Progress 04/01/13 to 03/31/18
Outputs
Target Audience:Entomologists and toxicologists working in government, industry, and academia. Changes/Problems:Some data still needs to be writen up and published. What opportunities for training and professional development has the project provided?Postdoctoral scientists have gained practical and theoretical experience working with mosquitoes, resistance, enzyme catalysis measurements, and insecticide discovery methods. How have the results been disseminated to communities of interest?Peer reviewed publication and meeting presentations to scientific audiences, the latter including representatives of international agrochemical companies. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Impact: New compounds with novel modes of action are needed to control insecticide-resistant insects. The compoundssynthesized over the life of this 5 year project period showed that bivalent molecules having two linked binding groups can inhibit acetylcholinesterase (AChE) selectively and have the expected neuro-excitatory effect on the insect central nervous system. Such compounds can have contact (topical) toxicity to mosquitoes, here demonstrated for the first time. Although paper contact activity was poor, stomach activity isalso possible, but was not tested. Other chemical series of carbamate inhibitors that were synthesized and tested included dimethylcarboxamides, isoxazol-3-yl dimethylcarbamates, trifluoromethyl ketones, and α,α,α,- trifluoroacetophenone oxime methylcarbamates. Besides mosquitoes, evaluations of these compounds were performed on Drosophila melanogaster, Blatella germanica, and the sandfly Phlebotomus papatasi, the latter species included pharmacological mapping of its resistance mutations (G119S, F290V and F331W) in genetically engineered enzymes. Some of the carbamate and bivalent compounds tested were able to effectively circumvent these major resistance mutations. However, potency for inhibition of human enzyme was often substantial for materials active against the resistant enzyme forms, so that aspect of selectivity remains to be conquered. AChE enzymes from agriculturally importance species we tested included the honey bee (Apis mellifera), Asian citrus psyllid (Diaphorina citri), and lepidopteran agricultural pests (Plutella xylostella and Ostrinia nubilalis). Although mosquito toxicity was good overall, the compounds were not that active on the agriculturally important species. This low activity may be an advantage for mosquito management; because it would result in few ancillary uses that should reduce overall selection for resistance in the field. The low honey bee enzyme activity for some of the compounds is also a welcome observation. Molecular modeling provided new vistas into the design of new AChE inhibitors across species by revealing possible binding poses between enzyme and inhibitor. Overall, these results represent a substantial increase in knowledge of the structural rangeof chemistries that can effectively block acetylcholinesterases and haveinsecticidal activity. Objective: 1. Assess the ability of novel anticholinesterases to block the activity of arthropod pest and non-target vertebrate enzymes, in vitro. 1) Major activities completed / experiments conducted: Given in annual reports. 2) Data collected: Given in annual reports. 3) Summary statistics and discussion of results: Given in annual reports. 4) Key outcomes or other accomplishments realized: For enzyme inhibition, three benchmarks must be met, including high potency on both the susceptible and resistant ACHE, as well as low potency on the human enzyme. Year in and year out, we have consistently achieved 2 out of the 3 benchmarks across several chemical series. The known mutations in AchE responsible for resistance are formidable, but we find are not insurmountable . Objective 2. Determine any lethal actions of active compounds, in vivo, and the involvement of any significant pharmacokinetic processes impacting toxicity. 1) Major activities completed / experiments conducted: Given in annual reports. 2) Data collected: Given in annual reports. 3) Summary statistics and discussion of results: Given in annual reports. 4) Key outcomes or other accomplishments realized: The small core carbamates defeat G119S mutation carrying An. gambiae and show little cross resistance in a strain that is essentially insensitive to conventional carbamates. Moreover, we have proved that inhibition of bivalent carbamates can be insecticidal. Injection studiesindicatedthat poor uptake via the cuticle is limiting toxicity of bivalent molecules. Since the calculated LogP value (measure of lipophilicity) for the lead bivalent is 1.2 (lower than most contact insecticides), perhaps adding hydrophobic groups may increase cuticular penetration. The central neuron recordings indicated that the nerve sheath was a penetration barrier to dimeric tacrines in the insect brain and that the cationic tacrine dimers do not readily cross the blood brain barrier, explaining the observed non-lethality to insects. Lack of activity on bees or agricultural pests may assist in resistance management with these compounds Objective 3. Model structure-activity relationships via correlation analysis of in vitro and in vivo data to aid in design of new molecules. 