Generation of Nitric Oxide and Possibly Nitroxyl by Nitrosation of Sulfohydroxamic Acids and Hydroxamic Acids

Diazeniumdiolates (NONOates) and sulfohydroxamic acids are chemical entities that spontaneously generate nitric oxide (NO) and nitroxyl (HNO), respectively, at physiological pH and temperature. By combining the functional aspects of the NONOates with the hydroxamic acids and sulfohydroxamic acids, hybrid NONOate-type compounds that could theoretically generate nitroxyl or nitric oxide can be rationalized. Although the instability of these compounds, viz., the N-nitrosohydroxamic acids and the N-nitrososulfohydroxamic acids, precluded their chemical characterization by actual isolation, their transient existence was deduced by identification of the products of their decomposition. Thus, treatment of benzohydroxamic acid (BHA) with limiting or excess nitrous acid (from NaNO(2) and H(3)PO(4)) gave rise to quantitative generation of N(2)O, possibly via HNO, based on the limiting reactant. Nitrosation of N-t-butyloxycarbonyl hydroxylamine gave similar results. The organic acid produced from BHA was identified as benzoic acid. No nitric oxide was detected from these reactions. In contrast, treatment of Piloty's acid (benzenesulfohydroxamic acid) and methanesulfohydroxamic acid (MSHA) with nitrous acid under the same conditions as above gave 36% of the theoretical quantity of NO from Piloty's acid and 47% of NO from MSHA, although finite quantities of HNO (measured as N(2)O) were also formed. The organic acid produced from Piloty's acid was identified by reverse-phase HPLC as the redox product, benzenesulfinic acid.     

Formation of nitroxyl and hydroxyl radical in solutions of sodium trioxodinitrate: effects of pH and cytotoxicity

Despite its negative redox potential, nitroxyl (HNO) can trigger reactions of oxidation. Mechanistically, these reactions were suggested to occur with the intermediate formation of either hydroxyl radical (.OH) or peroxynitrite (ONOO-). In this work, we present further experimental evidence that HNO can generate.OH. Sodium trioxodinitrate (Na2N2O3), a commonly used donor of HNO, oxidized phenol and Me2SO to benzene diols and.CH3, respectively. The oxidation of Me2SO was O2-independent, suggesting that this process reflected neither the intermediate formation of ONOO- nor a redox cycling of transition metal ions that could initiate Fenton-like reactions. In solutions of phenol, Na2N2O3 yielded benzene-1,2-diol and benzene-1,4-diol at a ratio of 2:1, which is consistent with the generation of free.OH. Ethanol and Me2SO, which are efficient scavengers of.OH, impeded the hydroxylation of phenol. A mechanism for the hydrolysis of Na2N2O3 is proposed that includes dimerization of HNO to cis-hyponitrous acid (HO-N=N-OH) with a concomitant azo-type homolytic fission of the latter to N2 and.OH. The HNO-dependent production of.OH was with 1 order of magnitude higher at pH 6.0 than at pH 7.4. Hence, we hypothesized that HNO can exert selective toxicity to cells subjected to acidosis. In support of this thesis, Na2N2O3 was markedly more toxic to human fibroblasts and SK-N-SH neuroblastoma cells at pH 6.2 than at pH 7.4. Scavengers of.OH impeded the cytotoxicity of Na2N2O3. These results suggest that the formation of HNO may be viewed as a toxicological event in tissues subjected to acidosis.     

Nitroxyl triggers Ca2+ release from skeletal and cardiac sarcoplasmic reticulum by oxidizing ryanodine receptors

