Glutamate Receptor-Cation Channel Complex: An Unexploited Target

Chapter 7

Glutamate Receptor-Cation Channel Complex: An Unexploited Target for Mosquito Control Downloaded by UNIV OF FLORIDA on November 30, 2017 | http://pubs.acs.org Publication Date (Web): October 24, 2017 | doi: 10.1021/bk-2017-1264.ch007

Aaron D. Gross,1 Rafique Islam,1 and Jeffrey R. Bloomquist* Neurotoxicology Laboratory, Emerging Pathogens Institute, Department of Entomology and Nematology, University of Florida, Gainesville, Florida 32610, United States 1Co-primary authors *E-mail: [email protected].

While the glutamate-gated chloride channel receptor is a known target for the avermectins and milbemycins, the depolarizing subtype of glutamate receptor (GluRd), which contains an intrinsic cation channel, remains unexploited for insect control. Using a headless larva assay and injection of adult mosquitoes (to enhance compound penetration), as well as adult feeding treatment, we show that agonists of GluRd induce paralysis/death of the yellow fever mosquito, Aedes aegypti. Adult injection revealed potent toxicity, with most LD50s between 4-20 ng/mg, and excellent structure-activity correlation to the headless larval paralysis assay. Most compounds showed mortality of ≤ 50% at 1 mg/ml by adult sugar water treatment, with β-methylamino-L-alanine (L-BMAA) the most potent. Surprisingly, there was little or no toxicity to adult Drosophila melanogaster in parallel feeding assays. Results from the different routes of exposure suggest that pharmacokinetic processes affected toxicity, and that glutamate agonists might be candidates for further study.

© 2017 American Chemical Society Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Downloaded by UNIV OF FLORIDA on November 30, 2017 | http://pubs.acs.org Publication Date (Web): October 24, 2017 | doi: 10.1021/bk-2017-1264.ch007

Introduction Mosquitoes are vectors of many debilitating and deadly diseases throughout the world. Chemical insecticides play an important role in the control of mosquito populations, and thereby help reduce the impact of vector-borne diseases. While significant strides have been made in the control of some mosquito-borne diseases, widespread insecticide resistance coupled with the recent outbreak of the Zika virus in the Americas (1) has highlighted the importance of chemical insecticides, and reinforces the urgent need to find new compounds for mosquito control. Part of this effort includes investigating underutilized targets to develop novel chemistry. L-Glutamic acid (Figure 1) is the primary mediator of both excitatory neuromuscular and central synaptic transmission in insects (2–6). It activates both ionotropic (iGluR) and metabotropic (mGluR) glutamate receptors. Two subtypes of insect iGluR have been described, which include the cys-loop inhibitory glutamate-gated chloride channels (H-receptors; GluRh), and the depolarizing cation channel (GluRd) complex (4, 5). While GluRh is the target of antiparasitic, miticidal, and insecticidal macrocyclic lactones (7), the GluRd has not been successfully exploited for insecticide development. Activation of GluRd generates an excitatory junction potential and triggers a postsynaptic depolarization that in muscle leads to contraction (4, 5). Subsequently, the receptor transitions to a non-conducting, persistent ligand-bound state (desensitized) (4, 6). Kinetically altered activation and/or desensitization of muscle excitatory GluRd receptors could underlie paralysis and/or death if induced by an insecticide. The pharmacology of GluRd has been studied in locusts (3, 4) and cockroaches (8–10), as well as in fruit fly larval muscle (11–13); however, no studies on the pharmacology or toxicology of GluRd in mosquitoes has been reported to date. The goal of the research presented here was to investigate the toxicity of glutamatergic agonists to mosquitoes. Compounds were chosen because they are are known agonists of insect or mammalian iGluR (Figure 1). We also evaluated the toxicity of a diuretic and antihypertensive benzothiadiazide (cyclothiazide), as well as the nootropic racetams (Figure 1). Racetams share a pyrrolidone ring as a structural motif, and are thought to affect mammalian iGluR by slowing or eliminating receptor desensitization, same as cyclothiazide (11–16). However, no information on their action has been reported in insects. Based on the results presented in this chapter, we propose that the insect GluRd could serve as an interesting target for the development of new insecticides or chemical probes of glutamate receptor function.

