Transpeptidase Inhibitor Research Articles

Drug Discovery in the Field of β-Lactams: An Academic Perspective.

β-Lactams are the most widely prescribed class of antibiotics that inhibit penicillin-binding proteins (PBPs), particularly transpeptidases that function in peptidoglycan synthesis. A major mechanism of antibiotic resistance is the production of β-lactamase enzymes, which are capable of hydrolyzing β-lactam antibiotics. There have been many efforts to counter increasing bacterial resistance against β-lactams. These studies have mainly focused on three areas: discovering novel inhibitors against β-lactamases, developing new β-lactams less susceptible to existing resistance mechanisms, and identifying non-β-lactam inhibitors against cell wall transpeptidases. Drug discovery in the β-lactam field has afforded a range of research opportunities for academia. In this review, we summarize the recent new findings on both β-lactamases and cell wall transpeptidases because these two groups of enzymes are evolutionarily and functionally connected. Many efforts to develop new β-lactams have aimed to inhibit both transpeptidases and β-lactamases, while several promising novel β-lactamase inhibitors have shown the potential to be further developed into transpeptidase inhibitors. In addition, the drug discovery progress against each group of enzymes is presented in three aspects: understanding the targets, screening methodology, and new inhibitor chemotypes. This is to offer insights into not only the advancement in this field but also the challenges, opportunities, and resources for future research. In particular, cyclic boronate compounds are now capable of inhibiting all classes of β-lactamases, while the diazabicyclooctane (DBO) series of small molecules has led to not only new β-lactamase inhibitors but potentially a new class of antibiotics by directly targeting PBPs. With the cautiously optimistic successes of a number of new β-lactamase inhibitor chemotypes and many questions remaining to be answered about the structure and function of cell wall transpeptidases, non-β-lactam transpeptidase inhibitors may usher in the next exciting phase of drug discovery in this field.

γ-glutamyl transpeptidase阻害剤における口腔粘膜創傷治癒への効果の検討

γ-glutamyl transpeptidase阻害剤における口腔粘膜創傷治癒への効果の検討

Involvement of γ-Glutamyl Transpeptidase in Ischemia/Reperfusion-Induced Cardiac Dysfunction in Isolated Rat Hearts.

GGsTop is a highly potent and specific, and irreversible γ-glutamyl transpeptidase (GGT) inhibitor without any influence on glutamine amidotransferases. The aim of the present study was to investigate the involvement of GGT in ischemia/reperfusion-induced cardiac dysfunction by assessing the effects of a treatment with GGsTop. Using a Langendorff apparatus, excised rat hearts underwent 40 min of global ischemia without irrigation and then 30 min of reperfusion. GGT activity was markedly increased in cardiac tissues exposed to ischemia, and was inhibited by the treatment with GGsTop. Exacerbation of cardiac functional parameters caused by ischemia and reperfusion, namely the reduction of left ventricular (LV) developed pressure and the maximum and negative minimum values of the first derivative of LV pressure, and the increment in LV end-diastolic pressure was significantly attenuated by GGsTop treatment. The treatment with GGsTop suppressed excessive norepinephrine release in the coronary perfusate, a marker for myocardial dysfunction, after ischemia/reperfusion. In addition, oxidative stress indicators in myocardium, including superoxide and malondialdehyde, after ischemia/reperfusion were significantly low in the presence of GGsTop. These observations demonstrate that enhanced GGT activity contributes to cardiac damage after myocardial ischemia/reperfusion, possibly via increased oxidative stress and subsequent norepinephrine overflow. GGT inhibitors have potential as a therapeutic strategy to prevent myocardial ischemia/reperfusion injury in vivo.