1) Major activities completed / experiments conducted: Given in annual reports. 2) Data collected: Modeling calculations are not data and are omitted from the report. 3) Summary statistics and discussion of results: Given in annual reports. 4) Key outcomes or other accomplishments realized: We have proved that inhibition of enzyme activity correlates with in vivo toxicity for our experimental molecules, which of course was observed with carbamates previously. The models can also explain resistance quite well for the mutants, usually a steric clash within the catalytic triad, that is defeated by small core carbamates, or bivalents, albeit via a different mechanism. They are also useful for generating hypotheses about ligand-enzyme interactions that can be tested by compound synthesis of novel analogs, in particular the design of future bivalent insecticides. Objective 4. Generate new molecules and optimize activity by appropriate chemical substitution and retesting. 1) Major activities completed / experiments conducted: Chemicals used in this project were either purchased commercially or were synthesized by Paul Carlier and his collaborators at the Dept. of Chemistry, Virginia Tech and are given in annual reports. 2) Data collected: NA 3) Summary statistics and discussion of results: NA 4) Key outcomes or other accomplishments realized: The compounds were used to explore insecticidal activity, as proposed. Methods for synthesizing the various types of molecules used in these studies over the past five years are now available for making additional analogs by our group and others, as the reactions are published in the scientific literature.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
P. R. Carlier, J. R. Bloomquist, M. M. Totrov, and J. Li (2017) Discovery of species-selective and resistance-breaking anticholinesterase insecticides for the malaria mosquito. Curr. Med. Chem. 24(27), 2946-2958 (invited).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
P. R. Carlier, J.R. Bloomquist, J. Li, and M. Totrov (2017) Lessons learned in the search for mosquitocidal AChE inhibitors having both target selectivity and resistance-breaking properties. National meeting of the American Chemical Society, Division of Agrochemicals, Washington D.C.
|
Progress 10/01/16 to 09/30/17
Outputs
Target Audience:Entomologists and insect toxicologists working in government, industry, and academia. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Postdoctoral scientists have gained practical and theoreticalexperience working with mosquitoes, resistance, enzyme catalysis measurements,and insecticidediscovery methods. How have the results been disseminated to communities of interest?Peer reviewed publication and meeting presentations to scientific audiences, the latter including representatives ofinternational agrochemical companies. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Impact: Insecticide resistance is an important problem mitigating effective chemical insect control. Work was done in collaboration with the USDA-ARS Knipling-Bushland U.S. Livestock Insects Research Laboratory in Kerrville, Texas, where the laboratory of Kevin Temeyer produced the mutant enzymes using standard molecular biological techniques. Pharmacological and substrate kinetics were then performed in the Bloomquist Lab. This study focused on the leishmaniasis sandfly vector, Phlebotomus papatasi (Scopoli). Our previous studies (Parasites & Vectors 2014, 7:577) found that an engineered mutation (G119S) in the acetylcholinesterase (AchE) of P. papatasi could readily occur from a single nucleotide transversion. Moreover, it gave strong resistance to standard organophosphorus and carbamate insecticides. In an elaboration of this approach, additional F290V and F331W orthologous mutations were made and tested. In general, enzymes carrying these 2 mutations showed less cross resistance than G119S. Enzyme kinetics analysis found that all the mutations reduced maximal catalytic rate and substrate affinity was also reduced by F290V and F331W. These findings suggest that the lowered cross resistance and impaired catalytic activity of F290V and F331W mutations could be less of a field resistance problem for anticholinesterases against P. papatasi. Objective: 1. Assess the ability of novel anticholinesterases to block the activity of arthropod pest and non-target vertebrate enzymes, in vitro. 1) Major activities completed / experiments conducted: IC50 values (molar concentration of inhibitor that blocks enzyme activity 50%) were determined for 12 compounds on F290V and F331W engineered mutants of P. papatasi AchE. Further comparative substrate kinetics of enzyme activity was analyzed for wild-type, G119S, F290V, and W331W enzymes. Many, if not all of the compounds have been run before on human AchE, and the studies published. 2) Data collected: Inhibitory potencyfor each compound with the three enzymes was determined experimentally as IC50 valuesby fitting thedata to a four parameter logistic equation as defined in PrismTMsoftware (GraphPad, San Diego, CA). Enzyme catalytic measurements with varying substrate concentrations (Km, substrate concentration at which catalysis is half-maximal; Vmax, maximal catalytic activity) were calculated from nonlinear regression using the Michaelis-Menten model in PrismTMsoftware (GraphPad, San Diego, CA). 