1. Search for mosquito controlling compounds DWFP-related work Kumudini M. Meepagala USDA-ARS-NPURU Oxford MS

2. Search for selective AChE inhibitors against mosquitoes

3. Role of acetylcholinesterase

4. Synthesis of acetylcholine

5. Search for selective AChE inhibitors as adulticides and repellents DEET as a neurotoxin- AChE inhibitor Not target specific Need a replacement

6. DEET toxic effects Active ingredient in insect repellent Deet is neurotoxin! The active ingredient in many insect repellents, deet, has been found to be toxic to the central nervous system. Researchers say that more investigations are urgently needed to confirm or dismiss any potential neurotoxicity to humans, especially when deet-based repellents are used in combination with other neurotoxic insecticides. Vincent Corbel from the Institut de Recherche pour le Développement in Montpellier, and Bruno Lapied from the University of Angers, France, led a team of researchers who investigated the mode of action and toxicity of deet (N,N-Diethyl-3-methylbenzamide). Corbel said, “We’ve found that deet is not simply a behavior-modifying chemical but also inhibits the activity of a key central nervous system enzyme, acetycholinesterase, in both insects and mammals”. Here's the source report: Deet inhibits cholinesterase: Evidence for inhibition of cholinesterases in insect and mammalian nervous systems by the insect repellent deet (BioMed Central)Deet inhibits cholinesterase: Evidence for inhibition of cholinesterases in insect and mammalian nervous systems by the insect repellent deet

7. What do we know Acetylcholinesterase (AChE) a serine hydrolase, vital for regulating cholinergic neurotransmission in mammals, birds, fish, and insects Many insecticides phosphorylate or carbamylate indiscriminately a ubiquitous catalytic serine residue at the active site of the enzyme Some drugs for Alzheimer’s disease are also AChE inhibitors. Galatamine does not kill mosquitoes. Galantamine is a competitive and reversible AChE inhibitor. It is believed that it works by enhancing cholinergic function by increasing the concentration of acetylcholinein the brain. Galantamine Caucasian snowdrops

8. Mode of action of DEET Mosquitoes smell and avoid the insect repellent DEET PNAS September 9, 2008 vol. 105 no. 36 “ The widely accepted hypothesis that DEET interferes with the detection of lactic acid has been challenged by demonstrated DEETinduced repellency in the absence of lactic acid. The most recent hypothesis suggests that DEET masks or jams the olfactory system by attenuating electrophysiological responses to 1-octen-3-ol. Our research shows that mosquitoes smell DEET directly and avoid it. “ Behavioural mode of action of deet : inhibition of lactic acid attraction. Medical and veterinary entomology (1999), 13(1), 97-100. “We could not confirm that 'deet' is a repellent of mosquitoes. In the absence of a host, deet was an attractant and in the presence of a host, it was an inhibitor of attraction. We determined that L-lactic acid, a component of human sweat that is an attractant to mosquitoes, is the target of this inhibition, implying that lactic acid may be a bottleneck in the behavioural cascade preceding blood-sucking.”

9. MOA of DEET ctd…. Model for the Mechanism of Acion of the Repellent DEET on Aedes Aegpti Journal of Medical Entomology Vol 18 no5, 357-361, 1981 “Interaction of DEET molecules with lipid portion of the cell membrane perturbs organization of the dendritic membranesin such a way that the normal responses to attractants are altered.”

