Lorraine M. Deck

Inhibition of lactate dehydrogenase A induces oxidative stress and inhibits tumor progression

As the result of genetic alterations and tumor hypoxia, many cancer cells avidly take up glucose and generate lactate through lactate dehydrogenase A (LDHA), which is encoded by a target gene of c-Myc and hypoxia-inducible factor (HIF-1). Previous studies with reduction of LDHA expression indicate that LDHA is involved in tumor initiation, but its role in tumor maintenance and progression has not been established. Furthermore, how reduction of LDHA expression by interference or antisense RNA inhibits tumorigenesis is not well understood. Here, we report that reduction of LDHA by siRNA or its inhibition by a small-molecule inhibitor (FX11 [3-dihydroxy-6-methyl-7-(phenylmethyl)-4-propylnaphthalene-1-carboxylic acid]) reduced ATP levels and induced significant oxidative stress and cell death that could be partially reversed by the antioxidant N-acetylcysteine. Furthermore, we document that FX11 inhibited the progression of sizable human lymphoma and pancreatic cancer xenografts. When used in combination with the NAD+ synthesis inhibitor FK866, FX11 induced lymphoma regression. Hence, inhibition of LDHA with FX11 is an achievable and tolerable treatment for LDHA-dependent tumors. Our studies document a therapeutical approach to the Warburg effect and demonstrate that oxidative stress and metabolic phenotyping of cancers are critical aspects of cancer biology to consider for the therapeutical targeting of cancer energy metabolism.

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.

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.

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.

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.

Isocoumarin-based inhibitors of pancreatic cholesterol esterase.

Pancreatic cholesterol esterase (CEase), which is secreted from the exocrine pancreas, is a serine hydrolase that aids in the bile salt-dependent hydrolysis of dietary cholesteryl esters and contributes to the hydrolysis of triglycerides and phospholipids. Additional roles for CEase in intestinal micelle formation and in transport of free cholesterol to the enterocyte have been suggested. There also are studies that point to a pathological role(s) for CEase in the circulation where CEase accumulates in atherosclerotic lesions and triggers proliferation of smooth muscle cells. Thus, there is interest in CEase as a potential drug target. 4-Chloro-3-alkoxyisocoumarins are a class of haloenol lactones that inhibit serine hydrolases and serine proteases and have the potential to be suicide inhibitors. In the present study, we have developed 3-alkoxychloroisocoumarins that are potent inhibitors of CEase. These inhibitors were designed to have a saturated cycloalkane ring incorporated into a 3-alkoxy substituent. The size of the ring as well as the length of the tether holding the ring was found to be important contributors to binding to CEase. 4-Chloro-3-(4-cyclohexylbutoxy)isocoumarin and 4-chloro-3-(3-cyclopentylpropoxy)isocoumarin were demonstrated to be potent reversible inhibitors of CEase, with dissociation constants of 11nM and 19nM, respectively. The kinetic results are consistent with predictions from molecular modeling.

A chemical analog of curcumin as an improved inhibitor of amyloid Abeta oligomerization.

Amyloid-like plaques are characteristic lesions defining the neuropathology of Alzheimers disease (AD). The size and density of these plaques are closely associated with cognitive decline. To combat this disease, the few therapies that are available rely on drugs that increase neurotransmission; however, this approach has had limited success as it has simply slowed an imminent decline and failed to target the root cause of AD. Amyloid-like deposits result from aggregation of the Aβ peptide, and thus, reducing amyloid burden by preventing Aβ aggregation represents an attractive approach to improve the therapeutic arsenal for AD. Recent studies have shown that the natural product curcumin is capable of crossing the blood-brain barrier in the CNS in sufficient quantities so as to reduce amyloid plaque burden. Based upon this bioactivity, we hypothesized that curcumin presents molecular features that make it an excellent lead compound for the development of more effective inhibitors of Aβ aggregation. To explore this hypothesis, we screened a library of curcumin analogs and identified structural features that contribute to the anti-oligomerization activity of curcumin and its analogs. First, at least one enone group in the spacer between aryl rings is necessary for measureable anti-Aβ aggregation activity. Second, an unsaturated carbon spacer between aryl rings is essential for inhibitory activity, as none of the saturated carbon spacers showed any margin of improvement over that of native curcumin. Third, methoxyl and hydroxyl substitutions in the meta- and para-positions on the aryl rings appear necessary for some measure of improved inhibitory activity. The best lead inhibitors have either their meta- and para-substituted methoxyl and hydroxyl groups reversed from that of curcumin or methoxyl or hydroxyl groups placed in both positions. The simple substitution of the para-hydroxy group on curcumin with a methoxy substitution improved inhibitor function by 6-7-fold over that measured for curcumin.

