Molecular basis for hycanthone drug action in schistosome parasites.

Hycanthone (HYC) is a retired drug formerly used to treat schistosomiasis caused by infection from Schistosoma mansoni and S. haematobium. Resistance to HYC was first observed in S. mansoni laboratory strains and in patients in the 1970s and the use of this drug was subsequently discontinued with the substitution of praziquantel (PZQ) as the single antischistosomal drug in the worldwide formulary. In endemic regions, multiple organizations have partnered with the World Health Organization to deliver PZQ for morbidity control and prevention. While the monotherapy reduces the disease burden, additional drugs are needed to use in combination with PZQ to stay ahead of potential drug resistance. HYC will not be reintroduced into the schistosomiasis drug formulary as a combination drug because it was shown to have adverse properties including mutagenic, teratogenic and carcinogenic activities. Oxamniquine (OXA) was used to treat S. mansoni infection in Brazil during the brief period of HYC use, until the 1990s. Its antischistosomal efficacy has been shown to work through the same mechanism as HYC and it does not possess the undesirable properties linked to HYC. OXA demonstrates cross-resistance in Schistosoma strains with HYC resistance and both are prodrugs requiring metabolic activation in the worm to toxic sulfated forms. The target activating enzyme has been identified as a sulfotransferase enzyme and is currently used as the basis for a structure-guided drug design program. Here, we characterize the sulfotransferases from S. mansoni and S. haematobium in complexes with HYC to compare and contrast with OXA-bound sulfotransferase crystal structures. Although HYC is discontinued for antischistosomal treatment, it can serve as a resource for design of derivative compounds without contraindication.

Lucanthone and its derivative hycanthone inhibit apurinic endonuclease-1 (APE1) by direct protein binding.

Lucanthone and hycanthone are thioxanthenone DNA intercalators used in the 1980s as antitumor agents. Lucanthone is in Phase I clinical trial, whereas hycanthone was pulled out of Phase II trials. Their potential mechanism of action includes DNA intercalation, inhibition of nucleic acid biosyntheses, and inhibition of enzymes like topoisomerases and the dual function base excision repair enzyme apurinic endonuclease 1 (APE1). Lucanthone inhibits the endonuclease activity of APE1, without affecting its redox activity. Our goal was to decipher the precise mechanism of APE1 inhibition as a prerequisite towards development of improved therapeutics that can counteract higher APE1 activity often seen in tumors. The IC(50) values for inhibition of APE1 incision of depurinated plasmid DNA by lucanthone and hycanthone were 5 µM and 80 nM, respectively. The K(D) values (affinity constants) for APE1, as determined by BIACORE binding studies, were 89 nM for lucanthone/10 nM for hycanthone. APE1 structures reveal a hydrophobic pocket where hydrophobic small molecules like thioxanthenones can bind, and our modeling studies confirmed such docking. Circular dichroism spectra uncovered change in the helical structure of APE1 in the presence of lucanthone/hycanthone, and notably, this effect was decreased (Phe266Ala or Phe266Cys or Trp280Leu) or abolished (Phe266Ala/Trp280Ala) when hydrophobic site mutants were employed. Reduced inhibition by lucanthone of the diminished endonuclease activity of hydrophobic mutant proteins (as compared to wild type APE1) supports that binding of lucanthone to the hydrophobic pocket dictates APE1 inhibition. The DNA binding capacity of APE1 was marginally inhibited by lucanthone, and not at all by hycanthone, supporting our hypothesis that thioxanthenones inhibit APE1, predominantly, by direct interaction. Finally, lucanthone-induced degradation was drastically reduced in the presence of short and long lived free radical scavengers, e.g., TRIS and DMSO, suggesting that the mechanism of APE1 breakdown may involve free radical-induced peptide bond cleavage.

Changes of mate occur in Schistosoma mansoni.

Male and female schistosomes are generally assumed to form stable monogamous pairs for the whole span of their long existence in the mammalian host. Recent evidence from mixed infections has shown that Schistosoma mansoni males can displace S. intercalatum males from their homologous partners, but no information exists about the existence of similar phenomena within a single schistosome species. Here, we determine whether male S. mansoni can displace males of the same species from pre-formed pairs in vivo. The availability of clear-cut genetic markers of drug resistance in schistosomes was exploited to show that hycanthone sensitive S. mansoni males can displace homospecific hycanthone resistant males from pre-formed pairs and vice versa. The frequency of changes is dependent on the magnitude of the excess single males competing with paired worms. The possible mechanics and the biological significance of mate changing are discussed.

