Selective and synergistic activity of L-S,R-buthionine sulfoximine on malignant melanoma is accompanied by decreased expression of glutathione-S-transferase

L-buthionine-S,R-sulfoximine (BSO) selectivley inhibits glutathione (GSH) synthesis. Malignant melanoma may be uniquely dependent on GSH and its linked enzymes, glutathione S-transferase (GST) and GSH-peroxidase, for metabolism of reactive orthoquinones and peroxides produced during melanin synthesis. We compared the in vitro effects of BSO on melanoma cell lines and fresh melanoma specimens (n = 118) with breast and ovarian cell lines and solid tumors (n = 244). IC50 values (microM) for BSO on melanoma, breast and ovarian tumor specimens were 1.9, 8.6, and 29, respectively. The IC90 for melanoma was 25.5 microM, a level 20-fold lower than steady state levels achieved clinically. The sensitivity of individual specimens of melanoma correlated with their melanin content (r = 0.63). BSO synergistically enhanced BCNU activity against melanoma cell lines and human tumors. We followed GSH levels, GST enzyme activity, GST isoenzyme profiles and mRNA levels after BSO. BSO (50 microM) treatment for 48 hr resulted in a 95% decrease in ZAZ and M14 melanoma cell line GSH levels, and a 60% decrease in GST enzyme activity. GST-mu protein and mRNA levels were significantly reduced in both cell lines. GST-pi expression was unaffected. These data suggest that BSO action on melanoma may be related to GSH depletion, diminishing the capacity to scavenge toxic metabolites produced during melanin synthesis. We report here for the first time that BSO enhancement of alkylator action may be related in part to down regulation of GST. BSO may be a clinically useful adjunct in the treatment of malignant melanoma.     

Leukotriene C4 is a tight-binding inhibitor of microsomal glutathione transferase-1. Effects of leukotriene pathway modifiers

Microsomal glutathione transferase-1 (MGST-1) is an abundant protein that catalyzes the conjugation of electrophilic compounds with glutathione, as well as the reduction of lipid hydroperoxides. Here we report that leukotriene C4 is a potent inhibitor of MGST-1. Leukotriene C4 was found to be a tight-binding inhibitor, with a Ki of 5.4 nM for the unactivated enzyme, and 9.2 nM for the N-ethylmaleimide activated enzyme. This is the first tight-binding inhibitor characterized for this enzyme. Leukotriene C4 was competitive with respect to glutathione and non-competitive toward the second substrate, CDNB. Analysis of stoichiometry supports binding of one molecule of inhibitor per homotrimer. Leukotrienes A4, D4, and E4 were much weaker inhibitors of the purified enzyme (by at least 3 orders of magnitude). Leukotriene C4 analogues, which have been developed as antagonists of leukotriene receptors, were found to display varying degrees of inhibition of MGST-1. In particular, the cysteinyl-leukotriene analogues SKF 104,353, ONO-1078, and BAYu9773 were strong inhibitors (IC50 values: 0.13, 3. 7, and 7.6 microM, respectively). In view of the partial structural similarity between MGST-1, leukotriene C4 synthase, and 5-lipoxygenase activating protein (FLAP), it was of interest that leukotriene C4 synthesis inhibitors (which antagonize FLAP) also displayed significant inhibition (e.g. IC50 for BAYx1005 was 58 microM). In contrast, selective 5-lipoxygenase inhibitors such as zileuton only marginally inhibited activity at high concentrations (500 microM). Our discovery that leukotriene C4 and drugs developed based on its structure are potent inhibitors of MGST-1 raises the possibility that MGST-1 influences the cellular processing of leukotrienes. These findings may also have implications for the effects and side-effects of drugs developed to manipulate leukotrienes.     

Catalytic contribution of flap-substrate hydrogen bonds in "HIV-1 protease" explored by chemical synthesis

An analogue of "HIV-1 protease" was designed in which the ability to donate important water-mediated hydrogen bonds to substrate was precisely and directly deleted. Chemical ligation of unprotected peptide segments was used to synthesize this "backbone-engineered" enzyme. The functionally relevant amide -CONH- linkage between residues Gly49-Ile50 in each flap of the enzyme was replaced by an isosteric thioester -COS- bond. The backbone-engineered enzyme had normal substrate specificity and affinity (Km). However, the catalytic activity (kcat) was reduced approximately 3000-fold compared to the native amide bond-containing enzyme. Inhibition by the reduced peptide bond substrate analogue MVT-101 was unaffected compared with native enzyme. By contrast, the normally tight-binding hydroxyethylamine inhibitor JG-365 bound to the backbone-engineered enzyme with an approximately 2500-fold reduction in affinity. The reduced catalytic activity of the -Gly49-psi(COS)-Ile50-backbone-engineered enzyme analogue provides direct experimental evidence to support the suggestion that backbone hydrogen bonds from the enzyme flaps to the substrate are important for the catalytic function of the HIV-1 protease.     

