Repositioning of the Anthelmintic Drugs Bithionol and Triclabendazole as Transthyretin Amyloidogenesis Inhibitors.

Transthyretin (TTR) is a causative protein of TTR amyloidosis (ATTR amyloidosis), a general term for diseases characterized by deposition of TTR amyloid fibrils in specific organs. ATTR amyloidosis can be ameliorated by stabilization of the TTR tetramer through the binding of small molecules. Here, we show that the clinical anthelmintic drugs bithionol (42) and triclabendazole (43) potently inhibit aggregation of the amyloidogenic variant V30M-TTR. A competitive binding assay using a fluorescence probe showed that the binding affinity of 42 with V30M-TTR was significantly higher than that of the first-in-class drug tafamidis (1), and the binding affinity of 43 was similar to that of 1. The crystallographic and thermodynamic analysis revealed that 42 efficiently occupied the halogen-binding grooves of TTR, resulting in the favorable binding entropy. Multifaceted in vitro studies of anthelmintic drugs have the potential to reposition these drugs as ATTR amyloidosis inhibitors.

Biosynthesis of N-Docosahexanoylethanolamine from Unesterified Docosahexaenoic Acid and Docosahexaenoyl-Lysophosphatidylcholine in Neuronal Cells.

We investigated the synthesis of N-docosahexaenoylethanolamine (synaptamide) in neuronal cells from unesterified docosahexaenoic acid (DHA) or DHA-lysophosphatidylcholine (DHA-lysoPC), the two major lipid forms that deliver DHA to the brain, in order to understand the formation of this neurotrophic and neuroprotective metabolite of DHA in the brain. Both substrates were taken up in Neuro2A cells and metabolized to N-docosahexaenoylphosphatidylethanolamine (NDoPE) and synaptamide in a time- and concentration-dependent manner, but unesterified DHA was 1.5 to 2.4 times more effective than DHA-lysoPC at equimolar concentrations. The plasmalogen NDoPE (pNDoPE) amounted more than 80% of NDoPE produced from DHA or DHA-lysoPC, with 16-carbon-pNDoPE being the most abundant species. Inhibition of N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD) by hexachlorophene or bithionol significantly decreased the synaptamide production, indicating that synaptamide synthesis is mediated at least in part via NDoPE hydrolysis. NDoPE formation occurred much more rapidly than synaptamide production, indicating a precursor-product relationship. Although NDoPE is an intermediate for synaptamide biosynthesis, only about 1% of newly synthesized NDoPE was converted to synaptamide, possibly suggesting additional biological function of NDoPE, particularly for pNDoPE, which is the major form of NDoPE produced.

Dissecting the Antenna in Human Glutamate Dehydrogenase: Understanding Its Role in Subunit Communication and Allosteric Regulation.

Glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of l-glutamate. While GDH is found in all living organisms, only that from animals is highly allosterically regulated by a wide array of metabolites. Because only animal GDH has a 50-residue antenna domain, we hypothesized that it was critical for allostery. To this end, we previously replaced the antenna with the loop found in bacteria, and the resulting chimera was no longer regulated by purine nucleotides. Hence, it seemed logical that the purpose of the antenna is to exert the subunit communication necessary for heterotrophic allosteric regulation. Here, we revisit the antenna deletion studies by retaining 10 more of the human GDH (hGDH) residues without adding the bacterial loop. Unexpectedly, the results were profoundly different than before. The basal activity of the mutant is only ∼13% of that of the wild type but ∼100 times more sensitive to all allosteric activators. In contrast, the mutant is still affected by all of the tested inhibitors to approximately the same degree. The resulting antenna-less mutant retained its negative cooperativity with respect to the coenzyme, again suggesting that intersubunit communication is intact. Finally, the mutant still exhibits substrate inhibition, albeit there are differences in the details. We present a model in which the majority of the antenna is not directly involved in allosteric regulation per se but rather may be responsible for improving enzymatic efficiency by acting as a conduit for substrate binding energy between subunits.

The inhibitors of soluble adenylate cyclase 2-OHE, KH7, and bithionol compromise mitochondrial ATP production by distinct mechanisms.

Soluble adenylate cyclase (sAC) is a non-plasma membrane-bound isoform of the adenylate cyclases signaling via the canonical second messenger, 3',5'-cyclic AMP (cAMP). sAC is involved in key physiological processes such as insulin release, sperm motility, and energy metabolism. Thus, sAC has attracted interest as a putative drug target and attempts have been made to develop selective inhibitors. Since sAC has a binding constant for its substrate, ATP, in the millimolar range, reductions in mitochondrial ATP production may be part of the mechanism-of-action of sAC inhibitors and the potential of these compounds to study the physiological outcomes of inhibition of sAC might be severely hampered by this. Here, we evaluate the effects of two commonly employed inhibitors, 2-OHE and KH7, on mitochondrial ATP production and energy metabolism. For comparison, we included a recently identified inhibitor of sAC, bithionol. Employing mitochondria isolated from mouse brain, we show that all three compounds are able to curb ATP production albeit via distinct mechanisms. Bithionol and KH7 mainly inhibit ATP production by working as a classical uncoupler whereas 2-OHE mainly works by decreasing mitochondrial respiration. These findings were corroborated by investigating energy metabolism in acute brain slices from mice. Since all three sAC inhibitors are shown to curb mitochondrial ATP production and affect energy metabolism, caution should be exercised when employed to study the physiological roles of sAC or for validating sAC as a drug target.

