Search for mosquito controlling compounds DWFP-

related work

Kumudini M. Meepagala

USDA-ARS-NPURU

Oxford MS

Search for selective AChE inhibitors against mosquitoes

Role of acetylcholinesterase

Synthesis of acetylcholine

Search for selective AChE inhibitors as adulticides and repellents

DEET as a neurotoxin- AChE inhibitor

Not target specific

Need a replacement

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)

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

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.”

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.”

Extracts tested for AChE inhibitory activity

Sample Name [mg/ml] Active Test 1 Active Test 2

AT05 27-1 AT05 EtoAc Leaves (Pure cpd) 1.0 No --

AT05 24 AT05 EtoAc Leaves (Pure cpd) 2.0 Yes Yes

AT05 27-2 AT05 EtoAc Leaves (Pure cpd) 1.0 Maybe No

P. trifoliata fruit Hex. Ext 5.0 Yes Maybe

E. runyonii leaf/stem EtoAc Ext 5.5 Maybe No

Z. hirsutum MeOH Ext 5.0 No --

E. runyonii leaf/stem MeOH Ext 7.0 No --

A. texana Hexane Ext 6.3 Yes YES ++

P. trifoliata Fruits Hex. Ext. 5.0 Yes Maybe

P. trifoliata EtoAc ext 5.0 Maybe No

P. trifoliata Root MeOH ext 5.0 No --

P. trifoliata Root Hexane ext 5.0 No --

H.parvifolia EtoAc Ext 5.4 Maybe Yes ++

H.parvifolia MeOH Ext. 22.8 Maybe Yes++

Galanthamine standard 1.0 Yes Yes

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.

Binding of AChE inhibitors

Dosage (mg/cm²) M-3 Proportion to

Days Protection DEET

SYN 3-39 0.75 1 0.05

SYN 3-113 0.75 2 1.00

SYN 3-127 0.75 1 0.50

SYN 3-131 0.75 1 0.50

SYN 131-1 0.75 6 3.00

DEET 0.75 2 1.00

Duration of Repellency for Compounds

SYN 3-3

9

SYN 3-1

25

SYN 3-1

27

SYN 3-1

31

SYN 3-1

43

SYN 3-1

13

SYN 3-1

45

DEET

SYN 131

-1

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

Day

s P

rote

ctio

n

Compound (0.75 mg/cm2)

DEET

SYN 131

-1

SYN 3-1

13

SYN 3-1

31

SYN 3-1

27

SYN 3-1

45

SYN 3-3

9

SYN 3-1

43

SYN 3-3

7

SYN 3-1

21

SYN 119

-1

SYN 3-2

5

SYN 3-1

25

SYN 2-5

SYN 3-1

05

SYN 3-1

11

SYN 3-1

15

Aceto

ne C

ontro

l

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

ME

D (

mg/

cm2

)

Compound

An. albimanus Ae. aegypti

Averaged minimum effective dosageof compounds

AChE assay

OH

+ CH3COOH

naphthyl acetate -naphthol

H3CO

OCH3

OH

H3CO OCH3

Azo dye (purple)

HO

N N N

NN N N 2Cl-+ +

acetylcholineseterase

O

O

CH3

N

Fast blue Salt B

TLC assay for AChE activity

OMe

OMe

NH

H

H

O

OMe

OMe

H

H

NH

O

N

N

AT-05-27-2

AT-05-24

O

NH

O

HO

CH3

HOCH3

CH3

OH

AT-05-27-1

Isolated from A. texana

Acid F OMe+ CH2OH CH2O G

Syn131-1More active and longer lasting than DEETIs the mode of action the same as DEET?No nitrogen groupCan bind to a Cys group in AChECan interact with serine group in the catalytic site

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.

Piperine 3.8 ± 4.8 CD

pelliotorin 92.5 ± 15.0 A

PIPER-syn 15 100.0 ± 0A

PIPER-syn 21 30.0 ± 26.0 BC

PIPER-syn 23 48.8 ± 20.6 B

PIPER-syn 25 33.8 ± 24.6 BC

PIPER-syn 7 36.3 ± 7.5 BC

UNTREATED 2.5 ± 5.0 D

Treatment @1%wt/wt mortality at day 21

O

O

C

O

NH

C

O

NH

Piper Syn-7

MeS

O

NH

Piper Syn-15

Piper Syn-21

O

HN

Piper Syn-23

HO

NHPiper Syn-25

C

O

NH

Cl

Piper -syn-35M. W.=210 C

O

NH

S

Piper -syn-37M.W. = 222

CH3

C

O

NH

Cl

Piper -syn-33M. W. = 254

O

O

C

O

NH

S

Piper -syn-39M. W. =266

O

O

CH3

HN

C

O

Cl

Piper-syn-41M. W. = 210

HN

C

O

S

Piper-syn-43M. W. = 222

CH3

Amides with insecticidal activity

CH3

H3C CH3

O

-turmeron

CH3

CH3

H3C CH3

O

-turmeron

O

H3C

CH3

H3C

O

CH3

HO

O

CH3

H3C

12

3

4 5

67

8

910

11

12

13

14

15

16

17

1819

20

Crispanone

Other natural products isolated with AChE inhibitory activity

Turmeric rootsCurcuma longa

Dill seedsAnethum graveolens

O

O

Br

OCH2CH3

O

PPh3

ACN or toluene

reflux 48 hr

+ Ph3P

OCH2CH3

O

Br -

30 min NaHCH2Cl2

O

OCH2CH3

1. NaOH/MeoH2. LiAlH4/dry ether3. PCC/CH2Cl2

O

O

O

HO

O

O

H

1. NaOH/MeoH2. Pd/C H23. LiAlH4/dry ether4. PCC/CH2Cl2

LDA/THF -780 C

triethyl-4-phosphonocrotonate

O

O

(CH2)5

O

OEt

O

O

(CH2)3

O

OEt

1. Hydrolysis2. Oxalylchloride3. i-butylamine or piperidine

R1

O

NH R2

O

NH

R1

R2

R1

O

N R2

O

N

57-1

57-2

Reaction scheme for synthesis of larvicides

Synthetic scheme for analogs with two carbons longer

O

O

Br (CH2)3

OCH2CH3

O

PPh3

ACN or toluene

reflux 48 hr

+ Ph3P (CH2)3

OCH2CH3

O

Br -

30 min NaHCH2Cl2

(CH2)3

O

OCH2CH3

1. NaOH/MeoH2. LiAlH4/dry ether3. PCC/CH2Cl2

O

O

(CH2)3

O

HO

O

(CH2)3

O

H

1. NaOH/MeoH2. Pd/C H23. LiAlH4/dry ether4. PCC/CH2Cl2

LDA/THF -780 C

triethyl-4-phosphonocrotonate

O

O

(CH2)8

O

OEt

O

O

(CH2)8

O

OEt

1. Hydrolysis2. Oxalylchloride3. i-butylamine or piperidine

R1

O

NH R2

O

NH

R1

R2

R1

O

N R2

O

N

No reaction

CSCo3reflux 24 hrtoluene

Larvae Assay against 1st intar larvae

0

20

40

60

80

100

120

8 ppm 2 ppm 0.5 ppm

Concentration

Mo

rtal

ity Indoxacarb

57-1

57-2

8 ppm 2 ppm 0.5 ppm

Indoxacarb 100 100 40

57-1 100 100 80

57-2 100 80 20

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.

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.