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Molecular Design and Semi-FieldPerformance of Highly Selective
Carbamates for Control of the MalariaMosquito, Anopheles gambiae
Jeff Bloomquist
Neurotoxicology Laboratory
Dept. of Entomology and Nematology
Emerging Pathogens Institute
University of Florida
Project Focused on Controlling Anopheles gambiae
"The World's Most Dangerous Animal"
M. P. Scott, Proc. Nat. Acad. Sci. U.S.A. 2007, 104, 11865
• >1 million deaths from malaria annually
• 100 children per hour in Africa
• 400 million cases of malaria annually
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 (Zaim et al., 2000).
• Emergence of pyrethroid resistance could render current ITNs ineffective
• We are developing new anticholinesterase insecticides
Bayer Environmental Science Public Health Journal, No. 18/2006
>>[Ach]
Convulsions
and
Death
Acetylcholinesterase (AChE)
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
Catalytic Triad
Peripheral Site
Anionic Site
OHS200
NHNH440
C
C
OH
E327
N
W279
N
W84
Acetylcholine
Acetate + choline
Conservation of the AChE Catalytic Site (Selectivity)
W84
S200
E327
H440ACh
A201
G118
G119
Choline-binding site (W84)
Oxy-anionhole (G118,
G119, A201)
Catalytic triad (S200, H440, E327)
2ace.pdbTcAChE
Dr. Dawn Wong
+O
SER 200
C NCH
3
HO
Carbamylation
Pi e-
Assess IC50s for Enzyme Inhibition in vitro (Ellman assay)
Anopheles gambiae homogenate (Ag hmg)(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 (h AChE)(Sigma)
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.
WHO Protocol
or
• 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.
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)
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
1.1 ug/cm2
(Propoxur)5.6 ug/cm2 11 ug/cm2
PRC331
PRC387
PRC388
PRC331 on Housefly AChE IC50 = 250 nM
266 533 286An.g. human An.g. human An.g. human
AChE Phylogenetics
Weill, Proc. Royal. Soc., 2002, 2692007.
A. gambiae
D. melanogasterM. domestica
40%identity
Why is PRC331
So Selective?
Black = Human(W84 Stable)
P446
Y449
Y337 Y328
W84 W84
C286
M438
D441
Blue = An. gambiae(W84 flexible)
Second Structural Class: *2-Substituted Carbamates
PRC337
*Patent Pending
O O
N H C H 3
O O
N H C H 3
P R C 391
P R C 40 7
P R C 408S S
R 1 R 2
R 1C H 3C H 3C H 2C H 3
R 2C H 3C H 2C H 3C H 2C H 3
R1 R2
Ag hmg Ag ace-1S Class Compound IC50 (nM) II PRC337 9550 124 (77x) 165 (58x) II PRC391 8110 109 (74x) nd II PRC407 3540 30 (118x) 27 (131x) II PRC408 3630 3 (1210x) 2.9 (1250x)
hAChE IC50 (nM) IC50 (nM)
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
Synergized Toxicity
Selected PRC331 for Semi-Field Studies
Developed in the 1970s as an insecticide "Terbam"Some toxicology information published (Toxline):Mouse LD50 (oral): 470 mg/kg20x less toxic than propoxurFiled a use patent for vector control
Compare to propoxur:
Mouse oral LD50 h/Ag AChE IC50 ratio
24 mg/kg 0.4
Semi-Field Testing in Kenya
ICIPE at Mbita Point, J. Githure
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
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
Pooled Toxicity Data
The treatment rate is based on lab toxicity data and is expected to be equitoxic to deltamethrin (25 mg/m2).
Little excito-repellency by PRC331 (not surprising).
Immediate and overall mortality is quite good up to 50 days after treatment.
Treatment
Entry rate (%)
Exit rate (%)
Immediate Mortality (%)
Overall Mortality (%)
Untreated bed net
29 ± 1.1 0.0 0.5 ± 0.2 2.71 ± 0.7
PRC 331 at 111mg/m 2
32 ± 1.0 0.0 76.66 ± 3.07 98.6 ± 0.7
Persistence of Toxicity
0
10
20
30
40
2 4 6 8 10 12 14 16 18 20 22 26 28 30 32 34 36 38 40 43 45 47 49
Days post-treatment
% m
osq
uit
oes
dea
d
Control PRC331-Hut 2PRC331-Hut 1
100
90
80
70
60
50
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.
Acknowledgments
Virginia TechMolsoft
Financial Supportby FNIH ICIPE