Evaluation of the cytotoxicity of the Bithionol - cisplatin combination in a panel of human ovarian cancer cell lines.

BACKGROUND: Combination drug therapy appears a promising approach to overcome drug resistance and reduce drug-related toxicities in ovarian cancer treatments. In this in vitro study, we evaluated the antitumor efficacy of cisplatin in combination with Bithionol (BT) against a panel of ovarian cancer cell lines with special focus on cisplatin-sensitive and cisplatin-resistant cell lines. The primary objectives of this study are to determine the nature of the interactions between BT and cisplatin and to understand the mechanism(s) of action of BT-cisplatin combination. METHODS: The cytotoxic effects of drugs either alone or in combination were evaluated using presto-blue assay. Cellular reactive oxygen species were measured by flow cytometry. Immunoblot analysis was carried out to investigate changes in levels of cleaved PARP, XIAP, bcl-2, bcl-xL, p21 and p27. Luminescent and colorimetric assays were used to test caspases 3/7 and ATX activity. RESULTS: The efficacy of the BT-cisplatin combination depends upon the cell type and concentrations of cisplatin and BT. In cisplatin-sensitive cell lines, BT and cisplatin were mostly antagonistic except when used at low concentrations, where synergy was observed. In contrast, in cisplatin-resistant cells, BT-cisplatin combination treatment displayed synergistic effects at most of the drug ratios/concentrations. Our results further revealed that the synergistic interaction was linked to increased reactive oxygen species generation and apoptosis. Enhanced apoptosis was correlated with loss of pro-survival factors (XIAP, bcl-2, bcl-xL), expression of pro-apoptotic markers (caspases 3/7, PARP cleavage) and enhanced cell cycle regulators p21 and p27. CONCLUSION: In cisplatin-resistant cell lines, BT potentiated cisplatin-induced cytotoxicity at most drug ratios via enhanced ROS generation and modulation of key regulators of apoptosis. Low doses of BT and cisplatin enhanced efficiency of cisplatin treatment in all the ovarian cancer cell lines tested. Our results suggest that novel combinations such as BT and cisplatin might be an attractive therapeutic approach to enhance ovarian cancer chemosensitivity. Combining low doses of cisplatin with subtherapeutic doses of BT can ultimately lead to the development of an innovative combination therapy to reduce/prevent the side effects normally occurring when high doses of cisplatin are administered.

Assessment of the antitumor potential of Bithionol in vivo using a xenograft model of ovarian cancer.

In terms of the concept of 'drug repurposing', we focused on pharmaceutical-grade Bithionol (BT) as a therapeutic agent against ovarian cancer. Our recent in-vitro study provides preclinical data suggesting a potential therapeutic role for BT against recurrent ovarian cancer. BT was shown to cause cell death by caspases-mediated apoptosis. The present preliminary study further explores the antitumor potential of pharmaceutical-grade BT in an in-vivo xenograft model of human ovarian cancer. Nude Foxn1 mice bearing SKOV-3 human ovarian tumor xenografts were treated with titrated doses of BT and the therapeutic efficacy of pharmaceutical BT was determined using bioluminescence imaging. BT-induced changes in cell proliferation and apoptosis were evaluated by Ki-67 immunochemical staining and TUNEL assay. The effect of BT on autotaxin levels in serum, ascitic fluid, and tumor tissue was assessed by colorimetric and western blot techniques. BT treatment did not show antitumor potential or enhanced survival time at any of the doses tested. No apparent signs of toxicity were observed with any of the doses tested. Immunohistological analysis of tumor sections did not indicate a significant decrease in cellular proliferation (Ki-67 assay). An increase in apoptosis (by TUNEL assay) was observed in all BT-treated mice compared with vehicle-treated mice. Although BT did not show significant antitumor activity in the present study, the ability of BT to induce apoptosis still makes it a promising therapeutic agent. Further confirmatory and optimization studies are essential to enhance the therapeutic effects of BT.

Bithionol inhibits ovarian cancer cell growth in vitro - studies on mechanism(s) of action.

