Development of Broad Spectrum Insecticides Using a Rational Design Approach

And Vector Genomic Data

Photos Courtesy of Tim Gathany, CDC Photoservices

~ 150 gene models

~ 5-10 genes

~ 5 genes

2-4 genesPrimary Screen• Assay Development• Preliminary Screening (~1-5K compounds)• Identification of “lead chemistries”

Target Validation• Gene Expression Profiling• Analysis of knock-down phenotype• Cell-based assays

Future Work• High-throughput screening (~100-300K

compounds)• In Silico Screening• Novel Vector Genome Sequencing

Target Identification • Genome Mining • Selection of genes with high “therapeutic index”

(TI)

1-3 prioritized targets

Pipeline

Target IdentificationGenome

Mining • Vector Genomes

Anopheles gambaieAedes aegyptiCulex pipiensIxodes scapularisPhelbotomus papatasiLutzomyia longipalpis

• High Priority Targets (Druggable)

G protein-coupled receptors (GPCRs)Ion Channels

• Multi-gene Family• Essential Functions• Highly Specific

GaGb

GgGDPGTP

Adenylyl CyclaseATP

cAMP

GPCRs• ~27% Current Drugs target

GPCRs• Amenable to Screening

Ca++

Ion Channels• Current Insecticide Targets

• Voltage Gated/Ligand Gated• Multi-Component/Multi-Protein• High-throughput

Electrophysiology

Target IdentificationGenome

Mining • Vector Genomes

Anopheles gambaieAedes aegyptiCulex pipiensIxodes scapularisPhelbotomus papatasiLutzomyia longipalpis

• High Priority Targets (Drugable)G protein-coupled receptors (GPCRs)Ion Channels

• BioinformaticsBlast-based Homology SearchesExisting Algorithms (QFC)Novel Algorithms Computational Modeling

Selection of Genes with High “Therapeutic Index”

~ 150 gene models

~ 5-10 genes

~ 5 genes

2-4 genesPrimary Screen• Assay Development• Preliminary Screening (~1-5K compounds)• Identification of “lead chemistries”

Target Validation• Gene Expression Profiling• Analysis of knock-down phenotype• Cell-based assays

Future Work• High-throughput screening (~100-300K

compounds)• In Silico Screening• Novel Vector Genome Sequencing

Target Identification • Genome Mining • Selection of genes with high “therapeutic index”

(TI)

1-3 prioritized targets

Pipeline

Target ValidationGene Expression Profiling (RT-

PCR) • Vector Species

Broad Spectrum Vector Specific

• Developmental Stages Adult

• Tissue Expression

Synganglion

“Loss of Function” Screening • RNAi – knock down

Adults (Cell lines) Lethal Effect or Paralysis

• FlyRNAi DatabaseLethal Phenotypes

“Gain of Function”

????

Cell-based Assays

• Expression• Functional

~ 150 gene models

~ 5-10 genes

~ 5 genes

2-4 genesPrimary Screen• Assay Development• Preliminary Screening (~1-5K compounds)• Identification of “lead chemistries”

Target Validation• Gene Expression Profiling• Analysis of knock-down phenotype• Cell-based assays

Future Work• High-throughput screening (~100-300K

compounds)• In Silico Screening• Novel Vector Genome Sequencing

Target Identification • Genome Mining • Selection of genes with high “therapeutic index”

(TI)

1-3 prioritized targets

Pipeline

Primary ScreenHeterologous Expression

Systems • Traditional Cloning/synthesis

• Mammalian, Insect, Yeast• Commericial & “Home Grown” vectors• Ca2+/cAMP

Libraries • LOPAC1280

• TimTec• Maybridge• Notre Dame-Miller Group• Industry

~ 150 gene models

~ 5-10 genes

~ 5 genes

2-4 genesPrimary Screen• Assay Development• Preliminary Screening (~1-5K compounds)• Identification of “lead chemistries”

Target Validation• Gene Expression Profiling• Analysis of knock-down phenotype• Cell-based assays

