ASHUTOSH FULAR M-5473 (Veterinary Parasitology)

Major Credit Seminar

on

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ESTERAES:A POTENTIAL TARGET FOR DRUG

DEVELPOMENT. . . Its ro l e i n in s e c t i c i d e

r e s i s tance

Arthropod Infestation in animals

• Vector borne diseases are an increasing cause of death and suffering worldwide.

• 80% of world cattle population at risk of TTBD .(Snelson ,1975).

• Animals with an average of 40 ticks/day could lose weight equivalent to 20 kg / year.

(Frisch et al.,2000).

• In India > 2000 crore annual loss due to TTBDs (Minijauw & Macleod, 2003).

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BACKGROUND

• Bluetongue results in annual losses of approximately $3 billion due to morbidity and mortality of animals, trade embargoes, and vaccination costs

(FAO;Osburn,2007)

• In 2007, the world market for insecticides was estimated to be in the order of $11 billion US.

Grube A, et.al.(2011,USEPA)

• Effectiveness of insecticides is decreasing, with over 500 documented instances of insecticide resistance

( Whalon, ME. et.al. (2008) Global Pesticide Resistance in Arthropods ,UK).

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BACKGROUND

Current drugs available in the market: (Ref. : Insecticide Mode of Action Technical Training Manual)

BASF

VARIOUS APPROACHES FOR INSECTICIDE DEVELOPMENT:

•Enzyme (Esterases,lipases,chitinases etc).

•Receptors (ACh , NACh , receptors on skin, chloride channel receptors etc).

•Hormones (Juvenile hormone mimics etc).

CLASS DRUGS EFFECTS

Organochlorines. DDT, aldrin, Biomagnification and residual

Organophosphates diazinon, malathion, and parathion

more toxic to vertebrates than the chlorinated hydrocarbons

Carbamates. carbaryl (Sevin), propoxur (Baygon®)

insects that developed resistance to chlorinated hydrocarbons and OPs.

Synthetic Pyrethroids.

fenvalerate, permethrin, tetramethrin

fast knockdown activity against flying insects and low mammalian toxicity.

Botanicals nicotine, rotenone

Insect Growth Regulators (IGR’s)

mithoprene,diflubenzuron advantage of insect-specific physiology

Nervous System of insects:-

Nervous System of insects:-

DJS

Effects on Nerves

• Hydrolase enzyme under the International Union of Biochemistry system

• Belong to the α/β hydrolase fold protein superfamily.

• This superfamily includes proteases, lipases, esterases, dehalogenases, peroxidases and epoxide hydrolases.

(Ollis et al., 1992; Nardini & Dijkstra 1999; Hotelier et al., 2004)

What are Esterases ?

•Catabolism of esters of higher fatty acids that proceeds actively in the flight muscles and enables insects to fly.

• Recent MS results have shown αE7 is expressed in the fat body lipid droplet proteome, implying a role in lipid or cholesterol metabolism.

(Beller et al., (2006) Mol Cell Proteomics)

•Implicated in the detoxification of organophosphate(OP) insecticides. (Jackson, 2013)

Function of esterases in insects:-

•Crucial functions in insect development and behaviour, such as odorant degradation

•Functions related to reproduction and digestion. (Biology of the Insect Midgut) (Labate et al.,1990)

•Many important molecules, such as pheromones and other semiochemicals, are types of esters that are hydrolysed by esterases.

• α/β hydrolase fold is composed of six α helices and eight β-sheets in a parallel orientation.

• Fold maintains the juxtaposition of the residues of the catalytic site in the 3-D structure.

• Residues belong to a conserved triad containing a nucleophilic residue (Ser, Cys or Asp), an acidic residue (Glu or Asp) and a His residue.

ESTERASES Super Family

The nucleophil ic elbow (C), i.e., a sharp turn where the nucleophile is located is the most conserved feature of the fold

• The ESTHER database was created around 1994 by Olis et al.

• For more information visit website http://bioweb.ensam.inra.fr/esther

• It is an annotated collection of all published information related to the genes that encode proteins of this superfamily.

ESTHER database

Mechanism of esterases interaction with substrate with ester bond

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R1 O

O

R2R1 OH

O

R2OH+esterase

acid alcohol

acidpart

alcoholpart

H2OO

Also can metabolize thiols, amides, and carbamates

Esterase Activity

•Different classification schemes developed over a period of time.

•A brief description of them are as follows:-

CLASSIFICATIONS OF ESTERASES

Conventional classifications

1.Aldridge 1953 – based on the interaction of esterases with OP molecules.

2.Enzyme Commission 1978:- NC-IUBMB classifies enzymes using a series of four numbers preceded by the letters EC (Enzyme Committee).Ex.3.1.1.1- Caboxylesterases

Modern classifications methods:-

1.Enzyme Committee 1989 - NC-IUBMB in 1989 (Walker 1993).

2.Inhibition criterion:- (Oakeshott et al. ,1993).

