The kinetic properties and sensitivities to inhibitors of lactate dehydrogenases (LDH1 and LDH2) from Toxoplasma gondii: comparisons with pLDH from Plasmodium falciparum.

Toxoplasma gondii differentially expresses two forms of lactate dehydrogenase in tachyzoites and bradyzoites, respectively, designated LDH1 and LDH2. Previously it was demonstrated that LDH1 and LDH2 share a unique structural feature with LDH from the malarial parasite Plasmodium falciparum (pLDH), namely, the addition of a five-amino acid insert into the substrate specificity loops. pLDH exhibits a number of kinetic properties that previously were thought to be unique to pLDH. In the present study, kinetic properties of LDH1 and LDH2 were compared with those of pLDH. LDH1 and LDH2 exhibit broader substrate specificity than pLDH. For both LDH1 and LDH2, 3-phenylpyruvate is an excellent substrate. For LDH2, 3-phenylpyruvate is a better substrate even than pyruvate. By comparison, pLDH does not utilize 3-phenylpyruvate. Both LDH1 and LDH2 can utilize the NAD analog 3-acetylpyridine adenine dinucleotide (APAD) efficiently, similar to pLDH. LDH1 and LDH2 are inhibited competitively by a range of compounds that also inhibit pLDH, including gossypol and derivatives, dihydroxynaphthoic acids, and N-substituted oxamic acids. The lack of substrate inhibition observed with pLDH is also observed with LDH2. By comparison, LDH1 differs from LDH2 in exhibiting substrate inhibition in spite of an identical residue (M163) at a cofactor binding site that is thought to be critical for production of substrate inhibition. For gossypol and gossylic iminolactone, but not the other gossypol derivatives tested, the in vitro inhibition of T. gondii LDH activity correlated with specific inhibition of T. gondii tachyzoite growth in fibroblast cultures.

Oxidative stress indices in IDDM subjects with and without long-term diabetic complications.

BACKGROUND: Numerous animal and population studies of diabetes have identified markers of oxidative stress. However, for most markers that have been measured the results are not consistent. In addition, it is less clear whether oxidative stress is related to the development of diabetic complications. The objective of this study was to evaluate a series of plasma markers and leukocyte markers to test the hypothesis that type 1 Insulin Dependent Diabetes Mellitus (IDDM) subjects experience oxidative stress. A related question was whether markers of oxidative stress are higher in IDDM subjects who have developed long-term complications. METHODS: The study population consisted of 22 IDDM subjects with diabetic complications and 22 IDDM subjects without complications, both groups matched by age and gender and with similar HbA1c levels, and 16 nondiabetic control subjects. Plasma levels of organoperoxides were determined by the ferrous oxidation/xylenol orange (FOX) assay, malondialdehyde by the thiobarbituric acid (TBARS) assay, and vitamin E by HPLC. Mononuclear cells and polymorphonuclear cells were analyzed for ascorbic acid by HPLC and for glutathione (GSH) by enzymatic recycling. In addition, GSH peroxidase, GSH transferase and glucose-6-phosphate dehydrogenase levels were determined in both cell fractions. RESULTS: Plasma organoperoxides were significantly elevated in the IDDM subjects compared to controls (p = 0.02) while TBARS and vitamin E levels were not significantly different. In the IDDM subjects, mononuclear cell levels of ascorbic acid were significantly lower (p < 0.02) and levels of GSH were lower, approaching significance (p = 0.07), compared to controls. Ascorbic acid and GSH levels in polymorphonuclear cells were not significantly different between IDDM subjects and controls, nor were enzyme levels different. In addition, the plasma and intracellular indices of oxidative status in IDDM subjects were not different when IDDM subjects with complications were compared to IDDM subjects without complications. CONCLUSION: Demonstration of oxidative stress in IDDM subjects depends upon which markers are measured. This is in agreement with previous studies of oxidative stress in various disease states including diabetes. Plasma levels of organoperoxides may be the most reliable indicators of oxidative stress. However, it is unclear whether elevated plasma organoperoxides indicate a generalized systemic stress or are produced in localized areas. By comparison, oxidative stress indices determined with isolated blood cells may provide a clearer picture. Depressed levels of ascorbic acid and GSH were observed only in mononuclear cells, which are mainly long-lived T lymphocytes. Mononuclear cells antioxidant status may reflect systemic oxidative stress. In this study, neither plasma markers nor intracellular markers of oxidative stress were different in IDDM subjects with long-term diabetic complications compared to subjects without complications.

