Prediction of the Hydrogen Peroxide–Induced Methionine Oxidation Propensity in Monoclonal Antibodies

Methionine oxidation in therapeutic antibodies can impact the product's stability, clinical efficacy, and safety and hence it is desirable to address the methionine oxidation liability during antibody discovery and development phase. Although the current experimental approaches can identify the oxidation-labile methionine residues, their application is limited mostly to the development phase. We demonstrate an in silico method that can be used to predict oxidation-labile residues based solely on the antibody sequence and structure information. Since antibody sequence information is available in the discovery phase, the in silico method can be applied very early on to identify the oxidation-labile methionine residues and subsequently address the oxidation liability. We believe that the in silico method for methionine oxidation liability assessment can aid in antibody discovery and development phase to address the liability in a more rational way.     

Hydrogen sulfide prodrugs—a review

Hydrogen sulfide (H₂S) is recognized as one of three gasotransmitters together with nitric oxide (NO) and carbon monoxide (CO). As a signaling molecule, H₂S plays an important role in physiology and shows great potential in pharmaceutical applications. Along this line, there is a need for the development of H₂S prodrugs for various reasons. In this review, we summarize different H₂S prodrugs, their chemical properties, and some of their potential therapeutic applications.KEY WORDS: Hydrogen sulfide (H₂S), Gasotransmitters, H₂S prodrugs, H₂S-hybrid nonsteroidal anti-inflammatory drugs, Controllable H₂S prodrugs, Hydrolysis-based H₂S prodrugs     

Hydrogen sulphide--a novel mediator of inflammation?

Hydrogen sulphide (H2S) is a naturally occurring gas synthesized from cysteine. It exhibits vasodilator activity (most probably by opening vascular smooth muscle K(ATP) channels), influences leucocyte chemotaxis and promotes vascular smooth muscle cell apoptosis. Increased biosynthesis of H2S has been demonstrated in animal models of septic/endotoxic and haemorrhagic shock, pancreatitis and carrageenan-evoked hindpaw oedema in the rat. In each case, pharmacological inhibition of H2S biosynthesis is anti-inflammatory.     

Hydrogen sulfide in inflammation: friend or foe?

Hydrogen sulfide (H(2)S), the gaseous mediator produced by various cells in our body, was recently discovered to play a major role in human physiology despite its toxic nature known for centuries. In addition to its pathophysiological relevance in cardiovascular and neuronal disorders, there is considerable interest in the significance of H(2)S in inflammation. A number of preclinical studies in our laboratory as well as by others, using H(2)S donors and inhibitors of its endogenous synthesis, have provided evidence for both pro- and anti-inflammatory character of H(2)S. But so far, there is a significant lack of support from relevant clinical studies. One of the major contentious issues being variable dose and sampling time, controversies exist on the precise friend or foe nature of this gaseous transmitter. However, it is well accepted that once a clearer picture of the whole story of H(2)S in inflammation emerges, potential for therapeutic manipulations in this field are immense. This review focuses on the intriguing effects of H(2)S in some of the inflammatory conditions such as acute pancreatitis, sepsis, burn injuries and local inflammation of the joints. Active research projects have been undertaken to elucidate the mechanisms of action of H(2)S in inflammation, including neurogenic inflammation and interaction with other biological mediators and pathways. The early and fragmentary evidence obtained holds promise for a successful drug intervention for these inflammatory diseases.     

Hydrogen sulfide attenuates epithelial–mesenchymal transition of human alveolar epithelial cells

We previously reported that the endogenous cystathionine γ-lyase (CSE)/hydrogen sulfide (H₂S) pathway is implicated in the pathogenesis of bleomycin-induced pulmonary fibrosis in rats, but the exact cellular mechanisms are not well characterized. Epithelial–mesenchymal transition (EMT), induced by transforming growth factor β1 (TGF-β1) in alveolar epithelial cells, plays an important role in the pathogenesis of pulmonary fibrosis. We studied whether H₂S could attenuate EMT in cultured alveolar epithelial cells and TGF-β1 treatment suppressed CSE expression in A549 cells. Inhibition of endogenous CSE by dl-propargylglycine led to spontaneous EMT, as manifested by decreased E-cadherin level, increased vimentin expression and fibroblast-like morphologic features. Exogenous H₂S applied to TGF-β1-treated A549 cells decreased vimentin expression, increased E-cadherin level and retained epithelial morphologic features. In addition, preincubation with H₂S decreased Smad2/3 phosphorylation in A549 cells stimulated by TGF-β1, and H₂S-inhibited alveolar EMT was mimicked by treatment with SB505124, a Smad2/3 inhibitor, but not pinacidil, an ATP-sensitive K⁺ channel (K_ATP) opener. H₂S serves a critical role in preserving an epithelial phenotype and in attenuating EMT in alveolar epithelial cells, mediated, at least in part, by decreased Smad2/3 phosphorylation and not dependent on K_ATP channel opening.     

