Michael Londershausen

Review: Approaches to New Parasiticides

Intensive use of parasiticides has sometimes led to severe resistance in arthropods and helminths of veterinary importance. In the context of the growing awareness of parasitic diseases, this has created a public demand for effective and safe control agents. During the last two decades considerable knowledge in parasite neurophysiology and endocrinology has accumulated which allows the development of new screening procedures and target-site-directed approaches for the discovery of new drugs. The suitability of this strategic approach is discussed on the basis of recent discoveries of new chemical and natural compounds. In particular, target sites such as GABA A receptors, muscarinic and nicotinic acetylcholine receptors, cuticle synthesis and degradation, ecdysteroid receptors, the calcium release channel and semiochemicals have been selected in order to demonstrate the current approaches to identify new chemical entities, biologically active against nematodes and arthropods.

Chitin metabolism: a target for drugs against parasites.

Chitin is an important component of the exoskeleton of arthropods and of the egg shell in nematodes, but it does not occur in vertebrates. Therefore, it represents a useful target for drugs against ectoparasitic crustaceans, insects and endoparasitic nematodes. In this review we describe the basic characteristics of chitin, chitin synthesis and degradation and the hormonal regulation of chitin metabolism. Substances interfering with chitin metabolism like benzoylphenyl-urea derivatives but also some recently detected compounds are described. The necessity for a more detailed understanding of chitin metabolism and the establishment of better model systems, like e.g. chitin producing insect cell lines, is stressed and some examples are given in this review.

Stability and Biological Activity of Cyfluthrin Isomers

A GC-MS method capable of completely separating the four pairs of diastereoisomers of cyfluthrin is presented and the method used to show that isomerisation of the cyfluthrin enantiomers occurs in methanol. This methanol-induced isomerisation could also be demonstrated by bioassays using water fleas. The biological activities of the various cyfluthrin isomers contained in the commercial products cyfluthrin and beta-cyfluthrin were assayed using several strains of lepidopteran larvae including Plutella xylostella, Heliothis virescens and Spodoptera frugiperda. With the susceptible strains, the efficiencies of the isomers mixtures of cyfluthrin and beta-cyfluthrin were shown to obey the rules of additivity. However, in tests with a resistant strain of P. xylostella originating from Thailand, the ‘inactive’ isomer III acted synergistically with the active isomer IV. Resistance factors in strains of H. virescens and P. xylostella were found to be higher with cis than with trans isomers. This probably contributes to the superior action of cyfluthrin and beta-cyfluthrin against various pests of agricultural importance since the commercial products contain a high content of trans isomers (‘high trans pyrethroids’).

Anthelmintic Actions of the Cyclic Depsipeptide PF1022A and its Electrophysiological Effects on Muscle Cells of Ascaris suum

The cyclic depsipeptide PF1022A, given orally to mice, showed very good anthelmintic activity against Heligmosomoides polygyrus and Heterakis spumosa at 50 mg kg -1 . In vitro, PF1022A was very active against Trichinella spiralis and had good activity against Nippostrongylus brasiliensis at 1 μg ml -1 . An 18-membered enniatin analogue, JES 1798, showed good activity only against N. brasiliensis at 10 μg ml -1 . The optical antipode of PF1022A had poor activity even at 100 μg ml -1 . The effects of PF1022A on the membrane potential and input conductance of somatic muscle of Ascaris suum were examined using a two-microelectrode current-clamp technique. PF1022A did not antagonize the effects of the selective nicotinic agonist levamisole. PF1022A and an analogue, JES 1798, but not the PF1022A antipode, produced a small time-dependent increase in input conductance associated with no potential change. The increase in input conductance did not occur in the Cl - -free bathing solution, suggesting that the increase in input conductance was mediated by Cl - ions. The addition of high concentrations of Ca 2+ to the preparation after the addition of PF1022A did not lead to production of Ca 2+ -activated Cl - channels, suggesting that its mode of action was not that of a Ca 2+ ionophore. The mechanism by which the cyclic depsipeptide might increase the Cl - conductance is discussed.

Ionophore and anthelmintic activity of PF 1022A, a cyclooctadepsipeptide, are not related

PF 1022A, a 24-membered cyclooctadepsipeptide and a potent anthelmintic drug, is active against nematodes but not against arthropods. Muscle cells of Ascaris suum generate autorhythmic spikes in electrophysiological control experiments. Exposure of the worm to PF 1022A leads to flaccid paralysis and in parallel to the disappearance of these spike events. Results of such experiments in vitro were compared with those from experiments using planar lipid bilayer membranes incorporating PF 1022A, a related linear octadepsipeptide and other cyclodepsipeptides. Whereas PF 1022A acts both as an ionophore in lipid bilayers, similar to other cyclodepsipeptides like valinomycin and enniatin A, and as a paralysing drug in worms, some of the series of depsipeptides examined have only an ion carrier function, while others exhibit only nematocidal activity. It is concluded that the ion carrier property of PF 1022A is not responsible for its paralyzing effect on nematodes and that there is a specific binding site for PF 1022A in nematodes. Binding may trigger the lethal reaction cascade, which is responsible for anthelmintic activity.

