Molecular Design and Semi-FieldPerformance of Highly Selective

Carbamates for Control of the MalariaMosquito, Anopheles gambiae

Jeff Bloomquist

Neurotoxicology LaboratoryDept. of Entomology and Nematology

Emerging Pathogens InstituteUniversity of Floridajbquist@epi.ufl.edu

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]

Convulsionsand

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

3H

O

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 391P R C 40 7P R C 408

S 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 (118 x) 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

osqu

itoes

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