The biological activity of nitric oxide (NO) and NO-donors has been extensively investigated yet few studies have examined those of nitroxyl (HNO) species even though both exist in chemical equilibrium but oxidize thiols by different reaction mechanisms: S-nitrosation versus disulfide bond formation. Here, sodium trioxodinitrate (Na2N2O3; Angeli's salt; ANGS) was used as an HNO donor to investigate its effects on skeletal (RyR1) and cardiac (RyR2) ryanodine receptors. At steady-state concentrations of nanomoles/L, HNO induced a rapid Ca2+ release from sarcoplasmic reticulum (SR) vesicles then the reducing agent dithiothreitol (DTT) reversed the oxidation by HNO resulting in Ca2+ re-uptake by SR vesicles. With RyR1 channel proteins reconstituted in planar bilayers, HNO added to the cis-side increased the open probability (Po) from 0.056+/-0.026 to 0.270+/-0.102 (P<0.005, n=4) then DTT (3 mM) reduced Po to 0.096+/-0.040 (P<0.01, n=4). In parallel experiments, the time course of HNO production from ANGS was monitored by EPR and UV spectroscopy and compared with the rate of SR Ca2+ release indicating that picomolar concentrations of HNO triggered SR Ca2+ release. Controls showed that the hydroxyl radical scavenger, phenol did not alter ANGS-induced SR Ca2+ release, indicating that hydroxyl radical production from ANGS did not account for Ca2+ release from the SR. The findings indicate that HNO is a more potent activator of RyR1 than NO and that HNO activation of RyRs may contribute to NO's activation of RyRs and to the therapeutic effects of HNO-releasing prodrugs in heart failure.     

N,O-diacylated-N-hydroxyarylsulfonamides: nitroxyl precursors with potent smooth muscle relaxant properties.

N,O-Diacylated-N-hydroxyarylsulfonamides are capable of slowly releasing nitroxyl (HNO) by simple, non-enzymatic hydrolysis in Krebs solution at 37 degrees C. Release of nitric oxide (NO) was not seen. These compounds were also found to elicit vasorelaxation in rabbit thoracic aorta in vitro, presumably as a result of their ability to release HNO. This effect was enhanced by the addition of superoxide dismutase (SOD). Thus, these results are consistent with previous work indicating that HNO is a potent vasorelaxant.     

Mass spectrometric analysis of nitroxyl-mediated protein modification: comparison of products formed with free and protein-based cysteines

Although biologically active, nitroxyl (HNO) remains one of the most poorly studied NO(x). Protein-based thiols are suspected targets of HNO, forming either a disulfide or sulfinamide (RSONH2) through an N-hydroxysulfenamide (RSNHOH) addition product. Electrospray ionization mass spectrometry (ESI-MS) is used here to examine the products formed during incubation of thiol proteins with the HNO donor, Angeli's salt (AS; Na2N2O3). Only the disulfide, cystine, was formed in incubates of 15 mM free Cys with equimolar AS at pH 7.0-7.4. In contrast, the thiol proteins (120-180 microM), human calbindin D(28k) (HCalB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and bovine serum albumin (BSA) gave four distinct types of derivatives in incubates containing 0.9-2.5 mM AS. Ions at M + n x 31 units were detected in the ESI mass spectra of intact HCalB (n = 1-5) and GAPDH (n = 2), indicating conversion of thiol groups on these proteins to RSONH2 (+31 units). An ion at M + 14 dominated the mass spectrum of BSA, and intramolecular sulfinamide cross-linking of Cys34 to one of its neighboring Lys or Arg residues would account for this mass increase. Low abundant M + 14 adducts were observed for HCalB, which additionally formed mixed disulfides when free Cys was present in the AS incubates. Cys149 and Cys153 formed an intramolecular disulfide in the AS/GAPDH incubates. Since AS also produces nitrite above pH 5 (HN2O3(-) --> HNO + NO2(-)), incubation with NaNO2 served to confirm that protein modification was HNO-mediated, and prior blocking with the thiol-specific reagent, N-ethylmaleimide, demonstrated that thiols are the targets of HNO. The results provide the first systematic characterization of HNO-mediated derivatization of protein thiols.     

Induction of DNA strand breakage and base oxidation by nitroxyl anion through hydroxyl radical production.

