Synthesis, Docking, Computational Studies, and Antimicrobial Evaluations of New Dipeptide Derivatives Based on Nicotinoylglycylglycine Hydrazide.

Within a series of dipeptide derivatives (5-11), compound 4 was refluxed with d-glucose, d-xylose, acetylacetone, diethylmalonate, carbon disulfide, ethyl cyanoacetate, and ethyl acetoacetate which yielded 5-11, respectively. The candidates 5-11 were characterized and their biological activities were evaluated where they showed different anti-microbial inhibitory activities based on the type of pathogenic microorganisms. Moreover, to understand modes of binding, molecular docking was used of Nicotinoylglycine derivatives with the active site of the penicillin-binding protein 3 (PBP3) and sterol 14-alpha demethylase's (CYP51), and the results, which were achieved via covalent and non-covalent docking, were harmonized with the biological activity results. Therefore, it was extrapolated that compounds 4, 7, 8, 9, and 10 had good potential to inhibit sterol 14-alpha demethylase and penicillin-binding protein 3; consequently, these compounds are possibly suitable for the development of a novel antibacterial and antifungal therapeutic drug. In addition, in silico properties of absorption, distribution, metabolism, and excretion (ADME) indicated drug likeness with low to very low oral absorption in most compounds, and undefined blood-brain barrier permeability in all compounds. Furthermore, toxicity (TOPKAT) prediction showed probability values for all carcinogenicity models were medium to pretty low for all compounds.

Diglycine Enables Rapid Intrabacterial Hydrolysis for Activating Anbiotics against Gram-negative Bacteria.

Antimicrobial drug resistance demands novel approaches for improving the efficacy of antibiotics, especially against Gram-negative bacteria. Herein, we report that conjugating a diglycine (GG) to an antibiotic prodrug drastically accelerates intrabacterial ester-bond hydrolysis required for activating the antibiotic. Specifically, the attachment of GG to chloramphenicol succinate (CLsu) generates CLsuGG, which exhibits about an order of magnitude higher inhibitory efficacy than CLsu against Escherichia coli. Further studies reveal that CLsuGG undergoes rapid hydrolysis, catalyzed by intrabacterial esterases (e.g., BioH and YjfP), to generate chloramphenicol (CL) in E. coli. Importantly, the conjugate exhibits lower cytotoxicity to bone marrow stromal cells than CL. Structural analogues of CLsuGG indicate that the conjugation of GG to an antibiotic prodrug is an effective strategy for accelerating enzymatic prodrug hydrolysis and enhancing the antibacterial efficacy of antibiotics.

ß-Klotho as a Negative Regulator of the Peptide Transporters PEPT1 and PEPT2.

BACKGROUND/AIMS: ß-Klotho, a transmembrane protein expressed in several tissues including the brain and the kidney, is critically important for inhibition of 1,25(OH)2D3 formation by FGF23. The extracellular domain of Klotho protein could be cleaved off, thus being released into blood or cerebrospinal fluid. Soluble klotho is a ß-glucuronidase participating in the regulation of several ion channels and carriers. The present study explored the effect of ß-Klotho protein on the peptide transporters PEPT1 and PEPT2. METHODS: cRNA encoding PEPT1 or PEPT2 was injected into Xenopus laevis oocytes and glycine-glycine (2 mM)-induced inward current (IGly) taken as measure of glycine-glycine transport. Measurements were made without or with prior 24 h treatment with soluble ß-Klotho protein (30 ng/ml) in the absence and presence of ß-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate (DSAL,10 µM). Ussing chamber experiments were employed to determine electrogenic peptide transport across intestinal epithelia of klotho deficient (kl-/-) and corresponding wild type (kl+/+) mice. RESULTS: IGly was observed in PEPT1 and in PEPT2 expressing oocytes but not in water injected oocytes. In both, PEPT1 and PEPT2 expressing oocytes IGly was significantly decreased by treatment with soluble ß-Klotho protein. As shown for PEPT1, ß-klotho protein decreased significantly the maximal transport rate without significantly modifying the affinity of the carrier. The effect of ß-Klotho on PEPT1 was reversed by DSAL. Intestinal IGly was significantly larger in kl-/- than in kl+/+ mice. CONCLUSION: ß-Klotho participates in the regulation of the peptide transporters PEPT1 and PEPT2.