3) Summary statistics and discussion of results: Both phenyl- and pyrazole-bearing carbamates showed IC50 values of around 1-2 µM and typical resistance ratios of 20 to 40-fold when tested against the F331W mutant. All differences between wild type and F331W were statistically significant by non-overlap of 95% confidence limits. For the F290V mutant, IC50 values and resistance ratios were generally similar. Overall, the F331W and F290V mutants had inhibitor sensitivities similar to those of the G119S mutant, except for substituted phenyl carbamates, such as propoxur and carbofuran. This class of chemistry had resistance ratios of 3,000- to 19,000-fold against G119S, much greater than the other compounds. Substrate kinetic analysis showed that Vmax was significantly reduced (ANOVA, p < 0.05) by all the mutants, with the G119S mutant showing the greatest reduction (90%), followed by F331W (78%) and F290V (53%). In contrast, Km was unchanged for G119S, and was increased 3-fold and 4-fold for F290V and F331W, respectively. 4) Key outcomes or other accomplishments realized: These enzyme studies advance knowledge on the impact of these AchE mutations on anticholinesterase sensitivity to insecticides, and allow predictions about their likely field impact on leishmaniasis control. The F290V and F331W mutants showed less cross resistance than G119S. Also, they shared, to a variable extent, compromised catalytic activity that should contribute to a fitness cost for carrying these mutations in resistant individuals. Objective 2. Determine any lethal actions of active compounds, in vivo, and the involvement of any significant pharmacokinetic processes impacting toxicity. 1) Major activities completed / experiments conducted: Given the great facility of evaluating compounds on genetically engineered enzymes, in vitro, no experiments in the past year were run on live insects because no colonies of sandflies are available that contain these mutations. 2) Data collected: NA 3) Summary statistics and discussion of results: NA 4) Key outcomes or other accomplishments realized: NA. Objective 3. Model structure-activity relationships via correlation analysis of in vitro and in vivo data to aid in design of new molecules. 1) Major activities completed / experiments conducted: None in the past year. 2) Data collected: NA 3) Summary statistics and discussion of results: NA 4) Key outcomes or other accomplishments realized: NA Objective 4. Generate new molecules and optimize activity by appropriate chemical substitution and retesting. 1) Major activities completed / experiments conducted: The compounds synthesized as probes of the resistant mutants have all been disclosed previously in reports, presentations, or published manuscripts. 2) Data collected: NA 3) Summary statistics and discussion of results: NA 4) Key outcomes or other accomplishments realized: NA
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
P. R. Carlier, J. R. Bloomquist, M. M. Totrov, and J. Li (2017) Discovery of species-selective and resistance-breaking anticholinesterase insecticides for the malaria mosquito. Curr. Med. Chem. 24(27), 2946-2958
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2017
Citation:
P. R. Carlier, J.R. Bloomquist, J. Li, and M. Totrov (2017) Lessons learned in the search for mosquitocidal AChE inhibitors having both target selectivity and resistance-breaking properties. National meeting of the American Chemical Society, Division of Agrochemicals, Washington D.C.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2017
Citation:
K. B. Temeyer, F. Tong, Q.-H. Chen, P. Carlier, A. A. P�rez de Le�n and J. R. Bloomquist (2017) Acetylcholinesterase of Phlebotomus papatasi (Scopoli): expression and biochemical properties of orthologous organophosphate resistance mutations from mosquitoes. Vector-Borne Disease Symposium, University of Texas, San Antonio, Texas.
|
Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Entomologists in academia, industry and government interested in insecticide development and resistance. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The following paper was presented at theNational Meeting of the American Chemical Society, Philadelphia, Pennsylvania: J. R. Bloomquist(speaker), J. Taylor-Wells, A. D. Gross, and P. R. Carlier (2016) Chemical control of mosquitoes by re-purposed and modified agricultural insecticides. How have the results been disseminated to communities of interest?Communicated to researchers in industry andacademia, as well as other interested parties via peer-reviewed publication and scientific meeting presentation. What do you plan to do during the next reporting period to accomplish the goals?Publish a comprehensive, invited review of our body of work on insecticidal anticholinesterases. Additional research wil focus on engineered mutants of sandfly (Phlebotomus papatasi) AChE and their sensitivity to a structurally varied series of inhibitors, and other related topics.
Impacts
What was accomplished under these goals?
