Selected gold compounds cause pronounced inhibition of Falcipain 2 and effectively block P. falciparum growth in vitro

A number of structurally diverse gold compounds were evaluated as possible inhibitors of Falcipain 2 (Fp2), a cysteine protease from P. falciparum that is a validated target for the development of novel antimalarial drugs. Remarkably, most tested compounds caused pronounced but reversible inhibition of Fp2 with K_i values falling in the micromolar range. Enzyme inhibition is basically ascribed to gold binding to catalytic active site cysteine. The same gold compounds were then tested for their ability to inhibit P. falciparum growth in vitro; important parasite growth inhibition was indeed observed. However, careful analysis of the two sets of data failed to establish any direct correlation between enzyme inhibition and reduction of P. falciparum growth suggesting that Fp2 inhibition represents just one of the various mechanisms through which gold compounds effectively antagonize P. falciparum replication.     

Mercury, silver, and gold inhibition of selenium-accelerated cysteine oxidation

In vivo, cysteine in proteins or glutathione is the major amino acid involved in sulfhydryl oxidation-reduction reactions. An in vitro model of cysteine oxidation accelerated by selenium compounds was used to study the interaction of selenocystine and sodium selenite with metal ions. The interaction of metal ions with selenium compounds inhibited cysteine oxidation. The ionic forms of three toxic soft-acid metals, mercury, silver, and gold, were the most effective inhibitors. The antiarthritic gold drugs, aurothiomalate and aurothioglucose, were of particular interest as they inhibit the activity of selenium-glutathione peroxidase. The effect of gold ligands on gold(I) inhibition of selenocystine-accelerated cysteine oxidation was tested. Sodium cyanide partially reversed inhibition and potassium iodide had no effect. Inhibition of selenium-accelerated oxidation-reduction reactions by soft-acid metal ions may be of biological relevance during toxicities or during antiarthritic gold therapy.     

Aromatic hydroxamic acids and hydrazides as inhibitors of the peroxidase activity of prostaglandin H2 synthase-2

The cyclooxygenase activity of the bifunctional enzyme prostaglandin H(2) synthase-2 (PGHS-2) is the target of non-steroidal anti-inflammatory drugs. Inhibition of the peroxidase activity of PGHS has been less studied. Using Soret absorption changes, the binding of aromatic hydroxamic acids to the peroxidase site of PGHS-2 was examined to investigate the structural determinants of inhibition. Typical of mammalian peroxidases, the K(d) for benzhydroxamic acid (42mM) is much greater than that for salicylhydroxamic acid (475microM). Binding of the hydroxamic acid tepoxalin (25microM) resulted in only minor Soret changes. However, tepoxalin is an efficient reducing cosubstrate, indicating that it is an alternative electron donor rather than an inhibitor of the peroxidase activity. Aromatic hydrazides are metabolically activated inhibitors of peroxidases. 2-Naphthoichydrazide (2-NZH) caused the time- and concentration-dependent inhibition of both PGHS-2 peroxidase and cyclooxygenase activities. H(2)O(2) was required for the inactivation of both PGHS-2 activities and indomethacin (which binds at the cyclooxygenase site) did not affect the peroxidase inhibitory potency of 2-NZH. A series of aromatic hydrazides were found to be potent inhibitors of PGHS-2 peroxidase activity with IC(50) values in the 6-100microM range for 13 of the 18 hydrazides examined. Selective inhibition of PGHS-2 over myeloperoxidase and horseradish peroxidase isozyme C was increased by certain ring substitutions. In particular, a chloro group para to the hydrazide moiety increased the PGHS-2 selectivity relative to both myeloperoxidase and horseradish peroxidase isozyme C.     

Inhibition of the biosynthesis of sterols by phenyl and phenolic compounds in rat liver

The presence of mitochondria increased the incorporation of [2-(14)C]mevalonate into sterols in a cell-free system from rat liver. Various phenyl and phenolic compounds inhibited the incorporation of mevalonate when added in vitro. p-Hydroxycinnamate, a metabolite of tyrosine, was the most powerful inhibitor among the compounds tested. Catechol, resorcinol and quinol were inhibitory at high concentrations. Organic acids lacking an aromatic ring were not inhibitory. Two hypocholesterolaemic drugs, Clofibrate (alpha-p-chlorophenoxyisobutyrate) and Clofenapate [alpha,4-(p-chlorophenyl)phenoxyisobutyrate], which are known to affect some step before the formation of mevalonate in the biosynthesis of cholesterol in vivo, showed inhibition at a step beyond the formation of mevalonate in vitro. The presence of the aromatic ring and the carboxyl group in a molecule appears to be necessary for the inhibition.     