Metabolomic analysis revealed glycylglycine accumulation in astrocytes after methionine enkephalin administration exhibiting neuron protective effects.

Owing to its unrevealed etiology, multiple sclerosis lacks specific therapies up to now. Experiential administration of methionine enkephalin (MENK) on mouse model improved disease manifestations to some extent. In order to gain more insight on the significance of MENK application, a capillary electrophoresis-mass spectrometry (CE-MS) technique was employed to profile intracellular metabolite fluctuation in 5 astrocytoma cell lines challenged by MENK. The processed data were first evaluated through a bioinformatic process to ensure their compatibility with the study aims and then subjected to multivariate analysis. The results indicated that MENK administration increased intracellular tyrosine, phenylalanine, methionine and glycylglycine. Exemplified by U87 cells, glycylglycine inhibited cell proliferation as well as MENK but it also decreased cell nitric oxide excretion which could not be evoked by MENK. The neuron protective effects were also mirrored by the increased expression of some genes related to remyelination. This study demonstrated CE-MS to be a promising tool for cell metabolomic analysis and benefited the therapeutic exploring of multiple sclerosis with respect to metabolism intervention.

Functional expression of novel peptide transporter in renal basolateral membranes.

We examined the peptide transport activity in renal basolateral membranes. [(14)C]glycylsarcosine (Gly-Sar) uptake in rat renal cortical slices was saturable and inhibited by excess dipeptide and aminocephalosporin cefadroxil. When several renal cell lines were screened for the basolateral peptide transport activity, Madin-Darby canine kidney (MDCK) cells were demonstrated to have the greatest transport activity. [(14)C]Gly-Sar uptake across the basolateral membranes of MDCK cells was inhibited by di- and tripeptide and decreased with decreases in extracellular pH from 7.4 to 5.0. The Michaelis-Menten constant value of [(14)C]Gly-Sar uptake across the basolateral membranes of MDCK cells was 71 microM. The basolateral peptide transporter in MDCK cells showed several different [(14)C]Gly-Sar transport characteristics in growth dependence, pH profile, substrate affinity, and sensitivities to chemical modifiers from those of the apical H(+)-peptide cotransporter of MDCK cells. The findings of the present investigation indicated that the peptide transporter was expressed in the renal basolateral membranes. In addition, from the functional characteristics, the renal basolateral peptide transporter was suggested to be distinguishable from known peptide transporters, i.e., H(+)-peptide cotransporters (PEPT1 and PEPT2) and the intestinal basolateral peptide transporter.

Comparative properties of two cysteine proteinases (gingipains R), the products of two related but individual genes of Porphyromonas gingivalis.

Proteolytic enzymes produced by Porphyromonas gingivalis are important virulence factors of this periodontopathogen. Two of these enzymes, referred to as arginine-specific cysteine proteinases (gingipains R), are the product of two related genes. Here, we describe the purification of an enzyme translated from the rgpB/rgp-2 gene (gingipain R2, RGP-2) and secreted as a single chain protein of 422 residues. The enzyme occurs in several isoforms differing in pI, molecular mass, mobility in gelatin zymography gels, and affinity to arginine-Sepharose. In comparison to the 95-kDa gingipain R1, a complex of catalytic and hemagglutinin/adhesin domains, RGP-2 showed five times lower proteolytic activity, although its activity on various P1-arginine p-nitroanilide substrates was generally higher. Gingipains R amidolytic activity, but not general proteolytic activity, was stimulated by glycyl-glycine. However, in cases of limited proteolysis, such as the inactivation of alpha-1-antichymotrypsin, glycyl-glycine potentiated inhibitor cleavage. In contrast, alpha-1-proteinase inhibitor was not inactivated by gingipains R and only underwent proteolytic degradation during boiling in reducing SDS-polyacrylamide gel electrophoresis treatment buffer. Similarly, native type I collagen was completely resistant to cleavage by gingipains but readily degraded after denaturation. Together, these data explain much of the controversy regarding gingipains structure and substrate specificity and indicate that these enzymes function as P. gingivalis virulence factors by proteolysis of selected target proteins rather than random degradation of host connective tissue components.