Nitroxyl anion (NO-), the one-electron reduction product of nitric oxide (NO*), has been reported to be formed under various physiological conditions and to be cytotoxic, although the mechanism responsible for the toxic effects has not been identified. We have studied the effects of NO- generated from Angeli's salt (sodium trioxodinitrate) or Piloty's acid (N-hydoxybenzenesulfonamide) on DNA strand breakage and DNA base oxidation in vitro. Induction of strand breakage was dose- and time-dependent upon incubation of plasmid pBR322 with Angeli's salt or Piloty's acid. Similarly, 8-oxo-2'-deoxyguanosine and malondialdehyde were formed when calf-thymus DNA or 2'-deoxyribose, respectively, were incubated with Angeli's salt. Electron acceptors (ferricyanide, 4-hydroxy-TEMPO), that convert NO to NO*, inhibited the reactions, indicating that NO , but not NO*, is responsible for the reactions. Furthermore, the reactions were also inhibited by the presence of hydroxyl radical (HO*) scavengers, antioxidants, metal chelators and superoxide dismutase and catalase, implying involvement of free HO*. These results suggest that NO- is a possible endogenous source of HO*, that may be formed either directly from the reaction product of NO- with NO* (N2O2*-) or indirectly through H2O2 formation. Thus NO may play an important role as a cause of diverse pathophysiological conditions such as inflammation and neurodegenerative diseases.     

Piloty’s acid derivative with improved nitroxyl-releasing characteristics

Recent studies have shown that nitroxyl (HNO) (¹HNO/³NO^?), which is the one-electron-reduced form of nitric oxide (NO), has unique biological activities, especially in the cardiovascular system, and HNO-releasing agents may have therapeutic potential. Since few HNO donors are available for use under physiological conditions, we synthesized and evaluated a series of Piloty’s acid (PA) derivatives and evaluated their HNO-releasing activity under physiological conditions. N-Hydroxy-2-nitrobenzenesulfonamide (17) was the most efficient HNO donor among our synthesized PA derivatives, including the lead compound, 2-bromo-N-hydroxybenzenesulfonamide (2). The high HNO-releasing activity is suggested to be due to electronic and steric effects. Compound 17 may be a useful tool for biological experiments.     

Pharmacokinetic studies of naproxen amides of some amino acid esters with promising colorectal cancer chemopreventive activity

Naproxen (nap) is belonging to Non-steriodal anti-inflammatory drugs (NSAIDs) group of drugs that characterized by their free carboxylic group. The therapeutic activity of nap is usually accompanied by GI untoward side effects. Recently synthesized naproxen amides of some amino acid esters prodrugs to mask the free carboxylic group were reported. Those prodrugs showed a promising colorectal cancer chemopreventive activity. The current study aims to investigate the fate and hydrolysis of the prodrugs kinetically in different pH conditions, simulated gastric and intestinal fluids with pHs of 1.2, 5.5 and 7.4 in vitro at 37 °C. The effect of enzymes on the hydrolysis of prodrugs was also studied through incubation of these prodrugs at 37 °C in human plasma and rat liver homogenates. The pharmacokinetic parameters of selected prodrugs and the liberated nap were studied after oral and intraperitoneal administration in male wistar rats. The results showed the hydrolysis of naproxen amides of amino acid esters to nap through two steps first by degradation of the ester moiety to form the amide of nap with amino acid and the second was through the degradation of the amide link to liberate nap. The two reactions were followed and studied kinetically where K₁ and K₂ (rate constants of degradation) is reported. The hydrolysis of prodrugs was faster in liver homogenates than in plasma. The relative bioavailability of the liberated nap in vivo was higher in case of prodrug containing ethyl glycinate moiety than that occupied l-valine ethyl ester moiety. Each of nap. prodrugs containing ethyl glycinate and l-valine ethyl ester moieties appears promising in liberating nap, decreasing direct GI side effect and consequently their colorectal cancer chemopreventive activity.     

Acyloxymethyl as a drug protecting group. Part 7: Tertiary sulfonamidomethyl ester prodrugs of benzylpenicillin: chemical hydrolysis and anti-bacterial activity

Tertiary sulfonamidomethyl esters of benzylpenicillin (4) were synthesised and evaluated as a new class of potential prodrugs for beta-lactam antibiotics. Their hydrolysis in aqueous buffers was studied by HPLC and reveal a U-shaped pH rate profile with a pH-independent process extending from ca. pH 2 to ca. pH 10. This pathway is characterised by kinetic data that are consistent with a unimolecular mechanism involving rate-limiting iminium ion formation and penicillinoate expulsion. Benzylpenicillin and the corresponding sulfonamide are the ultimate products detected and isolated, indicating that beta-lactam ring opening is much slower than ester hydrolysis. As expected from the high reactivity, benzylpenicillin esters (4) displayed similar in vitro antibacterial activity to benzylpenicillin itself. Compared to the benzylpenicillin derivatives, sulfonamidomethyl esters of benzoic, clofibric and valproic acids display a much higher stability, giving rise to a Br?nsted beta1g value of -0.96 and suggesting that tertiary sulfonamidomethyl esters may be useful prodrugs for carboxylic acid drugs with pKa > 4.     

