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C. elegans and the Pharmaceutical Industry

C. elegans and the Pharmaceutical Industry. Brief overview of C. elegans. 2. C. elegans as model in biomedical research. 3. C. elegans in the drug development line. 4. Examples of applications: Ion-gated channel Prozac. 1. Brief Overview of C. elegans. C.elegans A free living

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C. elegans and the Pharmaceutical Industry

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  1. C. elegans and the Pharmaceutical Industry • Brief overview of C. elegans. 2. C. elegans as model in biomedical research. 3. C. elegans in the drug development line. 4. Examples of applications: Ion-gated channel Prozac

  2. 1. Brief Overview of C. elegans

  3. C.elegans A free living 1mm nematode “SPECIFICATIONS” • ~ 50 hours life cycle (egg to egg; facultative hermaphrodite) • Ease of Culture (Grows on E.coli lawns at 20oC) • Power of Genetics (Screen for Supressor, Complementation, etc.) • Strains Can Be Frozen • 959 Cells (302 neurons; ~7000 synapses) • Complete Cell Lineage Characterized (from zygote to adult) • 100 Mb Genome, >99.9% Sequenced (6 chromosomes) • ~ 19 000 Genes (~ 5000 essential genes)

  4. The C. elegans Life Cycle

  5. Consideration Regarding the C. elegans Genome -100 Mb, 19 000 genes, ~5 000 essential. - About 70% of human genes have a clear C. elegans homolog (this includes human disease genes, of course) -Human genes can often rescue the worm mutant.

  6. 2. C. elegans as Model in Biomedical Research Validated C. elegans disease models • CNS • Depression, Psychosis • Parkinson’s, Alzheimer’s • Pain • Metabolic • Type II diabetes • Obesity • Other • Cardiovascular (arrhythmia) • Oncology • Muscle disease

  7. Conserved pathways Example: diabetes C. elegans Human Insulin Insulin, IGF1 DAF-2/insulin receptor Insulin receptor, IGF1 receptor AGE-1 PI3 Kinase DAF-18 PTEN SHIP2 PDK-1 PDK AKT-1, AKT-2 AKT/PKB DAF-16 FKHR, FKHRL1, AFX Growth, dauer bypass Growth, survival

  8. tryptophan tryptophan hydroxylase tph-1 L-AAAH cofactor synthesis cat-4 5-OH-tryptophan L-AAAD bas-1 5-OH-tryptamine (5HT) cat-1vesicular monoamine transporter 5HT receptor 5HT 5HT reuptake 5HT 5HT receptor Conserved pathways Example: serotonergic synapse

  9. C.elegans Living test tube experiments in disease relevant genetic backgrounds experiments in complex multi-cellular physiology amenable to high throughput screening in an entire animal model 3. C. elegans in the Drug Development Line. Versus cells and other model organisms

  10. d e V G e n Moving directly to genes and compounds effective in human disease and agricultural pest control

  11. d e V G e n • Uses C. elegans to: • Identify/Validate targets. • Assay development and screening of compounds. • Other Companies • - Exelixis Pharmaceuticals • - Axys Pharmaceuticals • - Cambria Biosciences • - Hoffmann-La Roche

  12. 4. Two Examples of Applications 4a. Voltage-gated channel (in vivo screening) 4b. Prozac (Mechanism of action study)

  13. 4a. C. elegans and Voltage-Gated Channels • Couple changes in membrane potential to cell behavior, including: neurotransmitter release, muscle contraction, gene expression. Many are interesting drug targets. • Several subtypes with different kinetics, pharmacology and tissue distribution.

  14. Pharyngeal Pumping Depends on Voltage-Gated Channels Mutants in Critical Voltage-Gated Channels Can Be Rescued with Corresponding Human Channel: “Humanization”

  15. Express human target in selected C. elegans physiology • Obtain quantitative phenotypic effect dependent on activity of human gene • Assay development into robust high throughput screen • Screen chemical library • Identify on target hits In vivo Screening for Ion Channel Modulators

  16. Humanized Worms (Mutant worms rescued with human gene can pump) (Mutant worms with non-functional pharynx would not uptake dye) human channel Ab staining dye-loaded intestine

  17. 100 pumping pumping Pumping µM 0.01 0.1 1 10 100 “Humanized” Worms to Screen for Inhibitors of a Human Voltage-Gated Channel Screen for worms that don’t load with dyes using worm sorter. (Amount of dye in gut correlates with drinking rate)

  18. Cell Sorting of Worms (Furlong et al (2001) Nature Biotech. 19:153-156) Can also be used to measure fluorescence in individual worms. Can be automated to sample 96-well plates.

  19. Fluorescence Measurement/Sorting of Worms Mixed Sorted GFP+ Worms • Some Uses: • Screen for drugs that inhibit voltage-gated channels (sample 96-well plates with test worms + compound). • Screen for drugs that turn on a target promoter. (sample 96-well plates with worms with GFP under target promoter + compound) 3) Screen for mutants able to pick up dyes.

  20. 4b. C. elegans and Prozac • Also known as fluoxetine. • One of the best-selling drug (2.5 B$ in 2000). • Acts as a selective serotonin reuptake inhibitor (SSRI) • Low levels of serotonin in brain = depression? • But Prozac acts immediately as SSRI, yet the mood takes weeks to improve. • Also, some side effects are not due to SSRI activity. • Could Prozac have other targets and effects?

  21. Identify a Compound-Induced Phenotype • Do Random Mutagenesis • Identify and molecularly clone mutant resistant to compound • Identify human homologue Prozac: What Genes Mediate its Side-Effects?

  22. High Concentrations of Prozac Cause Acute Response in C. elegans 1mg/ml Prozac, 20min Control - ”Nose” hypercontraction - Paralysis - Sudden egg-laying

  23. The Logic of this Paper Fluoxetine-Resistant Mutants in C. elegans Define a Novel Family of Transmembrane Proteins Choy, R. K. M. and Thomas, J. H. Molec. Cell 4:143-152 1. Nose-contraction in response to Prozac doesn’t depend on serotonin. 2. Isolating C. elegans mutants resistant to the non-serotonin effects of Prozac and cloning the genes mutated could elucidate the non-SSRI effects of Prozac (side-effects, long-term effects). 3. This could provide new pharmacological targets.

  24. Screen for Mutants Resistant to Nose-Contraction by Prozac 0.5% EMS, 4hrs Recover many L4s F1 animals are m/+ 25% of F2s are m/m for each mutation. Incubate F2s 20 min in 1mg/ml Prozac. Pick animals with no nose contraction.

  25. Nose Resistant to Fluoxetine mutants (15 mutants found, affecting 7 genes) Note: none have obvious neuromuscular defects

  26. Cloning C. elegans Mutants Map the gene genetically. Align genetic and physical maps. Clone by rescue using candidate DNA region.

  27. NRF-6 and NDG-4 form a Novel Transmembrane Family (GM06434 is from Drosophila)

  28. Dendogram Hydrophobicity Profiles Note: worms have lots of members in this new protein family

  29. Blast Search: No Vertebrate Homolog of Nrf-6 There seems to be no vertebrate homolog...

  30. Study Guide • You should know: • The key features of C. elegans • Life cycle • Size, genome, cell number, etc. • How to use C. elegans to screen for compounds that modulate voltage-gated channels. • Why humanize? • Why use a cell sorter? • How to use C. elegans to identify genes through which a drug acts (e.g. Prozac).

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