Materials and Methods Chemicals The following test compounds were obtained from Sigma-Aldrich (St. Louis, MO, USA): L-glutamic acid (≥99%), kainic acid (≥99%), L-aspartic acid (≥98%), N-methyl-D-aspartic acid (NMDA; ≥98%), domoic acid (≥90%), β-methylamino-L-alanine (L-BMAA; ≥97%), S(-)-willardiine, 112 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


(±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA; ≥90%), cyclothiazide (>95%), piracetam (>90%), pramiracetam (>98%), and rolipram (>98%). Quisqualic acid (>99) and aniracetam (>99%) were purchased from TOCRIS Bioscience (Ellisville, MO, USA). Phenylpiracetam (>98%) was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Propoxur (≥99%) was obtained from Fluka (Morris Plains, NJ, USA). The majority of these compounds are water soluble, and therefore, were dissolved in water or buffer, except aniracetam and cyclothiazide, which were dissolved in DMSO (final DMSO concentration was 0.1%).

Figure 1. Chemical structures of glutamatergic agonists, cyclothiazide, and racetams. 113 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Insects Aedes aegypti mosquitoes were obtained from the United States Department of Agriculture – Agricultural Research Service, Center for Medical, Agricultural, and Veterinary Entomology (USDA-ARS, CMAVE), Gainesville, FL, USA as 3rd or 4th instar larvae, and reared as previously described (17). Briefly, the larvae were held in tap water and fed a diet that consisted of 3-parts liver powder (MP Biomedical, Solon, OH, USA) to 2-parts Brewer’s yeast (MP Biomedical). Adult mosquitoes were provided a cotton ball soaked with 10% sugar water for sustenance. Mosquitoes were maintained at 28°C, relative humidity >60%. Adult female mosquitoes that were 1-5 days post-emergent were used for feeding and injection studies. Oregon-R, a susceptible strain of Drosophila melanogaster has been maintained in culture at the University of Florida (Gainesville, FL, USA) since 2009. Flies were reared in plastic vials on artificial media that was purchased from Carolina Biological Supply (Burlington NC, USA). Adult females that were 1-2 weeks old were used for feeding experiments. Toxicity and Paralysis Bioassays Oral toxicity of glutamatergic compounds was assessed against adult female Ae. aegypti and D. melanogaster. Mosquitoes were anaesthetized on ice, transferred to a holding container, and starved for 6 hr. D. melanogaster were also starved for 6 hr and anaesthetized using carbon dioxide before being transferred to a test tube. Compounds were delivered by dissolving in 10% sugar water and 1 mL was applied to a cotton ball that stoppered a glass vial or test tube that held adult insects. Mortality of the test compounds was determined after 48 hr of exposure. A headless larval bioassay (18) was employed to examine the rapid paralytic activity against fourth-instar Ae. aegypti larvae, because pilot studies with L-aspartic acid showed it was inactive against intact larvae. Briefly, fourth-instar Ae. aegypti larvae were decapitated by pulling off the head using forceps. Decapitated larvae were placed into 5 mL of mosquito physiological saline, which was composed of (mM) sodium chloride (154), calcium chloride (1.4), potassium chloride (2.7), and HEPES (1.2); pH was adjusted to 6.9 (19). Motor responses to manual probing of the headless larvae were monitored for five hours post-decapitation. At this time, if the larva displayed little or no movement after being gently probed with a needle, it was classified as paralyzed. Control paralysis under identical conditions was 1 were L-BMAA, quisqualic acid, and willardiine (Table 1). The paralytic effect of the putative GluRd desensitization blockers (cyclothiazide and racetams) was also investigated against fourth-instar headless larvae (Table 2). Piracetam and pramiracetam had the lowest PC50 values ( 10 ng/mg, but ≤ 100 ng/mg. Finally, the least active compounds showed an LD50 value > 100 ng/mg. Five out of the nine-glutamatergic agonists were in the high activity category (LD50 ≤ 10 ng/mg) when injected into adult female Ae. aegypti. The five compounds were essentially equipotent, and included L-BMAA, quisqualic acid, domoic acid, kainic acid, and aspartic acid. The moderately active glutamatergic agonists were AMPA, willardiine, and NMDA. L-Glutamic acid was the least active glutamatergic agonist when injected into adult female Ae. aegypti (Table 3). 116 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Table 2. Paralytic Activity of Cyclothiazide and Racetam Compounds against Headless Fourth-Instar Ae. aegypti Larvae Drug aniracetam cyclothiazide phenyl-piracetam