Chemotherapeutic potential of diazeniumdiolate-based aspirin prodrugs in breast cancer

Diazeniumdiolate-based aspirin prodrugs have previously been shown to retain the anti-inflammatory properties of aspirin while protecting against the common side effect of stomach ulceration. Initial analysis of two new prodrugs of aspirin that also release either nitroxyl (HNO) or nitric oxide (NO) demonstrated increased cytotoxicity toward human lung carcinoma cells compared to either aspirin or the parent nitrogen oxide donor. In addition, cytotoxicity was significantly lower in endothelial cells, suggesting cancer-specific sensitivity. To assess the chemotherapeutic potential of these new prodrugs in treatment of breast cancer, we studied their effect both in cultured cells and in a nude mouse model. Both prodrugs reduced growth of breast adenocarcinoma cells more effectively than the parent compounds while not being appreciably cytotoxic in a related nontumorigenic cell line (MCF-10A). The HNO donor also was more cytotoxic than the related NO donor. The basis for the observed specificity was investigated in terms of impact on metabolism, DNA damage and repair, apoptosis, angiogenesis and metastasis. The results suggest a significant pharmacological potential for treatment of breast cancer.     

Ingress and reactive chemistry of nitroxyl-derived species within human cells

The mechanisms that control the biological signaling and toxicological properties of the nitrogen oxide species nitroxyl (HNO) are largely unknown. The ingress and intracellular reactivity of nitroxyl-derived species were examined using Angeli's salt (AS), which decomposes initially to HNO and nitrite at physiologic pH. Exposure of 4,5-diaminofluorescein (DAF) to AS resulted in fluorescent product formation only in the presence of molecular oxygen. Kinetic analysis and the lack of signal from a nitric oxide (NO)-sensitive electrode suggested that these processes did not involve conversion of HNO to NO. On an equimolar basis, bolus peroxynitrite (ONOO(-)) exposure generated only 15% of fluorescent product formation observed from AS decomposition. Moreover, infusion of synthetic ONOO(-) at a rate comparable to AS decomposition resulted in only 4% of the signal. Quenching of AS-mediated product formation within intact human MCF-7 breast carcinoma cells containing DAF by addition of urate to buffer suggested involvement of an oxidized intermediate formed from reaction between HNO and oxygen. Conversely, intact cells competitively sequestered the HNO-derived species from reaction with DAF in solution. These data show this intermediate to be a long-lived diffusible species. Relative product yield from intracellular DAF was decreased 5- to 8-fold when cells were lysed immediately prior to AS addition, consistent with the partitioning of HNO and/or derived species into the cellular membrane, thereby shielding these reactive intermediates from either hydrolysis or cytoplasmic scavenger pools. These findings establish that oxygen-derived species of nitroxyl can readily penetrate and engage the intracellular milieu of cells and suggest this process to be independent of NO and ONOO(-) intermediacy. The substantial facilitation of oxygen-dependent nitroxyl chemistry by intact lipid bilayers supports a focusing role for the membrane in modulation of cellular constituents proteins by this unique species.     

Detection of nitroxyl (HNO) by membrane inlet mass spectrometry

Membrane inlet (or introduction) mass spectrometry (MIMS) was used to detect nitroxyl (HNO) in aqueous solution for the first time. The common HNO donors Angeli's salt (AS) and Piloty's acid (PA), along with a newly developed donor, 2-bromo-N-hydroxybenzenesulfonamide (2-bromo-Piloty's acid, 2BrPA), were examined by this technique. MIMS experiments revealed that under physiological conditions 2BrPA is an essentially pure HNO donor, but AS produces a small amount of nitric oxide (NO). In addition, MIMS experiments also confirmed that PA is susceptible to oxidation and NO production, but that 2BrPA is not as prone to oxidation.     