Effects of iron chelates on the transferrin-free culture of rat dermal fibroblasts through active oxygen generation.

Effects of nonchelating and chelating agents at 10 mM on the serum-free culture of rat dermal fibroblasts were investigated. A strong iron-chelating agent, iminodiacetic acid (IDA), and a weak one, dihydroxyethylglycine (DHEG), decreased iron permeation into preconfluent fibroblasts. A weak iron-chelating agent, glycylglycine (GG), a nonchelating agent, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and human apotransferrin (10 micrograms/ml) increased the permeation with time. Iron may be essential for survival of fibroblasts because subconfluent fibroblasts exposed to 100 microM FeSO4 in combination with transferrin, HEPES, or GG significantly decreased to release lactate dehydrogenase into the medium. Superoxide dismutase and dimethyl sulfoxide blocked the enzyme release, suggesting that superoxide and hydroxyl radical induce cellular damage but hydrogen peroxide (H2O2) generated by superoxide dismutation does not. GG significantly reduced H2O2 cytotoxicity. DHEG acted as a potent promoter of the iron-stimulated cellular damage if ascorbate or H2O2 was added to the medium. FeSO4 and FeCl3 (50 to 100 microM) individually combined with IDA maximally promoted fibroblast proliferation. Ascorbate increased formation of thiobarbituric acid-reactive substances from deoxyribose in the medium supplemented with FeSO4 and either IDA or DHEG. Conversely, ascorbate decreased the formation in the medium with FeSO4 and with or without other agents. Fibroblast proliferation may thus be stimulated through the active oxygen generation mediated by a redox-cycling between Fe3+ and Fe2+, which are dissolved in the medium at a high concentration, rather than through delivery of iron into the cells.

Alteration in gamma-glutamyl transpeptidase activity and messenger RNA of human prostate carcinoma cells by androgen.

Concentrations of the synthetic androgen R1881 that correspond to physiologically relevant concentrations of 5alpha-dihydrotestosterone are capable of altering the activity of gamma-glutamyl transpeptidase (GGT) in human prostate carcinoma cells. GGT activity of the androgen-responsive prostate cancer cell line LNCaP increases >50% above that of the control after a 72-h exposure to 1 nM R1881. This elevation in GGT activity occurs as early as 48 h after treatment and is maintained for at least 96 h. Loss of glutathione (GSH) from media and accumulation of intracellular GSH of cells pretreated with 1 nM R1881 occur at a higher rate than in control cells, suggesting that a greater rate of GSH salvage is associated with the increased GGT activity. Immunohistochemical staining detects an increase in GGT-positive staining in cells treated with 1 nM R1881 for 72 h. Steady-state mRNA levels for GGT are elevated above those of the control 24-72 h after treatment. R1881 has no effect on the GGT activity of the androgen-independent prostate cell line DU145. Growth of LNCaP but not DU145 cells is inhibited by 1 nM R1881 compared to that of the control. Inhibitors of GGT activity, acivicin and serine-borate, are capable of dampening or blocking the effect of R1881 on growth. Growth of LNCaP cells treated with 1 nM R1881 plus 100 mM glycylglycine, a stimulator of GGT activity, is inhibited to a greater extent than the growth of LNCaP cells treated with R1881 alone. These data demonstrate that androgens can elevate GGT activity and increase GGT mRNA and protein levels in human prostate carcinoma cells. In addition, compounds able to alter GGT activity are capable of altering androgen-related growth effects.

Drug conjugate of doxorubicin with glutathione is a potent reverser of multidrug resistance in rat hepatoma cells.

