Transcript Molecular Design of Selective Anticholinesterases for

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
[email protected]
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
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
Acetylcholinesterase (AChE)
Convulsions
and
Death
>>[Ach]
Bayer Environmental Science Public Health Journal, No. 18/2006
Acetylcholinesterase
(AChE)
Acetylcholine
Peripheral Site
Proven, but nonselective
target site with current
compounds
N
W279
Numerous gene
sequences available
>100 x-ray crystal
structures with docked
ligands (1 from insect)
E327
OH
C
C
H440
N
W84
NH
OH
Catalytic Triad
S200
N
Anionic Site
Acetate + choline
Possesses a gorge with
conserved catalytic and
also peripheral sites
Conservation of the AChE
Catalytic Site (Selectivity)
Catalytic triad
(S200, H440, E327)
A201
S200
G119
E327
G118
H440
ACh
SER 200
N
2ace.pdb
TcAChE
+
O
C
Oxy-anion
hole (G118,
G119, A201)
CH3
H
Pi
e-
Dr. Dawn Wong
O
Carbamylation
Choline-binding site (W84)
W84
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
WHO Protocol
• 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.
or
– 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 parentheses
Minimum dosage for 100% lethality at 24 hr-WHO paper assay
PRC
331
An.g.
human
266
1.1 ug/cm2
(Propoxur)
PRC
387
An.g.
human
533
5.6 ug/cm2
PRC331 on Housefly AChE IC50 = 250 nM
PRC
388
An.g.
human
286
11 ug/cm2
AChE Phylogenetics
A. gambiae
40%
identity
D. melanogaster
M. domestica
Weill, Proc. Royal.
Soc., 2002, 269
2007.
C286
Y337
Y328
Why is
PRC331
So
Selective?
Blue = An. gambiae
(W84 flexible)
P446
Black = Human
(W84 Stable)
M438
W84 W84
D441
Y449
Second Structural Class:
*2-Substituted Carbamates
NHCH
O
NHCH
3
O
S
O
3
O
P RC 391
S
P RC 407
P RC 408
R
1
R
R
R1
1
CH
3
R
R2
2
CH
3
CH
CH CH
3
2
3
CH CH CH CH
2
3
2
3
2
P RC 337
Class Compound
II
PRC337
II
PRC391
II
PRC407
II
PRC408
*Patent Pending
hAChE
IC50 (nM)
9550
8110
3540
3630
Ag hmg
IC50 (nM)
124 (77x)
109 (74x)
30 (118 x)
3 (1210x)
Ag ace-1S
IC50 (nM)
165 (58x)
nd
27 (131x)
2.9 (1250x)
Performed Topical TmtsSome Have Low Activity in
WHO Paper Assay (1 hr Exposure)
LD50
More active
topically
Suggests
pen/metab are
impacting activity
Mitigated by
formulation
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/kg
20x less toxic than propoxur
Filed a use patent for vector control
Compare to propoxur:
Mouse oral LD50
24 mg/kg
h/Ag AChE IC50 ratio
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
Entry rate Exit rate Immedia te
Treatme nt
(%)
(%)
Mortality (%)
Untreated
29 ± 1.1
0.0
0.5 ± 0.2
bed net
PRC 331 at
111mg/m 2
32 ± 1.0
0.0
76.66 ± 3.07
Overall
Mortality (%)
2.71 ± 0.7
98.6 ± 0.7
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.
Persistence of Toxicity
100
% mosquitoes dead
90
80
70
60
50
40
30
20
10
0
Days post-treatment
Control
PRC331-Hut 1
PRC331-Hut 2
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 Tech
Financial Support
by FNIH
Molsoft
ICIPE