BACKGROUND: Drug resistance is a cause of ovarian cancer recurrence and low overall survival rates. There is a need for more effective treatment approaches because the development of new drug is expensive and time consuming. Alternatively, the concept of 'drug repurposing' is promising. We focused on Bithionol (BT), a clinically approved anti-parasitic drug as an anti-ovarian cancer drug. BT has previously been shown to inhibit solid tumor growth in several preclinical cancer models. A better understanding of the anti-tumor effects and mechanism(s) of action of BT in ovarian cancer cells is essential for further exploring its therapeutic potential against ovarian cancer. METHODS: The cytotoxic effects of BT against a panel of ovarian cancer cell lines were determined by Presto Blue cell viability assay. Markers of apoptosis such as caspases 3/7, cPARP induction, nuclear condensation and mitochondrial transmembrane depolarization were assessed using microscopic, FACS and immunoblotting methods. Mechanism(s) of action of BT such as cell cycle arrest, reactive oxygen species (ROS) generation, autotaxin (ATX) inhibition and effects on MAPK and NF-kB signalling were determined by FACS analysis, immunoblotting and colorimetric methods. RESULTS: BT caused dose dependent cytotoxicity against all ovarian cancer cell lines tested with IC50 values ranging from 19 µM - 60 µM. Cisplatin-resistant variants of A2780 and IGROV-1 have shown almost similar IC50 values compared to their sensitive counterparts. Apoptotic cell death was shown by expression of caspases 3/7, cPARP, loss of mitochondrial potential, nuclear condensation, and up-regulation of p38 and reduced expression of pAkt, pNF-κB, pIκBα, XIAP, bcl-2 and bcl-xl. BT treatment resulted in cell cycle arrest at G1/M phase and increased ROS generation. Treatment with ascorbic acid resulted in partial restoration of cell viability. In addition, dose and time dependent inhibition of ATX was observed. CONCLUSIONS: BT exhibits cytotoxic effects on various ovarian cancer cell lines regardless of their sensitivities to cisplatin. Cell death appears to be via caspases mediated apoptosis. The mechanisms of action appear to be partly via cell cycle arrest, ROS generation and inhibition of ATX. The present study provides preclinical data suggesting a potential therapeutic role for BT against recurrent ovarian cancer.

An improved thyroid hormone reporter assay to determine the thyroid hormone-like activity of amiodarone, bithionol, closantel and rafoxanide.

A number of environmental chemicals have been reported to exhibit thyroid hormone-like activity. Since thyroid hormones play a crucial role in development, it is important to identify chemicals in the environment that are capable of endocrine disruption of thyroid hormone homeostasis. In order to detect thyroid hormone-like activity, the growth of pituitary cell lines has been commonly used as a sensitive marker, albeit with limited specificity to thyroid hormones. Reporter gene assays using the thyroid hormone responsive element (TRE) connected to the luciferase reporter gene have also been developed. Thus far however, this type of assay appears to have limited sensitivity compared to cell growth assays. In the present study, we developed a highly sensitive TRE reporter gene assay by using a pituitary cell line, MtT/E-2, and by culturing cells in a serum-free medium. Our assay was developed in order to detect T3 activity at a concentration of 10(-11)M. This assay identified thyroid hormone-like activity from the antiarrhythmic drug, amiodarone, and from three anti-parasitic drugs, bithionol, closantel and rafoxanide, all commonly used in veterinary medicine. Thyroid hormone-like activity of these compounds was further confirmed by the induction of BCL3 gene expression in MtT/E-2, which is known to be regulated by thyroid hormones. Our improved assay was proved to be a sensitive tool for assessing thyroid hormone-like activity of environmental chemicals.

Phosphorylation of histone H2AX is a powerful tool for detecting chemical photogenotoxicity.

Several light-absorbing chemicals are known to show phototoxic effects involving many kinds of DNA damage, and are suspected of initiating skin cancer. In this study, we clarified that phosphorylated histone H2AX (γ-H2AX) (phosphorylated histone H2AX), which was produced with the induction of DNA double-strand breaks, is a sensitive photogenotoxic marker. The immortal human keratinocyte line HaCaT was treated with a library of 11 chemicals (including known strong and weak phototoxic chemicals, and nonphototoxic chemicals) and/or UVA exposure. γ-H2AX was generated after treatments with all phototoxic chemicals and UVA. The limit of detection using γ-H2AX was 100-1,000 times lower than that using cell viability and DNA gel electrophoresis. γ-H2AX was not generated following treatments with nonphototoxic chemicals and UVA. These results indicated that γ-H2AX is a powerful tool for detecting chemical photogenotoxicity.

Novel inhibitors complexed with glutamate dehydrogenase: allosteric regulation by control of protein dynamics.