Busulfan-glutathione conjugation catalyzed by human liver cytosolic glutathione S-transferases

We have examined the catalytic activity of glutathione S-transferases (GST) in the conjugation of busulfan with glutathione (GSH) in human liver cytosol, purified human liver GST, and cDNA-expressed GST-alpha 1-1. Human liver microsomes and cytosol were incubated with 40 microM busulfan and 1 mM GSH. Cytosol catalyzed the formation of the GSH-busulfan tetrahydrothiophenium ion (THT+) in a concentration-dependent manner, whereas microsomes lacked activity. The total and spontaneous rates of THT+ formation increased with pH (pH range, 6.50-7.75), with the maximum difference at pH 7.4. Due to the limited aqueous solubility of busulfan, a K(m) for busulfan was not determined. The intrinsic clearance (Vmax/K(m)) of busulfan conjugation was 0.167 microliter/min/mg with 50-1200 microM busulfan and 1 mM GSH. GSH Vmax and K(m) for busulfan conjugation were 30.6 pmol/min/mg and 312 microM, respectively. Ethacrynic acid (0.03-15 microM) inhibited cytosolic busulfan-conjugating activity with 40 microM busulfan and 1 mM GSH. Enzyme-mediated THT+ formation was decreased 97% by 15 microM ethacrynic acid with no effect on the spontaneous reaction. In incubations with affinity-purified liver GST and GST-alpha 1-1, the intrinsic clearance for busulfan conjugation was 0.87 and 2.92 microliters/min/mg, respectively. Busulfan is a GST substrate with a high K(m) relative to concentrations achieved clinically (1-8 microM).     

Inhibition of N-nitrosodimethylamine demethylase in rat and human liver microsomes by isothiocyanates and their glutathione, L-cysteine, and N-acetyl-L-cysteine conjugates

Natural and synthetic isothiocyanates and their conjugates were examined for their inhibitory effects toward rat and human liver microsomal N-dimethylnitrosoamine demethylase (NDMAd) activity using a radiometric NDMAd assay. Substrate concentrations of 30 and 60 microM were used to probe the activity of cytochrome P4502E1 isozyme through the alpha-hydroxylation of NDMA. It was found that alkyl isothiocyanates such as sulforaphane and allyl isothiocyanate displayed very weak inhibition, whereas the arylalkyl isothiocyanates such as benzyl and phenethyl isothiocyanate showed significant inhibition toward rat liver NDMAd activity with IC50 values of 9.0 and 8.3 microM, respectively. More interestingly, glutathione conjugates of benzyl, phenethyl, and 6-phenylhexyl isothiocyanates all inhibited NDMAd at the comparable concentrations. In the phenethyl isothiocyanate conjugates series, there exist marked differences in their inhibitory activity; i.e., its conjugates with L-cysteine (IC50 = 4.3 microM) and with glutathione (IC50 = 4.0 microM) are more potent than its conjugate of N-acetylcysteine (IC50 = 24.0 microM). The same trend was also observed for the human liver microsomal NDMAd activity. The half-lives of these conjugates were determined in the presence of other free thiols from L-cysteine or glutathione using an HPLC system. It was shown that isothiocyanates are released from their conjugates and react with the free thiols present in the solution. The longer half-life of N-acetylcysteine conjugate of phenethyl isothiocyanate as compared to the other conjugates is consistent with its lower inhibitory activity. The inhibition of NDMAd, and therefore cytochrome P4502E1, by isothiocyanate conjugates is most likely due to the action of the free isothiocyanates released from the conjugates. Since cytochrome P4502E1 and other isozymes play important roles in the activation of the tobacco-specific nitrosoamines, these results provide a basis for investigating the potential of isothiocyanate conjugates as chemopreventive agents.     