A recent study has suggested that degraded adducts smaller than 2 kDa in molecular weight of bovine serum albumin (BSA)-conjugated doxorubicin (DXR) (BSA-DXR) might exhibit cytotoxicity against multidrug resistant (MDR) cells. To investigate this notion further, intracellular accumulation and cytotoxicity of DXR coupled to several small peptides, such as glycylglycine (diGly), glycylglycylglycine (triGly), reduced glutathione (GSH) and oxidized glutathione (GSSG), were investigated using DXR-sensitive (AH66P) and DXR-resistant (AH66DR) rat hepatoma cell lines. Against both AH66P and AH66DR cells, diGly-conjugated DXR (diGly-DXR) and triGly-conjugated DXR (triGly-DXR) demonstrated the same cytotoxic activity as DXR, and the accumulation of both conjugates in the two cell lines was almost similar to that of DXR. After treatment of AH66DR cells with 5 microM verapamil [an inhibitor of P-glycoprotein (Pgp)], the intracellular levels of diGly-DXR and triGly-DXR were markedly increased and consequent cytotoxicity was improved. On the other hand, GSH-conjugated DXR (GSH-DXR) showed 9- and 7.5-fold more cytotoxic activity than BSA-DXR against AH66P and AH66DR cells, respectively. GSH-DXR accumulated rapidly in AH66DR cells, probably by the same mechanism as in AH66P cells, because the treatment of AH66DR cells with verapamil did not cause a significant increase in the intracellular drug level as compared with that in cells treated without verapamil. The levels of cytotoxicity and accumulation of GSSG-DXR were the same as those of BSA-DXR for both cell lines. These results indicate that GSH-DXR exerts potent cytotoxicity against both cell lines among the peptide DXR conjugates examined because of the rapid uptake and high accumulation of GSH-DXR similar to that of DXR without efflux.

Protective properties of amino acids in liver preservation: effects of glycine and a combination of amino acids on anaerobic metabolism and energetics.

BACKGROUND/AIMS/METHODS: In this study, we investigated the hepatoprotective effects of three storage solutions containing glycine (180 mM), glycylglycine (180 mM), and a mixture of 20 amino acids (combined concentration of 180 mM) on energy metabolism and levels of glucose and lactate (as an index of glycolytic flux) in rat livers. All effects were compared to those of livers flushed/stored with a modified University of Wisconsin solution. RESULTS: Glycine-treatment showed no improvement in liver energetics (ATP, ADP, AMP) and lactate accumulation; this solution had the lowest buffering capacity of the four tested (approximately 30% of the University of Wisconsin solution). The glycylglycine solution had the highest buffering capacity of the four solutions tested (including University of Wisconsin solution). Complete titration of the glycine-, combined amino acids-, and University of Wisconsin solutions (from 8.0 to pH = 6.0) resulted in a minor decrease in glycylglycine buffer pH; pH dropped by 0.2 pH units. In glycylglycine-treated livers, energetics showed an improvement over the first 1 h cold storage; ATP and 'energy charge' values remained high and ADP levels (and consequently total adenylate contents) were 0.7-2.4 micro mol/g greater than livers stored in University of Wisconsin solution. A 2-fold increase in lactate accumulation suggested that the improvement in liver energetics for the glycylglycine buffer was due to maintained flux through glycolysis brought about by enhanced buffering capacity. The solution containing a combination of amino acids exhibited maximum maintenance of liver energetics via increased glycolytic flux, despite its slightly inferior buffering capacity (85% of University of Wisconsin solution). ATP levels were maintained over the first 2 h storage and ADP levels (and consequently, total adenylate contents) were 1.2-2.1 micro mol/g greater than University of Wisconsin solution-treated livers during the entire 24 h storage period. Energy charge values for livers treated with the combination of amino acids were also significantly higher than with glycine-, glycylglycine- and University of Wisconsin solution-treatment; even at 24 h, energy charge was 0.36 (comparable to only 4 h storage in University of Wisconsin solution). CONCLUSIONS: Our data suggest that a combination of amino acids may be required for maximum protection of the liver, and furthermore there may be several independent mechanisms, including buffering capacity, responsible for cytoprotection of the liver during cold storage.

Dialysis solutions buffered with lactate or bicarbonate: in vitro comparison of two dialysis solutions on human peritoneal cell growth from ESRD and non-ESRD patients.