Therapeutic Effect of GGsTop, Selective Gamma-glutamyl Transpeptidase Inhibitor, on a Mouse Model of 5-Fluorouracil-induced Oral Mucositis

Oral mucositis (OM) induced by cancer chemotherapy has a high incidence and serious symptoms, which often force chemotherapy to be stopped. GGsTop is a newly-discovered gamma-glutamyl transpeptidase (GGT) inhibitor. Previous research suggested that inhibition of GGT suppressed reactive oxygen species and induced the production of collagen and elastin. We hypothesized that GGsTop could safely treat OM. A mouse model of OM was treated with GGsTop and ulcer area, weight, and white blood cell count were determined. The treatment effect was also evaluated by hematoxylin-eosin and collagen staining. The therapeutic effect of GGsTop was better than that of an existing drug and may be safely used in combination with chemotherapy. Furthermore, GGsTop promoted collagen production in oral mucosa. GGsTop treated OM quickly and safely. GGsTop is highly valuable for use as a treatment for OM.

Isolation and Identification of Three γ-Glutamyl Tripeptides and Their Putative Production Mechanism in Aged Garlic Extract.

We analyzed aged garlic extract (AGE) to understand its complex sulfur chemistry using post-column high-performance liquid chromatography with an iodoplatinate reagent and liquid chromatography high resolution mass spectrometry (LC-MS). We observed unidentified peaks of putative sulfur compounds. Three compounds were isolated and identified as γ-glutamyl-γ-glutamyl- S-methylcysteine, γ-glutamyl-γ-glutamyl- S-allylcysteine (GGSAC) and γ-glutamyl-γ-glutamyl- S-1-propenyl-cysteine (GGS1PC) by nuclear magnetic resonance and LC-MS analysis based on comparisons with chemically synthesized reference compounds. GGSAC and GGS1PC were novel compounds. Trace amounts of these compounds were detected in raw garlic, but the contents of these compounds increased during the aging process. Production of these compounds was inhibited using a γ-glutamyl transpeptidase (GGT) inhibitor in the model reaction mixtures. These findings suggest that γ-glutamyl tripeptides in AGE are produced by GGT during the aging process.

Functional characterisation of glutathione export from the rat lens

In this study, we have investigated whether the lens was capable of exporting the antioxidant glutathione. Pairs of rat lenses were cultured in isosmotic artificial aqueous humour for one, two, three, or six hours in low oxygen conditions (90% N2, 5% CO2, 5% O2), and reduced glutathione (GSH) and oxidised glutathione (GSSG) levels measured in the media and lenses. We show that the rat lens is capable of releasing ∼5 nmol GSH for each time point suggesting that GSH release is regulated since it does not appreciably increase over time. We also demonstrated that the predominant form of glutathione released was the reduced form. We next cultured lenses in the absence or presence of acivicin, a γ-glutamyl transpeptidase (GGT) inhibitor, and found that GSH levels were significantly increased (p < 0.001) in the presence of this inhibitor, which indicated that GSH released by the lens undergoes degradation into its constituent amino acids. GSH release was significantly decreased (p < 0.001) in the presence of 100 μM MK571, a multidrug resistance-associated protein (Mrp) inhibitor, suggesting that Mrp transporters mediate GSH efflux from the lens. Culturing lenses in low (10 μM) or high (70 μM) concentrations of H2O2 for one hour significantly increased total glutathione levels (p < 0.05) relative to controls, due to the increased release of GSSG. Our results show that in response to oxidative stress, the rat lens is able to release GSH or GSSG, thereby serving to maintain lens redox state or potentially influence the redox state of nearby tissues.

Synthesis and evaluation of the inhibitory activity of the four stereoisomers of the potent and selective human γ-glutamyl transpeptidase inhibitor GGsTop