Glutathione-doxorubicin conjugate expresses potent cytotoxicity by suppression of glutathione S-transferase activity: comparison between doxorubicin-sensitive and -resistant rat hepatoma cells

The cytotoxic mechanism of a conjugate of doxorubicin (DXR) and glutathione (GSH) via glutaraldehyde (GSH-DXR) was investigated using DXR-sensitive (AH66P) and -resistant (AH66DR) rat hepatoma cells. GSH-DXR accumulated in AH66DR cells as well as in AH66P cells without efflux by P-gp and exhibited the potent cytocidal activity against both cells compared with DXR. To examine whether thiol from GSH-DXR affected the expression of cytotoxicity, two conjugates of DXR, with modified peptides containing alanine or serine substituted for cysteine in GSH were prepared and their cytotoxicities determined. Substitution of these amino acids for cysteine resulted in an approximately two- to fourfold reduction in cytotoxic activity against both cell lines compared with the effect of GSH-DXR. Depletion of intracellular GSH by treatment of both cells with buthionine sulphoximine did not change the cytotoxic activity of DXR, BSA-DXR or GSH-DXR. By co-treating the cells with tributyltin acetate, an inhibitor of glutathione S-transferase (GST), and either DXR, BSA-DXR or GSH-DXR, the cytotoxicity was markedly increased. Interestingly, GSH-DXR showed non-competitive inhibition of GST activity and its IC50 value was 1.3 microM. These results suggested that the inhibition of GST activity by GSH-DXR must be an important contribution to the expression of potent cytotoxicity of the drug.     

A new approach to the design of novel inhibitors of Na+,K+-ATPase: 17alpha-substituted seco-D 5beta-androstane as cassaine analogues

A new three-dimensional model for the relative binding mode of cassaine 1 and digitoxigenin 2 at the digitalis receptor site is proposed on the basis of the structural and conformational similarities among 1, 2 and its 14,15-seco analogues 3 and 4. Accordingly, the speculation that also 17alpha-substituted derivatives of the digitalis 5beta,14beta-androstane skeleton could efficiently bind to the Na+,K+-ATPase receptor is put forward and verified through the synthesis of some related compounds. The binding affinity shown by 2-(N,N-dimethylamino)ethyl 3beta, 14-dihydroxy-5beta,14beta-androstane-17alpha-acrylate 6 (IC50 = 5.89 microM) and, much more significantly, by the corresponding 14, 15-seco-14-oxo derivative 9 (IC50 = 0.12 microM) substantiates the new hypothesis and opens new prospects to the design of novel inhibitors of Na+,K+-ATPase as potential positive inotropic compounds.     

Haloenol lactone is a new isozyme-selective and active site-directed inactivator of glutathione S-transferase

A haloenol lactone derivative has been synthesized and found to be an isozyme-selective and active site-directed inactivator of glutathione S-transferase (GST). Preincubation of the haloenol lactone (100 microM) with murine Alpha, Mu, or Pi GST isozyme (1.0 microM) at pH 6.5, 37 degrees C resulted in time-dependent loss of enzyme activity with highly selective inhibition of the Pi isozyme (t1/2, approximately 2 min). In a separate experiment, a 10-fold excess of the lactone was incubated with GST-Pi isozyme at 37 degrees C for 3 h, followed by dialysis against Nanopure water. GST activity lost upon incubation with the lactone could not be restored by exhaustive dialysis, and only 8% of enzyme activity for the modified GST remained relative to the control that was treated identically except the lactone was omitted from the incubation. Both control and modified GST were characterized using electrospray ionization mass spectrometry. No native GST (23,478 Da) was observed in the spectrum of modified GST. Instead, protein incubated with the lactone exhibited an increase in molecular mass of 230 Da relative to control GST. The lactone (100 microM) was incubated with GST Pi isozyme (1.0 microM) in the presence of the competitive inhibitor S-hexylglutathione (10 microM), which suppressed time-dependent inhibition of GST by the lactone. The results suggest that this haloenol lactone is an irreversible and active site-directed inhibitor of GST that appears to inhibit the enzyme through two consecutive steps of nucleophilic attack.     