A gold-containing drug against parasitic polyamine metabolism: the X-ray structure of trypanothione reductase from Leishmania infantum in complex with auranofin reveals a dual mechanism of enzyme inhibition

Auranofin is a gold(I)-containing drug in clinical use as an antiarthritic agent. Recent studies showed that auranofin manifests interesting antiparasitic actions very likely arising from inhibition of parasitic enzymes involved in the control of the redox metabolism. Trypanothione reductase is a key enzyme of Leishmania infantum polyamine-dependent redox metabolism, and a validated target for antileishmanial drugs. As trypanothione reductase contains a dithiol motif at its active site and gold(I) compounds are known to be highly thiophilic, we explored whether auranofin might behave as an effective enzyme inhibitor and as a potential antileishmanial agent. Notably, enzymatic assays revealed that auranofin causes indeed a pronounced enzyme inhibition. To gain a deeper insight into the molecular basis of enzyme inhibition, crystals of the auranofin-bound enzyme, in the presence of NADPH, were prepared, and the X-ray crystal structure of the auranofin-trypanothione reductase-NADPH complex was solved at 3.5 ? resolution. In spite of the rather low resolution, these data were of sufficient quality as to identify the presence of the gold center and of the thiosugar of auranofin, and to locate them within the overall protein structure. Gold binds to the two active site cysteine residues of TR, i.e. Cys52 and Cys57, while the thiosugar moiety of auranofin binds to the trypanothione binding site; thus auranofin appears to inhibit TR through a dual mechanism. Auranofin kills the promastigote stage of L. infantum at micromolar concentration; these findings will contribute to the design of new drugs against leishmaniasis.     

Gold(III) compounds as anticancer agents: relevance of gold-protein interactions for their mechanism of action

Gold(III) compounds constitute an emerging class of biologically active substances, of special interest as potential anticancer agents. During the past decade a number of structurally diverse gold(III) complexes were reported to be acceptably stable under physiological-like conditions and to manifest very promising cytotoxic effects against selected human tumour cell lines, making them good candidates as anti-tumour drugs. Some representative examples will be described in detail. There is considerable interest in understanding the precise biochemical mechanisms of these novel cytotoxic agents. Based on experimental evidence collected so far we hypothesize that these metallodrugs, at variance with classical platinum(II) drugs, produce in most cases their growth inhibition effects through a variety of "DNA-independent" mechanisms. Notably, strong inhibition of the selenoenzyme thioredoxin reductase and associated disregulation of mitochondrial functions were clearly documented in some selected cases, thus providing a solid biochemical basis for the pronounced proapoptotic effects. These observations led us to investigate in detail the reactions of gold(III) compounds with a few model proteins in order to gain molecular-level information on the possible interaction modes with possible protein targets. Valuable insight on the formation and the nature of gold-protein adducts was gained through ESI MS (electrospray ionization mass spectrometry) and spectrophotometric studies of appropriate model systems as it is exemplified here by the reactions of two representative gold(III) compounds with cytochrome c and ubiquitin. The mechanistic relevance of gold(III)-induced oxidative protein damage and of direct gold coordination to protein sidechains is specifically assessed. Perspectives for the future of this topics are briefly outlined.     

Scavenger effect of flavonols on HOCl-induced luminol chemiluminescence.