Future Work• High-throughput screening (~100-300K

compounds)• In Silico Screening• Novel Vector Genome Sequencing

Target Identification • Genome Mining • Selection of genes with high “therapeutic index”

(TI)

1-3 prioritized targets

Pipeline

Prioritizing Targets:Tier 1 & Tier 2 Arthropod GPCRs

GPCR Receptor

Family

Predicted Receptor

Class

Name/Number Receptors Identified in GenomeAnopheles gambiae1

Aedes aegypti2 Ixodes scapularis3

Tier 1 – Immediate ScreeningDopamine Biogenic

Aminedop1, dop2, dop3 dop1, dop2, dop3 dop1, dop2, dop3

Muscarinic acetylcholine

“ “ mAcR1, mAcR2, mAcR3

mAcR1, mAcR2, mAcR3

mAcR1, mAcR2, mAcR3

Octopamine “ “ oar1, oar2, oar3, oar4

oar1, oar2, oar3, oar4

oar1, oar2, oar3, oar4

Serotonin “ “ 5ht1, 5ht2, 5ht3, 5ht4, 5ht5

5ht1, 5ht2, 5ht3, 5ht4, 5ht5

5ht1, 5ht2, 5ht3, 5ht4, 5ht5

Tier 2 – Functional AnalysisHistamine Biogenic

aminehis1 his1 his1

Calcitonin Peptide cal1, cal2, cal3 cal1, cal2, cal3 cal1, cal2, cal3

SIFamide “ “ sif1, sif2 sif1, sif2 sif1, sif2Opioid “ “ opr1 opr1 opr1Metabotropic glutamate

Amino acid mgl1, mgl2, mgl3, mgl4,mgl5

mgl1, mgl2, mgl3, mgl4, mgl5

mgl1, mgl2, mgl3, mgl4, mgl5

GABAB “ “ gbb1, gbb2, gbb3 gbb1, gbb2, gbb3 gbb1, gbb2, gbb3Latrophilin Neurotoxin cir1 cir1 cir1

[1. Hill et al., 2002; 2. Nene et al., 2007; 3. Hill et al., unpublished]

Exon 1 (315 bp) Exon 2 (129 bp) Exon 3 (258 bp) Exon 4 (537 bp)36628 bp 133823 bp 86933 bp

EST

Exon 1 (1255 bp) Exon 2 (84 bp) Exon 3 ( 71 bp)54361 bp 482 bp 86303 bp

EST

Contig 1 Contig 2

EST

1552 bp37873 bp

18033 bp 13023 bp 118 bpExon 1 (109 bp) Exon 2 (252 bp) Exon 3 (223 bp) Exon 4 (86 bp) Exon 5 (200 bp) Exon 6 (143 bp) Exon 8 (211 bp)Exon 7 (108 bp)

Intron30527 bp

Putative first exon, found using two 7TM regions from trace file

GPRd

op1

GPRd

op3

Identification of Aedes Dopamine Receptors

Initiation methionine Stop codon 7TM region

GPRd

op2

Dop1 Dop2 Dop3

2kb

1kb

0.3kb

Detection of Aedes GPRdop transcripts

splice variants

Target Validation

• Jason Meyer

• Cate Hill

Acknowledgements

Cate Hill Charles Buck

Val WattsLarisa

Avramova

Ed RowtonDept. of

Entomology

Mary Ann McDowell

Frank CollinsJesus IsaguirreScott EmrichMarv Miller

FundingIndiana 21C Res. Technol.

FundPurdue Disc. Park Seed

GrantTATRC/DoD

Nialah Orr

Special ThanksGabriella ZollnerKendra Lawrence

Russ Coleman

Aim 2: Target Validation (Developing Strength)

RT-PCR• Confirm annotation & transcripts (tissues/stages)• Guide cloning efforts• HTP proteomics?

RNAi• Only approach to investigate phenotype• Many limitations

Biochemical Assay

Dop1 Dop2 Dop3

2kb

1kb

0.3kb

Detection of Aedes GPRdop transcripts

splice variants

F, forskolin; D, dopamine; S, serotonin; O, octopamine; T, tyramine