In order to evaluate the injury to the mesothelial cell layer during long-term peritoneal dialysis (PD), a dialysis solution (solution A), buffered with bicarbonate, stabilized with 10 mmol/L glycylglycine, and sterilized by filtration (0.22 micron double filtration, pH = 7.4), was compared to traditional heat sterilized lactate solution (solution B) on human mesothelial cell cultures. The respective effects of both solutions were evaluated on first passage cells by 3H thymidine incorporation after 72-, 96-, 120-, and 144-h contact. Mesothelial cells to be cultured were obtained from the omentum biopsies of 7 end-stage renal disease (ESRD) patients (during first peritoneal catheter placement) and from 7 non-ESRD patients undergoing abdominal surgery. Solution A (diluted 1/5) induced a time-dependent stimulation of growth in 6 cases of ESRD patient cell cultures, and inhibition occurred only in 1 case. Stimulation was also observed in 5 non-ESRD patient cell cultures, and no effect occurred in 2 cases. Solution B inhibited growth in all the cultures except in 1 case of an ESRD patient in which no effect was observed. This study shows that solution A induced mesothelial cell proliferation, while an inhibitory effect of solution B was observed. No significant differences were observed between the sensitivity of mesothelial cells from ESRD and non-ESRD patients. Further analysis will be carried out to identify precisely the cause of the differences observed: buffer or glycylglycine by themselves and/or glucose by-products.

Gamma-glutamyl transferase ectoactivity in the intact rat liver: effect of chronic alcohol consumption.

The localization of gamma-glutamyl transferase (GGT) in the intact rat liver was studied by a new approach in which the chromogenic gamma-glutamyl donor substrate of GGT gamma-glutamyl-p-nitroanilide is perfused through the portal vein to yield p-nitroaniline, which is monitored spectrophotometrically. GGT activity was markedly increased by the gamma-glutamyl acceptors glycyl-glycine, cystine and methionine, following Michaelis-Menten kinetics. Infusion of glutathione (GSH), the natural substrate of GGT, was shown to markedly reduce or to abolish the formation of p-nitroaniline without entering the liver cells, indicating the existence of a GGT ectoactivity accessible to the sinusoidal circulation. This ectoenzyme was shown to remove significant amounts of GSH from the circulation, amounting, in the naive rat, to 20-25% of the net rate at which GSH is contributed by the liver into the circulation. Chronic alcohol consumption is known to increase hepatic GGT activity, although the biological significance of such an effect remains unknown. Present studies show that chronic administration of alcohol to rats leads to a significant (40-75%) increase in hepatic GGT ectoactivity. GGT ectoactivity significantly correlates with total liver GGT, both in control and alcohol-treated animals (r = .76 and r = .90, respectively). Livers of alcohol-fed rats showed an increased (80-110%) capacity to remove circulating GSH which strongly correlated with total liver GGT (r = .96; p less than 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)

Glutathione mutagenesis in Salmonella typhimurium TA100: dependence on a single enzyme, gamma-glutamyltranspeptidase.

Glutathione was mutagenic in Salmonella typhimurium strain TA100 in the presence of purified mammalian gamma-glutamyltranspeptidase. Glutathione disulfide, gamma-glutamyl glutamic acid, and S-methyl-glutathione were not mutagenic under the same conditions. Glutathione-mediated, gamma-glutamyltranspeptidase-dependent mutagenesis of TA100 cells was inhibited by serine-borate complex, a known gamma-glutamyltranspeptidase inhibitor, and potentiated by glycylglycine, a known gamma-glutamyltranspeptidase enhancer. It is concluded that this enzyme is necessary and sufficient to activate glutathione to a mutagen.

Cytotoxicity of S-(1,2-dichlorovinyl)glutathione and S-(1,2-dichlorovinyl)-L-cysteine in isolated rat kidney cells.

