Andreas Turberg

High-resolution structures of a chitinase complexed with natural product cyclopentapeptide inhibitors: Mimicry of carbohydrate substrate

Over the past years, family 18 chitinases have been validated as potential targets for the design of drugs against human pathogens that contain or interact with chitin during their normal life cycles. Thus far, only one potent chitinase inhibitor has been described in detail, the pseudotrisaccharide allosamidin. Recently, however, two potent natural-product cyclopentapeptide chitinase inhibitors, argifin and argadin, were reported. Here, we describe high-resolution crystal structures that reveal the details of the interactions of these cyclopeptides with a family 18 chitinase. The structures are examples of complexes of a carbohydrate-processing enzyme with high-affinity peptide-based inhibitors and show in detail how the peptide backbone and side chains mimic the interactions of the enzyme with chitooligosaccharides. Together with enzymological characterization, the structures explain why argadin shows an order of magnitude stronger inhibition than allosamidin, whereas argifin shows weaker inhibition. The peptides bind to the chitinase in remarkably different ways, which may explain the differences in inhibition constants. The two complexes provide a basis for structure-based design of potent chitinase inhibitors, accessible by standard peptide chemistry.

Structure of argifin, a new chitinase inhibitor produced by Gliocladium sp.

A new chitinase inhibitor, named argifin, was isolated from the cultured broth of a fungal strain Gliocladium sp. FTD-0668. Argifin was a water-soluble cyclic pentapeptide, and its structure was elucidated as cyclo(Nω-(N-methylcarbamoyl)-l-arginyl-N-methyl-l-phenylalanyl-β-l-aspartyl-β-l-aspartyl-d-alanyl). The IC50 value of argifin against blowfly (Lucilia cuprina) chitinase was 3.7 μM.

Predictive 3D modelling of the interactions of pyrethroids with the voltage-gated sodium channels of ticks and mites

BACKGROUND The pyrethroid insecticides are a very successful group of compounds that target invertebrate voltage-gated sodium channels and are widely used in the control of insects, ticks and mites. It is well established that some pyrethroids are good insecticides whereas others are more effective as acaricides. This species specificity is advantageous for controlling particular pest(s) in the presence of another non-target invertebrate, for example controlling the Varroa mite in honeybee colonies. RESULTS We applied in silico techniques to compare the voltage-gated sodium channels of insects versus ticks and mites and their interactions with a range of pyrethroids and DDT analogues. We identified a single amino acid difference within the pyrethroid binding pocket of ticks/mites that may have significant impact on the effectiveness of pyrethroids as acaricides. Other individual amino acid differences within the binding pocket in distinct tick and mite species may provide a basis for future acaricidal selectivity. CONCLUSIONS Three-dimensional modelling of the pyrethroid/DDT receptor site has led to a new hypothesis to explain the preferential binding of acaricidal pyrethroids to the sodium channels of ticks/mites. This is important for understanding pyrethroid selectivity and the potential effects of mutations that can give rise to resistance to pyrethroids in commercially-important pest species.

The synergistic action of imidacloprid and flumethrin and their release kinetics from collars applied for ectoparasite control in dogs and cats.

BackgroundThe control of tick and flea burdens in dogs and cats has become essential to the control of important and emerging vector borne diseases, some of which are zoonoses. Flea worry and flea bite hypersensitivity are additionally a significant disease entity in dogs and cats. Owner compliance in maintaining the pressure of control measures has been shown to be poor. For these reasons efforts are continuously being made to develop ectoparasiticides and application methods that are safe, effective and easy to apply for pet owners. A new polymer matrix collar has recently been developed which is registered for 8 months use in cats and dogs. The basic properties of this collar have been investigated in several in vitro and in vivo studies.MethodsThe effects of imidacloprid, flumethrin and the combination were evaluated in vitro by means of whole cell voltage clamp measurement experiments conducted on isolated neuron cells from Spodoptera frugiperda. The in vitro efficacy of the two compounds and the combination against three species of ticks and their life stages and fleas were evaluated in a dry surface glass vial assay. The kinetics of the compounds over time in the collar were evaluated by the change in mass of the collar and measurement of the surface concentrations and concentrations of the actives in the collar matrix by HPLC. Hair clipped from collar treated dogs and cats, collected at various time points, was used to assess the acaricidal efficacy of the actives ex vivo.ResultsAn in vitro isolated insect nerve model demonstrated the synergistic neurotoxic effects of the pyrethroid flumethrin and the neonicotinoid imidacloprid. An in vitro glass vial efficacy and mortality study against various life stages of the ticks Ixodes ricinus, Rhipicephalus sanguineus and Dermacentor reticulatus and against the flea (Ctenocephalides felis) demonstrated that the combination of these products was highly effective against these parasites. The release kinetics of these actives from a neck collar (compounded with 10% imidacloprid and 4.5% flumethrin) was extensively studied in dogs and cats under laboratory and field conditions. Acaricidal concentrations of the actives were found to be consistently released from the collar matrix for 8 months. None of the collar studies in dogs or cats were associated with any significant collar related adverse event.ConclusionHere we demonstrated the synergism between the pyrethroid flumethrin and the neonicotinoid imidacloprid, both provided in therapeutically relevant doses by a slow release collar matrix system over 8 months. This collar is therefore a convenient and safe tool for a long-term protection against ectoparasites.

Verticilide, a new ryanodine-binding inhibitor, produced by Verticillium sp. FKI-1033.

A new ryanodine-binding inhibitor, verticilide, was isolated from the cultured broth of a fungus, Verticillium sp. FKI-1033. It is a 24-membered ring cyclic depsipeptide, its structure being elucidated as cyclo[(2R)-2-hydroxyheptanoyl-N-methyl- L-alanyl]4. Verticilide inhibited ryanodine binding to ryanodine receptors in the cockroach at an IC50 value of 4.2 μM, whereas inhibition against mouse ryanodine receptors was weak (IC50=53.9 μM).

Action of Brassinosteroids on the Epithelial Cell Line from Chironomus tentans

Abstract The two brassinosteroids, 22S′,23S′-homobrassinolide and 22S′,23S′-homocastasterone are weak competitors of the binding of [3H]ponasterone A to the intracellular ecdysteroid receptor from the epithelial cell line from Chironom us tentans. The relative affinities to the ecdysteroid receptor are 0.001 for both brassinosteroids as compared to 20-OH-ecdysone and 0.1 in comparison to ecdysone. Both substances exert morphological effects similar to those observed with 20-OH-ecdysone. Like moulting hormones both brassinosteroids inhibit chitin synthesis. However, these effects were observed only at rather high concentrations (10-5 to 10-4 m) which were cytotoxic for 22 S′,23 S′-homobrassinolide.

Association of Neonicotinoid Insensitivity with a Conserved Residue in the Loop D Binding Region of the Tick Nicotinic Acetylcholine Receptor

Neonicotinoid insecticides target nicotinic acetylcholine receptors (nAChR) in the nervous system of insects but are largely ineffective against ticks. This study aimed to identify the molecular basis for this insensitivity. A homology model of the nAChR binding domain was generated on the basis of the crystal structure of an acetylcholine-binding protein with the insecticide imidacloprid bound. We hypothesized that tick β-subunits would differ at a critical residue (Arg81) in their D loops. To test this, we sequenced nAChR genes from five tick species and found that instead of the conserved arginine found in insects, a glutamine was present in all the tick sequences.

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.

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.