Discovery of substituted maleimides as liver X receptor agonists and determination of a ligand-bound crystal structure

Substituted 3-(phenylamino)-1H-pyrrole-2,5-diones were identified from a high throughput screen as inducers of human ATP binding cassette transporter A1 expression. Mechanism of action studies led to the identification of GSK3987 (4) as an LXR ligand. 4 recruits the steroid receptor coactivator-1 to human LXR alpha and LXRP with EC(50)s of 40 nM, profiles as an LXR agonist in functional assays, and activates LXR though a mechanism that is similar to first generation LXR agonists.

Antitumour prodrug development using cytochrome P450 (CYP) mediated activation

An ideal cancer chemotherapeutic prodrug is completely inactive until metabolized by a tumour-specific enzyme, or by an enzyme that is only metabolically competent towards the prodrug under physiological conditions unique to the tumour. Human cancers, including colon, breast, lung, liver, kidney and prostate, are known to express cytochrome P450 (CYP) isoforms including 3A and 1A subfamily members. This raises the possibility that tumour CYP isoforms could be a focus for tumour-specific prodrug activation. Several approaches are reviewed, including identification of prodrugs activated by tumour-specific polymorphic CYPs, use of CYP-gene directed enzyme prodrug therapy and CYPs acting as reductases in hypoxic tumour regions. The last approach is best exemplified by AQ4N, a chemotherapeutic prodrug that is bioreductively activated by CYP3A. This study shows that freshly isolated murine T50/80 mammary carcinoma and RIF-1 fibrosarcoma 4-electron reduces AQ4N to its cytotoxic metabolite, AQ4 (T50/80 K-m = 26.7 mu M, V-max = 0.43 mu M/mg protein/min; RIF-1 K-m = 33.5 mu M, V-max = 0.42 mu M/mg protein/min) via AQM, a mono-N-oxide intermediate (T50/80 K-m = 37.5 mu M; V-max = 1.4 mu M/mg protein/min; RIF-1 K-m = 37.5 mu M; V-max = 1.2 mu M/mg protein/min). The prodrug conversion was dependent on NADPH and inhibited by air or carbon monoxide. Cyp3A mRNA and protein were both present in T50/80 carcinoma grown in vivo (RIF-1 not measured). Exposure of isolated tumour cells to anoxia (2 h) immediately after tumour excision increased cyp3A protein 2-3-fold over a 12 h period, after which time the cyp protein levels returned to the level found under aerobic conditions. Conversely, cyp3A mRNA expression showed an initial 3-fold decrease under both oxic and anoxic conditions; this returned to near basal levels after 8-24 h. These results suggest that cyp3A protein is stabilized in the absence of air, despite a decrease in cyp3A mRNA. Such a 'stabilization factor' may decrease cyp3A protein turnover without affecting the translation efficiency of cyp3A mRNA. Confirmation of the CYP activation of AQ4N bioreduction was shown with human lymphoblastoid cell microsomes transfected with CYP3A4, but not those transfected with CYP2B6 or cytochrome P450 reductase. AQ4N is also reduced to AQ4 in NADPH-fortified human renal cell carcinoma (K-m = 4 mu M, V-max = 3.5 pmol/mg protein/min) and normal kidney (K-m = 4 mu M, V-max = 4.0 pmol/mg protein/min), both previously shown to express CYP3A. Germane to the clinical potential of AQ4N is that although both normal and tumour cells are capable of reducing AQ4N to its cytotoxic species, the process requires low oxygen conditions. Hence, AQ4N metabolism should be restricted to hypoxic tumour cells. The isoform selectivity of AQ4N reduction, in addition to its air sensitivity, indicates that AQ4N haem coordination and subsequent oxygen atom transfer from the active-site-bound AQ4N is the likely mechanism of N-oxide reduction. The apparent increase in CYP3A expression under hypoxia makes this a particularly interesting application of CYPs for tumour-specific prodrug activation.

Genetic diversity of liver flukes (Fasciola hepatica) from Eastern Europe.

The genetic diversity of liver fluke populations in three different countries from Eastern Europe (Greece, Bulgaria, and Poland) in comparison with available data from other countries was determined. Specifically, SNPs from regions of two nuclear genes, 28S rDNA, ß-tubulin 3 and an informative region of the mitochondrial genome were examined. Two major lineages for the 28S rDNA gene based on the highly polymorphic 105th nucleotide position were found. These lineages were widely and almost equally spread not only through the countries studied but also in other investigated geographical areas. Two basic lineages and additional haplotypes were defined for the mtDNA gene region, consisting of the cytochrome c oxidase subunit III gene, transfer RNA histidine gene and cytochome b gene. The basic lineages were observed within Greek, Bulgarian, and Polish Fasciola hepatica populations but the distribution of additional haplotypes differed between the populations from the three countries. For the ß-tubulin 3 gene multiple polymorphic sites were revealed but no explicit clades. The SNPs were spread unequally in all studied geographical regions with an evident distinction between the Greek and Polish specimens. Additional genotypes for the 28S rDNA region as well as haplotypes of the mtDNA region that were typical for the Greek or Polish populations were observed. Significant polymorphisms for ß-tubulin 3 gene were displayed with decreasing percentage of presence within populations from Greece to Poland. There was an amino acid substitution in ß-tubulin 3 protein found only among Polish specimens. It is hypothesized that genotypic differences between Greek, Bulgarian, and Polish liver fluke populations are due to territorial division and genetic drift in past epochs.

