C. elegans and the Pharmaceutical Industry 1.Brief overview of C. elegans. 2. C. elegans as model in...
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Transcript of C. elegans and the Pharmaceutical Industry 1.Brief overview of C. elegans. 2. C. elegans as model in...
C. elegans and the
Pharmaceutical Industry
1. 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 channelProzac
1. Brief Overview of C. elegans
“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)
C.elegansA free living
1mm nematode
The C. elegans Life Cycle
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.
Validated C. elegans disease models
• CNS• Depression, Psychosis• Parkinson’s, Alzheimer’s• Pain
• Metabolic • Type II diabetes• Obesity
• Other• Cardiovascular (arrhythmia)• Oncology• Muscle disease
2. C. elegans as Model in Biomedical Research
Conserved pathwaysExample: diabetes
DAF-18
Growth, dauer bypass
C. elegans
Insulin
DAF-2/insulin receptor
AGE-1
AKT-1, AKT-2
DAF-16
PTEN
Growth, survival
Human
Insulin, IGF1
Insulin receptor, IGF1 receptor
PI3 Kinase
AKT/PKB
FKHR, FKHRL1, AFX
PDK-1 PDKSHIP2
Conserved pathwaysExample: serotonergic synapse
5HT receptor
5HT receptor
5HTreuptake
5HT
5HT
tryptophan
5-OH-tryptophan
5-OH-tryptamine (5HT)
tryptophan hydroxylase tph-1L-AAAH cofactor synthesis cat-4
L-AAAD bas-1
cat-1 vesicularmonoamine transporter
Versus cells and other model organisms
experiments in disease relevant genetic backgrounds
experiments in complex multi-cellular physiology
amenable to high throughput screening in an entire animal model
C.elegansLiving test tube
3. C. elegans in the Drug Development Line.
d e V G e n
Moving directly to genes and compoundseffective in human disease and agricultural pest control
Uses C. elegans to:
-Identify/Validate targets.-Assay development and screening of compounds.
Other Companies- Exelixis Pharmaceuticals- Axys Pharmaceuticals- Cambria Biosciences- Hoffmann-La Roche
d e V G e n
4. Two Examples of Applications
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
4a. Voltage-gated channel(in vivo screening)
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
4b. Prozac(Mechanism of action study)
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.
Pharyngeal Pumping Depends on Voltage-Gated Channels
Mutants in Critical Voltage-Gated Channels Can BeRescued with Corresponding Human Channel: “Humanization”
In vivo Screening for Ion Channel Modulators
Express human target in selected C. elegans physiology
Assay development into robust high throughput screen
Obtain quantitative phenotypic effect dependent on activity of human gene
Screen chemical library
Identify on target hits
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
pumpingpumping
0.01 0.1 1 10 100
Pum
pin
g100
µM
Screen for worms that don’t load with dyes using worm sorter.(Amount of dye in gut correlates with drinking rate)
“Humanized” Worms to Screen for Inhibitors of a Human Voltage-Gated Channel
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.
Mixed Sorted GFP+ Worms
Some Uses:
1) Screen for drugs that inhibit voltage-gated channels(sample 96-well plates with test worms + compound).
2) 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.
Fluorescence Measurement/Sorting of Worms
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
• 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?
4b. C. elegans and Prozac
Prozac: What Genes Mediate its Side-Effects?
Identify a Compound-Induced Phenotype
Identify and molecularly clone mutant resistant to compound
Do Random Mutagenesis
Identify human homologue
Control 1mg/ml Prozac, 20min
High Concentrations of Prozac CauseAcute Response in C. elegans
- ”Nose” hypercontraction- Paralysis
- Sudden egg-laying
The Logic of this PaperFluoxetine-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’tdepend 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.
Screen for Mutants Resistant to Nose-Contraction by Prozac
0.5% EMS, 4hrsRecover 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.
Nose Resistant to Fluoxetine mutants(15 mutants found, affecting 7 genes)
Note: none have obvious neuromuscular defects
Cloning C. elegans Mutants
1. Map the gene genetically.2. Align genetic and physical maps.
3. Clone by rescue using candidate DNA region.
NRF-6 and NDG-4 form a Novel Transmembrane Family
(GM06434 is from Drosophila)
Hydrophobicity Profiles Dendogram
Note: worms have lots of members in this new protein family
Blast Search: No Vertebrate Homolog of Nrf-6
There seems to be no
vertebrate homolog...
Study Guide
You should know:
1. The key features of C. elegans• Life cycle• Size, genome, cell number, etc.
2. How to use C. elegans to screen for compounds that modulate voltage-gated channels.• Why humanize?• Why use a cell sorter?
3. How to use C. elegans to identify genes through which a drug acts (e.g. Prozac).