The authors have read the journal’s policy and have the following conflicts: Dr. Vernon M. Bowles is the founder and a shareholder of the funder of this research Hatchtech Pty Ltd. Dr. Bowles is an inventor on the following patents owned by Hatchtech Pty Ltd: Methods and Compositions for Controlling Ectoparasites WO/2005/007188, Composition and Methods for Controlling Infestation PCT/IB2007/003226 and Pediculicidal Composition U.S. Provisional Application no. 61/659,30. There are no other patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: BB MV AV RB PB. Performed the experiments: BB MV AV AW CP. Analyzed the data: BB MV AW. Contributed reagents/materials/analysis tools: RB VB. Wrote the paper: BB MV VB PB.
Insecticide resistance has limited the number of available chemical options for insect pest control. Hence there is a need for new chemistries with novel modes of action. Here we investigate the mode of action for an insecticide that has not yet been released for commercial use. The ovicidal, larvacidal and adulticidal effects of 5,5′-dimethyl -2, 2′-dipyridyl (termed Ha44), which is being developed as a treatment for head lice, were evaluated in the
Insects represent, by far, the largest number of species in the animal kingdom. Therefore, it is not surprising that in situations where there is competition for resources some insect species have a major detrimental effect on human life. Pest insects can threaten agriculture, human and animal health through direct damage or as vectors for diseases. For several decades insecticides have been the mainstay in attempts to control insect pests, however, the strong natural selection imposed by the widespread use of insecticides has resulted in the evolution of insecticide resistance in at least 500 species
Human head lice, (
5,5′-dimethyl-2,2′-bipyridyl (termed Ha44) is a heterocyclic organic molecule predicted to chelate heavy metal ions and thereby interact with a range of targets that require heavy metal ions as co-factors within the insect. Ha44 has recently completed a Phase 2 b clinical trial in subjects with head lice, which demonstrated safety and efficacy (unpublished data). This paper analyses the biological activity and mode of action of this compound.
Using both feeding and contact assays in the
All flies were raised on standard semolina medium, whereas embryos were reared on apple juice plates (160 ml H2O, 1.75 g brewer’s yeast, 5 g agar, 6.5 g sugar, 13 g glucose, 50 ml apple juice, 1.5 ml 10% methylparaben solution). The
Copper, zinc, iron(II) and iron(III) were supplied by Sigma as CuSO4.5H2O; ZnCl2; FeSO4.7H2O and FeCl3.6H2O, respectively and dissolved in distilled H2O prior to usage (100 mM).
All experiments were set up to include no less than four technical replicates and were repeated at least three times (n ≥4). Results were statistically analysed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA). For the calculation of the LD-50 and LD-90 values at a 95% significance level and for drafting the dose response curves, the program (PreProbit V1.6.3) was used. The calculation of the dosage-mortality curve was undertaken according to the method described.
Adult flies (
To study the effect of Ha44 throughout embryonic development, the same setup was used as described above. However, for this assay embryos were collected at different time points after laying (2, 4, 6, 8, 10, 14, 16 and 20 hours) and lined up on food plates containing a lethal dose of Ha44 (1 mM) for the remaining time. After 24 hours of incubation at 29°C unhatched embryos were dechorionated (50% bleach; 1 minute). The time of developmental arrest was estimated by imaging under a light microscope.
Using the same methodology as described for ‘
For each of seven concentrations of Ha44, 250 first instar larvae were collected and placed on food The LD-50 and LD-90 values were calculated by counting the number of pupae and adults after 10–14 days.
Contact assay
Sexually mature non-virgin females (4 days post-eclosion) were exposed in a contact assay over a period of 24 hours to different doses of Ha44. Glass scintillation vials and plugs were coated with 300 µl of acetone containing different concentrations of the compound. After evaporation of the liquid by rolling, the vials were plugged with a coated piece of cotton wool. The cotton wool plugs were wrapped into parafilm® after being soaked in 5% sucrose and prior to exposure to acetone. After evaporation of the acetone, the parafilm® was punctured multiple times with a needle to allow the flies to access the sucrose liquid. Subsequently, 20 flies were placed in each vial. 24 hours later, mortality was scored and compared to controls. The LD-50 and LD-90 values were calculated and expressed in mM/vial.