Classification Sulphydryl reagents

OPs Carbamate eserine sulphate

Preference

Acetylesterases, Aliphatic

Arylesterases, Aromatic

Carboxylesterase Aliphatic esters

Cholinesterases, Charged substrates

Inhibitors used

3. Substrate and electrophoretic mobility criterion –

•Enzyme alleles categorised based on electrophoresis under non-denaturing conditions, (Hemingway & Karunaratne ,1998).

• Does not have systematic value because 20-30% of the existing variability is hidden within the different alleles (Thorpe 1982, 1983)

•Different esterase isoforms based on substrate specificity. Mackness et al.,(1987)

4. Phylogenetic/genomic criterion –

•Use of nucleotide sequence similarity of carboxylesterase-encoding genes as a criterion to distinguish amongst mammalian carboxylesterase isozymes . Satoh and Hosokawa (1995).

•For insects--14 clades (A-N). Based on catalytic competence and cellular localisation.

Oakeshott et al., (2005)

•Mutant enzymes that confer insecticide resistance occur in all three classes.

(metabolic insecticide resistance genes)

(metabolic insecticide resistance genes)

(target side insecticide resistance)

• In genera Anopheles and Aedes, genes from clades A and F (juvenile hormone esterase and other secreted esterases) were up-regulated in insecticide-resistant populations . (Saavedra-Rodriguez et al.,2011).

BUT……

• Despite malathion resistance in anopheles, no significant increased esterases activity were found. Hemingway (2000)

• Monitoring of insecticide resistance of Aedes aegypti populations in Brazil. Montella et al.,(2007)

BUT . . .

ESTERASES

Important group of enzymes

PROS CONS

Are

OP’s. . . As a curse to insects

OPs inhibit acetylcholinesterase(AChE) at cholinergic synapses in the central and peripheral

nervous systems, which leads to endless nerve signaltransduction and death.

Marrs TC (1993) Organophosphate poisoning. Pharmacol Ther

But as a boon to insects. . . CARBOXYESTERASES

However, α-CBEs confer a significant protective effect owing to their ability to bind and slowly hydrolyze OPs.

Birner-Gruenberger R, et al. ,(2012) Insect Biochem Mol Biol

•Derived from metabolic detoxification can develop in natural populations in three different ways:

(Hemingway et al.,1998).

1.Gene amplification.

detected in both Diptera and Hemiptera (Bass & Field 2011).

INSECTICIDE RESISTANCE -As a curse to us

2.The up-regulation of gene expression detected in the orders Hemiptera, Hymenoptera, Lepidoptera and Diptera ` (Li et al., 2007, Bass & Field ,2011).

3.Genetic mutations in coding regions:-

• Detected in Diptera, Hymenoptera and Lepidoptera (Li et al.,2007, Hotelier et al.,2010).

• A single amino acid substitution converts a carboxylesterase to an organophosphorus hydrolase in the blowfly Lucilia cuprina. (Newcomb et al., (1997)

Despite the importance of OP’s and the spread of insect-borne diseases,

the molecular basis for the ability of -αcarboxylesterases to confer OP resistance to

insects is poorly understood.

REF: Jacksona,(2013). Structure and function of an insect α-carboxylesterase associated with insecticide resistance, PNAS

Immense losses due to insects globally.

Nervous system targetted insecticides.

Esterases are essence of insects and so important target for insecticide development.

Almost all drugs have ester bonds.

Insecticide resistance monitoring programs are of critical importance if chemical controls continue to be used as a part of integrated insect management strategies.

CONCLUSIONS

FUTURE PERSPECTIVES FOR DRUG DEVELOPMENT:-

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REFERENCES

• Aharoni A, Gaidukov L, Khersonsky O, Gold SM, Roodveldt C, Tawfik DS 2005. The ‘evolvability’ of promiscuous protein functions. Nat Genet 37: 73-76.

• Aldridge WN 1953a. Serum esterases. I. Two types of esterase (A and B) hydrolysing p-nitrophenyl acetate, propionate and butyrate and a method for their determination. Biochem J 53: 110-117.

• Hemingway J, Ranson H 2000. Insecticide resistance in insect vectors of human disease. Annu Rev Entomol 45: 371-391.

• Liu N, Xu Q, Li T, He L, Zhang L 2009. Permethrin resistance and target site insensitivity in the mosquito Culex quinquefasciatus in Alabama. J Med Entomol 46: 1424-1429

• Montella ,I R , Schama, R, Valle, D(2012).The classification of esterases: an important gene family involved in insecticide resistance - A Review; Mem Inst Oswaldo Cruz, Vol. 107(4): 437-449.

• Oakeshott JG, Horne I, Sutherland TD, Russel RJ 2003. The genomic of insecticide resistance. Genome Biol 4: 202

• Yan,S , Cui,F and Qiao,C (2009) ; Structure, Function and Applications of Carboxylesterases from Insects for Insecticide Resistance. Protein & Peptide Letters

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