15N-tracer and NMR studies on the pathway of denitrification. Evidence against trioxodinitrate but for nitroxyl as an intermediate

The effects of four species of denitrifying bacteria on the conversion of [15N]nitrite to trioxodinitrate (HN2O3-) and N2O and of trioxodinitrate to N2O were studied. For all species, the N2O produced in the presence of [15N]nitrite and trioxodinitrate was isotopically randomized throughout the period of incubation and was not composed at the outset predominantly of 14N2O or 14N2O plus 15N2O. The N2O produced was also heavily enriched in 15N at times when the trioxodinitrate pool was only weakly enriched in 15N. By 15N NMR, the N(2) position, but not the N(1) position, of trioxodinitrate was found to become progressively labeled with 15N during incubation with [15N]nitrite. These results argue that (a) the N-N bond of trioxodinitrate is not preserved in its conversion to N2O, (b) trioxodinitrate can be neither a free nor enzyme-bound intermediate in denitrifying bacteria, and (c) the pathways from nitrite and trioxodinitrate involve a common mononitrogen intermediate. The conclusion that this intermediate is probably nitroxyl (HNO), at least with Paracoccus denitrificans and Pseudomonas stutzeri, provides indirect evidence that N-N bond formation in denitrification can occur through the dimerization of nitroxyl.     

Penetration of ascorbic acid into bilayer phospholipid membranes

Using the EPR method, the temperature dependencies of the rates of ascorbic acid-induced reduction of nitroxyl radicals carrying the nitroxyl fragment in different positions of the fatty acid chain [N(4-methylidene++-1-oxyl-2,2,5,5-tetramethyl-3-imidazolidine hydrazine)]myristic acid (I) and 1-oxyl-2,2-dimethyloxazolidine derivatives of 5-ketostearic (II) and 12-ketostearic (III) acids incorporated into egg phosphatidylcholine liposomal membranes were studied. The reduction rates, activation energy and shape of kinetic curves were found to be dependent on the mode of liposome preparation (ultrasonication or reverse phase evaporation), label type and chemical composition of the membrane (with regard to the presence or absence of stearic acid). The coefficients of partition and diffusion of ascorbic acid through the membrane lipid bilayer were calculated from the rates of transbilayer (flip-flop) diffusion of I and ascorbate penetration inside the liposomes containing Fremi salt nitroxyl radical. The experimental results formed the basis for a hypothesis on the dependence of the rate of membrane-embedded spin probe reduction on the ascorbate distribution pattern inside the lipid bilayer.     

Effect of nitroxyl on the hamster retinal nitridergic pathway

There is a growing body of evidence on the role of nitric oxide (NO) in retinal physiology. Recently, interest has developed in the functional role of an alternative redox form of NO, namely nitroxyl (HNO/NO(-)), because it is formed by a number of diverse biochemical reactions. The aim of the present report was to comparatively analyze the effect of HNO and NO on the retinal nitridergic pathway in the golden hamster. For this purpose, sodium trioxodinitrate (Angeli's salt) and diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA/NO) were used as HNO and NO releasers, respectively. Angeli's salt and DEA/NO significantly decreased nitric oxide synthase activity. In addition, Angeli's salt (but not DEA/NO) significantly decreased l-arginine uptake. DEA/NO significantly increased cGMP accumulation at low micromolar concentrations, while Angeli's salt affected this parameter with a threshold concentration of 200muM. Although Angeli's salt and DEA/NO significantly diminished reduced glutathione and protein thiol levels in a similar way, DEA/NO was significantly more effective than AS in increasing S-nitrosothiol levels. None of these compounds increased retinal lipid peroxidation. These results suggest that HNO could regulate the hamster retinal nitridergic pathway by acting through a mechanism that only partly overlaps with that involved in NO response.     