2-Amino-4-{[3-(carboxymethyl)phenoxy](methoxy)phosphoryl}butanoic acid (GGsTop) is a potent, highly selective, nontoxic, and irreversible inhibitor of γ-glutamyl transpeptidase (GGT). GGsTop has been widely used in academic and medicinal research, and also as an active ingredient (Nahlsgen) in commercial anti-aging cosmetics. GGsTop consists of four stereoisomers due to the presence of two stereogenic centers, i.e., the α-carbon atom of the glutamate mimic (l/d) and the phosphorus atom (RP/SP). In this study, each stereoisomer of GGsTop was synthesized stereoselectively and their inhibitory activity against human GGT was evaluated. The l- and d-configurations of each stereoisomer were determined by a combination of a chiral pool synthesis and chiral HPLC analysis. The synthesis of the four stereoisomers of GGsTop used chiral synthetic precursors that were separated by chiral HPLC on a preparative scale. With respect to the configuration of the α-carbon atom of the glutamate mimic, the l-isomer (kon=174M−1s−1) was ca. 8-fold more potent than the d-isomer (kon=21.5M−1s−1). In contrast, the configuration of the phosphorus atom is critical for GGT inhibitory activity. Based on a molecular modeling approach, the absolute configuration of the phosphorus atom of the active GGsTop isomers was postulated to be SP. The SP-isomers inhibited human GGT (kon=21.5–174M−1s−1), while the RP-isomers were inactive even at concentrations of 0.1mM.

An improved synthesis of the potent and selective γ-glutamyl transpeptidase inhibitor GGsTop together with an inhibitory activity evaluation of its potential hydrolysis products

A previously reported synthetic route to 2-amino-4-{[3-(carboxymethyl)phenoxy](methoxy)phosphoryl}butanoic acid (GGsTop), a potent, highly selective, non-toxic, and irreversible inhibitor of γ-glutamyl transpeptidase (GGT) was substantially improved. This route furnishes GGsTop in four steps with an overall yield of 32% from inexpensive starting materials, i.e., the yield is increased approximately sixfold relative to the previous protocol. The synthesis and inhibitory activity evaluation of potential hydrolysis products of GGsTop clearly demonstrated that GGsTop is the active inhibitor, and the conceivable hydrolysis products barely affect the activity of human GGT.

Lipid II overproduction allows direct assay of transpeptidase inhibition by β-lactams.

Peptidoglycan is an essential crosslinked polymer that surrounds bacteria and protects them from osmotic lysis. Beta-lactam antibiotics target the final stages of peptidoglycan biosynthesis by inhibiting the transpeptidases that crosslink glycan strands to complete cell wall assembly. Characterization of transpeptidases and their inhibition by beta-lactams has been hampered by lack of access to substrate. We describe a general approach to accumulate Lipid II in bacteria and to obtain large quantities of this cell wall precursor. We demonstrate utility by isolating Staphylococcus aureus Lipid II and reconstituting the synthesis of crosslinked peptidoglycan by the essential penicillin-binding protein 2, PBP2, which catalyzes both glycan polymerization and transpeptidation. We also show that we can compare the potencies of different beta-lactams by directly monitoring transpeptidase inhibition. The methods reported here will enable a better understanding of cell wall biosynthesis and facilitate studies of next-generation transpeptidase inhibitors.

A new CysLT1 and CysLT2 receptors-mediated anaphylaxis guinea pig model

Although the effectiveness of CysLT1 receptor antagonists on asthma has been clinically established, the effects of CysLT2 receptor antagonists are still unclear. The purpose of this study was to develop a new CysLT1 and CysLT2 receptors-mediated anaphylaxis guinea pig model using S-hexyl GSH, a γ-glutamyl transpeptidase (GTP) inhibitor, to suppress conversion of LTC4 to LTD4. Actively sensitized guinea pigs were challenged with OVA in the absence or presence of S-hexyl GSH, and survival rate following anaphylactic response was monitored. OVA-induced fatal anaphylaxis in the absence of S-hexyl GSH was almost completely inhibited by montelukast, a CysLT1 receptor antagonist, but not by the CysLT2 receptor antagonist BayCysLT2RA. However, under treatment with S-hexyl-GSH, the inhibitory effect of motelukast was dramatically diminished, whereas that of BayCysLT2RA was markedly increased. The dual CysLT1/2 receptor antagonist ONO-6950 effectively inhibited anaphylactic response in both S-hexyl GSH-treated and non-treated animals. LC/MS/MS analysis revealed that S-hexyl GSH treatment actually inhibited LTC4 metabolism in the blood and lung tissues. Using S-hexyl GSH, we developed a novel CysLT1 and CysLT2 receptors-mediated anaphylaxis guinea pig model that can be useful for not only screening both CysLT2 and CysLT1/2 receptors antagonists, but also for functional analysis of CysLT2 receptors.