10. Extracts tested for AChE inhibitory activity Sample Name[mg/ml]Active Test 1Active Test 2 AT05 27-1 AT05 EtoAc Leaves (Pure cpd)1.0No-- AT05 24 AT05 EtoAc Leaves (Pure cpd)2.0Yes AT05 27-2 AT05 EtoAc Leaves (Pure cpd)1.0MaybeNo P. trifoliata fruit Hex. Ext5.0YesMaybe E. runyonii leaf/stem EtoAc Ext5.5MaybeNo Z. hirsutum MeOH Ext5.0No-- E. runyonii leaf/stem MeOH Ext7.0No-- A. texana Hexane Ext6.3YesYES ++ P. trifoliata Fruits Hex. Ext.5.0YesMaybe P. trifoliata EtoAc ext5.0MaybeNo P. trifoliata Root MeOH ext5.0No-- P. trifoliata Root Hexane ext5.0No-- H.parvifolia EtoAc Ext5.4MaybeYes ++ H.parvifolia MeOH Ext.22.8MaybeYes++ Galanthamine standard1.0Yes

11. Irreversible inhibitors of AChE Organophosphate insecticides disable the catalytic serine residue of acetylcholinesterase (AChE). Because these agents also affect vertebrate AChEs, and hence toxic to humans. A cysteine residue (Cys) is present at the active site in mosquitoes but not in mammals that serve as a target for insect-selective pesticides. Aphids also have two different AChEs (termed AP and AO), and only AP-AChE carries the unique Cys. Methanethiosulfonate-containing small molecule that, at 6.0 mM, irreversibly inhibits 99% of all AChE activity extracted from the greenbug aphid (Schizaphis graminum) without any measurable inhibition of the human AChE. Reactivation studies using β-mercaptoethanol confirm that the irreversible inhibition resulted from the conjugation of the inhibitor to the unique Cys. These results suggest that AO-AChE does not contribute significantly to the overall AChE activity in insects. Thus this unique Cys may be a viable target for species selective AChE inhibitors.

12. Binding of AChE inhibitors

13. Dosage (mg/cm²)M-3Proportion to Days ProtectionDEET SYN 3-39 0.7510.05 SYN 3-113 0.7521.00 SYN 3-127 0.7510.50 SYN 3-131 0.7510.50 SYN 131-1 0.7563.00 DEET 0.75 21.00

14. Duration of Repellency for Compounds

15. Averaged minimum effective dosage of compounds

16. AChE assay

17. TLC assay for AChE activity Isolated from A. texana

18. Acid F OMe+ CH2OH CH2O G Syn131-1 More active and longer lasting than DEET Is the mode of action the same as DEET? No nitrogen group Can bind to a Cys group in AChE Can interact with serine group in the catalytic site

19. Artemisia ludoviciana oils from Aromagen These essential oils have shown activity against aphids and fireants We are in the process of isolating these compounds in sufficient quantities.

20. Piperine3.8 ± 4.8 CD pelliotorin92.5 ± 15.0 A PIPER-syn 15100.0 ± 0A PIPER-syn 2130.0 ± 26.0 BC PIPER-syn 2348.8 ± 20.6 B PIPER-syn 2533.8 ± 24.6 BC PIPER-syn 736.3 ± 7.5 BC UNTREATED2.5 ± 5.0 D Treatment @1%wt/wt mortality at day 21

21. Amides with insecticidal activity

22. Other natural products isolated with AChE inhibitory activity Turmeric roots Curcuma longa Dill seeds Anethum graveolens

23. Reaction scheme for synthesis of larvicides

24. Synthetic scheme for analogs with two carbons longer

25. 8 ppm 2 ppm 0.5 ppm Indoxacarb100 40 57-1100 80 57-21008020

26. Future work Microplate assay using Elman’s method to evaluate AChE activity using mosquito homogenate. Some of this work will be carried out in Dr. Bloomquist’s lab in Gainesville Isolation of the constituents in the essential oil samples from Aromagen. Complete the synthesis of lavicide analogs and assay them for repellency, lavicide activity and AChE activity.

27. Acknowledgements Drs. Jimmy Becnel,Uli Bernier and Julia Pridgeon for assays. Dr. Jeff Bloomquist for suggestions with microplate mosquito AChE assay procedure. Jason Martin and Jennifer Cox for technical help with synthesis and isolation. George Sturtz and Dr.Charles Burandt for plant materials DWFP for funding. NCNPR for NMR facility.