Further evidence for distinct reactive intermediates from nitroxyl and peroxynitrite: effects of buffer composition on the chemistry of Angeli's salt and synthetic peroxynitrite

The nitroxyl (HNO) donor Angeli's salt (Na(2)N(2)O(3); AS) is cytotoxic in vitro, inducing double strand DNA breaks and base oxidation, yet may have pharmacological application in the treatment of cardiovascular disease. The chemical profiles of AS and synthetic peroxynitrite (ONOO(-)) in aerobic solution were recently compared, and AS was found to form a distinct reactive intermediate. However, similarities in the chemical behavior of the reactive nitrogen oxide species (RNOS) were apparent under certain conditions. Buffer composition was found to have a significant and unexpected impact on the observed chemistry of RNOS, and varied buffer conditions were utilized to further distinguish the chemical profiles elicited by the RNOS donors AS and synthetic ONOO(-). Addition of HEPES to the assay buffer significantly quenched oxidation of dihydrorhodamine (DHR), hydroxylation of benzoic acid (BA), and DNA damage by both AS and ONOO(-), and oxidation and nitration of hydroxyphenylacetic acid by ONOO(-). Additionally, H(2)O(2) was produced in a concentration-dependent manner from the interaction of HEPES with both the donor intermediates. Interestingly, clonogenic survival was not affected by HEPES, indicating that H(2)O(2) is not a contributing factor to in vitro cytotoxicity of AS. Variation in RNOS reactivity was dramatic with significantly higher relative affinity for the AS intermediate toward DHR, BA, DNA, and HEPES and increased production of H(2)O(2). Further, AS reacted to a significantly greater extent with the unprotonated amine form of HEPES while the interaction of ONOO(-) with HEPES was pH-independent. Addition of bicarbonate only altered ONOO(-) chemistry. This study emphasizes the importance of buffer composition on chemical outcome and thus on interpretation and provides further evidence that ONOO(-) is not an intermediate formed between the reaction of O(2) and HNO produced by AS.     

Synthesis and immunological activity of water-soluble thalidomide prodrugs

A series of new water-soluble thalidomide prodrugs was prepared. All compounds were derivatized on the nitrogen of the glutarimide ring. Esters of natural amino acids and succinic acid derivatives have been introduced by reaction with the hydroxymethyl thalidomide 2. Nicotinic acid derivatives were prepared from halomethyl derivatives. Additionally, a methoxymethyl derivative and a carboxymethyl derivative were prepared directly from thalidomide. Most compounds showed a very large increase in water solubility compared to thalidomide itself (0.012mg/mL). The amorphous hydrochlorides of the N-methylalanine ester 8, valine ester 9, and glycylglycine ester 10, respectively, were the most soluble compounds showing solubility greater than 300mg/mL, which equals an increase greater than 15,000-fold. The lipophilicity of the prodrugs has been determined by their HPLC capacity factors k'. The stability of selected compounds was determined. The hydrolysis rates follow pseudo-first order kinetics. In order to assess the immunological activity, the prodrugs were tested using tumor necrosis factor-alpha and interleukin-2 inhibition assays. Selected compounds were additionally investigated on their abililty to inhibit the local Shwartzman reaction, an assay to determine the vascular permeability. The prodrugs retained high effectiveness in the inhibition of TNF-alpha release. Our results indicated that the more stable prodrugs exhibited higher activity in the immunological assays. Some compounds showed higher activity than thalidomide itself, suggesting a high affine binding to the pharmacophore. In conclusion, the prodrugs exhibited high water solubility and high activity and might therefore be used in therapeutic applications.     

Angeli盐对二氢硫辛酰胺脱氢酶的可逆性灭活作用(英文)