Ingestion assay
Sexually mature females (4 days post-eclosion) were fed different concentrations of Ha44 over a period of 48 hours. Flies were transferred into small vials (2 cm in height with a diameter of 4 mm) of which the lid held 500 µl of food containing different concentrations of Ha44 (10 flies/cage). Mortality was scored after 48 hours and the LD-50 and LD-90 values were calculated.
Activation of Meprin
Recombinant mouse meprin 1A was activated according to the manufacturer’s instructions. Briefly, inactive meprin in activation buffer (Tris pH 7.5 [50 mM], NaCl [150 mM] 0.05% Brij-35) was incubated in the presence of 0.01% trypsin for 2 hours at 37°C, after which trypsin activity was inhibited by the addition of AEBSF to a final concentration of 1 mM.
Gelatin zymography
Samples of active meprin 1A (100 ng) in non-reducing SDS-PAGE buffer were resolved through 10% SDS-PAGE gel containing 0.1% gelatin (Sigma Aldrich). After electrophoresis, marker lanes were cut off and fixed in 50% methanol (v/v) and 10% (v/v) acetic acid in deionised water. The remaining gel was washed twice in Triton-X (2.5%; Sigma Aldrich) in deionised water. Individual lanes were cut from the gel and were incubated overnight at 37°C in a buffer consisting of Tris pH 8.0 (0.1 M), NaCl (0.2 M) and Triton-X (0.02%; v/v) in the presence or absence of Ha44. Gel strips were then fixed, as described above, and stained with Coomassie Brilliant Blue R-250 (0.1% [w/v]; Sigma Aldrich) for 2 hours, prior to destaining in 10% methanol (v/v) and 10% (v/v) acetic acid in deionised water.
Azocasein assay
Activity of meprin 1A in the presence of Ha44, TPEN and EDTA was measured using an azocasein assay as previously described
Freshly laid embryos were exposed to food containing a lethal dose of Ha44 and a 1 mM concentration of one of the four metals [Cu, Zn, Fe(II) and Fe(III)] or a mix of all four metals (0.25 mM/metal). After 24 hours, the number of hatched embryos was counted and compared to three controls: (
A similar method was used to study the effect of metals on larvae exposed to a lethal dose of Ha44. First instar larvae were placed on plates containing Ha44 and a single metal or a mix of the four metals. Subsequently, the number of pupae and successfully eclosed adults were counted and compared to controls in which larvae were not exposed to the compound and/or metal(s).
The
By means of a contact assay, freshly eclosed virgin male and female flies were exposed to a sub-lethal dose of Ha44 (0.5 mM) for 72 hours. Subsequently, virgins were mated to unexposed sexually mature flies and the eggs and progeny were counted and compared to controls.
The effect of Ha44 on mating behaviour of male flies was studied by means of a contact assay. Sexually mature males were exposed to a lethal dose of Ha44 (4 mM) for 3 hours. Next, the exposed flies were placed with virgin females (1∶1 ratio) in small vials containing food. Courting and mating behaviour of the effected males (wing movement, circling the female and mounting) were scored during the first hour and the couples were left to mate for three days onwards. The embryos were collected every 12 hours. Hatching numbers were counted and compared to controls.
Exposing 0–2 hour embryos to a concentration of Ha44 (0–0.1 mM) over a period of 24 hours had no significant effect on hatching (
Concentration of Ha44 (mM) to which 0–2 hours old embryos were exposed for 24 hours at 29°C (100 eggs/concentration). For each concentration the average mortality (percentage) is presented±S.E. The legend below the graph displays the LD-50 and −90 values (mM) for Ha44 at a 95% significance level according to Probit.