Impact: New compounds with novel modes of action are needed to control insecticide-resistant insects. The compoundssynthesized and tested this past year proved that bivalent molecules having two linked binding groupscan have contact (topical) toxicity to mosquitoes by blocking the enzyme acetylcholinesterase (AChE). Although paper contact activity was poor, stomach activity isalso possible, but was not tested. These results are a substantial change of knowledge in the structural rangeof chemistries that can effectively block acetylcholinesterase and haveinsecticidal activity. Objective: 1. Assess the ability of novel anticholinesterases to block the activity of arthropod pest and non-target vertebrate enzymes, in vitro. 1) Major activities completed / experiments conducted: A series of 8 phthalimide-pyrazolebivalent carbamates was tested for inhibitory activity against susceptible and carbamate-resistant (G119S) acetylcholinestrease, as well as recombinanthuman enzyme to check for selectivity. 2) Data collected: Inhibition kinetics foreachcompound with the threeenzymes was determined experimentally (kiexpressed asmM-1min-1). 3) Summary statistics and discussion of results: The most active compound was (1-(3-(1,3-dioxoisoindolin-2-yl)propyl)-1H-pyrazol-4-yl methylcarbamate), which had a chain length of three carbons, good mosquito anticholinesterase activity (Susceptible ki = 951 + 39; Resistant ki = 17 + 0.7), and ca. 5-fold selectivity compared to human AChE (ki = 174 + 7). The enzyme inhibition observed for the best compound is in the range of that of the commercial carbamate insecticides propoxur(Susceptible ki= 323+8; Resistant ki= 0.04+0.005)and terbam(Susceptible ki= 1710+20; Resistant ki= 0.65+0.06), although the new compound also is less affected by the G119S mutation, itis less selective than propoxur or terbam, which are19- and 14-fold selective, respectively). 4) Key outcomes or other accomplishments realized: The enzyme inhibition observed for the best compund is in the range of that of the commercial carbamate insecticides propoxurand terbam.It is less affected by the G119S mutation that gives high resistance to other carbamates, butitis less selective than the standards compared to human AChE. Objective 2. Determine any lethal actions of active compounds, in vivo, and the involvement of any significant pharmacokinetic processes impacting toxicity. 1) Major activities completed / experiments conducted: A series of 8 bivalent phthalimide-pyrazolecarbamates was tested for lethality by topical application of acetone solutions to mosquitoes, as well as in the WHO contact paper assay. Both susceptible G3 and resistant Akron strains of Anopheles gambiaewere used. The best compound, as noted above for the enzyme assays was also tested by injection to see if the cuticle was a significant barrier to penetration. 2) Data collected: All but two of the eight compoundshad little toxicity in single concentration screensby topical application, and none had more than 16% toxicity at 1 mg/ml in the WHO paper assay. Inthe paperassay, propoxur LC50 is ca. 0.04 mg/ml 3) Summary statistics and discussion of results: The most active compound was (1-(3-(1,3-dioxoisoindolin-2-yl)propyl)-1H-pyrazol-4-yl methylcarbamate), which had anLD50= 63 ng/female (95% CL = 55-72). This toxicity was less than propoxur (LD50= 3.2ng/female)and terbam (LD50= 4.5ng/female).The experimental bivalent showedonly 6-fold cross resistance in the Akron strain ofAnopheles gambiae, whereaspropoxur and terbamshowed 50- to 60-fold resistance. When injected,(1-(3-(1,3-dioxoisoindolin-2-yl)propyl)-1H-pyrazol-4-yl methylcarbamate) had anLD50= 0.24ng/female (95% CL = 0.16-0.3), a 263-fold increase in toxicity. 4) Key outcomes or other accomplishments realized: The experimental bivalent carbamate was 15- to 20-fold less active than standards on susceptible mosquitoes, and showed much less cross resistance. Injection studiesindicatedthat poor uptake via the cuticle is limiting toxicity of bivalent molecules. Objective 3. Model structure-activity relationships via correlation analysis of in vitro and in vivo data to aid in design of new molecules. 1) Major activities completed / experiments conducted: The most active bivalent carbamate,(1-(3-(1,3-dioxoisoindolin-2-yl)propyl)-1H-pyrazol-4-yl methylcarbamate, was dockedinto ahomology model of AgAChE usingMolsoftICM software (Molsoft, San Diego, CAUSA). 2) Data collected: NA 3) Summary statistics and discussion of results: The modelsuggeststhatthe most active bivalentcompoundprobably interactswith both the catalytic and peripheral sites on AgAChE, as intended, but in a new way. In thecatalytic site, carbamate interacts with theoxyanion hole, forming hydrogen bondsto backbone N-H groups of residues G118and A200. These interactions position the carbamatefor the nucleophilic attack on the S199hydroxyl, and the pyrazole nitrogen formsa hydrogen bond to Y121 sidechain. Thephthalimido moiety is mostly occupyingthe 'gorge' that connects peripheral andcatalytic sites, making a hydrogen bondto Y328 and contacts with Y121 and Y332side-chains as well as V71 and D72 backbone.It does not reach as far as the keyW280 peripheral site side-chain, which represents anovel mode of interaction mode AchE. 4) Key outcomes or other accomplishments realized: This model can be used in the design of future bivalent phthalimide-pyrazolecarbamate insecticides. Objective 4. Generate new molecules and optimize activity by appropriate chemical substitution and retesting. 1) Major activities completed / experiments conducted: Eightbivalent phthalimide-pyrazolecarbamates weresynthesized, but no futher analogs are planned at present, due to the low contact activity on paper. 2) Data collected: NA 3) Summary statistics and discussion of results: NA 4) Key outcomes or other accomplishments realized: Methods for synthesizing this type of molecule are now available for making additional analogs by our group and others, as the method is published in the scientific literature.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
J. M. Mutunga, Q.-H. Chen, D. M. Wong, P. C-H. Lam, J. Li, M. M. Totrov, A. D. Gross, P. R. Carlier, and J. R. Bloomquist (2016) Bivalent carbamates as novel control agents of the malaria mosquito, Anopheles gambiae. (invited) Chimia 70, 704-708.
