1 / 23

Jeff Bloomquist Neurotoxicology Laboratory Dept. of Entomology and Nematology

Molecular Design and Semi-Field Performance of Highly Selective Carbamates for Control of the Malaria Mosquito, Anopheles gambiae. Jeff Bloomquist Neurotoxicology Laboratory Dept. of Entomology and Nematology Emerging Pathogens Institute University of Florida jbquist@epi.ufl.edu.

tomai
Download Presentation

Jeff Bloomquist Neurotoxicology Laboratory Dept. of Entomology and Nematology

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Molecular Design and Semi-FieldPerformance of Highly SelectiveCarbamates for Control of the MalariaMosquito, Anopheles gambiae Jeff Bloomquist Neurotoxicology Laboratory Dept. of Entomology and Nematology Emerging Pathogens Institute University of Florida jbquist@epi.ufl.edu

  2. Project Focused on Controlling Anopheles gambiae"The World's Most Dangerous Animal" • >1 million deaths from malaria annually • 100 children per hour in Africa • 400 million cases of malaria annually M. P. Scott, Proc. Nat. Acad. Sci.U.S.A.2007, 104, 11865

  3. Background • Insecticide treated nets (ITNs) are the major component of the "Roll Back Malaria Partnership "http://www.rbm.who.int/ • Pyrethroids are the only insecticides currently recommended for use on nets (Zaimet al., 2000). • Emergence of pyrethroid resistance could render current ITNsineffective • We are developing new anticholinesterase insecticides

  4. Acetylcholinesterase (AChE) Convulsions and Death >>[Ach] Bayer Environmental Science Public Health Journal, No. 18/2006

  5. O H N H O H Acetylcholine Acetylcholinesterase(AChE) Proven, but nonselective target site with current compounds Numerous gene sequences available >100 x-ray crystal structures with docked ligands (1 from insect) Possesses a gorge with conserved catalytic and also peripheral sites Peripheral Site N W279 E327 C C W84 H440 N S200 N Catalytic Triad Anionic Site Acetate + choline

  6. SER 200 O CH 3 C N H O Conservation of the AChE Catalytic Site (Selectivity) Catalytic triad (S200, H440, E327) A201 S200 Oxy-anionhole (G118, G119, A201) G119 E327 G118 H440 ACh 2ace.pdbTcAChE Dr. Dawn Wong + Pi e- Carbamylation Choline-binding site (W84) W84

  7. Assess IC50s for Enzyme Inhibition in vitro (Ellman assay) Anopheles gambiae homogenate (Aghmg)(G3 strain, female, not blood-fed) Recombinant wild type AgAChE (Ag ace1-S)in Dros. S2 cell lysate Recombinant resistant (G119S) AgAChE (Ag ace1-R)in Dros. S2 cell lysate Recombinant human AChE (hAChE)(Sigma)

  8. WHO Protocol Mosquito Toxicity • Batches of 25 non-blood fed female A. gambiae. • Transfer to exposure tube. • 1-hr exposure to treated filter paper (180 cm2) • Check for mortality at 10-minute intervals. • Transfer back to holding tube for 24 hours. • Provide sugar water. • Record final mortality. • Treat topically in 0.2 ul of ETOH, with or without synergists • Transfer back to holding tube for 24 hours. • Provide sugar water. • Record final mortality. or

  9. Inhibitor Design Goals • Achieve high (>100- to 1000-fold) target selectivity for inhibition of AgAChE relative to humanAChE. • Achieve contact toxicity for Anopheles gambiae>propoxur, a standard carbamate insecticide, and minimize mammalian contact toxicity • Achieve potent inhibition of the resistant enzyme (G119S, AKRON strain)

  10. Re-Screened Some Known 3-Substituted Carbamates IC50s in nM; human/An.g.selectivities in parenthesesMinimum dosage for 100% lethality at 24 hr-WHO paper assay PRC331 PRC387 PRC388 An.g. human An.g. human An.g. human 266 533 286 1.1 ug/cm2 (Propoxur) 5.6 ug/cm2 11 ug/cm2 PRC331 on Housefly AChE IC50 = 250 nM

  11. AChEPhylogenetics A. gambiae 40% identity D. melanogaster M. domestica Weill, Proc. Royal. Soc., 2002, 269 2007.

  12. Why is PRC331 So Selective? C286 Blue = An. gambiae (W84 flexible) Y337 Y328 P446 Black = Human (W84 Stable) M438 W84 W84 D441 Y449

  13. R1 R2 O O O O S S N H C H N H C H 3 3 R R 1 2 R R 1 2 C H C H P R C 3 3 7 P R C 3 9 1 3 3 C H C H C H P R C 4 0 7 3 2 3 C H C H C H C H P R C 4 0 8 2 3 2 3 Second Structural Class: *2-Substituted Carbamates h A C h E Ag h m g Ag ace - 1S IC ( n M) C l ass IC ( n M) IC ( n M) C o m p o u n d 50 50 50 II PRC337 9550 124 (77 x ) 165 (58 x ) II PRC391 8110 109 (74 x ) n d II PRC407 3540 30 (118 x ) 27 (131 x ) II PRC408 3630 3 (1210 x ) 2.9 (1250x) *Patent Pending

  14. Performed Topical Tmts-Some Have Low Activity in WHO Paper Assay (1 hr Exposure) More active topically Suggests pen/metab are impacting activity Mitigated by formulation LD50

  15. Synergized Toxicity

  16. Selected PRC331 for Semi-Field Studies Developed in the 1970s as an insecticide "Terbam" Some toxicology information published (Toxline): Mouse LD50 (oral): 470 mg/kg 20x less toxic than propoxur Filed a use patent for vector control Compare to propoxur: Mouse oral LD50 h/AgAChE IC50ratio 24 mg/kg 0.4

  17. Semi-Field Testing in Kenya ICIPE at Mbita Point, J. Githure

  18. Inside The Huts: Treated, Baited Bednets MMX traps without suction are baited with human-worn socks underneath treated bednets. Mosquitoes are released and dead ones counted at regular intervals

  19. Semi-Field Trial Methods • Nets treated by soaking in an ethanolic solution of PRC331 • 200 female non-blood fed An. gambiae released each night. • Each dawn mosquitoes in the hut collected • Scored dead or alive (early mortality) • Live ones held for another 24 hrs to score delayed mortality • Mosquitoes not entering the hut were collected via backpack aspirator

  20. Pooled Toxicity Data The treatment rate is based on lab toxicity data and is expected to be equitoxicto deltamethrin (25 mg/m2). Little excito-repellency by PRC331 (not surprising). Immediate and overall mortality is quite good up to 50 days after treatment.

  21. D a y s p o s t - t r e a t m e n t Persistence of Toxicity 100 90 80 70 60 % mosquitoes dead 50 4 0 3 0 2 0 1 0 0 6 8 2 4 10 12 14 16 18 20 22 26 28 30 32 34 36 38 40 43 45 47 49 Control PRC331-Hut 1 PRC331-Hut 2

  22. Conclusions • We identified five selective 3-substituted carbamates (IC50 ratios 38-130) with toxicity similar to propoxur. • The 2-Substituted cmpds are even more selective (>1000-fold), toxicity impacted by pharmacokinetics. • Activity in semi-field tests with PRC331 is encouraging. • Have commercial interest: lab testing by BASF and Bayer CropScience, as well as full scale field tests with PRC331 in commercially formulated bednets by Vestergaard-Frandsen in Viet Nam.

  23. Acknowledgments Molsoft Virginia Tech Financial Support by FNIH ICIPE

More Related