The anthelmintic action of praziquantel

Although fairly expensive (around US$4.00 per single dose) praziquantel is now the most favoured drug against all forms of schistosomiasis, and against many other helminth infections. It is now marketed by four companies: E. Merck and Bayer (F.R.G.), Ames-Myers (USA), and Shin-Poon Pharmaceuticals (S. Korea). Administration of praziquantel typically causes paralysis of susceptible worms, or damage to their tegument, making them more vulnerable to host enzymes or antibody-dependent immune effector mechanisms. Other effects may also be involved. Here, Bill Harnett reviews the range of anthelmintic effects displayed by this remarkable drug.     

Drug interactions with Bacillus anthracis topoisomerase IV: biochemical basis for quinolone action and resistance

Bacillus anthracis, the causative agent of anthrax, is considered a serious threat as a bioweapon. The drugs most commonly used to treat anthrax are quinolones, which act by increasing the levels of DNA cleavage mediated by topoisomerase IV and gyrase. Quinolone resistance most often is associated with specific serine mutations in these enzymes. Therefore, to determine the basis for quinolone action and resistance, we characterized wild-type B. anthracis topoisomerase IV, the GrlA(S81F) and GrlA(S81Y) quinolone-resistant mutants, and the effects of quinolones and a related quinazolinedione on these enzymes. Ser81 is believed to anchor a water-Mg(2+) bridge that coordinates quinolones to the enzyme through the C3/C4 keto acid. Consistent with this hypothesized bridge, ciprofloxacin required increased Mg(2+) concentrations to support DNA cleavage by GrlA(S81F) topoisomerase IV. The three enzymes displayed similar catalytic activities in the absence of drugs. However, the resistance mutations decreased the affinity of topoisomerase IV for ciprofloxacin and other quinolones, diminished quinolone-induced inhibition of DNA religation, and reduced the stability of the enzyme-quinolone-DNA ternary complex. Wild-type DNA cleavage levels were generated by mutant enzymes at high quinolone concentrations, suggesting that increased drug potency could overcome resistance. 8-Methyl-quinazoline-2,4-dione, which lacks the quinolone keto acid (and presumably does not require the water-Mg(2+) bridge to mediate protein interactions), was more potent than quinolones against wild-type topoisomerase IV and was equally efficacious. Moreover, it maintained high potency and efficacy against the mutant enzymes, effectively inhibited DNA religation, and formed stable ternary complexes. Our findings provide an underlying biochemical basis for the ability of quinazolinediones to overcome clinically relevant quinolone resistance mutations in bacterial type II topoisomerases.     

Therapy and anthelmintic resistance

Treatment against nematode parasites in sheep and goats is reviewed. The main risk factors for parasitic infection in these hosts are briefly outlined. The mechanism of action of the most important chemical groups (imidazothiazoles and pyrimidines, benzimidazoles/ pro-benzimidazoles and macrocyclic lactones) to which the modern anthelmintic drugs belong are illustrated and discussed with particular emphasis on possible selection of anthelmintic resistance. The need for strategic integrated control based also on the epidemiological patterns of parasitism, the composition and the production of the herd (milk, meat, wool) and the potency of the drugs are discussed. The importance of diet and potential alternative control measures (nematophagus fungi and natural compounds such as tannins) are illustrated.     

Toxoplasma gondii: the biochemical basis of resistance to emimycin

Emimycin was a potent and selective inhibitor of the growth and nucleic acid synthesis of Toxoplasma gondii in human fibroblasts. An emimycin-resistant mutant of T. gondii lost the pyrimidine salvage enzyme uracil phosphoribosyltransferase, the same enzyme absent in parasites resistant to fluorodeoxyuridine. The mutant resistant to emimycin was completely cross-resistant to fluorodeoxyuridine. Emimycin was as good a substrate as uracil for the uracil phosphoribosyltransferase of T. gondii. [3H]Emimycin supplied in the medium of cultures with actively growing intracellular parasites was converted to emimycin riboside-5'-phosphate in the soluble pool of T. gondii. All other emimycin analogs of uracil-containing nucleotides were also formed but little emimycin riboside diphosphate-N-acetylhexosamine was found. [3H]Emimycin was not converted to analogs of the cytidine nucleotides. When intracellular T. gondii were treated with a concentration of [3H]emimycin that partially inhibited parasite RNA synthesis, much less [3H]emimycin was incorporated into RNA than would be predicted by the amount of intracellular [3H]emimycin riboside triphosphate.     