O6-methylguanine and O6-methylguanine-DNA [corrected] methyltransferase activity in tissues of BDF-1 mice treated with antiparasitic drugs.

Levels of the DNA promutagenic methylation damage, O6-methylguanine (O6-MeG) and the activity of the O6-methylguanine-DNA methyltransferase (MGMT), the enzyme responsible for repairing O6-MeG, were measured at various time intervals in tissues of BDF-I mice administered a single therapeutic dose of the antischistosomal agents hycanthone, oxaminiquine and metrifonate. Hycanthone increased O6-MeG in the liver-DNA after 6 h, then decreased by 3-fold after 48 h. Lower levels of the adduct and a slower rate of formation were found in the intestine and bladder. MGMT activities were significantly lower in the liver (74%) and bladder (25%) compared to control animals after 6 h, then restored by 48 h. Oxaminiquine increased O6-MeG in all tissues, but spleen, after 6 h and persisted only in the bladder after 48 h. Liver and bladder tissues of these animals exhibited a pattern of alteration in the MGMT activity similar to that observed for hycanthone. Metrifonate induced a profile of O6-MeG comparable to that of oxaminiquine but the levels of the adduct were about 2-fold lower. Hepatic MGMT in these animals was significantly lower (approximately 38%) than the control values after 6 h, then restored by 48 h. A significant negative correlation was obtained between O6-MeG and MGMT activity in the liver (r=- 0.85), intestine (r=- 0.62) and bladder (r=- 0.59). These results demonstrate that treatment with antischistosomal agents may lead to the formation of promutagenic alkylation damage in the tissue DNA and alterations in the DNA repair capacity.

Modulation of mitomycin C mutagenicity on Saccharomyces cerevisiae by glutathione, cytochrome P-450, and mitochondria interactions.

It is well established that most anticancer drugs also have mutagenic effects and require metabolic activation before exerting their mutagenic/antiblastic activity. Antitumoral compound effects strongly depend on the biochemical/physiological conditions of the tumoral cells, and especially on the activation of specific drugs metabolizing enzymes and on respiration. We examined the mitomycin C-induced mutagenic effects on the D7 strain of Saccharomyces cerevisiae and on its derivative mitochondrial mutant p degrees at different contents of glutathione and cytochrome P-450, molecules able to activate/detoxicate xenobiotics. The mutagenic activity of the drug was evaluated as frequency of mitotic gene conversion and reversion in different physiological conditions. The highest frequencies of reversion and especially of gene conversion were observed at the highest cytochrome P-450 contents in the D7 strain with a further increase at high glutathione level. In the respiratory-deficient strain, the highest frequency of convertants was shown at low glutathione level and lack of cytochrome P-450. These results suggest the relevance of mitochondrial functionality for the expression of genotoxic activity of this anticancer drug.

Enzymatic basis for the lack of oxamniquine activity in Schistosoma haematobium infections.

The notion that oxamniquine is active against Schistosoma mansoni but inactive against S. haematobium was confirmed using in vitro cultures of adult worms. Since oxamniquine and hycanthone have been shown to become effective upon activation by a schistosome enzyme, enzymatic tests were carried out to detect possible differences between the enzyme of S. mansoni and that of S. haematobium. It was found that the S. mansoni enzyme could activate hycanthone and, to a lesser extent, oxamniquine. The S. haematobium enzyme, on the other hand, was capable of activating hycanthone but virtually incapable of activating oxamniquine. It is concluded that the different activity of oxamniquine in the two species is due to differences in the drug-activating enzyme.

Molecular analysis of the TK locus in L5178Y large and small colony mouse lymphoma cell mutants induced by hycanthone methanesulfonate.