Hydrogen sulphide: an endogenous stimulant of capsaicin-sensitive primary afferent neurons?

Hydrogen sulphide (H(2)S) is a gas best known for its rotten egg smell. The toxic effects of high concentrations of H(2)S have been extensively investigated. It is known that H(2)S is generated in mammalian systems, but little is known of its effects in physiological concentrations. In the present issue of this journal, Patacchini et al. present evidence that H(2)S stimulates capsaicin-sensitive primary afferent neurons to release tachykinins in the rat urinary bladder. The possible significance of this finding is discussed in this commentary.     

Hydrogen sulfide： third gaseous transmitter, but with great pharmacological potential

Hydrogen sulfide （H2S）, which is well known traditionally as a toxic gas, has been proven to be produced endogenously by 3 enzymes in mammalian tissues and plays important roles in physiological and pathophysiological conditions. In the central nervous system, H2S functions as not only a neuromodulator, but also a neuroprotectant against oxidative stress. In the cardiovascular system, H2S relaxes vascular smooth muscles by the activation of KATP channels and inhibits smooth muscle cell proliferation via the mitogen-activated protein kinase signaling pathway. These effects are important for maintaining blood pressure and preventing vessel structural remodeling, and identifies H2S as an important factor in the development of some vascular diseases, such as hypertension. H2S also shows cardioprotective effects in ischemic myocardium and septic and endotoxin shock. Recent studies have demonstrated a new mechanism to explain the motor effect of H2S on the rat detrusor muscle, which is through the activation of the capsaicin-sensitive primary neuron. This review focuses on the recent research achievements on H2S and discloses the great potential of H2S as the third gaseous transmitter in cardiac protection.     

Hydrogen peroxide mobilizes Ca^2＋ through two distinct mechanisms in rat hepatocytes

Aim： Hydrogen peroxide （H2O2） is produced during liver transplantation. Ischemia/reperfusion induces oxidation and causes intracellular Ca^2＋ overload, which harms liver cells. Our goal was to determine the precise mechanisms of these processes. Methods： Hepatocytes were extracted from rats. Intracellular Ca^2＋ concentrations （[Ca^2＋]i）, inner mitochondrial membrane potentials and NAD（P）H levels were measured using fluorescence imaging. Phospholipase C （PLC） activity was detected using exogenous PIP2. ATP concentrations were measured using the luciferin-luciferase method. Patch-clamp recordings were performed to evaluate membrane currents.
Results： H2O2 increased intracellular Ca^2＋ concentrations （[Ca^2＋]i） across two kinetic phases. A low concentration （400 μmol/L） of H2O2 induced a sustained elevation of [Ca^2＋]i that was reversed by removing extracellular Ca^2＋. H2O2 increased membrane currents consistent with intracellular ATP concentrations. The non-selective ATP-sensitive cation channel blocker amiloride inhibited HRO2-induced membrane current increases and [Ca^2＋]i elevation. A high concentration （1 mmol/L） of H2O2 induced an additional transient elevation of [Ca^2＋]i, which was abolished by the specific PLC blocker U73122 but was not eliminated by removal of extracellular Ca^2＋. PLC activity was increased by 1 mmol/L H2O2but not by 400 μmol/L H2O2.
Conclusions： H2O2 mobilizes Ca^2＋ through two distinct mechanisms. In one, 400 μmol/L H2O2-induced sustained [Ca^2＋]i elevation is mediated via a Ca^2＋ influx mechanism, under which H2O2 impairs mitochondrial function via oxidative stress, reduces intracellular ATP production, and in turn opens ATP-sensitive, non-specific cation channels, leading to Ca^2＋ influx. In contrast, 1 mmol/L H2O2-induced transient elevation of [Ca^2＋]i is mediated via activation of the PLC signaling pathway and subsequently, by mobilization of Ca^2＋ from intracellular Ca^2＋ stores.     