GGsTop, a novel and specific γ-glutamyl transpeptidase inhibitor, protects hepatic ischemia-reperfusion injury in rats.

Ischemia-reperfusion (IR) injury is a major clinical problem and is associated with numerous adverse effects. GGsTop [2-amino-4{[3-(carboxymethyl)phenyl](methyl)phosphono}butanoic acid] is a highly specific and irreversible γ-glutamyl transpeptidase (γ-GT) inhibitor. We studied the protective effects of GGsTop on IR-induced hepatic injury in rats. Ischemia was induced by clamping the portal vein and hepatic artery of left lateral and median lobes of the liver. Before clamping, saline (IR group) or saline containing 1 mg/kg body wt of GGsTop (IR-GGsTop group) was injected into the liver through the inferior vena cava. At 90 min of ischemia, blood flow was restored. Blood was collected before induction of ischemia and prior to restoration of blood flow and at 12, 24, and 48 h after reperfusion. All the animals were euthanized at 48 h after reperfusion and the livers were harvested. Serum levels of alanine transaminase, aspartate transaminase, and γ-GT were significantly lower after reperfusion in the IR-GGsTop group compared with the IR group. Massive hepatic necrosis was present in the IR group, while only few necroses were present in the IR-GGsTop group. Treatment with GGsTop increased hepatic GSH content, which was significantly reduced in the IR group. Furthermore, GGsTop prevented increase of hepatic γ-GT, malondialdehyde, 4-hydroxynonenal, and TNF-α while all these molecules significantly increased in the IR group. In conclusion, treatment with GGsTop increased glutathione levels and prevented formation of free radicals in the hepatic tissue that led to decreased IR-induced liver injury. GGsTop could be used as a pharmacological agent to prevent IR-induced liver injury and the related adverse events.

Discovery of a New Class of Sortase A Transpeptidase Inhibitors to Tackle Gram-Positive Pathogens: 2-(2-Phenylhydrazinylidene)alkanoic Acids and Related Derivatives.

A FRET-based random screening assay was used to generate hit compounds as sortase A inhibitors that allowed us to identify ethyl 3-oxo-2-(2-phenylhydrazinylidene)butanoate as an example of a new class of sortase A inhibitors. Other analogues were generated by changing the ethoxycarbonyl function for a carboxy, cyano or amide group, or introducing substituents in the phenyl ring of the ester and acid derivatives. The most active derivative found was 3-oxo-2-(2-(3,4dichlorophenyl)hydrazinylidene)butanoic acid (2b), showing an IC50 value of 50 µM. For a preliminary assessment of their antivirulence properties the new derivatives were tested for their antibiofilm activity. The most active compound resulted 2a, which showed inhibition of about 60% against S. aureus ATCC 29213, S. aureus ATCC 25923, S. aureus ATCC 6538 and S. epidermidis RP62A at a screening concentration of 100 µM.

Effects of acivicin on growth, mycotoxin production and virulence of phytopathogenic fungi.

Acivicin is an inhibitor of γ-glutamyl transpeptidase and glutamine amidotransferase. When grown on a synthetic minimal agar medium, acivicin strongly inhibited the growth of Magnaporthe oryzae and Alternaria brassicicola, and to a lesser extent, Botrytis cinerea. However, only partial or marginal growth inhibition was observed with regard to Fusarium sporotrichioides and Fusarium graminearum. The growth retardation caused by acivicin was significantly alleviated by cultivating the fungus on a nutrient-rich medium. The inhibition of M. oryzae growth caused by 1 μmol l(-1) of acivicin on minimal agar medium was subdued by the addition of specific single amino acids, including His, a branched-chain amino acid (Leu, Ile or Val), an aromatic amino acid (Trp, Tyr or Phe), Met or Gln, at a concentration of 0·4 mmol l(-1). Trichothecene production by F. graminearum in trichothecene-inducing liquid medium was reduced significantly in the presence of acivicin despite its inability to inhibit growth in the trichothecene-inducing liquid medium. Foliar application of conidia in the presence of acivicin reduced the severity of rice blast disease caused by M. oryzae. These results suggest the usefulness of this modified amino acid natural product to mitigate agricultural problems caused by some phytopathogenic fungi. Significance and impact of the study: Fusarium head blight or scab disease and rice blast, caused by Fusarium graminearum and Magnaporthe oryzae, respectively, are major diseases of cereal crops that cause a significant loss of yield and deterioration in the quality of the grain. The present study investigated the effects of acivicin, a glutamine amino acid analog, on the physiology of various phytopathogenic fungi. Application of acivicin to a fungal culture and conidial suspension reduced mycotoxin production by the wheat scab fungus and the severity of rice blast, respectively. These results suggest the possibility that acivicin may serve as a lead compound to develop agricultural chemicals for the control of some plant diseases.