S-(1,2-Dichlorovinyl)glutathione (DCVG) and S-(1,2-dichlorovinyl)-L-cysteine (DCVC) produced time- and concentration-dependent cell death in isolated rat kidney proximal tubular cells. AT-125 blocked and glycylglycine potentiated DCVG toxicity, indicating that metabolism by gamma-glutamyltransferase is required. S-(1,2-Dichlorovinyl)-L-cysteinylglycine, a putative metabolite of DCVG, also produced cell death, which was prevented by 1,10-phenanthroline, phenylalanylglycine, and aminooxyacetic acid, inhibitors of aminopeptidase M, cysteinylglycine dipeptidase, and cysteine conjugate beta-lyase, respectively. Aminooxyacetic acid and probenecid protected against DCVC toxicity, indicating a role for metabolism by cysteine conjugate beta-lyase and organic anion transport, respectively. DCVC produced a small decrease in cellular glutathione concentrations and did not change cellular glutathione disulfide concentrations or initiate lipid peroxidation. DCVC caused a large decrease in cellular glutamate and ATP concentrations with a parallel decrease in the total adenine nucleotide pool; these changes were partially prevented by aminooxyacetic acid. Both DCVG and DCVC inhibited succinate-dependent oxygen consumption, but DCVC had no effect when glutamate + malate or ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine were the electron donors. DCVC inhibited mitochondrial, but not microsomal, Ca2+ sequestration. These alterations in mitochondrial function were partially prevented by inhibition of DCVG and DCVC metabolism and were strongly correlated with decreases in cell viability, indicating that mitochondria may be the primary targets of nephrotoxic cysteine S-conjugates.

Effect of dipolar ions on the entropy-driven polymerization of tobacco mosaic virus protein.

The effect of the dipolar ions, glycine, glycylglycine, and glycylglycylglycine on the polymerization of tobacco mosaic virus (TMV) protein has been studied by the methods of light scattering and ultracentrifugation. All three dipolar ions promote polymerization. The major reaction in the early stage is transition from the 4 S to the 20 S state. As in the absence of dipolar ions, the polymerization is enhanced by an increase in temperature; it is endothermic and therefore entropy-driven. The effect of the dipolar ions can be understood in terms of their action as salting-out agents; they increase the activity coefficient of TMV A protein, the 4 S material, and thus shift the equilibrium toward the 20 S state. The salting-out constants, K, for the reaction in 0.10 ionic strength phosphate buffer at pH 6.7 was found by the light scattering method to be 1.6 for glycine, 2.5 for glycylglycine, and 2.5 for glycylglycylglycine. A value of 2.7 was obtained by the ultracentrifugation method for glycylglycine in phosphate buffer at 0.1 ionic strength and pH 6.8 at 10 degrees C. For both glycine and glycylglycine, K increases when the ionic strength of the phosphate buffer is decreased. This result suggests that electrolytes decrease the activity coefficient of the dipolar ions, a salting-in phenomenon. However, the salting-in constants evaluated from these results are substantially higher than those previously determined by solubility measurements. The effect of glycine and glycylglycine on polymerization was studied at pH values between 6.2 and 6.8. The effectiveness of both dipolar ions is approximately 50% greater at pH 6.8 than at pH 6.2. The variation of the extent of polymerization with pH in the presence of the dipolar ions is consistent with the interpretation that approximately one hydrogen ion is bound for half of the polypeptide units in the polymerized A protein.

Isolation and properties of gamma-glutamyltranspeptidase from Morris hepatoma 5123D.

gamma-Glutamyltranspeptidase was purified 600-fold from Morris hepatoma 5123D by six-step procedure. Its apparent molecular weight estimated by centrifugation in sucrose gradient with Triton X-100 amounts to 108 000. Some dipeptides particularly glycylglycine and several amino acids considerably increase the enzyme activity but L-serine with borate decreases it. Usually transfer activity of the enzyme towards gamma-L-glutamyl substrates was much higher than hydrolytic. The best substrate for the hepatoma enzyme is reduced glutathione.

Alpha-aminoisobutyric acid transport into human glia and glioma cells in culture.

The AIB transport into human glia and glioma cells in culture has been studied. Because of the high affinity of AIB to the plastic culture dishes, a special washing technique had to be developed. With this technique, it was possible to perform transport experiments in a single plate containing about one million cells. The cells were viable, intact and adhered to the supporting medium throughout the experiment. The AIB transport into both types of cells was Na+-dependent and showed saturation kinetics when the small component of the transport due to diffusion had been subtracted. The AIB transport capacity of neoplastic glioma cells was 3.6 times higher than that of glia cells. This difference was related to the Vmax-values for the two types of cells. The apparent Km-values were the same. Inhibition experiments with other amino acids support the view that AIB is transported via System A in both glia and glioma cells. Sulfhydryl reagents (ethacrynic acid and NEM) and cytochalasin B clearly inhibited the AIB transport into glia cells whereas the effect on glioma cells was minimal.