Selective active site inhibitors of human lactate dehydrogenases A4, B4, and C4.

Human lactate dehydrogenases (LDH-A4, -B4, and -C4) are highly homologous with 84-89% sequence similarities and 69-75% amino acid identities. Active site residues are especially conserved. Gossypol, a natural product from cotton seed, is a non-selective competitive inhibitor of NADH binding to LDH, with K(i) values of 1.9, 1.4, and 4.2 microM for LDH-A4, -B4, and -C4, respectively. However, derivatives of gossypol and structural analogs of gossypol in the substituted 2,3-dihydroxy-1-naphthoic acid family exhibited markedly greater selectivity and, in many cases, greater potency. For gossypol derivatives, greater than 35-fold selectivity was observed. For dihydroxynaphthoic acids with substituents at the 4- and 7-positions, greater than 200-fold selectivity was observed. Inhibition was consistently competitive with the binding of NADH, with dissociation constants as low as 30 nM. By comparison, a series of N-substituted oxamic acids, which are competitive inhibitors of the binding of pyruvate to LDH, exhibited very modest selectivity. These results suggest that substituted dihydroxynaphthoic acids are good lead compounds for the development of selective LDH inhibitors. Selective inhibitors of LDH-C4 targeted to the dinucleotide fold may hold promise as male antifertility drugs. Selective inhibitors of LDH-A4 and -B4 may be useful for studies of lactic acidemia associated with ischemic events. More broadly, the results raise the question of the general utility of drug design targeted at the dinucleotide binding sites of dehydrogenases/reductases.

Metabolism of the 2-oxoaldehyde methylglyoxal by aldose reductase and by glyoxalase-I: roles for glutathione in both enzymes and implications for diabetic complications.

Numerous physiological aldehydes besides glucose are substrates of aldose reductase, the first enzyme of the polyol pathway which has been implicated in the etiology of diabetic complications. The 2-oxoaldehyde methylglyoxal is a preferred substrate of aldose reductase but is also the main physiological substrate of the glutathione-dependent glyoxalase system. Aldose reductase catalyzes the reduction of methylglyoxal efficiently (k(cat)=142 min(-1) and k(cat)/K(m)=1.8x10(7) M(-1) min(-1)). In the presence of physiological concentrations of glutathione, methylglyoxal is significantly converted into the hemithioacetal, which is the actual substrate of glyoxalase-I. However, in the presence of glutathione, the efficiency of reduction of methylglyoxal, catalyzed by aldose reductase, also increases. In addition, the site of reduction switches from the aldehyde to the ketone carbonyl. Thus, glutathione converts aldose reductase from an aldehyde reductase to a ketone reductase with methylglyoxal as substrate. The relative importance of aldose reductase and glyoxalase-I in the metabolic disposal of methylglyoxal is highly dependent upon the concentration of glutathione, owing to the non-catalytic pre-enzymatic reaction between methylglyoxal and glutathione.

Gossypol: prototype of inhibitors targeted to dinucleotide folds.

Gossypol, a disesquiterpene from cottonseed, exhibits multiple biological properties, including male antifertility activity and anticancer activity. Gossypol also inhibits the growth of numerous parasitic organisms and shows antiviral activity against a number of enveloped viruses, including the AIDS virus. Derivatives of gossypol, in which the aldehyde functional groups that contribute to toxicity have been modified, retain or even show enhanced biological activity. Ring substituted 2,3-dihydroxy-1-naphthoic acids, which are structural analogs of gossypol, share with gossypol the ability to complex with dehydrogenases at the dinucleotide fold (Rossmann fold) with selectivity, suggesting that gossypol may be considered the prototype of a new class of drugs targeted to dehydrogenases. Most of the biological activities of gossypol and related compounds may result from inhibition of dehydrogenases.