二氢硫辛酰胺脱氢酶(dihydrolipoamide dehydrogenase,DLDH)是线粒体3个α-酮酸脱氢酶复合物(丙酮酸脱氢酶复合物、α-酮戊二酸脱氢酶复合物、支链氨基酸脱氢酶复合物)的关键成分,属于吡啶依赖性二硫化物氧化还原酶类,对活性氮自由基(reactive nitrogen species,RNS)和活性氧自由基(reactive oxygenspecies,ROS)造成的氧化修饰非常敏感。本研究探索由Angeli盐所产生的RNS对DLDH的修饰作用及机制。将大鼠脑线粒体分离,与不同浓度的Angeli盐作用,应用分光光度计、蓝色胶、基于二维电泳的蛋白质组学等手段,测定DLDH酶活性。结果显示,Angeli盐呈浓度依赖性方式灭活DLDH,过氧亚硝酸盐在同样条件下对DLDH酶活性无抑制作用,说明Angeli盐对DLDH的作用可能是非随机的。由于Angeli盐在生理pH条件下可分解为硝基阴离子(nitroxyl anion,HNO)和一氧化氮(nitric oxide,NO),故进一步分析了Angeli盐对DLDH的灭活作用是否由HNO引起,结果证实确实如此。最后,二维电泳Western blot结果显示,Angeli盐对DLDH的灭活伴随着DLDH蛋白质的S-亚硝基硫醇形成,提示S-亚硝基硫醇化可能是导致DLDH酶失活的原因。综上,本研究为研究Angeli盐灭活DLDH的机制提供了新证据。     

Cathepsin B is a differentiation-resistant target for nitroxyl (HNO) in THP-1 monocyte/macrophages

We previously showed that the one-electron reduction product of nitric oxide (NO), nitroxyl (HNO), irreversibly inhibits the proteolytic activity of the model cysteine protease papain. This result led us to investigate the differential effects of the nitrogen oxides, such as nitroxyl (HNO), NO, and in situ-generated peroxynitrite on cysteine modification-sensitive cellular proteolytic enzymes. We used Angeli's salt, diethylaminenonoate (DEA/NO), and 3-morpholinosydnoniminehydrochloride (SIN-1), as donors of HNO, NO, and peroxynitrite, respectively. In this study we evaluated their inhibitory activities on the lysosomal mammalian papain homologue cathepsin B and on the cytosolic 26S proteasome in THP-1 monocyte/macrophages after LPS activation or TPA differentiation. HNO-generating Angeli's salt caused a concentration-dependent (62 +/- 4% at 316 muM) inhibition of the 26S proteasome activity, resulting in accumulation of protein-bound polyubiquitinylated proteins in LPS-activated cells, whereas neither DEA/NO nor SIN-1 showed any effect. Angeli's salt, but not DEA/NO or SIN-1, also caused (94 +/- 2% at 316 muM) inhibition of lysosomal cathepsin B activity in LPS-activated cells. Induction of macrophage differentiation did not significantly alter the inhibitory effect of HNO on lysosomal cathepsin B activity, but protected the proteasome from HNO-induced inhibition. The protection awarded by macrophage differentiation was associated with induction of the GSH synthesis rate-limiting enzyme gamma-glutamylcysteine synthetase, as well as with increased intracellular GSH. In conclusion, HNO abrogates both lysosomal and cytosolic proteolysis in THP-1 cells. Macrophage differentiation, associated with upregulation of antioxidant defenses such as increased cellular GSH, does not protect the lysosomal cysteine protease cathepsin B from inhibition.     

Synthesis and evaluation of amino acid esters of 6-deoxypenciclovir as potential prodrugs of penciclovir

The amino acid ester derivatives of 6-deoxypenciclovir, 11-20, were synthesized as potential prodrugs of penciclovir, and were evaluated for their oral penciclovir bioavailability in mice and rats. Esterification of 6-deoxypenciclovir with N-carbobenzyl-oxyglycine, -L-alanine, -L-valine, -L-leucine, or -L-isoleucine (3.75equiv.) using conventional coupling method (DCC/DMAP) afforded the mono-O-ester derivatives 1-5 in 47-55% yields as a mixture of two diastereomers along with the di-O-ester derivatives 6-10 in 20-29% yields. Reductive cleavage of carbobenzyloxy (Cbz) group (10% Pd/C, 1 atmosphere of H2, room temperature in methanol) followed by subsequent treatment of the resulting free amine with methanolic HCI solution provided the mono-O-ester derivatives 11-15 as di-HCl salt in 51-98% yields and the di-O-ester derivatives 16-20 as tri-HCl salt in 65 98% yields. Of the prodrugs tested in mice and rats, 6-deoxypenciclovir O-L-valinate (13), O-L-isoleucinate (15), and O,O-di-glycinate (16) showed significantly higher urinary recovery of penciclovir compared with that of penciclovir, but those are somewhat lower than that of famciclovir.