|
Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Entomologists and toxicologists working in government, industry, and academia. Changes/Problems:No changes/problems for the past year, but fewer novel compounds will be synthesized in 2016 due to lack of funds. What opportunities for training and professional development has the project provided?Graduate students and postdoctoral scientists have gained experience working with mosquitoes, resistance, and insecticide discovery methods. How have the results been disseminated to communities of interest?Peer reviewed publication and meeting presentations to scientific audiences, the latter including representatives of international agrochemical companies. What do you plan to do during the next reporting period to accomplish the goals?We will study experimental compounds on tick and sandfly enzymes to asses activity on resistance mutations, and continue to study the mode of action of materials we have in hand. Analysis of physiological responses to insecticides based upon target site resistance mechanisms in the Akron strain will also be performed in the coming year.
Impacts
What was accomplished under these goals?
Three new classes of experimental chemistry were assessed for activity at the enzyme level of susceptible and resistant mosquitoes, as well as recombinant human enzyme. Three favorable qualities are needed for a new commercial compound: i) high enzyme inhibition potency (Objective 1) and in vivotoxicity (Objective 2) againstsusceptible insects, ii)high enzyme inhibition potency andin vivotoxicity against resistantinsects, and iii) low potency against human enzyme as a biomarker for human safety. No molecular modeling was attemptedthis year and pharmacokineticfactors apperently distorted the correlation between enzyme inhibition potency and insect toxicity(Objective 3). Nonetheless,a number of analogs of each series was synthesized to explore structure-activity relationships (Objective 4). For the compunds tested this year, we have been able to achievegood activity against both susceptible and resistant insects, but not acceptablemammalian safety. We investigatedisoxazol-3-yl dimethylcarbamatesand the corresponding 3-oxoisoxazole-2(3H)-dimethylcarboxamidesfor their ability to inhibitacetylcholinesterase (AChE) and causemosquitocidal effect. In both chemical series, compounds were identifiedwith excellent contact toxicity to the wildtype (WT) susceptible G3strain and theresistant (Akron) strain ofAn. gambiae.The latter strain carriesthe Glycine toSerine muttion at position 119 (G119S), a resistantmutant formof AChE, as well as target site resistance to pyrethroids (kdr). Compounds possessing good to excellent toxicity to Akron strain mosquitoes inhibited the G119S mutant ofAn. gambiaeAChE (AgAChE)at least 10- to 600-fold greater than that of the commercial insecticide propoxur, a compound that does not kill Akron mosquitoes at the highest concentration tested. On average, inactivation of wild typeAgAChE by dimethylcarboxamideswas 10- to 20-fold faster than that of the corresponding isoxazol-3-yl dimethylcarbamates. Unfortunately, human/An. gambiaeAChE inhibition selectivities of these compounds were low, suggesting the need for additional structural modification for acceptable human safety. Continuing this work on additional chemical series, some experimental trifluoromethyl ketones were synthesized that demonstrated remarkable volatility in microtiter plate assays, but in some cases also potent (1-100 nM) inhibition of WTAgAChE, andonlyweak inhibition of resistant mutant G119SAgAChE. The corresponding fluoromethyl ketoneswere less active, being submicromolar to micromolar inhibitorsof WTAgAChE, but again only weakly inhibited G119S AgAChE. Interestingly, difluoromethyl ketone inhibitorshad single digit nanomolar inhibition of WTAgAChE, and in some casesexcellent potency against G119SAgAChE. Approach to steady-state inhibition was quite slow with these molecules. The slow, tightbinding toG119SAgAChE appears to resut from a balance of steric size and electrophilicity. However, toxicities of some potent enzyme inhibitors, when tested on adultAn. gambiaein tarsal contact, fumigation, and injection assays showed lower than expected activity. It is assumed that pharmacokinetic factors are responsible. Likewise, aseries of substituted α,α,α,- trifluoroacetophenone oxime methylcarbamates were evaluated for enzyme inhibition potency and toxicity against G3 and Akron mosquitoes. The compound bearing an unsubstituted phenyl ring showed the greatest toxicity to mosquitoes of both strains. Low cross resistance in Akron was retained by all analogs in the series. Kinetic analysis of acetylcholinesterase activity and its inhibition by insecticides in the G3 strain showed inactivation rate constants greater than that of propoxur, and against Akron enzyme inactivation