3-Alkyl-6-chloro-2-pyrones: selective inhibitors of pancreatic cholesterol esterase.

A series of 3-alkyl-6-chloro-2-pyrones with cyclohexane rings tethered to the 3-position was synthesized. The tether ranged from 0 to 4 methylene units. Inhibition of pancreatic cholesterol esterase by this series of pyrones was markedly dependent upon the length of the tether. Dissociation constants as low as 25 nM were observed for 6-chloro-3-(1-ethyl-2-cyclohexyl)-2-pyranone. This class of cholesterol esterase inhibitors functioned as simple competitive inhibitors of substrate binding rather than as suicide substrates or active site inactivators. Trypsin and chymotrypsin were not strongly inhibited by this class of pyrones. Selectivities for cholesterol esterase were greater than 10(3). This is in contrast to 3-aryl-6-chloro-2-pyrones which are nonselective, irreversible inactivators of serine hydrolases. Thus, replacement of the 3-aryl group by an appropriately tethered 3-alkyl ring can produce highly selective inhibitors of cholesterol esterase. A second series of halogen-containing esters was prepared in which cholesterol was esterified with alpha-haloacyl halides. These haloesters were simple substrates of cholesterol esterase with no evidence of irreversible inactivation.

Selective inhibitors of human lactate dehydrogenases and lactate dehydrogenase from the malarial parasite Plasmodium falciparum.

Derivatives of the sesquiterpene 8-deoxyhemigossylic acid (2, 3-dihydroxy-6-methyl-4-(1-methylethyl)-1-naphthoic acid) were synthesized that contained altered alkyl groups in the 4-position and contained alkyl or aralkyl groups in the 7-position. These substituted dihydroxynaphthoic acids are selective inhibitors of human lactate dehydrogenase-H (LDH-H) and LDH-M and of lactate dehydrogenase from the malarial parasite Plasmodium falciparum (pLDH). All inhibitors are competitive with the binding of NADH. Selectivity for LDH-H, LDH-M, or pLDH is strongly dependent upon the groups that are in the 4- and 7-positions of the dihydroxynaphthoic acid backbone. Dissociation constants as low as 50 nM were observed, with selectivity as high as 400-fold.

Z-2 microsatellite allele is linked to increased expression of the aldose reductase gene in diabetic nephropathy.

Epidemiological studies support the hypothesis that genetic factors modulate the risk for diabetic nephropathy (DN). Aldose reductase (ALDR1), the rate-limiting enzyme in the polyol pathway, is a potential candidate gene. The present study explores the hypothesis that polymorphisms of the (A-C)n dinucleotide repeat sequence, located 2.1 kb upstream of the transcription start site, modulate ALDR1 gene expression and the risk for DN. We conducted studies at two different institutions, the University of New Mexico Health Sciences Center (UNMHSC), and the Istituto Scientifico H San Raffaele (HSR). There were four groups of volunteers at UNMHSC: group I, normal subjects; group II, patients with insulin-dependent diabetes mellitus (IDDM) without DN; group III, IDDM with DN; and group IV, nondiabetics with kidney disease. At HSR we studied volunteers in groups I, II, and III. ALDR1 genotype was assessed by PCR and fluorescent sequencing of the (A-C)n repeat locus, and ALDR1 messenger ribonucleic acid (mRNA) was measured by ribonuclease protection assay in peripheral blood mononuclear cells. At UNMHSC we identified 10 alleles ranging from Z-10 to Z+8. The prevalence of the Z-2 allele among IDDM patients was increased in those with DN. Sixty percent of group III and 22% of group II were homozygous for Z-2. Moreover, 90% and 67% of groups III and II, respectively, had 1 or more copy of Z-2. In contrast, among nondiabetics, 19% of group IV and 3% of group I were homozygous for Z-2, and 69% and 32%, respectively, had 1 copy or more of Z-2. Among diabetics, homozygosity for the Z-2 allele was associated with renal disease [odds ratio (OR), 5.25; 95% confidence interval, 1.71-17.98; P = 0.005]. ALDR1 mRNA levels were higher in patients with DN (group III; 0.113 +/- 0.050) than in group I (0.068 +/- 0.025), group II (0.042 +/- 0.020), or group IV (0.015 +/- 0.011; P < 0.01). Among diabetics, ALDR1 mRNA levels were higher in Z-2 homozygotes (0.098 +/- 0.06) and Z-2 heterozygotes (0.080 +/- 0.04) than in patients with no Z-2 allele (0.043 +/- 0.02; P < 0.05). In contrast, among nondiabetics, ALDR1 mRNA levels in Z-2 homozygotes (0.034 +/- 0.04) and Z-2 heterozygotes (0.038 +/- 0.03) were similar to levels in patients without a Z-2 allele (0.047 +/- 0.03; P = NS). At HSR we identified eight alleles ranging from Z- 12 to Z+2. The prevalence of the Z-2 allele was higher in group III than in group II. In group III, 43% of the patients were homozygous for Z-2, and 81% had one copy or more of the Z-2 allele. In contrast, in group II, 4% were homozygous for Z-2, and 36% had one copy or more of the Z-2 allele. IDDM patients homozygous for Z-2 had an increased risk for DN compared with those lacking the Z-2 allele (OR, 18; 95% confidence interval, 2-159). IDDM patients who had one copy or more of Z-2 had increased risk (OR, 7.5; 95% confidence interval, 1.9-29.4) for DN compared with those without the Z-2 allele. These results support our hypothesis that environmental-genetic interactions modulate the risk for DN. Specifically, the Z 2 allele, in the presence of diabetes and/or hyperglycemia, is associated with increased ALDR1 expression. This interaction may explain the observed association between the Z-2 allele and DN.