Hydrogen sulphide inhibits carbachol-induced contractile responses in β-escin permeabilized guinea-pig taenia caecum

Hydrogen sulphide (H(2)S) is an endogenous mediator producing a potent relaxation response in vascular and non-vascular smooth muscles. While ATP-sensitive potassium channels are mainly involved in this relaxant effect in vascular smooth muscle, the mechanism in other smooth muscles has not been revealed yet. In the present study, we investigated how H(2)S relaxes non-vascular smooth muscle by using intact and β-escin permeabilized guinea-pig taenia caecum. In intact tissues, concentration-dependent relaxation response to H(2)S donor NaHS in carbachol-precontracted preparations did not change in the presence of a K(ATP) channel blocker glibenclamide, adenylate cyclase inhibitor SQ-22536, guanylate cyclase inhibitor ODQ, protein kinase A inhibitor KT-5720, protein kinase C inhibitor H-7, tetrodotoxin, apamin/charybdotoxin, NOS inhibitor L-NAME and cyclooxygenase inhibitor indomethacin. We then studied how H(2)S affected carbachol- or Ca(2+)-induced contractions in permeabilized tissues. When Ca(2+) was clamped to a constant value (pCa6), a further contraction could be elicited by carbachol that was decreased by NaHS. This decrease in contraction was reversed by catalase but not by superoxide dismutase or N-acetyl cysteine. The sarcoplasmic reticulum Ca(2+)-ATPase pump inhibitor, cyclopiazonic acid, also decreased the carbachol-induced contraction that was further inhibited by NaHS. Mitochondrial proton pump inhibitor carbonyl cyanide p-trifluromethoxyphenylhydrazone also decreased the carbachol-induced contraction but this was not additionally changed by NaHS. The carbachol-induced Ca(2+) sensitization, calcium concentration-response curves, IP(3)- and caffeine-induced contractions were not affected by NaHS. In conclusion, we propose that hydrogen peroxide and mitochondria may have a role in H(2)S-induced relaxation response in taenia caecum.     

Hydrogen sulfide: from the smell of the past to the mediator of the future?

Gases such as nitric oxide and carbon monoxide play important roles both in normal physiology and in disease. In recent years, interest has been directed towards other naturally occurring gases, notably hydrogen sulfide (H₂S), which is both a potent vasodilator and a mediator of long-term potentiation in the brain. This article focuses on recent work that suggests a role for H₂S, and perhaps other gases, in the CNS and cardiovascular system.     

Effect Of Benzyl Alcohol on Recombinant Human Interleukin-1 Receptor Antagonist Structure and Hydrogen–Deuterium Exchange

Benzyl alcohol, a preservative commonly added to multidose therapeutic protein formulations, can accelerate aggregation of recombinant human interleukin-1 receptor antagonist (rhIL-1ra). To investigate the interactions between benzyl alcohol and rhIL-1ra, we used nuclear magnetic resonance to observe the effect of benzyl alcohol on the chemical shifts of amide resonances of rhIL-1ra and to measure hydrogen–deuterium exchange rates of individual rhIL-1ra residues. Additionof 0.9% benzyl alcohol caused significant chemical shifts of amide resonances for residues 90–97, suggesting that these solvent-exposed residues participate in the binding of benzyl alcohol. In contrast, little perturbation of exchange rates was observed in the presence of either sucrose or benzyl alcohol. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:4215–4224, 2011     

Rapid Prediction of Deamidation Rates of Proteins to Assess Their Long-Term Stability Using Hydrogen Exchange–Mass Spectrometry

Deamidation is an important degradation pathway for proteins. Estimating deamidation propensities is essential for predicting their long-term stability. However, predicting deamidation rates in folded proteins is challenging because higher-order structure has a significant and unpredictable effect on deamidation. Here, we investigated the correlation between amide hydrogen exchange (HX) and deamidation to assess the potential of using hydrogen exchange–mass spectrometry (HX-MS) to rapidly predict deamidation propensity. Maltose-binding protein and a structurally less stable mutant, W169G, were stored in the dark at pH 7.0 at 23 ± 2°C for 1 year. Deamidation at each asparagine site was measured using liquid chromatography–mass spectrometry after trypsin digestion. Deamidation rates at each deamidation site were determined based on first-order kinetics. HX rates at the deamidation sites were determined before storage using the shortest peptic peptide containing each site using conventional bottom-up HX-MS at pD 7.0 at 25°C. We observed a power law correlation between deamidation half-life and HX half-life for the NG sites with measurable kinetics. For NA sites, slow deamidation was only observed at 2 sites located in rapidly exchanging regions. Our findings demonstrate that HX-MS can be used to reliably and rapidly rank deamidation propensity in folded proteins.     

Hydrogen bonding between the 17β-substituent of a neurosteroid and the GABAA receptor is not obligatory for channel potentiation

Background and purpose:

Potentiating neurosteroids are some of the most efficacious modulators of the mammalian GABA_A receptor. One of the crucial interactions may be between the C20 ketone group (D-ring substituent at C17) of the neurosteroid, and the N407 and Y410 residues in the M4 domain of the receptor. In this study, we examined the contribution of hydrogen bonding between 17β-substituents on the steroid D-ring and the GABA_A receptor to potentiation by neurosteroids.