The effect of exposure time in relation to the Ha44 concentration was determined by exposing embryos to different concentrations of Ha44 over varying time points: from 10 minutes up to four hours (
Concentration of Ha44 (mM) to which 0–2 hour old embryos were exposed over different periods of 10 minutes to 4 hours at 29°C (100 eggs/condition). For each concentration/exposure time the average mortality (percentage) is presented (n = 4)±S.E. and corresponds to the number of non-hatched embryos after 24 hours. Possible significant differences between the four exposure times were assessed using a two-way ANOVA model combined with a Bonferroni post hoc test (** = p<0.001; *** = p<0.0001).
Embryos were exposed to a lethal dose of Ha44 (1 mM) at different time points during development over a period of 24 hours. The non-hatched embryos were then dechorionated and the time of developmental arrest was visualized. Ha44 was able to arrest development in early stages of embryogenesis (Stage 5: blastoderm stage), at 12–15 hours (Stages 8–10: germ band elongation) and at a very late stage (Stages 16–17) near the point of hatching (data not shown).
Next, we studied the ability of different metals [Cu, Zn, Fe(II) and Fe(III)] to reverse the lethal effect of Ha44 on developing embryos. As shown in
Freshly laid embryos (0–2 hours) were exposed to a lethal dose of Ha44 in the absence/presence of metals [Cu, Zn, Fe(II) and Fe(III)] at different concentrations (mM). Possible significant differences in potency between the different metals were assessed using a two-way ANOVA model combined with a Bonferroni post hoc test (*** = p<0.0001, n.s. = not significant).
In vitro potency of Ha44 compared to other metal chelators
Zymography was used to determine the capacity of Ha44 to inhibit the Zn dependent metallo-protease (meprin-1A) which has a similar protease domain to previously described hatching enzymes (
As illustrated in
In vivo potency of Ha44 and other metal chelators on hatching
Freshly laid embryos were exposed to different concentrations of Ha44 and two other commercially available metal chelators (TPEN and EDTA). Only Ha44 could prevent hatching in embryos at a relatively low dose (0.25 mM;
First instar larvae were exposed to a range of concentrations of Ha44 and the effects on development, pupation and eclosion determined. As illustrated in
Concentration of Ha44 (mM) to which first instar larvae were exposed in function of mortality (50 larvae/condition). For each concentration the average mortality (percentage) is presented±S.E. The legend below the graph displays the LD-50 and −90 values (mM) for Ha44 at a 95% significance level according to Probit.
We studied the ability of different metals [Cu, Zn, Fe(II) and Fe(III)] to inhibit the insecticidal effect of Ha44 on developing larvae. As shown in
First instar larvae laid were exposed to a lethal dose of Ha44 in the absence/presence of metals (1 mM) [Cu, Zn, Fe(II), Fe(III) and a combination]. A non-parametrical, Mann Whitney test (One-tailed) was used to prove significant differences in the inhibitory potency between the metals (grey and white bars) and the control condition (Ha44, black bar) (** p<0.01)±S.E.
The metallothionein promoter (MtnB) is activated by high intracellular concentrations of heavy metals. Here MtnB was used to drive the expression of the enhanced yellow fluorescence reporter protein (eYFP). Copper or zinc induced strong expression throughout the midgut of third instar larvae (
Larvae were reared on normal food (NF) or food containing 1 mM Cu or Zn. Expression of eYFP was inhibited by addition of 0.1 mM Ha44. Red arrows indicate regions with lower eYFP expression. Pictures of every condition were taken with light (top) and fluorescence filter (bottom).
Sexually mature adult flies were exposed to a range of concentrations of Ha44 over a period of 24 hours and the numbers of survivors were counted. We compared the dose response when Ha44 was administered via either contact or ingestion. Using a two-way ANOVA (using a Bonferroni correction for multiple comparisons) we demonstrated significant differences in LD-50 values for these two methods of exposure (
Concentration of Ha44 (mM) to which adult flies were exposed in function of survival after 24 hours. The average mortality (percentage) is presented±S.E. for each concentration and both experimental conditions; contact (green line) and ingestion (red line). The legend below the graph displays the LD-50 and −90 values in mM for Ha44 for both conditions (red and green) at a 95% significance level according to Probit.