Inactivation of glutathione reductase by 4-hydroxynonenal and other endogenous aldehydes.

4-Hydroxynonenal, a product of oxidative degradation of unsaturated lipids, is an endogenous reactive alpha,beta-unsaturated aldehyde with numerous biological activities. 4-Hydroxynonenal rapidly inactivated glutathione reductase in an NADPH-dependent reaction. Inactivation appears to involve the initial formation of an enzyme-inactivator complex, K(D) = 0.5 microM, followed by the inactivation reaction, k = 1.3 x 10(-2) min(-1). alpha,beta-Unsaturated aldehydes such as acrolein, crotonaldehyde, and cinnamaldehyde also inactivated glutathione reductase, although rates varied widely. Inactivation of glutathione reductase by alpha,beta-unsaturated aldehydes was followed by slower NADPH-independent reactions that led to formation of nonfluorescent cross-linked products, accompanied by loss of lysine and histidine residues. Other reactive endogenous aldehydes such as methylglyoxal, 3-deoxyglucosone, and xylosone inactivated glutathione reductase by an NADPH-independent mechanism, with methylglyoxal being the most reactive. However, 2-oxoaldehydes were much less effective than 4-hydroxynonenal. Inactivation of glutathione reductase by these 2-oxoaldehydes was followed by slower reactions that led to the formation of fluorescent cross-linked products over a period of several weeks. These changes were accompanied by loss of arginine residues. Thus, the sequence of events is different for inactivation and modification of glutathione reductase by alpha,beta-unsaturated aldehydes compared with 2-oxoaldehydes with respect to kinetics, NADPH requirements, fluorescence changes, and loss of amino acid residues. The ability of 4-hydroxynonenal at low concentrations to inactivate glutathione reductase, a central antioxidant enzyme, suggests that oxidative degradation of unsaturated lipids may initiate a positive feedback loop that enhances the potential for oxidative damage.

Substrate and cofactor specificity and selective inhibition of lactate dehydrogenase from the malarial parasite P. falciparum.