Pharmacology of anthelmintic resistance.

Anthelmintic resistance has grown from a curiosity to an important economic problem in several animal industries and is now set to threaten the control of human parasites. The pharmacology of anthelmintics and anthelmintic resistance has been studied most extensively in the nematode parasites of sheep. Here, Nick Sangster and Jenny Gill discuss this veterinary experience, summarizing the progress made in understanding anthelmintic resistance and highlighting the tools available for research.     

The biochemical basis of mitochondrial diseases

Disfunctioning of human mitochondria is found in a rapidly increasing number of patients. The mitochondrial system for energy transduction is very vulnerable to damage by genetic and environmental factors. A primary mitochondrial disease is caused by a genetic defect in a mitochondrial enzyme or translocator. More than 60 mitochondrial enzyme deficiencies have been reported. Secondary mitochondrial defects are caused by lack of compounds to enable a proper mitochondrial function or by inhibition of that function. This may result from malnutrition, circulatory or hormonal disturbances, viral infection, poisoning, or an extramitochondrial error of metabolism. Once mitochondrial ATP synthesis decreases, secondary mitochondrial lesions may be generated further, due to changes in synthesis and degradation of mitochondrial phospholipids and proteins, to mitochondrial antibody formation following massive degradation, to accumulation of toxic products as excess acyl-CoA, to the depletion of Krebs cycle intermediates, and to the increase of free radical formation and lipid peroxidation.     

The biochemical basis for resistance to adenine arabinoside in a mutant of Toxoplasma gondii.

We have previously reported the isolation and preliminary characterization of a mutant of Toxoplasma gondii that was resistant to adenine arabinoside. Fiftyfold higher concentrations of adenine arabinoside were required to inhibit the growth of the resistant parasite in human fibroblast cultures. To determine the enzymic basis for resistance, we measured the kinases and deaminases that act on adenosine or deoxyadenosine. All of these enzymic activities were found in uninfected human fibroblast cells. The mutant and wild type parasite proved to have similar activities of adenosine deaminase, deoxyadenosine deaminase, and deoxyadenosine kinase. However, the adenine arabinoside resistant mutant had less than 0.1% of the adenosine kinase activity observed in the wild type T. gondii. The mutant parasite is presumably resistant because without adenosine kinase to phosphorylate adenine arabinoside it cannot carry out the first step in the conversion of the analogue to adenine arabinoside triphosphate, the active form. A mutant of 3T6 (mouse) cells previously selected for a loss of adenosine kinase also proved to be resistant to adenine arabinoside.     

Density dependence and the spread of anthelmintic resistance

Variation in the strength of selection pressures acting upon different subpopulations may cause density-dependent regulatory processes to act differentially on particular genotypes and may influence the rate of selection of adaptive traits. Using host-helminth parasite systems as examples, we investigate the impact of different positive and negative density dependence on the potential spread of anthelmintic resistance. Following chemotherapy, the negative density-dependent processes restricting parasite population growth will be relaxed, increasing the genetic contribution of resistant parasites to the next generation. Simple deterministic models of directly transmitted nematodes that merge population dynamics and genetics show that the frequency of drug-resistant alleles may increase faster in species whose population size is down-regulated by density-dependent parasite fecundity than in species with density-dependent establishment or parasite mortality. A genetically structured population dynamics model of an indirectly transmitted nematode is used to highlight how population regulation will influence the resistance allele frequency in different parasite lifestages. Results indicate that surveys aimed at monitoring the evolution of drug resistance should consider carefully which life stage to sample, and the time following treatment samples should be collected. Anthelmintic resistance offers a good opportunity to apply fundamental evolutionary and ecological principles to the management of a potentially crucial public health problem.     