Mammalian glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of l-glutamate to 2-oxoglutarate using NAD(P)(+) as coenzyme. Unlike its counterparts from other animal kingdoms, mammalian GDH is regulated by a host of ligands. The recently discovered hyperinsulinism/hyperammonemia disorder showed that the loss of allosteric inhibition of GDH by GTP causes excessive secretion of insulin. Subsequent studies demonstrated that wild-type and hyperinsulinemia/hyperammonemia forms of GDH are inhibited by the green tea polyphenols, epigallocatechin gallate and epicatechin gallate. This was followed by high throughput studies that identified more stable inhibitors, including hexachlorophene, GW5074, and bithionol. Shown here are the structures of GDH complexed with these three compounds. Hexachlorophene forms a ring around the internal cavity in GDH through aromatic stacking interactions between the drug and GDH as well as between the drug molecules themselves. In contrast, GW5074 and bithionol both bind as pairs of stacked compounds at hexameric 2-fold axes between the dimers of subunits. The internal core of GDH contracts when the catalytic cleft closes during enzymatic turnover. None of the drugs cause conformational changes in the contact residues, but all bind to key interfaces involved in this contraction process. Therefore, it seems likely that the drugs inhibit enzymatic turnover by inhibiting this transition. Indeed, this expansion/contraction process may play a major role in the inter-subunit communication and allosteric regulation observed in GDH.

Infection with Fasciola hepatica.

Fascioliasis, caused by the liver fluke Fasciola hepatica, is an infection that occurs worldwide, although humans are accidental hosts. F. hepatica infection comprises two stages, hepatic and biliary, with different signs and symptoms. Stool examination and ELISA can be used for the initial diagnosis. Radiographic techniques, such as computerised tomography and ultrasonography, as well as magnetic resonance imaging, are used widely for confirmation and follow-up of the disease. Invasive techniques, such as percutaneous cholangiography, endoscopic retrograde cholangiography and liver biopsy, may aid in the diagnosis but are not essential. Triclabendazole is recommended as the first-line agent for the treatment of F. hepatica infection, with bithionol as an alternative.

Compartive studies on the effect of bithionol, praziqantel and triclabendazole in rabbit's fascioliasis. 1. Parasitological studies.

Rabbits were infected with 30 metacercariae each. They were then divided into four batches each of two groups. One batch served as controls and the others were treated each with a drug. The first group from each batch was treated when worms were still immature, the other group was treated after maturity. The effect of the drugs was monitored by: (1) Stool examination for eggs and studying the percent of egg reduction. (2) Worm recovery at autopsy and calculating the percent of the drug efficacy. (3) Culturing some eggs and following their embryonation and hatching. Results pointed out that Triclabendazole was the best fasciolicide followed by Bithionol. Praziquantel had a negligible effect.

Trichomonas species: homocysteine desulphurase and serine sulphydrase activities.

Homocysteine desulphurase (EC 4.4.1.2) and serine sulphydrase (EC 4.2.1.22) activities in various lines of Trichomonas vaginalis, both metronidazole resistant and sensitive, and other trichomonad species were assessed. T. vaginalis contained the highest homocysteine desulphurase and serine sulphydrase activities of all the species. Although the levels of the enzyme activity in T. vaginalis isolates differed, no correlation between the activities and sensitivity to metronidazole was apparent. T. vaginalis homocysteine desulphurase catalysed both the hydrolysis of homocysteine to hydrogen sulphide, ammonia, and 2-oxoacid, and an exchange reaction between homocysteine and 2-mercaptoethanol. Homocysteine desulphurase was detected as a single enzyme band on isoelectric focusing, whereas several isoenzymes of serine sulphydrase were found. There were large differences in serine sulphydrase isoenzyme patterns between T. vaginalis lines and between species. Several isoenzymes were amplified in cells grown with 10(-5) M DL-propargylglycine for 24 hr. T. vaginalis homocysteine desulphurase and serine sulphydrase activities were inhibited by bithionol, hexachlorophene, and dichlorophene. These compounds also inhibited growth in vitro of T. vaginalis at concentrations similar to those that inhibited the enzymes.

The induction of photocontact sensitivity in guinea pigs without UVB radiation.

Allergic photocontact sensitization could be induced in guinea pigs with 3,3',4',5-tetrachlorosalicylanilide (TCSA), 3,4',5-tribromosalicylanilide (TBS), and bithionol using pretreatment with sodium lauryl sulfate (SLS) and long-wave ultraviolet (UVA) radiation. Mid-wave ultraviolet below 320 nm (UVB) was not necessary for the induction of sensitization. Combined use of SLS pretreatment with UVA radiation resulted in more effective sensitization than combined UVB and UVA radiation. Higher sensitization rates to TCSA and TBS were achieved by allowing rest periods between each of 5 2-hr exposures to UVA than by daily 1-hr exposures for 10 consecutive days. The opposite result was obtained with bithionol. Although UVB has been customarily used in the past for induction of photosensitivity, its role is only to irritate the site of induction.