Interference of S-alkyl derivatives of glutathione with brain ionotropic glutamate receptors

The effects of glutathione, glutathione sulfonate and S-alkyl derivatives of glutathione on the binding of glutamate and selective ligands of ionotropic N-methyl-D-aspartate (NMDA) and non-NMDA receptors were studied with mouse synaptic membranes. The effects of glutathione and its analogues on 45Ca2+ influx were also estimated in cultured rat cerebellar granule cells. Reduced and oxidized glutathione, glutathione sulfonate, S-methyl-, -ethyl-, -propyl-, -butyl- and -pentylglutathione inhibited the Na+-independent binding of L-[3H]glutamate. They strongly inhibited also the binding of (S)-2-amino-3-hydroxy-5-[3H]methyl-4-isoxazolepropionate [3H]AMPA (IC50 values: 0.8-15.9 microM). S-Alkylation of glutathione rendered the derivatives unable to inhibit [3H]kainate binding. The NMDA-sensitive binding of L-[3H]glutamate and the binding of 3-[(R)-2-carboxypiperazin-4-yl][1,2-(3)H]propyl-1-phosphonate ([3H]CPP, a competitive antagonist at NMDA sites) were inhibited by the peptides at micromolar concentrations. The strychnine-insensitive binding of the NMDA coagonist [3H]glycine was attenuated only by oxidized glutathione and glutathione sulfonate. All peptides slightly enhanced the use-dependent binding of [3H]dizocilpine (MK-801) to the NMDA-gated ionophores. This effect was additive with the effect of glycine but not with that of saturating concentrations of glutamate or glutamate plus glycine. The glutamate- and NMDA-evoked influx of 45Ca2+ into cerebellar granule cells was inhibited by the S-alkyl derivatives of glutathione. We conclude that besides glutathione the endogenous S-methylglutathione and glutathione sulfonate and the synthetic S-alkyl derivatives of glutathione act as ligands of the AMPA and NMDA receptors. In the NMDA receptor-ionophore these glutathione analogues bind preferably to the glutamate recognition site via their gamma-glutamyl moieties.     

Renovasculopathies of hypertension in Mexico City

The effect of pH and acyl-CoA chain length on the conversion of the malonyl-CoA-sensitive carnitine palmitoyltransferase (CPT-I/CPTo) to a high-affinity, malonyl-CoA-inhibited state using a particle derived from rat heart mitochondria was determined. Preincubation with malonyl-CoA for one minute in the absence of acyl-CoA substrate lowers the IC50 for malonyl-CoA from 2 microM, 14 microM, and 15 microM at pH 7.4 to 15 nM, 14 nM, and 14 nM for decanyl-, lauryl-, and palmitoyl-CoA, respectively. Reducing the pH to 7.1 and 6.8 had little effect on the transition to the high affinity, malonyl-CoA-inhibited state. Preincubation of malonyl-CoA with the acyl-CoA, but not with L-carnitine, prevented the transition to the high affinity, malonyl-CoA-inhibited state.     

DNA-Directed alkylating agents. 7. Synthesis, DNA interaction, and antitumor activity of bis(hydroxymethyl)- and bis(carbamate)-substituted pyrrolizines and imidazoles

A series of bis(hydroxymethyl)-substituted imidazoles, thioimidazoles, and pyrrolizines and related bis(carbamates), linked to either 9-anilinoacridine (intercalating) or 4-(4-quinolinylamino)benzamide (minor groove binding) carriers, were synthesized and evaluated for sequence-specific DNA alkylation and cytotoxicity. The imidazole and thioimidazole analogues were prepared by initial synthesis of [(4-aminophenyl)alkyl]imidazole-, thioimidazole-, or pyrrolizine dicarboxylates, coupling of these with the desired carrier, and reduction to give the required bis(hydroxymethyl) alkylating moiety. The pyrrolizines were the most reactive alkylators, followed by the thioimidazoles, while the imidazoles were unreactive. The pyrrolizines and some of the thioimidazoles cross-linked DNA, as measured by agarose gel electrophoresis. Strand cleavage assays showed that none of the compounds reacted at purine N7 or N3 sites in the gpt region of the plasmid gpt2Eco, but the polymerase stop assay showed patterns of G-alkylation in C-rich regions. The corresponding thioimidazole bis(carbamates) were more selective than the bis(hydroxymethyl) pyrrolizines, with high-intensity bands at 5'-NCCN, 5'-NGCN and 5'-NCGN sequences in the PCR stopping assay ( indicates block sites). The data suggest that these targeted compounds, like the known thioimidazole bis(carbamate) carmethizole, alkylate exclusively at guanine residues via the 2-amino group, with little or no alkylation at N3 and N7 guanine or adenine sites. The cytotoxicities of the compounds correlated broadly with their reactivities, with the bis(hydroxymethyl)imidazoles being the least cytotoxic (IC50s >1 microM; P388 leukemia) and with the intercalator-linked analogues being more cytotoxic than the corresponding minor-groove-targeted ones. This was true also for the more reactive thioimidazole bis(carbamates) (IC50s 0.8 and 11 microM, respectively), but both were more active than the analogous "untargeted" carmethizole (IC50 20 microM). The bis(hydroxymethyl)pyrrolizine analogues were the most cytotoxic, with IC50s as low as 0.03 microM.     

