Development of Broad Spectrum Insecticides Using a Rational Design Approach

1. Development of Broad Spectrum Insecticides Using a Rational Design Approach And Vector Genomic Data Photos Courtesy of Tim Gathany, CDC Photoservices

2. Pipeline Target Identification • Genome Mining • Selection of genes with high “therapeutic index” (TI) ~ 150 gene models • Target Validation • Gene Expression Profiling • Analysis of knock-down phenotype • Cell-based assays ~ 5-10 genes ~ 5 genes • Primary Screen • Assay Development • Preliminary Screening (~1-5K compounds) • Identification of “lead chemistries” 2-4 genes 1-3 prioritized targets • Future Work • High-throughput screening (~100-300K compounds) • In Silico Screening • Novel Vector Genome Sequencing

3. Target Identification Genome Mining • Vector Genomes Anopheles gambaie Aedesaegypti Culexpipiens Ixodesscapularis Phelbotomuspapatasi Lutzomyialongipalpis • High Priority Targets (Druggable) G protein-coupled receptors (GPCRs) Ion Channels • Multi-gene Family • Essential Functions • Highly Specific

4. GPCRs • ~27% Current Drugs target GPCRs • Amenable to Screening ATP Adenylyl Cyclase GDP GTP cAMP Gb Ga Gg Ca++

5. Ion Channels • Current Insecticide Targets • Voltage Gated/Ligand Gated • Multi-Component/Multi-Protein • High-throughput Electrophysiology

6. Target Identification Genome Mining • Vector Genomes Anopheles gambaie Aedesaegypti Culexpipiens Ixodesscapularis Phelbotomuspapatasi Lutzomyialongipalpis • High Priority Targets (Drugable) G protein-coupled receptors (GPCRs) Ion Channels • Bioinformatics Blast-based Homology Searches Existing Algorithms (QFC) Novel Algorithms Computational Modeling Selection of Genes with High “Therapeutic Index”

7. Pipeline Target Identification • Genome Mining • Selection of genes with high “therapeutic index” (TI) ~ 150 gene models • Target Validation • Gene Expression Profiling • Analysis of knock-down phenotype • Cell-based assays ~ 5-10 genes ~ 5 genes • Primary Screen • Assay Development • Preliminary Screening (~1-5K compounds) • Identification of “lead chemistries” 2-4 genes 1-3 prioritized targets • Future Work • High-throughput screening (~100-300K compounds) • In Silico Screening • Novel Vector Genome Sequencing

8. Target Validation Gene Expression Profiling (RT-PCR) • Vector Species Broad Spectrum Vector Specific • Developmental Stages Adult Cell-based Assays • Expression • Functional • Tissue Expression Synganglion “Loss of Function” Screening “Gain of Function” ???? • RNAi – knock down Adults (Cell lines) Lethal Effect or Paralysis • FlyRNAi Database Lethal Phenotypes

9. Pipeline Target Identification • Genome Mining • Selection of genes with high “therapeutic index” (TI) ~ 150 gene models • Target Validation • Gene Expression Profiling • Analysis of knock-down phenotype • Cell-based assays ~ 5-10 genes ~ 5 genes • Primary Screen • Assay Development • Preliminary Screening (~1-5K compounds) • Identification of “lead chemistries” 2-4 genes 1-3 prioritized targets • Future Work • High-throughput screening (~100-300K compounds) • In Silico Screening • Novel Vector Genome Sequencing

10. Primary Screen Heterologous Expression Systems • Traditional Cloning/synthesis • Mammalian, Insect, Yeast • Commericial & “Home Grown” vectors • Ca2+/cAMP Libraries • LOPAC1280 • TimTec • Maybridge • Notre Dame-Miller Group • Industry

11. Pipeline Target Identification • Genome Mining • Selection of genes with high “therapeutic index” (TI) ~ 150 gene models • Target Validation • Gene Expression Profiling • Analysis of knock-down phenotype • Cell-based assays ~ 5-10 genes ~ 5 genes • Primary Screen • Assay Development • Preliminary Screening (~1-5K compounds) • Identification of “lead chemistries” 2-4 genes 1-3 prioritized targets • Future Work • High-throughput screening (~100-300K compounds) • In Silico Screening • Novel Vector Genome Sequencing

12. Prioritizing Targets: Tier 1 & Tier 2 Arthropod GPCRs [1. Hill et al., 2002; 2. Nene et al., 2007; 3. Hill et al., unpublished]

13. EST Identification of Aedes Dopamine Receptors Exon 2 (129 bp) Exon 3 (258 bp) Exon 1 (315 bp) Exon 4 (537 bp) 36628 bp 133823 bp 86933 bp GPRdop1 Exon 1 (1255 bp) Exon 2 (84 bp) Exon 3 ( 71 bp) 86303 bp 482 bp 54361 bp GPRdop2 EST Contig 1 Contig 2 Exon 1 (109 bp) Exon 2 (252 bp) Exon 3 (223 bp) Exon 5 (200 bp) Exon 6 (143 bp) Exon 7 (108 bp) Exon 8 (211 bp) Exon 4 (86 bp) 37873 bp 1552 bp 18033 bp 13023 bp 118 bp Intron 30527 bp GPRdop3 EST Putative first exon, found using two 7TM regions from trace file Initiation methionine Stop codon 7TM region

14. Target Validation Detection of Aedes GPRdop transcripts Dop1 Dop2 Dop3 2kb splice variants 1kb 0.3kb • Jason Meyer • Cate Hill

15. Acknowledgements Mary Ann McDowell Frank Collins Jesus Isaguirre Scott Emrich Marv Miller Funding Indiana 21C Res. Technol. Fund Purdue Disc. Park Seed Grant TATRC/DoD Cate Hill Charles Buck Val Watts Larisa Avramova Special Thanks Gabriella Zollner Kendra Lawrence Russ Coleman Ed Rowton Dept. of Entomology Nialah Orr

16. Dop1 Dop2 Dop3 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 Detection of Aedes GPRdop transcripts 2kb splice variants 1kb 0.3kb F, forskolin; D, dopamine; S, serotonin; O, octopamine; T, tyramine