Hypochlorous acid (HOCl), the main product of the myeloperoxidase system, is a strong oxidant and a potent chlorinating agent, which can damage host tissues. In the present work, the scavenger effect of three aglycone flavonols (myricetin, quercetin and kaempferol) and of the natural glycoside flavonol, rutin, was studied towards HOCl using luminol-dependent chemiluminescence (CL). At 1 micro mol/L fi nal concentration, rutin was the most powerful scavenger of HOCl with an inhibitory luminol oxidation of 91.4% +/- 3.2%. Quercetin, kaempferol and myricetin inhibited the luminol-dependent CL at the same concentration only by 75.9% +/- 3.4%, 57.7% +/- 5.3% and 43.3% +/- 3.5%, respectively. With increasing concentration of these flavonols, a dose-dependent inhibition of luminol CL was observed. In order to prove to what extent flavonols scavenge HOCl, their concentrations that gave 50% inhibition of luminescence (IC50) were compared to IC50 values of the sulphur-containing compounds N-acetyl cysteine (NAC) and taurine. The scavenging activities of compounds tested decrease in the order: rutin > NAC > quercetin > kaempferol > taurine. The present study revealed that rutin was the most effective scavenger agent.     

Interaction of Gold(I) with the Active Site of Selenium-Glutathione Peroxidase

Gold(I) thioglucose in the presence of excess glutathione (GSH) leads to strong and reversible inhibition of selenium-glutathione peroxidase (EC 1.11.19) around neutral pH. Binding at equilibrium and competition studies demonstrated that the most reduced form of the active site selenocysteine is the only binding site for gold(I) Steady-state kinetics indicate that gold(I) forms a dead-end complex with glutathione peroxidase in competition with the reduction of hydroperoxide. The apparent K₁ is 2 3 μM at pH 7.6,37°C and 1 mM GSH. Theoretical models of inhibition were assessed by the use of linear least-squares fitting to a generalized integrated rate equation. The results are consistent with trapping of gold(I) at the active site in the form of a mixed bidentate selenolato-thiolate complex involving GSH and the active site selenocysteine. The kinetics of inhibition imply that the resting form of glutathione peroxidase in the presence of excess GSH is also within the enzyme cycle. This rules out the existence of selenium(+lV) species in the redox cycle of the active site when t-butylhydroperoxide is used as a substrate. Electronic properties of selenium and gold as well as a large relief of inhibition by selenocysteine suggest that a very stable interaction should be obtained between Se(-II) and gold(I) through covalent bonding. These results suggest that glutathione peroxidase could be a target of gold drugs used in the treatment of rheumatoid arthritis.     

Gold(III)-Dithiocarbamato Peptidomimetics in the Forefront of the Targeted Anticancer Therapy: Preclinical Studies against Human Breast Neoplasia

Since the serendipitous discovery of cisplatin, platinum-based drugs have become well-established antitumor agents, despite the fact that their clinical use is limited by many severe side-effects. In order to both improve the chemotherapeutic index and broaden the therapeutic spectrum of current drugs, our most recent anti-neoplastic agents, Au(III) complexes, were designed as carrier-mediated delivery systems exploiting peptide transporters, which are up-regulated in some cancers. Among all, we focused on two compounds and tested them on human MDA-MB-231 (resistant to cisplatin) breast cancer cell cultures and xenografts, discovering the proteasome as a major target both in vitro and in vivo. 53% inhibition of breast tumor growth in mice was observed after 27 days of treatment at 1.0 mg kg⁻¹ d⁻¹, compared to control. Remarkably, if only the most responsive mice are taken into account, 85% growth inhibition, with some animals showing tumor shrinkage, was observed after 13 days. These results led us to file an international patent, recognizing this class of gold(III) peptidomimetics as suitable candidates for entering phase I clinical trials.     

Thiol-containing molecules interact with the myeloperoxidase/H2O2/chloride system to inhibit LDL oxidation

Oxidized low-density lipoproteins (LDL) accumulate in the vascular wall and promote a local inflammatory process contributing to the progression of atheromatous plaque. The key role of myeloperoxidase (MPO) in this process has been documented and the enzyme has been involved in the oxidative modification of apolipoprotein B-100 in the intima and at the surface of endothelial cells. As the inhibition of this last phenomenon could be of relevance in pharmacological interventions, thiol-containing molecules such as glutathione, captopril, and N-acetylcysteine (NAC) and its lysinate salt (NAL) were tested in this system and their properties were compared with those of flufenamic acid (control). This last compound already demonstrated an inhibition of the production of HOCl by MPO and a more intense inhibition of MPO activity than glutathione, NAC, NAL, and captopril. However, NAC and NAL inhibited the oxidative modification of LDL more intensively than captopril and glutathione whereas flufenamic acid had no comparable inhibiting effect. This could be related to the presence of LDL close to the catalytic site of the enzyme. NAC and NAL therefore appeared as the most efficient inhibitors probably as a consequence of their relatively small size. The relevance of such effects has to be documented by in vivo studies.     

