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.

Activity of various amphiphilic agents in reversing multidrug resistance of L 1210 cells.

Several compounds (bamipine, chlorphenoxamine, estracyt, hycanthone, quinidine, quinine, tamoxifen, trifluoperazine and verapamil) have a common basic structure with the following features: lipophilic aromatic ring system; linked chain hydrophilic N-alkyl group. They are used medically for varying diseases. Their activity in reversing multidrug-resistance (MDR) with other compounds (diethylstilbestrol, beta-estradiol, methylbiguanide, methylpiperazine, testosterone) lacking one of these chemical features is compared. The in vitro test system we used was the nucleoside incorporation assay using parental L 1210 ascites tumor cells and a doxorubicin resistant subline, which expresses the MDR phenotype. The substances lacking one of these features were not effective in reversing the MDR whereas all other tested substances demonstrated modulating potential in the MDR resistant L 1210 cells.