Experimental approach:

Whole-cell and single-channel recordings were made from HEK 293 cells transiently expressing wild-type and mutant α1β2γ2L GABA_A receptors.

Key results:

A steroid with a 17β-carbonitrile group (3α5α18nor17βCN) was a potent and efficacious potentiator of the GABA_A receptor. Potentiation was also shown by a cyclosteroid in which C21 and the C18 methyl group of (3α,5α)-3-hydroxypregnan-20-one are connected within a six-membered ring containing a double bond as a hydrogen bond acceptor (3α5αCDNC12), a steroid containing a 17β-ethyl group on the D-ring (3α5α17βEt) and a steroid lacking a 17β-substituent on the D-ring (3α5α17H). Single-channel kinetic analysis indicates that the kinetic mechanism of action is the same for the neurosteroid 3α5αP, 3α5α18nor17βCN, 3α5αCDNC12, 3α5α17βEt and 3α5α17H. Interestingly, 3α5α17βEt, at up to 3 µM, was incapable of potentiating the α1N407A/Y410F double mutant receptor.

Conclusions and implications:

Hydrogen bonding between the steroid 17β-substituent and the GABA_A receptor is not a critical requirement for channel potentiation. The α1N407/Y410 residues are important for neurosteroid potentiation for reasons other than hydrogen bonding between steroid and receptor.     

Hydrogen sulfide-releasing aspirin suppresses NF-κB signaling in estrogen receptor negative breast cancer cells in vitro and in vivo

Hormone-dependent estrogen receptor positive (ER+) breast cancers generally respond well to anti-estrogen therapy. Unfortunately, hormone-independent estrogen receptor negative (ER-) breast cancers are aggressive, respond poorly to current treatments and have a poor prognosis. New approaches and targets are needed for the prevention and treatment of ER- breast cancer. The NF-κB signaling pathway is strongly implicated in ER- tumor genesis, constituting a possible target for treatment. Hydrogen sulfide-releasing aspirin (HS-ASA), a novel and safer derivative of aspirin, has shown promise as an anti-cancer agent. We examined the growth inhibitory effect of HS-ASA via alterations in cell proliferation, cell cycle phase transitions, and apoptosis, using MDA-MB-231 cells as a model of triple negative breast cancer. Tumor xenografts in mice, representing human ER- breast cancer, were evaluated for reduction in tumor size, followed by immunohistochemical analysis for proliferation, apoptosis and expression of NF-κB. HS-ASA suppressed the growth of MDA-MB-231 cells by induction of G(0)/G(1) arrest and apoptosis, down-regulation of NF-κB, reduction of thioredoxin reductase activity, and increased levels reactive oxygen species. Tumor xenografts in mice, were significantly reduced in volume and mass by HS-ASA treatment. The decrease in tumor mass was associated with inhibition of cell proliferation, induction of apoptosis and decrease in NF-κB levels in vivo. HS-ASA has anti-cancer potential against ER- breast cancer and merits further study.     

Hydrogen Bonding Potential as a Determinant of the in Vitro and in Situ Blood–Brain Barrier Permeability of Peptides

With the exception of various central nervous system (CNS)-required nutrients for which specific, saturable transport systems exist, the passage of most water-soluble solutes through the blood–brain barrier (BBB) is believed to depend largely on the lipid solubility of the solutes. Most peptides, therefore, do not enter the CNS because of their hydrophilic character. Recently, utilizing homologous series of model peptides and Caco-2 cell monolayers as a model of the intestinal mucosa, it was concluded that the principal determinant of peptide transport across the intestinal cellular membrane is the energy required to desolvate the polar amide bonds in the peptide (P. S. Burton et al., Adv. Drug Deliv. Rev. 7:365–386, 1991). To determine whether this correlation can be extended to the BBB, the permeabilities of the same peptides were determined using an in vitro as well as an in situ BBB model. The peptides, blocked on the N- and C-terminal ends, consisted of D-phenylalanine (F) residues: AcFNH₂, AcF₂NH₂, AcF₃NH₂, AcF₂(NMeF)NH₂, AcF(NMeF)₂NH₂, Ac(NMeF)₃NH₂, and Ac(NMeF)₃NHMe. A good correlation among the permeabilities of these model peptides across the bovine brain microvessel endothelial cell (BBMEC) monolayers, an in vitro model of the BBB, and their permeabilities across the BBB in situ was observed (r = 0.928, P < 0.05). The permeabilities of these peptides did not correlate with the octanol–buffer partition coefficients of the peptides (r = 0.389 in vitro and r = 0.155 in situ; P < 0.05). However, correlations were observed between the permeabilities of these peptides and the number of potential hydrogen bonds the peptides can make with water (r = 0.837 in vitro and r = 0.906 in situ; P < 0.05), suggesting that desolvation of the polar bonds in the molecule is a determinant of permeability. Consistent with this, good correlations were found between the permeabilities of these peptides and their partition coefficients between heptane–ethylene glycol (r = 0.981 in vitro and r = 0.940 in situ ; P < 0.05) or the differences in partition coefficients between octanol–buffer and isooctane–buffer (logPC) (r = 0.961 in vitro and r = 0.962 in situ; P < 0.05), both of which are experimental estimates of hydrogen bond or desolvation potential. These results suggest that the permeability of peptides through the BBB is governed by the same physicochemical parameter (hydrogen bonding potential) as their permeability through the intestinal mucosa.     