Potential Rôle of Hares in the Spread of Liver Fluke in the Netherlands.

Hares (Lepus europeanus) sharing pasture with cattle from six locations in the Netherlands were examined for the presence of liver fluke (Fasciola hepatica) and shown to have prevalences of infection ranging from zero to 41%. The mitochondrial haplotypes of liver flukes present in the hare populations were determined and compared with those found in cattle from a farm where triclabendazole resistance has been reported. Phylogenetic analysis indicated that the flukes present in the hares belonged to the same clades as those present in the cattle. A consideration of the life cycle of the liver fluke and the seasonal breeding pattern and ecology of hares supports the suggestion that hares may act as a refugia for liver fluke and as a vector for the spread of drug-resistant genotypes.

Comparative proteome analysis of triclabendazole response in the liver fluke, Fasciola hepatica.

Control of Fasciola hepatica infections of livestock in the absence of vaccines depends largely on the chemical triclabendazole (TCBZ) because it is effective against immature and adult parasites. Overdependence on a single drug and improper application is considered a significant factor in increasing global reports of fluke resistant to TCBZ. The mode(s) of action and biological target(s) of TCBZ are not confirmed, delaying detection and the monitoring of early TCBZ resistance. In this study, to further understand liver fluke response to TCBZ, the soluble proteomes of TCBZ-resistant and TCBZ-susceptible isolates of F. hepatica were compared with and without in vitro exposure to the metabolically active form of the parent drug triclabendazole sulphoxide (TCBZ-SO), via two-dimensional gel electrophoresis (2-DE). Gel image analysis revealed proteins displaying altered synthesis patterns and responses both between isolates and under TCBZ-SO exposure. These proteins were identified by mass spectrometry supported by a F. hepatica expressed sequence tag (EST) data set. The TCBZ responding proteins were grouped into three categories; structural proteins, energy metabolism proteins, and “stress” response proteins. This single proteomic investigation supported the reductionist experiments from many laboratories that collectively suggest TCBZ has a range of effects on liver fluke metabolism. Proteomics highlighted differences in the innate proteome profile of different fluke isolates that may influence future therapy and diagnostics design. Two of the TCBZ responding proteins, a glutathione transferase and a fatty acid binding protein, were cloned, produced as recombinants, and both found to bind TCBZ-SO at physiologically relevant concentrations, which may indicate a role in TCBZ metabolism and resistance.

Multiple origins of European populations of giant liver fluke Fascioloides magna (Trematoda: Fasciolidae), an invasive liver parasite of ruminants

The giant liver fluke, Fascioloides magna, liver parasite of free-living and domestic ruminants of Europe and North America, was analysed in order to determine the origin of European populations and to reveal the biogeography of this originally North American parasite on the European continent. The previously selected variable fragments of the mitochondrial cytochrome c oxidase subunit I (cox1; 384 bp) and nicotinamid dehydrogenase subunit I (nad1; 405 bp) were applied as a tool. The phylogenetic trees and haplotype networks were constructed and the level of genetic structuring was evaluated using population genetic tools. In F. magna individuals originating from all European natural foci (Italy, Czech Republic, Danube floodplain forests) and from four of five major North American enzootic areas, 16 cox1 and 18 nad1 haplotypes were determined. The concatenated sequence set produced 22 distinct haplotypes. The European fluke populations were less diverse than those from North America in that they contained proportionately fewer haplotypes (8), while more substantial level of genetic diversity and higher number of haplotypes (15) were recorded in North America. Only one haplotype was shared between the European (Italy) and North American (USA/Oregon and Canada/Alberta) flukes supporting a western North American origin of the Italian F. magna population. Haplotypes found in Italy were distinct from those determined in the remaining European localities what indicates that introduction of F. magna onto the European continent is a result of more than one event. In Czech focus, a south-eastern US origin of giant liver fluke was revealed. Identical haplotypes, common for parasites from Czech Republic and from expanding focus of Danube floodplain forests, implies introduction of F. magna to the Danube region from an already established Czech focus.