Inhibition of pancreatic cholesterol esterase reduces cholesterol absorption in the hamster

BackgroundPancreatic cholesterol esterase has three proposed functions in the intestine: 1) to control the bioavailability of cholesterol from dietary cholesterol esters; 2) to contribute to incorporation of cholesterol into mixed micelles; and 3) to aid in transport of free cholesterol to the enterocyte. Inhibitors of cholesterol esterase are anticipated to limit the absorption of dietary cholesterol.ResultsThe selective and potent cholesterol esterase inhibitor 6-chloro-3-(1-ethyl-2-cyclohexyl)-2-pyrone (figure 1, structure 1) was administered to hamsters fed a high cholesterol diet supplemented with radiolabeled cholesterol ester. Hamsters were gavage fed 3H-labeled cholesteryl oleate along with inhibitor 1, 0–200 micromoles. Twenty-four hours later, hepatic and serum radioactive cholesterol levels were determined. The ED50 of inhibitor 1 for prevention of the uptake of labeled cholesterol derived from hydrolysis of labeled cholesteryl oleate was 100 micromoles. The toxicity of inhibitor 1 was investigated in a 30 day feeding trial. Inhibitor 1, 100 micromoles or 200 micromoles per day, was added to chow supplemented with 1% cholesterol and 0.5% cholic acid. Clinical chemistry urinalysis and tissue histopathology were obtained. No toxicity differences were noted between control and inhibitor supplemented groups.ConclusionsInhibitors of cholesterol esterase may be useful therapeutics for limiting cholesterol absorption.

17-β-Hydroxysteroid dehydrogenase type 1: computational design of active site inhibitors targeted to the Rossmann fold

17-beta-Hydroxysteroid dehydrogenase type 1 (17betaHSD1), also called estradiol dehydrogenase, catalyzes the NADPH-dependent reduction of the weak estrogen, estrone, into the more potent estrogen, 17-beta-estradiol. 17betaHSD1 is an attractive drug target in hormone-sensitive breast cancer. Past efforts to develop selective inhibitors of 17betaHSD1 have focused on design of substrate analogs. It is challenging to develop steroid analogs that are devoid of any undesired biological activity. 17betaHSD1 is a member of the short-chain dehydrogenase/reductase (SDR) superfamily that includes many hydroxysteroid dehydrogenases. Members of the SDR family bind NAD(P)(H) in a motif that is a modified Rossmann fold. We demonstrated previously that the Rossmann folds of classical dehydrogenases can be selectively inhibited by derivatives and analogs of the natural product gossypol. In this study, we have addressed the question whether the modified Rossmann fold in 17betaHSD1 is a target for identification of lead compounds for structure-based drug design. 17betaHSD1 was purified from human placenta. 17betaHSD1 is inhibited by derivatives of gossypol with dissociation constants as low as 2 microM. Inhibition is competitive with the binding of cofactor. Molecular modeling studies using the published coordinates of human 17betaHSD1 suggest that these inhibitors occupy the modified Rossmann fold at the nicotinamide end of the dinucleotide-binding site, extending towards the substrate site. A computational approach was used to design potential new inhibitors of 17betaHSD1. The results suggest not only that derivatives of gossypol represent attractive lead compounds for structure-based drug design but also suggest that appropriate incorporation of a substrate analog into the design of these Rossmann fold inhibitors may provide pan-active site inhibitors that span the cofactor and substrate site, potentially offering specificity and increased potency.