rate constants similar to that of aldicarb. However, inactivation rate constants against recombinant human AChE were essentially identical to that of the G3 strain. Thus, the acetophenone oxime carbamates described here, though potent insecticides that control resistant Akron mosquitoes, require further structural modification to attain acceptable selectivity and human safety. Structure-activity analysis of carbamates inAnopheles gambiaewere extended to other insects (Objectives 1 and 2). Experimental carbamates were potent inhibitors of mosquito acetylcholinesterases,with IC50values in the nanomolar range. Similar potencies were observed forMusca domestica(house fly) andDrosophila melanogaster(friut fly) enzymes. Althoughmeta-substituted carbamates were potentinhibitors, twoortho-substituted carbamates displayed poor enzyme inhibition (IC50≥ 1 micromolar) inhoney bee (Apis mellifera), Asian citrus psyllid (Diaphorina citri), and lepidopteran agriculturalpests (Plutella xylostellaandOstrinia nubilalis). Enzyme inhibition results were confirmed bytoxicity studies in caterpillars, where the new carbamates were 2- to 3-fold less toxic thanpropoxur and up to 10-fold less active than bendiocarb, indicating little utility of these compoundsfor crop protection. The experimental carbamates were broadly active against mosquito species butnot agricultural pests, which should mitigate selection for mosquito insecticide resistance byreducing agricultural uses of these compounds. Studies on honey bee enzyme identified carbamates with low activity, which should provide a good margin of pollinator safety, although other species of pollinators were not tested. Another major area of research characterized resistance mechanisms in the AkronstrainofAn.gambiae,andalso fits under Objectives 1 and 2. Although this strainhas been studied using molecular diagnostics, quantitativeinformation on it sensitiviety to insecticides is not found in the literature. Here we report resistance in the Akronstrain to pyrethroids and DDT (13-fold to 35-fold at the LD50level compared to the susceptible G3 strain), but surprisingly little topical resistance to etofenprox, a compound sometimes described as a "pseudo-pyrethroid." As mentioned above, there was also strong resistance to topically-applied commercial carbamates (45-fold to 81-fold), except for the oximes aldicarb and methomyl. Biochemical assays showed enhanced cytochrome P450 monooxygenase and carboxylesterase activity, but not that of glutathione-?S-transferase.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2015
Citation:
J. R. Bloomquist, A. D. Gross, D. R. Swale, F. Tong, and T. D. Anderson (2015) Resistance characteristics of Anopheles gambiae Akron strain mosquitoes and resistance-breaking trifluoromethylphenyloxime carbamates. National meeting of the American Mosquito Control Association, New Orleans, Louisiana.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
E. Camerino, D. M. Wong, Fan Tong, F. K�rber, A. D. Gross, R. Islam, E. Viayna, J. M. Mutunga, J. Li, M. M. Totrov, J. R. Bloomquist, and P. R. Carlier (2015) Difluoromethyl ketones: potent inhibitors of wild type and carbamate-insensitive G119S mutant Anopheles gambiae acetylcholinesterase. Bioorg. Med. Chem. Lett. 25, 4405-4411.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
A. Verma, D. M. Wong, R. Islam, F. Tong, M. Ghavami, J. M. Mutunga, C. Slebodnicka, J. Li, E. Viayna, P. C.-H. Lam, M. M. Totrov, J. R. Bloomquist, and P. R. Carlier (2015) 3-Oxoisoxazole-2(3H)-carboxamides and isoxazol-3-yl carbamates: Resistance-breaking acetylcholinesterase inhibitors targeting the malaria mosquito, Anopheles gambiae. Bioorg. Med. Chem. 23, 1321-1340.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
J. M. Mutunga, T. D. Anderson, D. T. Craft, A. D. Gross, D. R. Swale, F. Tong, D. M. Wong, P. R. Carlier, and J. R. Bloomquist (2015) Carbamate and pyrethroid resistance in the Akron strain of Anopheles gambiae. Pestic. Biochem. Physiol. 121, 116-121.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
D. R. Swale, P. R. Carlier, J. A. Hartsel, M. Ma, J. R. Bloomquist (2015) Mosquitocidal carbamates with low toxicity to agricultural pests: An advantageous property for insecticide resistance management. Pest. Manag. Sci. 71, 1158-1164.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2015
Citation:
J. R. Bloomquist, F. Tong, and A. D. Gross (2015) Impact of synergists on insecticide resistance in Anopheles gambiae. National Meeting of the Society of Vector Ecology, Albuquerque, New Mexico.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2015
Citation:
J. R. Bloomquist, A. D. Gross, D. R. Swale, F. Tong, and T. D. Anderson (2015) Resistance mechanisms in the Akron strain of Anopheles gambiae isolated in Benin, West Africa. Twelfth (12th) Arbovirus Surveillance and Mosquito Control Workshop, St. Augustine, Florida.