Stereoselective conjugation of prostaglandin A2 and prostaglandin J2 with glutathione, catalyzed by the human glutathione S-transferases A1-1, A2-2, M1a-1a, and P1-1

Prostaglandins containing an alpha,beta-unsaturated keto group, such as prostaglandin A2 (PGA2) and prostaglandin J2 (PGJ2), inhibit cell proliferation. These cyclopentenone prostaglandins may be conjugated with GSH chemically or enzymatically via glutathione S-transferases, and this has been suggested to result in inhibition of the antiproliferative mode of action. In the present study, the role of the major human GSTs in the conjugation of PGA2 and PGJ2 with GSH was investigated with purified enzymes, i.e., the Alpha-class enzymes GST A1-1 and GST A2-2, the Mu-class enzyme GST M1a-1a, and the Pi-class enzyme GST P1-1. The GSH conjugates were separated from the parent compound by HPLC and identified by fast atom bombardment mass spectrometry and 1H-NMR. Two GSH conjugates were found for both PGA2 and PGJ2, the R- and S-GSH conjugates of both prostaglandins. Incubation experiments with PGA2 and PGJ2 (70-600 microM) clearly showed the role of individual GSTs in the conjugation of PGA2 and PGJ2. Compared to the chemical reaction, enzyme activities towards PGA2 were up to 5.4 times as high (GSTA1-1) at the lowest concentration (70 microM), while at the highest concentration (600 microM) enzyme activities were up to 3.0 times as high (GST P1-1). For PGJ2, enzyme activities were up to 4.3 (GSTM1a-1a, 70 microM) and up to 3.1 (GSTM1a-1a, 600 microM) times as high. As expected, similar amounts of the R- and S-conjugates of both prostaglandins were found in the chemical reaction. Striking stereoselectivities in conjugating activities were observed for GST A1-1 and GST P1-1. GST A1-1 favors the formation of the R-GSH conjugates of both prostaglandins. GST P1-1 showed a clear selectivity with regard to the formation of the S-GSH conjugate of PGA2. However, this selectivity was not found for the formation of the S-GSH conjugate of PGJ2. GSTM1a-1a showed no stereoselectivity with regard to the GSH conjugation of both PGA2 and PGJ2. GSTA2-2 only showed some minor formation of the R-GSH conjugate of PGJ2. The possible implications of the observed stereoselectivity on the effects of PGA2 and PGJ2 are discussed.     

Synthesis of structural analogues of leukotriene B4 and their receptor binding activity

Structural analogues of leukotriene B4 (LTB4) were designed based on the plausible conformation of LTB4 (1). Joining C-7-C-9 of the conformer A or B into an aromatic ring system led to the discovery of benzene analogues 2, 4 and 6a. Joining C-4-C-9 of the conformer C or D into an aromatic ring system led to the discovery of analogues 3, 5 and 7. The compounds examined in this study were evaluated as to their inhibition of [3H] LTB4 binding to human neutrophils, and by a secondary intact human neutrophil functional assay for agonist/antagonist activity. The first analogues prepared, compounds 2-7, demonstrated moderate potency in the LTB4 receptor binding assay. The modification of these compounds by the introduction of another substituent into the aromatic ring produced a marked increase in receptor binding (28c, IC50 = 0.020 microM; 38c, IC50 = 0.020 microM; 52a, IC50 = 0.020 microM; 52b, IC50 = 0.018 microM). Most of these structural analogues of LTB4 demonstrated agonist activity. Of the analogues prepared in this study, only compound 57 demonstrated weak LTB4 receptor antagonist activity, at 10 microM.     