piracetam pramiracetam rolipram

PC50, ppm1

Slope ± SE

χ2 value (df)

150 (74 – 400)

0.50 ± 0.08

7.98 (30)

34 (22-51)

1.24 ± 0.20

15.06 (15)

136 (101– 201)

2.28 ± 0.49

9.58 (19)

9 (6 – 13)

0.85 ± 0.09

13.73 (38)

10 (3-26)

0.87 ± 0.19

1.70 (5)

50 (36 – 72)

1.71 ± 0.24

11.21 (17)

Concentration that resulted in 50% larval paralysis in headless fourth-instar Ae. aegypti. Data are presented as the PC50 with the 95% confidence interval in parentheses, 5 hr postdecapitation. 1

Piracetam was the most toxic of the glutamatergic desensitization modulators when injected into adult mosquitoes. Moderately toxic compounds included rolipram and pramiracetam. Finally, aniracetam and phenyl-racetam were the least active (Table 4). Propoxur was again used as a benchmark for comparing relative toxicity, and had an injected LD50 of 0.14 (0.1-0.2) ng/mg (20). Propoxur was 29-fold more toxic than L-BMAA and piracetam, which were the most toxic glutamatergic agonist or modulator, respectively. The slope values for injected glutamatergic agonists/modulators into adult female mosquitoes were more steep (values greater than 2; Tables 3 and 4) when compared to headless larval slope values. A linear regression correlation analysis was performed between toxicities in the fourth-instar headless larval assay and by intrathoracic injection of adult female mosquitoes (Figure 2). When all glutamatergic and racetam compounds where included in linear regression, a correlation coefficient (R2) of 0.70 was obtained (dotted line, Figure 2). However, the R2 increased to 0.97 when kainic acid was excluded from the regression analysis, which had variable results between the two bioassays (solid line, Figure 2). 117 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Table 3. Intrathoracic Injection and Oral Toxicity of Glutamatergic Compounds against Adult Female Ae. aegypti LD50 (ng/mg)1

Slope ± SE

χ2 value (df)

Oral toxicity2

AMPA

12 (9 – 19)

2.80 ± 0.57

0.88 (2)

50%

L-aspartic acid

7 (6 – 10)

3.26 ± 0.59

0.58 (2)

0%

L-BMAA

4 (3 – 5)

3.73 ± 0.79

0.11 (2)

61 (40-79)

domoic acid

5 (4 – 7)

3.28 ± 0.64

1.43 (2)

294 (167-340)

L-glutamic acid

371 (304 – 473)

2.63 ± 0.41

6.09 (10)

20%

kainic acid

7 (6 – 10)

3.26 ± 0.59

0.58 (2)

40%

98 (70 – 153)

2.45 ± 0.55

0.55 (2)

10%

quisqualic acid

4 (3 – 6)

3.19 ± 0.65

0.79 (2)

7%

willardiine

82 (61 – 116)

2.91 ± 0.62

0.46 (2)

20%


Drug

NMDA

1 Mortality 24 hr post-injection, given as LD 50 with 95% confidence intervals in parentheses. 2 Oral toxicity of glutamatergic compounds determined at 48 hr and given as the LC50 in ppm (95% confidence intervals) or the percentage of mortality at 1 mg/mL.