|
Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Entomologists in academia, industry and government interested in insecticide development and resistance. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The following paper, which contained some of the data in our published paper was presented at the National Meeting of the Society of Vector Ecology, San Antonio, Texas (speaker underlined). K. B. Temeyer, A. P. Tuckow, J. R. Bloomquist, and A. A. Pérez de León. 2014. Expression and biochemical properties of a recombinant acetylcholinesterase 1 of the sand fly, Phlebotomus papatasi (Scopoli) insensitive to organophosphate inhibition. How have the results been disseminated to communities of interest? Communicated to interested parties via peer-reviewed publication and scientific meeting. What do you plan to do during the next reporting period to accomplish the goals? Continue synthesis and testing of new carbamates to try and increase activity against resistant enzyme forms. We will also investigate other chemistries that work on different targets as an alternative to anticholinesterases.
Impacts
What was accomplished under these goals?
Insecticide resistance is a threat to continued effective and economical control of insect pests, including biting midges such as Phlebotomus papatasi. Thus, new compounds are urgently needed for disease vector and agricultural insect control. This past year's research was a study into the molecular basis of insecticide resistance, using the enzyme acetylcholinesterase (AChE) and its interaction with establsihed and novel synthetic compounds as inhibitors. Previous expression of recombinant P. papatasi acetylcholinesterase (PpAChE1) revealed 85% amino acid sequence identity to mosquito AChE and identified synthetic carbamates that effectively inhibited PpAChE1 with improved specificity for arthropod AChEs compared to mammalian AChEs. We hypothesized that the G119S mutation causing high level resistance to carbamate insecticides in mosquitoes may occur in PpAChE1 and reduce its sensitivity to inhibition. Accordingly, targeted mutagenesis introduced the G119S orthologous substitution in PpAChE1 cDNA. Recombinant PpAChE1 enzymes containing or lacking the G119S mutation were expressed in the baculoviral system (done by Temeyer and Tucknow, Kerrville TX). Biochemical assays were conducted to determine altered catalytic properties and inhibitor sensitivity resulting from the G119S substitution (done by Tong and Bloomquist, Univ. of FL). Novel inhibitors were synthesized by Chen and Carlier (Virginia Tech). A molecular homology model was constructed to examine the possible structural interference with docking of inhibitors of different classes (done by Totrov, Molsoft LLC in CA). Genetic tests were conducted to determine if the G119S orthologous codon existed in polymorphic form in a laboratory colony of P. papatasi (done by Temeyer and Tucknow, Kerrville TX). Recombinant PpAChE1 containing the G119S substitution exhibited altered biochemical properties, and reduced inhibition by compounds that bind to the acylation site on the enzyme (with the exception of eserine). Less resistance was directed against bivalent or peripheral site inhibitors, in good agreement with modeled inhibitor docking. Eserine appeared to be a special case capable of inhibition in the absence of covalent binding at the acylation site. Genetic tests did not detect the G119S mutation in a laboratory colony of P. papatasi, but did reveal that the G119S codon existed in polymorphic form (GGA + GGC). The finding of G119S codon polymorphism in a laboratory colony of P. papatasi suggests that a single nucleotide transversion (GGC to AGC) may readily occur, causing rapid development of resistance to organophosphate and phenyl-substituted carbamate insecticides under strong selection. Careful management of pesticide use in IPM programs is important to prevent or mitigate development and fixation of the G119S mutation in susceptible pest populations. Availability of recombinant AChEs enables identification of novel inhibitory ligands with improved efficacy and specificity for AChEs of arthropod pests.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
K. B. Temeyer, F. Tong, A. P. Tuckow, Q-H. Chen, P. R. Carlier, A. A. Perez de Leon, J. R. Bloomquist. 2014. Acetylcholinesterase of the sand fly, Phlebotomus papatasi (Scopoli): construction, expression and biochemical properties of the G119S orthologous mutant. Parasites Vectors 7, 577 (doi: 10.1186/s13071-014-0577-4).