Synthesis of a radiolabeled enniatin cyclodepsipeptide [3H‐methyl]JES 1798

For receptor binding studies and the elucidation of the mode of action of the potent anthelmintic compound JES 1798 a tritium labeled compound at very high specific activity was necessary. Tritium was introduced by methylation of the N-demethyl precursor JES 2314. The identity of [N-methyl- 3 H]JES 1798 was determined by mass spectrometry. After synthesis and purification, 535 μg [N-methyl- 3 H]JES 1798 were available at a specific activity of 84 Ci/mmol (3.11 TBq/mmol) as determined by mass spectrometry. The total activity was 80 mCi (2.96 GBq). Radiolabeled JES 1798 showed an efficient and specific binding to a membrane fraction from Ascaris suum. Displacement by unlabeled JES 1798 was half-maximal at about 0.72 ± 0.06 μM. Different known enniatins also competed for the [ 3 H]JES 1798-binding in the Ascaris suum membrane preparation. In vitro comparison of JES 1798 with enniatin A, A 1 , B and B 1 or beauvericin revealed that enniatin A showed an anthelmintic activity against Nippostrongylus brasiliensis, Trichinella spiralis and Heterakis spurnosa at a concentration of 5 μg/ml, whereas enniatins A 1 , B and B 1 had an activity at concentrations between 1 and 100 μg/ml. On the other hand beauvericin and JES 1798 exerted their anthelmintic activities at 100 μg/ml and therefore possess minor anthelmintic potency in vitro as compared to the natural occurring enniatins

Characterization and Inhibitor Studies of Chitinases from a Parasitic Blowfly (Lucilia cuprina), a Tick (Boophilus microplus), an Intestinal Nematode (Haemonchus contortus) and a Bean (Phaseolus vulgaris)

The molecular weight pattern and the stage-specific activities of chitinases from the blowfly Lucilia cuprina, the tick Boophilus microplus and the intestinal nematode Haemonchus contortus were examined. Chitinolytic enzymes could be detected in all parasite species tested, but the activity was different between the stages. Highest chitinolytic titers were found in blowfly pupae (83 kDa, 118 kDa), hatching larvae of ticks (58 kDa, 94 kDa) and nematode eggs (43 kDa). Leaves from ethylene-treated bean plants Phaseolus vulgaris expressed two basic Class I chitinases (Ia, Ib) of 34 kDa, differing in their amino acid sequences at residue 33 and 34 (Ia : glycine, proline ; Ib : lysine, aspartic acid). Inhibitor studies with blowfly pupae revealed that allosamidin (IC 50 = 0.32 (±0.02) μM) was by far the best inhibitor when compared with various amino sugar derivatives. This compound also inhibited chitinases from tick larvae (IC 50 = 0.69(±0.10) μM) and nematode eggs (IC 50 = 0.048(±0.0045) μM) specifically. Whereas Class Ia chitinase from bean leaves was inhibited only up to 18% by 10 μM allosamidin, it had an IC 50 of 1(±0.14) μM for the Ib type, which is the first plant chitinase described to be highly sensitive to allosamidin.

Characterization of cytoplasmic ecdysteroid receptors in the hypodermis of the crayfish, Orconectes limosus

[3H]Ecdysone, [3H]20-OH-ecdysone and [3H]ponasterone A were specifically bound by the 120 000 g supernatant of the homogenate from crayfish hypodermis. According to the HPLC test, the bound hormone was not metabolized. Analysis of the cytosol receptor by sucrose-density-gradient centrifugation, gel filtration and enzymatic degradation revealed the protein character of the receptor, a molecular weight of 70 000-80 000 D, and 5S. The binding capacity was rapidly lost after treatment with N-ethyl-maleimide or heating for 10 min at 60 degrees C. Optimal conditions for the demonstration of the receptor were incubation at 0-8 degrees C for 30 min and a salt concentration of 120 mM. A sequence of moulting hormones, and steroid hormones without moulting hormone activity were tested for ligand specificities. Estimation KD values yielded 3-6 x 10(-11) M for ponasterone A, 2-3 x 10(-9) M for 20-OH-ecdysone and 2 x 10(-8) M for ecdysone.

Synthesis of a radiolabeled cyclodepsipeptide [3H-methyl]PF1022A

For receptor binding studies and the elucidation of the mode of action of the potent anthelmintic compound PF1022A a tritium labeled compound with very high specific activity was necessary. Tritium was introduced into the compound by methylation of the [bis-N-demethyl]precursor of PF1022A (PF1022-219). The identity of [bis-N-methyl- 3 H]PF1022A was determined by LC/MS. After synthesis and purification, 88.9 μg [bis-N-methyl- 3 H]PF1022A were available showing a specific activity of 162 Ci/mmol (5,99 TBq/mmol) determined by mass spectrometry. The total activity was 15 mCi (555 MBq). Radiolabeled PF1022A showed an efficient and specific binding to a membrane fraction from Ascaris suum. Displacement by unlabeled PF1022A was half-maximal at about 40 nM. At 100-fold higher concentrations the biologically much less effective optical antipodean (PF1022-001) competed for maximal 40% of the [ 3 H]PF1022A-binding in the Ascaris suum membrane preparation. In-vitro comparison of PF1022A with its optical antipodean revealed a more than 100-fold higher anthelmintic activity of PF1022A against Heterakis spumova, Nippostrongylus brasiliensis and Trichinella spiralis.

Activity of novel juvenoids on arthropods of veterinary importance.

Different juvenile hormone mimics were compared regarding their efficacy against larval and pupal stages of the diptera Aedes aegypti, Musca domestica, and Lucilia cuprina as well as the cat flea Ctenocephalides felis. Structure-activity relationship of a series of new diphenylethers was analyzed and compared to juvenoids like methoprene, hydroprene, and fenoxycarb. Selected compounds were taken for a competition binding assay and their effect on JH-degradation by esterases was examined. The results indicate that neither high affinity to JH-binding proteins nor interference with JH breakdown are responsible for the extraordinary efficacy of the diphenylethers investigated. Two superior diphenylethers, however, proved to be more resistant to UV-radiation than methoprene.