To study the short-term effect of Ha44 on mating behaviour, sexually mature males were exposed to Ha44 [4 mM] for 3 hours after which they were each placed with a single female. Contrary to the controls, the exposed males displayed no courting or mating behaviour (wing movement, circling the female) in the following 24 hours. However, 48 hours after exposure males did start to display these behaviours and viable offspring were produced from the matings (data not shown).
The long-term effect of Ha44 on fertility was studied on adult flies by means of contact assays. Freshly eclosed virgin male and female flies that had been exposed sub-lethal dose of Ha44 (0.5 mM) for 72 hours and mated to unexposed sexually mature flies did not show a significant decrease in fertility compared to a controls (data not shown).
The need to identify new safe and effective insecticides with novel modes of action represents a constant challenge as insect pests have demonstrated a remarkable ability to become resistant to many of the currently available insecticides. This situation is equally true for head lice where resistance has been reported to a number of the commonly used actives. At the present time, the compound 5,5′-dimethyl-2,2′-bipyridyl is being investigated as a potential new therapeutic treatment for head lice control, recently completing a Phase 2 b clinical trial (Clinical trials.gov identifier: NCT01336647). Further research identifying the mode of action and the potential for resistance development with this compound may be useful in assessing the efficacy of this insecticide in the longer term. This study describes the insecticidal activity of the metal chelating compound 5,5′-dimethyl-2,2′-bipyridyl (Ha44, MWt 184) against all life stages of
Ha44 was also shown to be lethal to larvae at a similar dose to that observed for embryos. However, it is worth noting that since the development of
By comparing the difference in the effects of the compound between contact and ingestion in adult flies, we observed that an eight-fold increase in concentration of compound was required to induce a lethal effect when the compound was mixed in food compared to when flies were exposed to the compound through contact only. The observed difference in potency indicates that Ha44 is more lethal when taken up via the cuticle than when taken up via the digestive tract. Possible reasons for this apparent difference in potency include metabolism of Ha44 or inactivation of the compound via binding of free bivalent metals present in the food. It was of interest that, males who were briefly exposed to the compound did not engage in courting behaviour or sexual intercourse until they recovered from the effects of exposure, approximately 48 hours later. Longer term exposure of adult flies to a sub-lethal concentration of the compound did not decrease fertility in males and females, both sexes recovered, after which they mated and produced viable and fertile offspring.
In order to more fully understand the nature of the interaction between Ha44 and different metal ions, experiments were conducted on both embryos and larvae to determine if the lethal effects of this compound could be reversed by the addition of specific metal ions. Data obtained indicated that indeed it was possible to reverse the lethal effect of Ha44 on
Previous research had indicated a potential role for metalloproteases in the hatching of lice
Of major concern in insect pest management is the occurrence of resistance imposed by the widespread use of an insecticide. In the case of head lice resistance to permethrin, pyrethrin and malathion has been described
The potential for Ha44 target site resistance to evolve is predicted to be low. Ha44 is a chelator of at least three different bivalent metals (Fe, Cu, Zn) and the activity of a vast number of proteins (many of which are required for viability) is dependent on these metals. Therefore, it is extremely unlikely that the lethality of Ha44 is caused by the loss of function of a single metalloprotein. Ha44 probably has a range of targets and it is unlikely that a mutation in a gene encoding any one of these targets could confer a significant level of resistance. A mutation in any one gene could, at best, only make one target insensitive to Ha44; all other potential target proteins would remain sensitive.
For most insecticides the highest levels of resistance are associated with target site insensitivity, which has been a major driver for the constant need to develop new insecticides to replace those that have been rendered ineffective via this mechanism
With existing options for the control of insect pests such as human head lice being diminished by resistance to current generation insecticides, new chemicals with novel targets need to be developed. To avoid the constant need to replace insecticides due to resistance there is a need to understand the mode of action of insecticides before they are deployed for insect pest control