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Progress 04/01/13 to 09/30/13
Outputs
Target Audience: Entomologists working in government, industry, and academia Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The following paper presented at a national meeting of the American Chemical Society contained discussion sof some of this data (speaker underlined). J. R. Bloomquist, J. Li, M. M. Totrov, P. R. Carlier. 2013. Inhibitors targeting acetylcholinesterase with high selectivity for arthropod disease vectors. International Award Symposium for Rene Feyereisen, National Meeting of the American Chemical Society, Indianapolis, IN. How have the results been disseminated to communities of interest? Communicated to interested parties via peer-reviewed publication. What do you plan to do during the next reporting period to accomplish the goals? Continue our characterization of dimeric tacrine effects in other species (e.g., ticks and sandfly) to learn more about arthropod acetylcholinesterases and use this information to design better insecticides. We will also investigate other chemistries as anticholinesterases, since although the tacrine dimers are interesting enzyme probes, they are not insecticidal.
Impacts
What was accomplished under these goals?
Insecticide resistance is a threat to continued effective and economical control of insect pests. Thus, new compounds are urgently needed for disease vector and agricultural insect control. This project was a study into the basic biology of insect acetylcholinesterase enzymes and their interaction with novel synthetic compounds. We selected the enzyme acetylcholinesterase as a model target site because it is an established insecticide target and much is known about its protein structure and interaction with chemical inhibitors. For example, this enzyme has two pharmacologically defined sites, the catalytic site and peripheral site, either of which can bind inhibitors. This study addressed the question of what happens to enzyme inhibition and toxicity when a ligand directed against each site are linked together into one molecule by using a simple carbon chain of varying length. Accordingly, a series of bis(n)-tacrines were used as pharmacological probes of the acetylcholinesterase (AChE) catalytic and peripheral sites of Blattella germanica and Drosophila melanogaster (organic chemical synthesis by Paul Carlier and Dawn Wong). Measurements were then made of enzyme activity in these species (James Mutunga and Troy Anderson). In general, the potency of bis(n)-tacrines was greater in D. melanogaster AChE (DmAChE, best IC50s were 2-10 nanomolar) than in B. germanica AChE (BgAChE, best IC50s were ca. 100 nanomolar). The change in potency with tether length was high in DmAChE and low in BgAChE, associated with 90-fold and 5.2-fold maximal potency gain, respectively, compared to the tacrine monomer. The optimal tether length for Blattella was 8 carbons and for Drosophila was 10 carbons. The two species differed by only about 2-fold in their sensitivity to tacrine monomer, indicating that differential potency occurred among dimeric bis(n)-tacrines due to structural differences in the peripheral site. Multiple sequence alignment (Dawn Wong) and in silico homology modeling (done by Polo Lam and Maxim Totrov) suggested that aromatic residues of DmAChE (W321, Y71, and Y324) forming a cation hydrophobic cage in the peripheral site confer higher affinity binding in D. melanogaster. Lack of same at the BgAChE peripheral site may account, at least in part, to the greater overall sensitivity of DmAChE to bis(n)-tacrines, as reflected by in vitro enzyme assay data. Despite being highly potent inhibitors in vitro, neither mortality nor signs of intoxication were observed following ingestion (2000 ppm, Drosophila) or injection (2000 ng, Blattella) bioassays (done by James Mutunga). It was hypothesized that lack of sufficient penetration into the central nervous system (CNS) was at least partly responsible for the lack of toxic effect. Accordingly, peripheral nerve firing rates after bis(4)-tacrine in both intact and transected CNS were determined with D. melanogaster larvae (done by DhanaRaj Boina). Unlike the intact preparations, the transected CNS had a disrupted barrier and therefore dimeric tacrines could penetrate the nervous system and elicit a higher frequency nerve firing response. This finding indicated that the sheath was a penetration barrier to dimeric tacrines in the insect brain and that the cationic tacrine dimers do not readily cross the blood brain barrier, explaining the observed non-lethality to insects. Although the bis(n)-tacrines were not good insecticide candidates, the information obtained in this study should aid in the design of selective bivalent ligands targeting insect pests and disease vectors. Such compounds, because they span 2 seperate binding domains, have the potential for high insect selectivity, and ability to mitigate target site resistance.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2013
Citation:
J. M. Mutunga, T. D. Anderson, D. Boina, P. R. Carlier, P. C.-H. Lam, M. M. Totrov, D. M. Wong, and J. R. Bloomquist. 2013. Neurotoxicology of bis(n)-tacrines on Blattella germanica and Drosophila melanogaster acetylcholinesterase. Arch. Insect Biochem. Physiol. 83(4), 180-194.
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