Fluorescence anisotropy-based measurement of Pseudomonas aeruginosa penicillin-binding protein 2 transpeptidase inhibitor acylation rate constants

High-molecular-weight penicillin-binding proteins (PBPs) are essential integral membrane proteins of the bacterial cytoplasmic membrane responsible for biosynthesis of peptidoglycan. They are the targets of antibacterial β-lactam drugs, including penicillins, cephalosporins, and carbapenems. β-Lactams covalently acylate the active sites of the PBP transpeptidase domains. Because β-lactams are time-dependent inhibitors, quantitative assessment of the inhibitory activity of these compounds ideally involves measurement of their second-order acylation rate constants. We previously described a fluorescence anisotropy-based assay to measure these rate constants for soluble constructs of PBP3 (Anal. Biochem. 439 (2013) 37–43). Here we report the expression and purification of a soluble construct of Pseudomonas aeruginosa PBP2 as a fusion protein with NusA. This soluble PBP2 was used to measure second-order acylation rate constants with the fluorescence anisotropy assay. Measurements were obtained for mecillinam, which reacts specifically with PBP2, and for several carbapenems. The assay also revealed that PBP2 slowly hydrolyzed mecillinam and was used to measure the rate constant for this deacylation reaction.

A microplate assay for the coupled transglycosylase–transpeptidase activity of the penicillin binding proteins; a vancomycin-neutralizing tripeptide combination prevents penicillin inhibition of peptidoglycan synthesis

A microplate, scintillation proximity assay to measure the coupled transglycosylase–transpeptidase activity of the penicillin binding proteins in Escherichia coli membranes was developed. Membranes were incubated with the two peptidoglycan sugar precursors UDP-N-acetyl muramylpentapeptide (UDP-MurNAc(pp)) and UDP-[3H]N-acetylglucosamine in the presence of 40μM vancomycin to allow in situ accumulation of lipid II. In a second step, vancomycin inhibition was relieved by addition of a tripeptide (Lys-d-ala-d-ala) or UDP-MurNAc(pp), resulting in conversion of lipid II to cross-linked peptidoglycan. Inhibitors of the transglycosylase or transpeptidase were added at step 2. Moenomycin, a transglycosylase inhibitor, had an IC50 of 8nM. Vancomycin and nisin also inhibited the assay. Surprisingly, the transpeptidase inhibitors penicillin and ampicillin showed no inhibition. In a pathway assay of peptidoglycan synthesis, starting from the UDP linked sugar precursors, inhibition by penicillin was reversed by a ‘neutral’ combination of vancomycin plus tripeptide, suggesting an interaction thus far unreported.