Feeding Bioassays and Aedes Aegypti and Drosophila melanogaster The oral toxicity of the glutamatergic compounds was investigated in adult female Ae. aegypti and D. melanogaster. Initially, the 48 hr toxicity of these compounds was measured at a high concentration (1 mg/mL) of the toxin delivered in 10% sugar water (w/v). D. melanogaster displayed little toxicity (≤ 30% mortality at 48 hr) for any glutamatergic compound, with the majority of the observed toxicities ranging from 0-10% (data not shown). For Ae. aegpyti, no or low mortality was observed for L-aspartic acid, NMDA, quisqualic acid, and willardiine at 1 mg/mL (mortality less than 50%; Table 3). For the remaining glutamatergic compounds tested L-BMAA was about 5-fold more active than domoic acid (Table 3). Three out of five racetams tested (piracetam, aniracetam, and phenyl-racetam) resulted in low mortality (< 30%) when tested at a high concentration of 1 mg/mL. Pramiracetam and rolipram had LC50 values of 389 and 314 ppm, respectively; however, they were still significantly less toxic than cyclothiazide (Table 4). For comparison, propoxur had an LC50 of 0.33 (0.27-0.38) ppm in this assay, making it 185-fold more toxic than L-BMAA. 118 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Table 4. Intrathoracic Injection (LD50) and Oral Toxicity of Cyclothiazide and Racetam Compounds against Adult Female Ae. aegypti Drug

LD50, ng/mg1

Slope ± SE

χ2 value (df)

Ae. aegypti oral toxicity 2

aniracetam

142 (101 – 247)

2.61 ± 0.63

0.0073 (2)

10%

cyclothiazide

14 (11 – 21)

2.33 ± 0.35

8.38 (10)

120 (83-167)

phenylpiracetam

171 (120-255)

2.28 ± 0.49

2.43 (2)

30%

piracetam

4 (3 – 5)

2.96 ± 0.66

0.51 (2)

0%

pramiracetam

76 (54 – 118)

2.16 ± 0.46

1.44 (2)

389 (251 – 502)

rolipram

50 (38 – 67)

2.88 ± 0.56

0.34 (2)

314 (226 – 368)

LD50 with 95% confidence interval in parentheses measured at 24 hr post-injection. 2 Oral toxicity (48 hr) of test compounds represented as the LC50 in ppm (95% confidence interval) or the percentage of mortality at 1 mg/mL. 1

Figure 2. Correlation of Ae. aegypti headless fourth-instar larval assay (x-axis) versus the intrathoracic injection of adult Ae. aegypti female mosquitoes (y-axis). Inclusion of all 15 of the tested glutaminergic compounds yielded the dotted regression line, with R2=0.70. Exclusion of kainic acid (KA) gave the solid regression line, with R2=0.97. 119 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Table 5. Paralytic Activity of Glutamatergic Compounds Mixed with 1 ppm Piracetam against Headless Fourth-Instar Ae. aegypti Larvae Calculated Additive PC50, ppm1

AR2

Actual PC50, ppm1

Actual SR3

AMPA

7 (2 – 16)

4.4

31 (14 – 72)

1.0

L-aspartic acid

3 (1 – 6)

4.0

10 (4 – 24)

1.2

L-BMAA

3 (1 – 7)

3.0

8 (2 – 18)

1.1

cyclothiazide

15 (8 – 25)

2.3

8 (3 – 20)

4.3

domoic acid

9 (3 – 26)

3.7

31 (11 – 83)