Litomosoides carinii: mode of action in vitro of benzothiazole and amoscanate derivatives with antifilarial activity

It is suggested that the recently developed benzothiazole and amoscanate derivatives with antifilarial activity exert their action in vitro by an inhibition of mitochondrial-derived respiration. It was confirmed that the drugs CGP 20376, 21835, 20308, 21306, and 6140 cause a rapid immobilization in vitro of the adult filarial worm, Litomosoides carinii, the time required being similar to rotenone at the same concentration. The other drugs investigated, CGPs 20309, 21833, 24589, 23518, and 13231, were also effective; however, they required much longer incubation times. Submitochondrial particles (SMP) were prepared from Ascaris muscle and rat liver. The concentration of drug causing 50% inhibition of respiration (IC50) was calculated. It was found that the drugs most rapidly inhibiting respiration have IC50s for NADH oxidase of less than 25 microM in both Ascaris and rat liver SMP. This effect on SMP respiration could be overcome by using succinate as a substrate, indicating the site of inhibition to be within complex I of the mitochondrial respiratory chain. Further experiments showed that whereas the respiratory chain's NADH:ferricyanide reductase was unaffected by these drugs, there were pronounced effects on both Ascaris and rat liver NADH:quinone reductase activity. This suggests that the inhibition within complex I occurs after the flavoprotein dehydrogenase, but before the site of the quinone reduction. The other compounds examined, which had a slower effect on motility, also showed inhibition of the NADH oxidase, but not to as great an extent as the aforementioned compounds. The compounds most active against motility were also most effective at inhibiting respiration in intact adult L. carinii. Analysis of the aerobic end products produced by L. carinii showed that acetate production was greatly reduced even in the presence of low concentrations of the drugs. There was also a slight decrease in lactate production. However, a direct effect on the glycolytic pathway was ruled out by two observations. One, that the production of lactate from cell-free extracts of L. carinii is unaffected by the presence of the drugs, and secondly, that a protozoan, Giardia lamblia, reliant on glycolysis for energy production, can survive for long periods of time in the presence of high concentrations of the drugs. A correlation can be observed between the time for immobilization of the filarial worm and the strength of inhibition of mitochondrial respiration. Therefore, it is suggested that, at least in vitro, the mechanism of toxicity of these antifilarials in L. carinii is due to the blocking of the respiratory chain at a site similar to that of rotenone.     

Comparative reactivities of various biological compounds with myeloperoxidase-hydrogen peroxide-chloride,and similarity of oxidant to hypochlorite

The reactivities of myeloperoxidase-H₂O₂-Cl^? and sodium hypochlorite with amino acids, uric acid, NADH, ascorbic acid, ADP, albumin, haemoglobin, α₁-antitrypsin and some hydroxyl radical scavengers have been compared. The ability of each compound to inhibit chlorination of monochlorodimedon by both oxidants was measured. Relative reaction rates varied over a range of 10⁵, but the reactivities of the two oxidants with each compound were very similar, from which it is concluded that the reactions of hypochlorite accurately reflect those of the myeloperoxidase system. Thiol compounds (cysteine and GSH) and methionine were more than 100-times more reactive than other amino acids, which had comparable reactivity to NADH and uric acid. Benzoate, dimethylsulphoxide and formate were very much less reactive. The significance of these reactions of myeloperoxidase in microbial killing and inflammation is discussed.     

Inhibition of peroxidase-catalyzed protein tyrosine nitration by antithyroid drugs and their analogues

