese data support the hypothesis that the 2-cyanopyridine moiety present in APA, but absent in ENZA or Analogue 1, may react with cysteine residues in proteins to form haptens that may trigger an immune response leading to the increased incidence of skin rash clinically observed in patients treated with APA, but not with ENZA. Editorial acknowledgementMedical writing and editorial assistance funded by Pfizer Inc. was provided by Ira Mills, PhD, and Michele Salernitano from Ashfield Healthcare Communications. Legal entity responsible for the studyPfizer Inc. and Astellas Pharma, Inc. FundingPfizer Inc. and Astellas Pharma, Inc. DisclosureM. Guha: Shareholder / Stockholder / Stock options, Employee: Pfizer Inc.. C. Ji: Shareholder / Stockholder / Stock options, Employee: Pfizer Inc.. X. Zhu: Shareholder / Stockholder / Stock options, Employee: Pfizer Inc.. J. Whritenour: Shareholder / Stockholder / Stock options, Employee: Pfizer Inc.. M. Hemkens: Shareholder / Stockholder / Stock options, Employee: Pfizer Inc.. S. Tse: Shareholder / Stockholder / Stock options, Employee: Pfizer Inc.. G.S. Walker: Shareholder / Stockholder / Stock options, Employee: Pfizer Inc.. E. Evans: Shareholder / Stockholder / Stock options, Employee: Pfizer Inc.. N.K. Khan: Shareholder / Stockholder / Stock options, Employee: Pfizer Inc.. M.B. Finkelstein: Shareholder / Stockholder / Stock options, Employee: Pfizer Inc.. E. Callegari: Shareholder / Stockholder / Stock options, Employee: Pfizer Inc.. R..S. Obach: Shareholder / Stockholder / Stock options, Employee: Pfizer Inc..