Identification and characterization of high and low affinity transport systems for reduced glutathione in liver cell canalicular membranes

Driving forces and substrate specificity for transport of reduced glutathione (GSH) across rat liver cell canalicular membrane were examined in vesicles isolated from this plasma membrane domain. In contrast to previous studies indicating a single saturable component of canalicular GSH transport, the present results demonstrate the presence of both high and low affinity components with apparent Km values of 0.24 +/- 0.04 and 17.4 +/- 2.1 mM and Vmax values of 0.09 +/- 0.01 and 2.3 +/- 0.3 nmol.mg-1.20 s-1, respectively. The Km values in two previously published reports are discordant, 0.33 versus 16 mM, but are comparable with the two transport components identified in the present study. To further characterize these GSH transport mechanisms, [3H]GSH uptake by canalicular vesicles was measured at concentrations of 50 microM, where transport is expected to occur largely on the high affinity component, and at 5 mM, where the low affinity system should predominate. Neither component of GSH transport was affected by ATP or a Na+ gradient, but both were stimulated by a valinomycin-induced membrane potential, indicating electrogenic transport pathways. The high affinity component was cis-inhibited by glutathione S-conjugates (1 mM), other gamma-glutamyl compounds (5 mM), and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (0.1 mM), whereas these agents had no effect on the low affinity component at similar inhibitor concentrations. Sulfobromophthalein (BSP, 0.1 mM) inhibited both GSH transport components. However, neither component was affected by taurocholate (0.5 mM) or L-glutamate (10 mM). The inhibition by S-butylglutathione, the GSH analogue ophthalmic acid, and by BSP was competitive in nature, although BSP also produced a slight decrease in Vmax, suggesting a mixed type of inhibition. Ophthalmic acid and some glutathione S-conjugates were also able to trans-stimulate high affinity GSH uptake. These results indicate the presence of at least two ATP-independent, electrogenic glutathione transport mechanisms on the canalicular membrane; the high affinity component may function to deliver some glutathione S-conjugates, gamma-glutamyl compounds, and other anions into bile, whereas the low affinity system probably functions as a high capacity transporter capable of delivering large amounts of GSH into bile.     

Uptake of L-glutamate into synaptic vesicles: competitive inhibition by dyes with biphenyl and amino- and sulphonic acid-substituted naphthyl groups

The specificity of the vesicular L-glutamate carrier was characterized using dyes with biphenyl and amino- and sulphonic acid substituted naphthyl groups, structurally similar to the specific vesicular L-glutamate inhibitor Evans Blue. The dye Trypan Blue was the most potent inhibitor; the IC50 value was determined to be 49 nM. Naphthol Blue Black, Reactive Blue 2, Benzopurpurin 4B, Ponceau SS, Direct Blue 71 and Acid red 114 were also highly potent inhibitors with IC50 values from 330 to 1670 nM (series 1). The dyes were competitive inhibitors of vesicular glutamate uptake, and acted therefore on the glutamate transporter. Their IC50 values for the vesicular uptake of gamma-aminobutyric acid (GABA) were all higher than 20 microM. They had no effect on synaptosomal uptake of glutamate. Furthermore, we have also found several other dyes with IC50 values for the vesicular uptake of glutamate ranging between 1 and 30 microM and for gamma-aminobutyric acid higher than 50 microM (series 2). The most potent inhibitor Trypan Blue contains a biphenyl group, linked by azo groups to side chains containing sulphonic, amino and/or hydroxyl groups coupled to a naphthalene ring system. Trypan Blue and Evans Blue are by molecular mechanics, shown to have planar structures with conjugated double bonds throughout the structure. The other dyes, which were less effective, had phenyl and/or naphthalene groups linked by an azo group. We have also tested a series of amino and/or hydroxyl naphthalene di-/sulphonic acids that correspond to the side chains of the most potent dyes, but they had no effect on glutamate nor on gamma-aminobutyric acid uptake. We conclude that the inhibitory action of these compounds is strictly dependent of the complete molecule.     