Multidrug resistance-associated proteins are involved in the transport of the glutathione conjugates of the ultimate carcinogen of benzo[a]pyrene in human Caco-2 cells

A wide variety of contaminants are ingested through food, among them the pro-carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BP) that is resorbed and partially metabolized in the enterocytes of the small intestine. Previous in vitro studies have revealed that BP phenols are excreted as Phase II metabolites including glucuronides and sulfates. This export is mediated by the breast cancer resistance protein (ABCG2). The ultimate carcinogenic Phase I BP metabolite anti-BP-7,8-dihydrodiol-9,10-epoxide (BPDE) can be detoxified by glutathione conjugate formation catalyzed by glutathione S-transferases. In the present study, differentiated human intestinal Caco-2 cells were used as a model for the human small intestine to investigate the detoxification of BPDE and excretion of stereoisomeric glutathione conjugates in the presence of an inhibitor of the glutathione-cleaving enzyme γ-glutamyl transpeptidase at the cell surface. The results indicate that the glutathione conjugates of BPDE are formed and excreted mainly to the apical and to a minor extent to the basolateral side of polarized Caco-2 monolayers. Inhibition studies revealed that the multidrug resistance-associated proteins (ABCCs) are involved in the transport of BPDE glutathione conjugates. Stable ABCC1, ABCC2 and ABCC3 knockdown cell lines were generated, thus making it possible to demonstrate that ABCC1 mediates the basolateral and ABCC2 the apical excretion of BPDE glutathione conjugates. In conclusion, the ultimate carcinogen BPDE is detoxified via glutathione conjugation and subsequently excreted by Caco-2 cells in both apical and basolateral directions. This finding is equivalent to a transport into feces as well as blood system in the in vivo situation.

Preventive Effect of GGsTop, a Novel and Selective γ-Glutamyl Transpeptidase Inhibitor, on Ischemia/Reperfusion-Induced Renal Injury in Rats

GGsTop [2-amino-4-{[3-(carboxymethyl)phenyl](methyl)phosphono}butanoic acid], is a novel, highly selective, and irreversible γ-glutamyl transpeptidase (GGT) inhibitor with no inhibitory activity on glutamine amidotransferases. In this study, we investigated the effects of treatment with GGsTop on ischemia/reperfusion-induced renal injury in uninephrectomized rats. Ischemic acute kidney injury (AKI) was induced by occlusion of the left renal artery and vein for 45 min followed by reperfusion 2 weeks after contralateral nephrectomy. Renal function in vehicle-treated AKI rats markedly decreased at 1 day after reperfusion. Treatment with GGsTop (1 and 10 mg/kg i.v.) 5 min before ischemia attenuated the ischemia/reperfusion-induced renal dysfunction in a dose-dependent manner. Histopathological examination of the kidney of AKI rats revealed severe renal damage, which was significantly suppressed by the GGsTop treatment. In renal tissues exposed to ischemia/reperfusion, GGT activity was markedly increased immediately after reperfusion, whereas renal superoxide production and malondialdehyde level were significantly increased 6 h after reperfusion. These alterations were abolished by the treatment with GGsTop. In addition, renal glutathione content was decreased by the 45-min ischemia, but its level was markedly elevated by the GGsTop treatment. Our results demonstrate that the novel and highly selective GGT inhibitor GGsTop prevents ischemia/reperfusion-induced AKI. The renoprotective effect of GGsTop seems to be attributed to the suppression of oxidative stress by inhibiting GGT activation, thereby preventing the degradation of glutathione.

Glutathione Transport Is a Unique Function of the ATP-binding Cassette Protein ABCG2

Glutathione (GSH) transport is vital for maintenance of intracellular and extracellular redox balance. Only a few human proteins have been identified as transporters of GSH, glutathione disulfide (GSSG) and/or GSH conjugates (GS-X). Human epithelial MDA1586, A549, H1975, H460, HN4, and H157 cell lines were exposed to 2',5'-dihydroxychalcone, which induces a GSH efflux response. A real-time gene superarray for 84 proteins found in families that have a known role in GSH, GSSG, and/or GS-X transport was employed to help identify potential GSH transporters. ABCG2 was identified as the only gene in the array that closely corresponded with the magnitude of 2',5'-dihydroxychalcone (2',5'-DHC)-induced GSH efflux. The role of human ABCG2 as a novel GSH transporter was verified in a Saccharomyces cerevisiae galactose-inducible gene expression system. Yeast expressing human ABCG2 had 2.5-fold more extracellular GSH compared with those not expressing ABCG2. GSH efflux in ABCG2-expressing yeast was abolished by the ABCG2 substrate methotrexate (10 microM), indicating competitive inhibition. In contrast, 2',5'-DHC treatment of ABCG2-expressing yeast increased extracellular GSH levels in a dose-dependent manner with a maximum 3.5-fold increase in GSH after 24 h. In addition, suppression of ABCG2 with short hairpin RNA or ABCG2 overexpression in human epithelial cells decreased or increased extracellular GSH levels, respectively. Our data indicate that ABCG2 is a novel GSH transporter.