Mouse lymphoma cells of the L5178Y TK + /-3.7.2C line were exposed to varying concentrations of the anti-schistosomal drug hycanthone methanesulfonate. The trifluorothymidine (TFT)-resistant cells fell into two classes based on colony size. Southern blot analyses were performed using NcoI-digested DNA from a number of large and small mutant colonies from each treatment group. Two different restriction fragment banding patterns were identified in these analyses, those colonies that contained the 6.4 kb NcoI restriction fragment and those that did not. A total of 471 mutant colonies were analyzed and 84.5% (398) of these colonies did not exhibit the 6.4 kb fragment. There did not appear to be a hycanthone methanesulfonate dose response effect in the number of colonies that did not contain the 6.4 kb fragment among the treated groups. In addition, 82% (154 out of 188) of spontaneous mutants did not contain the 6.4 kb fragment. The results imply that greater than 80% of all spontaneous mutations found in the mouse lymphoma assay regardless of colony size do not contain the 6.4 kb fragment and each may be considered to be a large scale mutation. In addition, greater than 80% of the hycanthone induced mutations in the mouse lymphoma assay do not contain the 6.4 kb fragment and thus may be considered to be a large scale mutation.

Extended-term cultures of human T-lymphocytes: a practical alternative to primary human lymphocytes for use in genotoxicity testing.

A simplified method, using recombinant interleukin-2, foetal bovine serum and freeze-killed feeder cells, has been developed for the mass culture of T-lymphocytes derived from human peripheral blood. In this protocol, bulk cultures can be cryopreserved approximately 8 days after initiation, and subsequent mass cultures generated a further week after recovery. At the end of this period, the lymphocytes have maintained a normal karyotype and cultures from different donors are very similar in terms of rate of cell division and expression of key antigenic markers. Background micronucleus frequencies and dose-responses for micronucleus induction by a reference clastogen, hycanthone, were also very similar in all the cultures examined. Such extended-term T-lymphocyte cultures are potentially valuable in genotoxicity testing, providing cells with the normal human karyotype which can be characterised and handled with the practical convenience of established rodent cell lines.

Drug resistance to schistosomicides and other anthelmintics of medical significance.

It is usual for people to be infected for some period in life with parasitic worms, which may cause morbidity or even kill. Anthelmintics are used for the treatment and control of the human helminthiases, since no vaccines are yet available. Despite the widespread use of these compounds, drug resistance has become apparent only with antischistosomal chemotherapy, in contrast to the situation with other anti-infective agents in human medicine and with veterinary anthelmintics, where resistance is widespread. This paper reviews research on drug resistance in human helminthiasis with emphasis on schistosomicidal drugs.

Evaluation of external-beam radiation therapy plus 5-fluorouracil (5-FU) versus external-beam radiation therapy plus hycanthone (HYC) in confined, unresectable pancreatic cancer.

From March 1981 to November 1987, 87 patients with histologically confirmed pancreatic adenocarcinoma, unresectable but confined to the pancreatic region, were randomized to two treatments. The standard treatment was 40-50 Gy external-beam radiation therapy (RT) to gross tumor plus potential microscopic tumor with a 5 Gy boost to gross tumor plus a 1.5-2.0 cm margin, using multiple fields and 5-fluorouracil (5-FU) 500 mg/m2/d intravenously by rapid infusion. The 5-FU was given each of the initial 3 days of each of three 20 Gy radiation courses. The experimental treatment used identical radiation fields, but the two Gy daily radiation fractions were administered in a continuous course to a total dose of 50 Gy. Hycanthone was administered 60 mg/m2 intravenously within 2 to 4 hr during each day of the 5-day course of infusions during the first and fifth weeks of radiation therapy. There was no statistically significant difference between treatment arms in survival (p = 0.82) or disease-free survival (p = 0.27). Seven percent of hycanthone-treated patients demonstrated hepatic toxicity which was usually mild in nature. There was, however, one death due to hepatic toxicity.

Schistosoma mansoni: genetic complementation analysis shows that two independent hycanthone/oxamniquine-resistant strains are mutated in the same gene.

Two drug-resistant strains of Schistosoma mansoni were compared in this study in order to decide whether they are both mutated in the same gene with respect to drug-sensitive schistosomes. One of the two strains was isolated in the laboratory, while the other one originated from a treated uncured patient and was subsequently drug selected in the laboratory. The approach consisted in a genetic complementation test performed essentially by crossing the two strains and assessing resistance in the progeny. Since no reappearance of drug sensitivity was detected in the progeny, it was concluded that the two strains failed to complement and were therefore mutated in the same gene. This finding suggests that a single step of drug activation operates in sensitive schistosomes and is ineffective in resistant worms.