The molecular basis of cytokinin action

Current understanding of cytokinin (CK) physiology at the cellular level results largely from the manipulation of endogenous CK levels by either application of exogenous CKs or the expression of CK biosynthetic transgenes, as well as the characterisation of single gene mutants. Cytokinins modulate changes in plant gene expression, which are in turn assumed to effect physiological and morphological changes with which CK action is associated. Presently, a major focus of investigation is elucidation of the biochemical events leading from the perception of CK to the manifestation of a response. Analysis of the expression patterns of CK-regulated genes and identification of their products provides one means of investigating CK action at the molecular level. Biochemical approaches have led to the identification of several soluble CK-binding proteins, although their functional roles in CK signalling largely remain uncertain. Conclusive identification of a bona fide CK receptor has yet to be achieved, although several potential candidates have been suggested. Pharmacological and molecular genetic strategies have implicated the involvement of signalling mechanisms likely to be involved in CK action. The apparent involvement of fluctuations in the concentration of intracellular Ca²⁺, changes in protein phosphorylation as well as DNA and/or protein methylation provide information concerning the types of proteins likely to be involved in the process. Dissection of CK signal transduction chains and elucidation of their interaction with other pathways that regulate plant growth and development is likely to be essential in understanding the mode of action of this poorly understood class of plant growth regulator. However, integration of this knowledge with an improved understanding of the mechanisms whereby overall hormone homeostasis is regulated at the metabolic level will be necessary for comprehensive appreciation of the influence of CKs on plant morphology and physiology.     

Behavioral fingerprints predict insecticide and anthelmintic mode of action

Novel invertebrate‐killing compounds are required in agriculture and medicine to overcome resistance to existing treatments. Because insecticides and anthelmintics are discovered in phenotypic screens, a crucial step in the discovery process is determining the mode of action of hits. Visible whole‐organism symptoms are combined with molecular and physiological data to determine mode of action. However, manual symptomology is laborious and requires symptoms that are strong enough to see by eye. Here, we use high‐throughput imaging and quantitative phenotyping to measure Caenorhabditis elegans behavioral responses to compounds and train a classifier that predicts mode of action with an accuracy of 88% for a set of ten common modes of action. We also classify compounds within each mode of action to discover substructure that is not captured in broad mode‐of‐action labels. High‐throughput imaging and automated phenotyping could therefore accelerate mode‐of‐action discovery in invertebrate‐targeting compound development and help to refine mode‐of‐action categories.     

Studies on the biochemical basis of susceptibility and resistance of the housefly to fluoroacetate

1. Administration of fluoroacetate to sensitive houseflies in amounts close to the L.D.₅₀ range (0.25–0.3 μg/fly) brought about a prompt elevation of their citrate content. With about 10-fold higher doses of fluoroacetate a concurrent increase of both citrate and pyruvate levels took place in the fly tissues.

2. Incubation of sarcosomes of the sensitive housefly strain in the presence of oxidizable substrates and fluoroacetate resulted in accumulation of citrate, inhibition of respiration and uncoupling of oxidative phosphorylation. The magnitude of the effects varied considerably with the different substrates used, being particularly pronounced with pyruvate and malate and inappreciable with succinate and -glycerophosphate.

3. The respiratory inhibition induced by a brief exposure in the cold of housefly sarcosomes to fluoroacetate, persisted after the sarcosomes had been washed free from fluoroacetate. The toxic effect of fluoroacetate on the respiratory chain could be prevented by an excess of simultaneously added acetate.

4. The susceptibility of the respiratory function of the sarcosomes to fluoroacetate inhibition was abolished by sonication. The unresponsiveness of the sonicated sarcosomes to fluoroacetate was attended by a loss of their respiratory chain phosphorylation activity.

5. Sarcosomes derived from a partially resistant housefly strain, when incubated in the presence of fluoroacetate, failed to accumulate citrate, but displayed the characteristic respiratory-inhibition response. Sarcosomes from a highly resistant strain showed no impairment of their functional capacity by fluoroacetate. However, all the different housefly strains tested proved to be equally sensitive to the deleterious effect of fluorocitrate on sarcosomal respiration.

6. The possible biochemical mechanisms underlying the toxicity of fluoroacetate in the housefly are considered with particular reference to the altered response of the target systems exhibited by the fluoroacetate-resistant strains.     

The molecular and biochemical basis of Duchenne muscular dystrophy.

Duchenne and Becker muscular dystrophy (DMD, BMD) have both been clinically recognized for over 100 years, yet throughout much of that time nothing beyond clinical evaluation and supportive care during the disease course was available to patients. The identification of the molecular basis of DMD/BMD in 1986 paved the way for extensive progress toward the understanding, diagnosis and treatment of this disease.