1.1

L-glutamic acid

96 (40 – 302)

3.2

164 (82 – 399)

1.9

kainic acid

89 (39 – 266)

2.3

42 (20 – 94)

4.9

NMDA

38 (17 – 87)

3.3

92 (45 – 211)

1.4

4 (1 – 8)

2.0

6 (3 – 13)

1.3

59 (41 – 86)

1.6

65 (35 – 135)

1.5


Compound

quisqualic acid willardiine

Calculated Additive and Actual PC50 values are given, with the 95% confidence interval in parentheses. 2 Additivity Ratio (AR) calculated by dividing the PC50 of the glutamatergic alone by the PC50 obtained when adding the 10% paralysis expected from piracetam (1 ppm). 3 Synergistic Ratio (SR) calculated by dividing the PC50 of the glutamatergic alone by the actual PC50 obtained when mixed with the glutamatergic with piracetam (1 ppm). 1

Synergism of Glutamatergic Compounds with Piracetam Since racetams reduce desensitization of mammalian iGluR (11–16), piracetam, the most active racetam in the headless larval assay, was further tested in this assay at 1 ppm in combination with other glutamatergic compounds to document any synergistic effects (Table 5). As part of the test for synergism, a theoretical additive PC50 value was calculated by probit analysis of the summed paralysis obtained with 1 ppm piracetam (10%) added to that obtained at each concentration of glutamatergic agonist alone, taken from Table 2. It was important to account for an additive effect of piracetam due to the low slope values observed in the headless larva assay (Table 1). 120 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

As described above, an additive effect of 1 ppm piracetam would be expected to decrease the PC50 of the agonists about 2-4 fold (Table 5) compared to that observed with agonist alone (Table 1). Cyclothiazide and kainic acid were the only two glutamatergic compounds tested where the SR was greater than the calculated AR, but given the extensive overlap in the 95% confidence limits, the difference was not statistically significant. SR values less than the AR indicate that the piracetam effect was less than additive, indicating some kind of antagonism was occurring with the majority of the glutamatergic agonists (Table 5).


Discussion The insect glutamatergic system plays an important role in the excitatory neuromuscular and central synaptic transmission, but it has not been a successful target of insecticide development. The lack of success at exploiting the GluRd has been attributed to a lack of chemistry that can penetrate the insect cuticle (21). In this chapter, we investigated the toxicity of known glutamatergic agonists and desensitization modulators in two mosquito life stages using bioassays that circumvented the cuticular barrier. In both headless larval and adult injection assays, fairly potent intrinsic activity of GluRd agonists was found. In the headless larval assay, we observed low slope values (< 1) in probit toxicity analysis (Tables 1 and 2). These shallow slopes suggest a slow rate of penetration of the test compounds through the cervical opening. As expected, the LD50 obtained by injecting test compounds into adult mosquitoes resulted in greater slope values and narrower 95% confidence limits. The high correlation coefficient for the toxicity data of headless larval and intrathoracic injection studies indicates a similar receptor type mediates toxicity in both life stages (Figure 2). The high potency of L-aspartic acid in larval and adult assays was unanticipated. This amino acid is known to stimulate insect muscle glutamate receptors, but usually requires higher concentrations than L-glutamate (22). However, it is worth noting that no information is available on relative potencies of these amino acids on mosquito receptors, nor their affinity for active transporters at either peripheral or central synapses that might impact toxicity. The low sensitivity of L-glutamic acid we observed in Ae. aegypti larvae and adults have previously been reported in other insects (21). When L-glutamic acid was injected into adult male Lucilia sericata, it resulted in an LD50 of 7800 ng/mg (23), which is much higher than the LD50 value we found in adult female Ae. aegypti (LD50 = 371 ng/mg). A previous review (24) indicated that variable concentrations of L-glutamic acid can be found in insect hemolymph and it discussed possible mechanisms that could serve to protect the neuromuscular junction from the circulating glutamic acid found in the hemolymph. Such mechanisms may include a diffusion barrier, which has been characterized as a connective tissue sheath surrounding muscle cells, as described in Schistocerca gregaria (6). This protective sheath acts as a barrier to glutamic acid, thereby protecting the nerve-muscle synapses. Additionally, glial cells and tracheal sheath cells may play a pivotal role in the uptake of glutamic acid at the neuromuscular junction, which has been described in cockroaches (24, 25). While barriers 121 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