Lactate dehydrogenase from the malarial parasite Plasmodium falciparum has many amino acid residues that are unique compared to any other known lactate dehydrogenase. This includes residues that define the substrate and cofactor binding sites. Nevertheless, parasite lactate dehydrogenase exhibits high specificity for pyruvic acid, even more restricted than the specificity of human lactate dehydrogenases M4 and H4. Parasite lactate dehydrogenase exhibits high catalytic efficiency in the reduction of pyruvate, kcat/Km = 9.0 x 10(8) min(-1) M(-1). Parasite lactate dehydrogenase also exhibits similar cofactor specificity to the human isoforms in the oxidation of L-lactate with NAD+ and with a series of NAD+ analogs, suggesting a similar cofactor binding environment in spite of the numerous amino acid differences. Parasite lactate dehydrogenase exhibits an enhanced kcat with the analog 3-acetylpyridine adenine dinucleotide (APAD+) whereas the human isoforms exhibit a lower kcat. This differential response to APAD+ provides the kinetic basis for the enzyme-based detection of malarial parasites. A series of inhibitors structurally related to the natural product gossypol were shown to be competitive inhibitors of the binding of NADH. Slight changes in structure produced marked changes in selectivity of inhibition of lactate dehydrogenase. 7-p-Trifluoromethylbenzyl-8-deoxyhemigossylic acid inhibited parasite lactate dehydrogenase, Ki = 0.2 microM, which was 65- and 400-fold tighter binding compared to the M4 and H4 isoforms of human lactate dehydrogenase. The results suggest that the cofactor site of parasite lactate dehydrogenase may be a potential target for structure-based drug design.

Increased levels of methylglyoxal-metabolizing enzymes in mononuclear and polymorphonuclear cells from insulin-dependent diabetic patients with diabetic complications: aldose reductase, glyoxalase I, and glyoxalase II--a clinical research center study.

Levels of aldose reductase, glyoxalase I, and glyoxalase II in mononuclear and polymorphonuclear cells from insulin-dependent diabetes mellitus (IDDM) patients with long term diabetic complications were compared to levels in IDDM patients without complications and to those in nondiabetic controls. Cells were isolated from 22 asymptomatic long term IDDM patients, 22 symptomatic IDDM patients, and 16 controls, using a double gradient centrifugation procedure. Aldose reductase was determined by Western blots using polyclonal antiserum to human aldose reductase purified from skeletal muscle. Glyoxalase I and glyoxalase II were determined spectrophotometrically. Aldose reductase in mononuclear cells from symptomatic IDDM patients is significantly elevated compared to that in asymptomatic IDDM patients (mean +/- SEM, 0.96 +/- 0.20 vs. 0.46 +/- 0.08 microgram/mg protein; P < 0.02). Aldose reductase was not detected in polymorphonuclear cells. Glyoxalase I in mononuclear and polymorphonuclear cells from symptomatic IDDM patients is significantly elevated compared to that in controls [mean for mononuclear cells, 0.46 +/- 0.03 vs. 0.37 +/- 0.03 mumol/min.mg (P < 0.05); mean for polymorphonuclear cells, 0.16 +/- 0.01 vs. 0.10 +/- 0.01 mumol/min.mg (P < 0.002)]. Glyoxalase II is significantly elevated only in polymorphonuclear cells from symptomatic IDDM patients compared to controls (mean, 0.13 +/- 0.01 vs. 0.063 +/- 0.016 mumol/min.mg; P < 0.005). Glutathione peroxidase and glutathione S-transferase were not significantly different in these populations. Aldose reductase, glyoxalase I, and glyoxalase II are involved in the metabolism of methylglyoxal, suggesting that methylglyoxal may play a role in the etiology of diabetic complications.

Synthesis and anti-HIV activity of 1,1'-dideoxygossypol and related compounds.

1,1'-Dideoxygossypol (DDG), 1,1'-dideoxygossylic acid (DDGA), 8-deoxyhemigossypol (DHG), and 8-deoxyhemigossylic acid (DHGA) were synthesized and tested for their ability to inhibit the replication of HIV in vitro. The EC50 for DDGA was < 1 microM, and its threshold cytotoxicity was approximately 20 microM. DDG was less effective than DDGA against HIV and showed considerable toxicity at 5 microM. DHGA was ineffective against HIV and had very low cytotoxicity. DHG showed some anti-HIV activity, but the threshold cytotoxicity was 5 microM. The dissociation constants for the binding of the four compounds to human serum albumin were determined by fluorescence quenching titrations, and all four were found to have much lower affinities for albumin than the parent compound gossypol.