Identification of the nonsubstrate steroid binding site of rat liver glutathione S-transferase, isozyme 1-1, by the steroid affinity label, 3beta-(iodoacetoxy)dehydroisoandrosterone

3beta-(Iodoacetoxy)dehydroisoandrosterone (3beta-IDA), an analogue of the electrophilic substrate, Delta5-androstene-3,17-dione, as well as an analogue of several other steroid inhibitors of glutathione S-transferase, was tested as an affinity label of rat liver glutathione S-transferase, isozyme 1-1. A time-dependent loss of enzyme activity is observed upon incubation of 3beta-IDA with the enzyme. The rate of enzyme inactivation exhibits a nonlinear dependence on 3beta-IDA concentration, yielding an apparent Ki of 21 microM. Upon complete inactivation of the enzyme, a reagent incorporation of approximately 1 mol/mol of enzyme subunit or 2 mol/mol of enzyme dimer is observed. Protection against inactivation and incorporation is afforded by alkyl glutathione derivatives and nonsubstrate steroid ligands such as 17beta-estradiol-3,17-disulfate but, surprisingly, not by Delta5-androstene-3,17-dione or any other electrophilic substrate analogues tested. These results suggest that the site of reaction is within the nonsubstrate steroid binding site of the enzyme, which is distinguishable from the electrophilic substrate binding site, near the active site of the enzyme. Two cysteine residues, Cys17 and Cys111, are modified in nearly equal amounts, despite an average reagent incorporation of 1 mol/mol enzyme subunit. Isolation of enzyme subunits indicates the presence of unmodified, singly labeled, and doubly labeled subunits, consistent with mutually exclusive modification of cysteine residues across enzyme subunits; i.e., modification of Cys111 on subunit A prevents modification of Cys111 on subunit B and similarly for Cys17. Molecular modeling analysis suggests that Cys17 and Cys111 are located in the nonsubstrate steroid binding site, within the cleft between the subunits of the dimeric enzyme.     

The effect of acylated polyamine derivatives on polyamine uptake mechanism, cell growth, and polyamine pools in Escherichia coli, and the pursuit of structure/activity relationships

Two acetyl analogues of spermidine and five analogues of spermine were used to determine the structural specificity of the polyamine transport system in Escherichia coli by measuring their ability to compete with [14C]putrescine or [14C]spermine for uptake, as well as to inhibit cell growth, and, finally, to affect the intracellular polyamine pools. Spermine uptake follows simple Michaelis-Menten kinetics (Kt = 24.58 +/- 2.24 microM). In contrast, the putrescine uptake system involves two saturable Michaelis-Menten carriers exhibiting different affinity towards putrescine (Kt = 3.63 +/- 0.43 microM, Kt' = 0.61 +/- 0.10 microM). From the Ki values, it is inferred that N1-5-amino-2-nitrobenzoylspermine is the most effective competitive inhibitor followed by N1-acetylspermine, and then N1,N12-diacetylspermine. N1-acetylspermidine and N8-acetylspermidine also inhibit competitively the uptake of spermine, the latter being the most effective inhibitor. In addition, the above-mentioned analogues inhibit identically one of the carriers of putrescine uptake, suggesting the existence of a common transporter for both putrescine and spermine. The order of analogue potency, regarding the other carrier of putrescine is as follows: N1,N12-diacetylspermine approximately N1-5-amino-2-nitro-benzoylspermine > N1-acetylspermine. Both N1-acetylspermidine (Ki = 753 +/- 25 microM, Ki' = 128 +/- 5 microM) and N8-acetylspermidine (Ki = 22.4 +/- 0.4 microM, Ki' = 279 +/- 3 microM) also cause competitive inhibition of putrescine uptake, however with inverse affinity towards the putrescine carriers. Neither N4,N9-diacetylspermine, nor N1,N4-bis(beta-alanyl)diaminobutane affect the uptake of any polyamine. Interestingly, none of the acetyl analogues of spermine has a measurable effect on cell growth and cellular polyamine pools, although some of them are accumulated in cells. Based on these findings, the relative significance of the primary and secondary amines and of the chain flexibility as determinants of cellular uptake are discussed.     

Extracellular reduced glutathione increases neuronal vulnerability to combined chemical hypoxia and glucose deprivation

In addition to its intracellular antioxidant role, reduced glutathione (GSH) is released by CNS cells and may mediate or modulate excitatory neurotransmission. Although extracellular GSH levels rise in the ischemic cortex, its effect on the viability of energy-compromised neurons has not been defined. In this study, we tested the hypothesis that exogenous GSH would increase the vulnerability of cultured cortical neurons to azide-induced chemical hypoxia combined with glucose deprivation. Thirty minutes azide exposure in a glucose-free buffer was tolerated by most neurons, with release of less than 10% of neuronal LDH over the subsequent 21-25 h. Concomitant treatment with 10-100 microM GSH increased cell death in a concentration-dependent fashion, to 71.6+/-5.1% of neurons at 100 microM; GSH alone was nontoxic. Injury was blocked by the selective N-methyl-d-aspartate (NMDA) antagonist MK-801 but not by the AMPA/kainate antagonist NBQX. The sulfhydryl reducing agent mercaptoethanol (10-100 microM) mimicked the action of GSH; however, the zinc chelator ethylenediaminetetraacetic acid (EDTA) was ineffective. Two GSH analogues that lack a sulfhydryl group, S-hexylglutathione (SHG) and oxidized glutathione (GSSG), were inactive per se but attenuated the effect of both GSH and mercaptoethanol. These results suggest that micromolar concentrations of GSH enhance neuronal loss due to energy depletion by altering the extracellular redox state, resulting in increased NMDA receptor activation.     