In this paper, we describe the effect of some commonly used thiourea-based antithyroid drugs and their analogues on the peroxidase-catalyzed nitration reactions. The nitration of bovine serum albumin (BSA) and cytochrome c was studied using the antibody against 3-nitro-l-tyrosine. This study reveals that the thione-based antithyroid drugs effectively inhibit lactoperoxidase (LPO)-catalyzed nitration of BSA. These compounds show very weak inhibition towards the nitration of cytochrome c. Some of these compounds also inhibit myeloperoxidase (MPO)-catalyzed nitration of l-tyrosine. A structure-activity correlation study on the peroxidase-catalyzed nitration of l-tyrosine reveals that the presence of thione/selone moiety is important for the inhibition. Although the presence of a free N-H group adjacent to CS moiety is necessary for most of the thiones to inhibit the LPO-catalyzed nitration, the corresponding selones do not require the presence of any free N-H group for their activity. Furthermore, experiments with different concentrations of H₂O₂ suggest that the antithyroid drugs and related thiones inhibit the nitration reaction mainly by coordinating to the Fe(III)-center of the enzyme active site as previously proposed for the inhibition of peroxidase-catalyzed iodination. On the other hand, the selenium compounds inhibit the nitration by scavenging H₂O₂ without interacting with the enzyme active site. This assumption is based on the observations that catalase effectively inhibits tyrosine nitration by scavenging H₂O₂, which is one of the substrates for the nitration. In contrast, superoxide dismutase (SOD) does not alter the nitration reactions, indicating the absence of superoxide radical anion (O₂^-) during the peroxidase-catalyzed nitration reactions.     

Phenylsulphonyl urenyl chalcone derivatives as dual inhibitors of cyclo-oxygenase-2 and 5-lipoxygenase

Two series of phenylsulphonyl urenyl chalcone derivatives (UCH) with various patterns of substitution were tested for their effects on nitric oxide (NO) and prostaglandin E2 (PGE2) overproduction in RAW 264.7 macrophages. None of the tested compounds reduced NO production more than 50% at 10 microM but most of them inhibited the generation of PGE2 with IC50 values under the micromolar range. Me-UCH 1, Me-UCH 5, Me-UCH 9, Cl-UCH 1, and Cl-UCH 9 were selected to evaluate their influence on human leukocyte functions and eicosanoids generation. These derivatives selectively inhibited cyclo-oxygenase-2 (COX-2) activity in human monocytes being Me-UCH 5 the most potent (IC50 0.06 microM). Selected compounds also reduced leukotriene B4 synthesis in human neutrophils by a direct inhibition of 5-lipoxygenase (5-LO) activity, with IC50 values from 0.5 to 0.8 microM. In addition, lysosomal enzyme secretion, such as elastase or myeloperoxidase as well as superoxide generation in human neutrophils were also reduced in a similar range. Our findings indicate that UCH derivatives exert a dual inhibitory effect on COX-2/5-LO activity. The profile and potency of these compounds may have relevance for the modulation of the inflammatory and nociceptive responses with reduction of undesirable side-effects associated with NSAIDs.     

The reactions of horseradish peroxidase, lactoperoxidase, and myeloperoxidase with enzymatically generated superoxide

The formation and decay of intermediate compounds of horseradish peroxidase, lactoperoxidase, and myeloperoxidase formed in the presence of the superoxide/hydrogen peroxide-generating xanthine/xanthine oxidase system has been studied by observation of spectral changes in both the Soret and visible spectral regions and both on millisecond and second time scales. It is tentatively concluded that in all cases compound III is formed in a two-step reaction of native enzyme with superoxide. The presence of superoxide dismutase completely inhibited compound III formation; the presence of catalase had no effect on the process. Spectral data which indicate differences in the decay of horseradish peroxidase compound III back to the native state in comparison with compounds III of lactoperoxidase and myeloperoxidase are also presented.     

Gold compounds as cysteine protease inhibitors: perspectives for pharmaceutical application as antiparasitic agents

Gold compounds form a new class of promising metal-based drugs with a number of potential therapeutic applications, particularly in the fields of anticancer and antimicrobial treatments. Previous research revealed that a group of structurally diverse gold compounds cause conspicuous inhibition of the protease activities of the human proteasome. Given the pharmacological importance of protease inhibition, the present study further explored whether these gold compounds might inhibit a few other proteases that are accepted druggable targets for disease treatment. In particular, four distinct cysteine proteases were considered here: cathepsin B and L that play a primary role in tumor-cell invasion and metastasis; rhodesain, the major cathepsin L-like cysteine protease of Trypanosoma brucei rhodesiense and CPB2.8ΔCTE, a Leishmania mexicana mature cysteine protease. Based on the encouraging results obtained for some of the tested gold compounds on the two parasitic cysteine proteases, especially against CPB2.8ΔCTE, with IC_50s in the micromolar range, we next evaluated whether those gold compounds might contrast effectively the growth of the respective protozoa and indeed important antiprotozoal properties were disclosed; on the other hand a certain lack of selectivity was highlighted. Also, no direct or clear correlation could be established between the in vitro antiprotozoal properties and the level of protease inhibition. The implications of these results are discussed in relation to possible pharmaceutical applications.     