might contribute to the low paralysis and toxicity we observed with L-glutamic acid in larval and adult mosquitoes, they would have to be permeable to other glutamatergic compounds having similar physical properties, suggesting that specific active transport is a more likely mechanism. The modest oral toxicity of glutamatergic compounds in Ae. aegypti and D. melanogaster (Tables 3 and 4) was disappointing. The most toxic compound via this route of administration was L-BMAA. Quisqualic (10) and domoic (9) acids were previously shown to inhibit peristaltic movements in the cockroach gut (10), but only domoic acid was orally toxic to Ae. aegypti in the present study. Overall, there was a species-specific oral toxicity of glutamatergic compounds favoring Ae. aegypti over D. melanogaster (Table 3 and 4). Additionally, L-BMAA is known to cause neurotoxicity from dietary exposure to this amino acid (26), and domoic acid ingestion is implicated as a cause of human amnesic shellfish poisoning (27). We find no reports on the injected toxicity of glutamatergic compounds into D. melanogaster to use for comparison with Ae. aegypti. However, domoic acid has been injected into adult male Peripleneta americana, resulting in an LD50 of 800 ng/insect, which was similar to allethrin (injected LD50 500 ng/insect) (9). Assuming that the average weight of an adult male P. americana is between 500-700 mg (weights obtained from 5 adult male cockroaches in our laboratory), domoic acid has an approximate LD50 between 1.1-1.6 ng/mg. This LD50 for domoic acid is not too dissimilar from that observed in adult female Ae. aegypti (5 ng/mg). Therefore, we hypothesize that species-selective toxicity may be induced by glutamatergic compounds, but its magnitude will differ by route of exposure. The racetam compounds (aniracetam, piracetam, pramiracetam, phenylpiracetam, and rolipram) are nootropics, cognitive enhancers that extend attention span and memory (28). Cyclothiazide (a diuretic and anti-hypertensive) and aniracetam have been reported to enhance glutamate-evoked currents, decrease receptor desensitization, and prolong synaptic current (14–16). Based on these previous findings, we hypothesized that piracetam could synergize or enhance the activity of glutamatergic agonists. Accordingly, synergism of glutamatergics by piracetam was examined in the headless larval assay at 1 ppm (Table 5). The lack of synergism observed between piracetam and agonists suggests that mosquito GluRd are pharmacologically different from mammalian iGluR, which may be beneficial in the future development of GluRd-selective insecticides. In conclusion, this study reports on the toxicity of glutamatergic agonists in Ae. aegypti. The insect GluRd is a physiological relevant receptor located in the central nervous system of insects, but also in the periphery, so it could serve as an excellent target for mosquito control. A drawback of the available lead compounds, some of which were studied in this chapter, is that they are highly polar and therefore have poor barrier penetration in insects. In addition, several of the compounds have deleterious effects in mammals from oral exposure, and selectivity would be an important factor in the development of any strategy for targeting the GluRd. The lack of synergism of racetams and agonists suggests further differences in the pharmacology of mammalian and insect iGluRs that will be the focus of future mode of action research.

122 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Acknowledgments This work was supported by USDA Specific Cooperative Agreement 58-0208-5-001 to J.R.B. as part of the Deployed War Fighter Research Program. The authors are thankful to Dr. Dan Kline (USDA-ARS, CMAVE) and his insectary staff for providing the mosquitoes used in this study.

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