Hydrogen sulphide protects mouse pancreatic β-cells from cell death induced by oxidative stress, but not by endoplasmic reticulum stress

BACKGROUND AND PURPOSE

Hydrogen sulphide (H₂S), a potentially toxic gas, is also involved in the neuroprotection, neuromodulation, cardioprotection, vasodilatation and the regulation of inflammatory response and insulin secretion. We have recently reported that H₂S suppresses pancreatic β-cell apoptosis induced by long-term exposure to high glucose. Here we examined the protective effects of sodium hydrosulphide (NaHS), an H₂S donor, on various types of β-cell damage.

EXPERIMENTAL APPROACH

Isolated islets from mice or the mouse insulinoma MIN6 cells were cultured with palmitate, cytokines (a mixture of tumour necrosis factor-α, interferon-γ and interleukin-1β), hydrogen peroxide, thapsigargin or tunicamycin with or without NaHS. We examined DNA fragmentation, caspase-3 and -7 activities and reactive oxygen species (ROS) production in the treated cells thereafter. Apoptotic cell death in isolated islets was also assessed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) method.

KEY RESULTS

NaHS suppressed DNA fragmentation and the activities of caspase-3 and -7 induced by palmitate, the cytokines or hydrogen peroxide. In contrast, NaHS failed to protect islets and MIN6 cells from apoptosis induced by thapsigargin and tunicamycin, both of which cause endoplasmic reticulum stress. NaHS suppressed ROS production induced by cytokines or hydrogen peroxide but it had no effect on ROS production in thapsigargin-treated cells. NaHS increased Akt phosphorylation in MIN6 cells treated with cytokines but not in cells treated with thapsigargin. Treatment with NaHS decreased TUNEL-positive cells in cytokine-exposed islets.

CONCLUSIONS AND IMPLICATIONS

H₂S may prevent pancreatic β-cells from cell apoptosis via an anti-oxidative mechanism and the activation of Akt signalling.     

Hydrogen sulfide protects against bleomycin-induced pulmonary fibrosis in rats by inhibiting NF-κB expression and regulating Th1/Th2 balance

Hydrogen sulfide (H₂S) displays vasodilative, anti-oxidative, anti-inflammatory and cytoprotective activities. The objective of this study was to evaluate the inhibitory effect of H₂S on bleomycin (BLM)-induced pulmonary fibrosis in rats and its possible mechanisms. Fifty-four pathogen-free Male Wistar rats were randomly divided into three groups: control, BLM and H₂S treated groups with 18 rats in each group. Each group was then divided into three subgroups based on time of study (7, 14 and 28 day). Pulmonary fibrosis model was established by a single intratracheal instillation of BLM A₅ (5 mg/kg). While control rats received saline, rats of the treated group simultaneously were administered intraperitoneal injections of NaHS (the H₂S donor, 28 μmol/kg) once daily. BLM induced pulmonary inflammation and fibrosis, increased lung hydroxyproline levels, lung index, total cell counts, neutrophils and eosinophils counts and expression of NF-κB p65 in lung tissue, decreased lymphocytes and macrophages counts. In addition, Th1 response is suppressed as shown by diminished IFN-γ in bronchoalveolar lavage fluid (BALF) after BLM exposure, and enhancement of Th2 response is marked by increased IL-4 in BALF. H₂S administration significantly attenuated these effects. The findings reveal the therapeutic potential of H₂S for BLM-induced pulmonary fibrosis in male rats, which were at least partly due to inhibition NF-κB p65 expression and regulation of Th1/Th2 balance.