Synthesis and evaluation of analogues of the partial agonist 6-(propyloxy)-4-(methoxymethyl)-beta-carboline-3-carboxylic acid ethyl ester (6-PBC) and the full agonist 6-(benzyloxy)-4-(methoxymethyl)-beta-carboline-3-carboxylic acid ethyl ester (Zk 93423) at wild type and recombinant GABAA receptors

A pharmacophore and an alignment rule have previously been reported for BzR agonist ligands. The design and synthesis of 6-(propyloxy)-4-(methoxymethyl)-beta-carboline-3-carboxylic acid ethyl ester (6-PBC, 24, IC50 = 8.1 nM) was based on this pharmacophore. When evaluated in vivo this ligand exhibited anticonvulsant/anxiolytic activity but was devoid of the muscle relaxant/ataxic effects of "classical" 1,4-benzodiazepines (i.e., diazepam). Significantly, 6-PBC 24 also reversed diazepam-induced muscle relaxation in mice. The 3-substituted analogues 40-46 and 48 of 6-PBC 24 and Zk 93423 27(IC50 = 1 nM) were synthesized and evaluated in vitro to determine what affect these modifications would have on the binding affinity at recombinant BzR subtypes. With the exception of the 3-amino ligands 40 and 41, all the beta-carbolines were found to exhibit high binding affinity at BzR sites. The 3-propyl ether derivative 45 was also evaluated in vivo and found to be devoid of any proconvulsant or anticonvulsant activity at doses up to 40 mg/kg. The 6-(1-naphthylmethyloxy) and 6-octyloxy analogues 25, 26, 28, and 29 of 6-PBC 24 were synthesized to further evaluate the proposed alignment of agonists vs inverse agonists in the pharmacophore of the BzR. In addition, ligands 26 and 29 were designed to probe the dimensions of lipophilic pocket L3 at the agonist site. The activity of 29 was evaluated in vivo; however, this analogue elicited no pharmacological effects at doses up to 80 mg/kg. These and other related beta-carbolines were also examined in five recombinant GABAA receptor subtypes. Ligands 52-61 all exhibited moderate to high affinity at GABAA receptors containing alpha1 subunits. These ligands will be useful in further defining the pharmacophore at alpha1 beta3 gamma2 receptors.     

Biochemical basis for a cholesterol-lowering activity of 2-[2"-(1",3"-dioxolane)]-2-methyl-4-(2'-oxo-1'-pyrrolidinyl)-6- nitro-2H-1-benzopyran (SKP-450), a novel antihypertensive agent

Administration (p.o.) of SKP-450, 2-[2"-(1",3"-dioxolane)]-2-methyl-4-(2'-oxo-1'-pyrrolidinyl)-6-nitro-2H- 1-benzopyran, a novel antihypertensive agent, to hypercholesterolemic Syrian hamsters led to a significant reduction in plasma lipids in a dose-dependent manner, i.e., a 10.8% to 29% reduction in low-density lipoprotein cholesterol at doses of 0.3 to 10 mg/kg of SKP-450. SKP-450 was found to specifically inhibit the hepatic microsomal lanosterol 14alpha-methyl demethylase (14alpha-DM) in a competitive manner (Ki:2.65 microM). Furthermore, a dose-dependent decrease in the 14alpha-DM activity by SKP-450 parallelled the cholesterol synthetic rate in vitro in both the rat hepatic S10 fractions (supernatants at 10,000 g; IC50:20 microM) and Chinese hamster ovary cells (IC50:23 microM). However, this phenomenon was not seen in AR45 cells, which are deficient in 14alpha-DM, suggesting that 14alpha-DM is the major target for the inhibitory action of SKP-450 in regard to cholesterol biosynthesis.