A peroxidase/phenolics/ascorbate system can scavenge hydrogen peroxide in plant cells

The function of a peroxidase/phenolics/ascorbic acid system in plant vacuoles has not yet been well elucidated. We wished to study the redox reactions among hydrogen peroxide, phenolics and ascorbic acid (AA) in the presence of horseradish peroxidase. Horseradish peroxidase oxidized rutin and chlorogenic acid (CGA), compounds present in many kinds of plant. The oxidation was inhibited by AA. As a result of the inhibition. AA was oxidized and when almost all of it had been oxidized, oxidation of the phenolics commenced. Monodehydroascorbic acid (MDA) radical was detected during the oxidation of AA, suggesting that the inhibition of oxidation of rutin and CGA was due to reduction of phenoxyl radicals by AA. By comparison of time courses of changes in levels of AA and MDA radicals, and by kinetic calculation, it is suggested that in addition to AA, MDA radicals may also reduce phenoxyl radicals. It is proposed that the peroxidase/phenolics/AA system can function as a hydrogen peroxide scavenging system.     

Effects of grapefruit juice on the multidrug transporter P-glycoprotein in the human proximal tubular cell line HK-2

The multidrug transporter MDR-1 P-glycoprotein (Pgp) has been recently pointed out as an important mechanism underlying chemical interaction between drugs and many commonly ingested substances, including grapefruit juice (GFJ). Modulation of intestinal Pgp dependent transport by GFJ may lead to changes in bioavailability of drugs that are substrates of Pgp itself, by affecting their presystemic clearance. Since other cellular sites expressing Pgp and devoted to drug disposition, like kidney proximal tubules, could be involved in these pharmacokinetic interactions, we investigated the effect of GFJ on the expression and activity of Pgp in the human immortalized tubular cell line HK-2. Two flavonoid compounds related to GFJ, kaempferol and naringenin, were also tested for their effects on HK-2 Pgp. HK-2 cells cultured for 4 days in the presence of GFJ, showed a dose-dependent decrease in Pgp immunoblottable amount as well as a decrease in MDR-1 mRNA level, as shown by western blot analysis and RT-PCR, respectively. Both kaempferol and naringenin were also able to significantly decrease Pgp immunoblottable amount. To test whether the downregulation of HK-2 Pgp due to GFJ exposition could influence the cell sensitivity to drugs that are transported by Pgp itself, HK-2 cells precultured with GFJ were exposed to scalar concentrations of Cyclosporin A or Vinblastine and cell viability examined 36 hours later. The cytotoxicity of both drugs was increased. The calcein-AM test in untreated cells showed that GFJ, kaempferol or naringenin inhibited Pgp activity. Downregulation of Pgp as well inhibition of its function by GFJ or its related components in tubular cells could have a role in changing disposition kinetics of some important therapeutic agents.     

Gold complexes as prospective metal-based anticancer drugs

Medical and therapeutic value of gold has been recognized thousands of years ago, but its rational use in medicine has not begun until the early 1920s. Cisplatin is one of the first metal-containing compounds with anti-cancer activity discovered in the 1960s. Despite the fact that cisplatin treatment is efficient for several types of solid tumors, its effectiveness is limited by toxic side effects and tumor resistance that often leads to the occurrence of secondary malignancies. Since gold(III) is isoelectronic with platinum(II) and tetracoordinate gold(III) complexes have the same square-planar geometries as cisplatin, the anticancer activity of gold(III) compounds has been investigated. Previous studies suggested that, in contrast to cisplatin, gold complexes target proteins but not DNA. Recently, we have investigated gold(III) dithiocarbamates for their anticancer activity and showed that their primary target is the proteasome. Treatment of human breast tumor-bearing nude mice with a gold(III) dithiocarbamate complex resulted in significant inhibition of tumor growth, associated with proteasome inhibition and massive apoptosis induction in vivo. Better understanding of physiological processing of gold compounds will provide a rational basis for their further development into novel anticancer drugs.