Formation of promutagenic methylation damage in tissue-DNA of mice treated with antischistosomal agents.

The existence of the promutagenic methylation damage O6-MedG has been measured at various time intervals in different tissue DNAs of mice received a single therapeutic dose of various antischistosomal agents (hycanthone, oxaminiquine and metrifonate). Liver-DNA exhibited the highest levels of O6-MedG in all treated animals while, spleen DNA contained the lowest. The three antischistosomal agents tested seemed to exert the peak concentrations of their alkylating metabolites over a period of several hours following the administration. In mice which had received hycanthone, liver-DNA contained readily detectable amounts of O6-MedG by 6 h post-treatment (0.089 mol O6-MedG/mol dG) and by the end of 48 h, this was decreased by about 3-fold to reach a level of 0.026 mumol/mol dG. In intestinal-DNA, however, O6-MedG was formed more slowly and contained about half the level of that found in the liver-DNA. In the tissue-DNA of animals which had received oxaminiquine, the highest level of O6-MedG was observed at 6 h after administration and at a 24-h time point, the adduct dramatically decreased in the liver and intestine-DNA to undetectable values. In neither tissues was there any evidence for O6-MedG accumulation in the DNA at the end of a 48-h post-treatment. A pattern of O6-MedG, almost similar to that of oxaminiquine, was also observed in tissue-DNA of mice pretreated with metrifonate. These results demonstrate that treatment with antischistosomal agents leads to the formation of highly promutagenic alkylated lesions in the tissue-DNA. The implication of such existence for antischistosomal-induced toxicity and carcinogenicity are discussed.

Preferential intercalation at AT sequences in DNA by lucanthone, hycanthone, and indazole analogs. A footprinting study.

DNAase I footprinting has been used to probe the DNA sequence selectivity of the antitumor intercalating agents lucanthone (1), hycanthone (2), 6-chlorolucanthone (7), and four indazole analogs (IA-3-IA-6). The latter have a benzothiopyranoindazole chromophore substituted with a diethylaminoethyl side chain identical to that attached to the thioxanthenone chromophore of compounds 1, 2, and 7. IA-3 and IA-5 are lucanthone analogs bearing a methyl group at position 4, whereas IA-4 and IA-6 are hycanthone analogs bearing a hydroxymethyl group. IA-3 and IA-4 have an additional chloro group at position 6. Studies employing the 160-bp tyrT DNA fragment as substrate to assay inhibition of DNAase I-mediated cleavage show that both lucanthone and hycanthone bind preferentially to AT sites. They discriminate against GC-rich sequences as well as short runs of a single base, which are often cut more readily in the presence of the drugs compared to the control. The indazole analogs exhibit more pronounced selectivity of binding to AT sequences and promote enhanced DNAase I cleavage both at GC-rich sequences and at homooligomeric runs of adenines or thymines. The results of further DNAase I cleavage inhibition assays, performed with three more restriction fragments having different base pair arrangements, are fully consistent with those obtained with the tyrT fragment. They reveal that the preferred binding sequences for lucanthone, hycanthone, and the indazole analogs are predominantly composed of alternating A and T residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Response of drug resistant isolates of Schistosoma mansoni to antischistosomal agents.

The susceptibility of four isolates of Schistosoma mansoni (BH, MAP, MPR-1 and K) to four multiple doses of anti-schistosomal agents (hycanthone, niridazole, oxamniquine, and praziquantel) were evaluated in infected female Swiss albino mice. These schistosomal isolates had been maintained in the laboratory without further drug pressure for 20 to 30 generations. Multiple dosage regimens were used for each drug against each isolate of S. mansoni to generate ED50 (effective dose 50%) values. Results demonstrated that the K isolate is resistant to niridazole, the MPR-1 isolate to oxamniquine, and the MAP isolate to both hycanthone and oxamniquine. The BH isolate was susceptible to all drugs and was used as the reference isolate. All isolates were susceptible to parziquantel. The significance of the difference in response of the MPR-1 and MAP isolates is discussed. These results confirm the resistance of these isolates of S. mansoni to three schistosomicides and demonstrate that the resistance of these isolates are stable over long periods of time without exposure to drugs.