Microplate Assay for Inhibitors of the Transpeptidase Activity of PBP1b of Escherichia coli

The transpeptidase (TP) activity of penicillin-binding proteins (PBPs), target of the beta-lactam antibiotics, is a well-validated antibacterial drug target. The TP activity of PBP1b converts un-cross-linked peptidoglycan to the cross-linked form. Directly measuring TP activity is difficult because cross-linked and un-cross-linked peptidoglycan have very similar chromatographic properties. The authors report a microdilution plate method to directly measure the TP enzyme activity, uncoupled from the transglycosylase (TG), for detection of TP inhibitors. Escherichia coli membranes were incubated with 100 mM ampicillin, followed by removal of unbound ampicillin. The substrate for the TP, un-cross-linked peptidoglycan, was prepared by incubating these membranes with peptidoglycan sugar precursors, 1 of which was radiolabeled. Subsequently, solubilized PBP1b was added and TP activity assayed. The cross-linked peptidoglycan formed was monitored by addition of wheat germ agglutinin scintillation proximity assay beads plus N-laurylsarcosine, which selectively captures cross-linked peptidoglycan. The PBP1bcatalyzed activity was inhibited by penicillin G but not by cephalexin or cephradine, which have higher affinity for PBP1a. Moenomycin, a TG inhibitor, also inhibited TP activity. Because this is a true enzyme assay, it has the potential to detect novel, non-beta-lactam TP inhibitors and could lead to the discovery of new antibacterial agents.

Elevated endogenous nitric oxide increases Ca 2+ flux via L-type Ca 2+ channels by S-nitrosylation in rat hippocampal neurons during severe hypoxia and in vitro ischemia

Nitric oxide (NO) mediates pathogenic changes in the brain subsequent to energy deprivation; yet the NO mechanism involved in the early events remains unclear. We examined the acute effects of severe hypoxia and oxygen–glucose deprivation (OGD) on the endogenous NO production and the NO-mediated pathways involved in the intracellular calcium ([Ca 2+] i) response in the rat hippocampal neurons. The levels of NO and [Ca 2+] i in the CA1 region of the slices rapidly elevated in hypoxia and were more prominent in OGD, measured by the electrochemical method and spectrofluorometry, respectively. The NO and [Ca 2+] i responses were enhanced by L-arginine and were reduced by NO synthase inhibitors, suggesting that the endogenous NO increases the [Ca 2+] i response to energy deprivation. Nickel and nifedipine significantly decreased the NO and [Ca 2+] i responses to hypoxia and OGD, indicating an involvement of L-type Ca 2+ channels in the NO-mediated mechanisms. In addition, the [Ca 2+] i responses were attenuated by ODQ or KT5823, inhibitors of the cGMP–PKG pathway, and by acivicin, an inhibitor of γ-glutamyl transpeptidase for S-nitrosylation, and by the thiol-alkylating agent N-ethylmaleimide (NEM). Moreover, L-type Ca 2+ currents in cultured hippocampal neurons with whole-cell recording were significantly increased by L-arginine and were decreased by L-NAME. Pretreatment with NO synthase inhibitors or NEM but not ODQ abolished the effect of L-arginine on the Ca 2+ currents. Also, vitamin C, which decomposes nitrosothiol but not disulfide by reduction, reversed the change in the Ca 2+ current with L-arginine. Taken together, the results suggest that an elevated endogenous NO production enhances the influx of Ca 2+ via the hippocampal L-type Ca 2+ channel by S-nitrosylation during an initial phase of energy deprivation.