Substrate specificity of human aldose reductase: identification of 4-hydroxynonenal as an endogenous substrate.

Aldose reductase, which catalyzes the reduction of glucose to sorbitol as part of the polyol pathway, has been implicated in the development of diabetic complications and is a prime target for drug development. However, aldose reductase exhibits broad specificity for both hydrophilic and hydrophobic aldehydes, which suggests that aldose reductase may also be a detoxification enzyme. Several series of structurally related aldehydes were compared as substrates in order to deduce the structural features that result in low Michaelis constants. Aldehydes that contain an aromatic ring are generally excellent substrates, consistent with crystallographic data which suggest that aldose reductase possesses a large hydrophobic substrate binding site. However, there is little discrimination among different aromatic aldehydes. In addition, small hydrophilic aldehydes exhibit low Km values if the alpha-carbon is oxidized. Analysis of the binding of NADPH by fluorescence quenching techniques indicates that aldose reductase exhibits higher affinity for NADPH than NADP, suggesting that this enzyme is normally primed for reductive metabolism. Thus aldose reductase appears to have evolved to catalyze the reduction of a very broad range of aldehydes. Structural features of substrates that bind to aldose reductase with low Km values were used to identify potential endogenous substrates. 4-Hydroxynonenal, a reactive alpha-beta unsaturated aldehyde produced during oxidative stress, is an excellent substrate (Km = 22 microM, kcat/Km = 4.6 x 10(6) M-1 min-1). Reductive metabolism of endogenous aldehydes in addition to glucose, catalyzed by aldose reductase, may play an important role in the development of diabetic complications.

Comparison of the antiviral activities of 3'-azido-3'-deoxythymidine (AZT) and gossylic iminolactone (GIL) against clinical isolates of HIV-1.

3'-Azido-3'-deoxythymidine (AZT) and gossylic iminolactone (GIL) were compared for antiviral activities in vitro against fresh clinical isolates of HIV-1 obtained from 19 subjects on AZT therapy. IC50 values for AZT ranged from 0.015 to 6.7 microM (447-fold range) while IC50 values for GIL ranged from 0.40 to 6.6 microM (16-fold range). There was no correlation between IC50 values for AZT and GIL, suggesting that the anti-HIV activity of GIL does not involve inhibition of reverse transcriptase.

Polyol and water accumulation in muscle of galactose-fed rats.

Skeletal muscle contains high levels of aldose reductase that catalyzes the reduction of galactose to the polyol galactitol. Galactitol and water were measured in muscle of rats fed a high galactose diet with or without addition of the aldose reductase inhibitor sorbinil. Galactitol, measured in isolated samples of muscle by HPLC, reached steady-state levels (5.9 +/- 1.0 mg/g tissue) within 3 days. Muscle water, determined in vivo by magnetic resonance imaging, increased (51 +/- 5%, P < 0.02) to steady-state levels within 7 days. Both the increased galactitol and water remained constant for the 4-month duration of this study. Aldose reductase activity also remained constant. Sorbinil prevented both the increase in galactitol and the increase in water. These results suggest that the increase in water is due to the osmotic effects of galactitol accumulation and demonstrate that galactitol and water accumulation neither up-regulate nor down-regulate aldose reductase expression in skeletal muscle.

Small molecule probes of glyoxalase I and glyoxalase II.

A number of synthetic tropolones and hydrophobic S-blocked glutathione analogues were investigated as potential inhibitors of glyoxalase I from Saccharomyces cerevisiae and glyoxalase II from bovine liver. Several tropolones containing a free C-2 hydroxy group were found to be potent inhibitors of glyoxalase I, whereas the glutathione conjugates were found to be modest to poor inhibitors of this enzyme. Most tropolones and glutathione conjugates, except 5-p-tolylazotropolone and S-carbobenzoxy-L-glutathione, were found to be poor inhibitors of glyoxalase II. A recent report on an extremely active glyoxalase system from Plasmodium falciparum suggested that several of the more potent inhibitors may have antimalarial properties. A number of these compounds in fact, exhibited antimalarial activity in the low micromolar range. Further studies are required to fully elucidate the mechanism(s) of the antimalarial properties of these compounds.