Schistosoma mansoni: hycanthone/oxamniquine resistance is controlled by a single autosomal recessive gene.

Individual schistosomes of an hycanthone/oxamniquine-sensitive strain were crossed with individual schistosomes of the opposite sex and belonging either to the same sensitive population or to a different strain which exhibited high resistance to the two drugs. Schistosome crosses were performed by transfer of single worm pairs into the mesenteric veins of mice and the drug sensitivity/resistance of individual progeny worms was assessed using an in vitro test. Drug resistance behaved as an autosomal recessive trait, as shown by the results of the F1 and F2 generation and of the backcrosses. Drug-resistant worms appeared to be slightly less viable than their sensitive counterpart at all stages of the life cycle. The results are relevant for an interpretation of drug resistance and drug mechanisms and the approach used in this study may be applicable to different genetic markers in schistosomes.

Sex differences in micronucleus induction with hycanthone methanesulfonate in bone marrow cells of mice.

The clastogenic effect of the antischistosomal drug hycanthone methanesulfonate was studied with the micronucleus test in mouse bone marrow cells. Male and female (102/El x C3H/El)F1 mice were treated with single i.p. injections. Bone marrow was sampled 18, 24 and 30 h after treatment with 100 mg/kg. The highest micronucleus yield occurred at 24 h. The dose response for micronucleus induction at 24 h after treatment was non-linear for doses between 5 and 300 mg/kg. The lowest effective dose was 5 mg/kg for females and 10 mg/kg for males. The experiments revealed a significantly higher sensitivity of female mice for the induction of micronuclei in polychromatic erythrocytes by hycanthone methanesulfonate. This result supports the recommendation to use both sexes for quantitative assessment of genotoxicity in the micronucleus test.

Hycanthone resistance in schistosomes correlates with the lack of an enzymatic activity which produces the covalent binding of hycanthone to parasite macromolecules.

Crude extracts of hycanthone sensitive Schistosoma mansoni incubated at 37 degrees C in the presence of ATP and Mg2+ induced the covalent binding of tritiated hycanthone (HC) to macromolecules. The same behavior was shown by the HC sensitive species, Schistosoma rodhaini, whereas two independently isolated HC resistant S. mansoni strains had no detectable activity. Sensitive male schistosomes had more activity than females or immature worms. Virtually no activity was present in mouse liver, in human liver, in HeLa cells or in the naturally resistant species Schistosoma japonicum. The activity was destroyed by boiling or by Proteinase K treatment. Covalent binding of tritiated HC to macromolecules could be inhibited by cold HC, oxamniquine or IA-4, while none of the in vitro ineffective analogs, like lucanthone, UK-3883 or 4-desmethyl lucanthone, were inhibitory. These results strongly support the previously advanced suggestion that HC is activated by enzymatic mechanisms which are present only in drug sensitive schistosomes.

Studies on carbohydrates extracted from native and chemically treated Biomphalaria alexandrina snails.

1. Carbohydrates were extracted from total tissue extracts of Biomphalaria alexandrina snails and were analyzed to their monosaccharides using GLC. 2. The snails were chemically treated with thioxanthone derivatives (compounds I, II, III) and the change in the monosaccharide constituents of their carbohydrates was investigated. 3. The isolated monosaccharides from native and chemically pretreated snails were injected into mice and their protective effects were examined after infection of mice with cercariae of Schistosoma mansoni. 4. The results showed that the main monosaccharides in carbohydrates of snails were galactose, glucose, fucose and mannose and that chemical treatment caused a drop in the galactose content. 5. Moreover, monosaccharide fractions from snails treated with compound III were the most effective in inducing protection against Schistosoma infection in mice.

Genetic complementation analysis of two independently isolated hycanthone-resistant strains of Schistosoma mansoni.

The objective of this study is to determine whether various hycanthone resistant strains of schistosomes which have been independently isolated are all affected in the same gene. A strain obtained from a Brazilian patient was compared with a strain of Puerto Rican origin selected in the laboratory. If the mutation conferring resistance involved two different genes, one would expect that progeny of a cross between the two strains would show complementation, i.e. it would be sensitive to the drug. We have performed such a cross and obtained F1 hybrid worms which were essentially all resistant, thus suggesting that the mutation conferring resistance in the two strains involves the same gene.