Transcript Document

C. elegans
and the
Pharmaceutical Industry
1.
2.
3.
4.
Brief overview of C. elegans.
C. elegans as model in biomedical research.
C. elegans in the drug development line.
Examples of applications:
Ion-gated channel
Prozac
1. Brief Overview of C. elegans
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)
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.
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
Conserved pathways
Example: diabetes
C. elegans
Human
Insulin
Insulin, IGF1
DAF-2/insulin receptor
Insulin receptor, IGF1 receptor
AGE-1
PDK-1
PI3 Kinase
PTEN
SHIP2
PDK
AKT-1, AKT-2
AKT/PKB
DAF-16
FKHR, FKHRL1, AFX
Growth, dauer bypass
Growth, survival
DAF-18
Conserved pathways
Example: serotonergic synapse
tryptophan
tph-1
L-AAAH cofactor synthesis
cat-4
tryptophan hydroxylase
5-OH-tryptophan
L-AAAD
bas-1
5-OH-tryptamine (5HT)
cat-1
5HT
5HT
reuptake
vesicular
monoamine transporter
5HT
5HT receptor
5HT receptor
3. C. elegans in the Drug Development Line.
Versus cells and other model organisms
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
deVGen
Moving directly to genes and compounds
effective in human disease and agricultural pest control
deVGen
Uses C. elegans to:
-Identify/Validate targets.
-Assay development and
screening of compounds.
Other Companies
- Exelixis Pharmaceuticals
- Axys Pharmaceuticals
- Cambria Biosciences
- Hoffmann-La Roche
4. Two Examples of Applications
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
4a. Voltage-gated channel
(in vivo screening)
QuickTime™ and a
TIFF (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
Quic kT ime™ and a YUV420 codec decompress or are needed to s ee this pi cture.
Mutants in Critical Voltage-Gated Channels Can Be
Rescued with Corresponding Human Channel: “Humanization”
In vivo Screening for Ion Channel Modulators
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
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
“Humanized” Worms to Screen for
Inhibitors of a Human Voltage-Gated Channel
100
Pumping
pumping
0.01
0.1
1
10
100
µM
Screen for worms that don’t load with dyes using worm sorter.
(Amount of dye in gut correlates with drinking rate)
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.
Fluorescence Measurement/Sorting of Worms
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.
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?
QuickTi me™ and a
T IFF (Uncompressed) decompressor
are needed to see thi s pi cture.
• 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?
Prozac: What Genes Mediate its Side-Effects?
Identify a Compound-Induced
Phenotype
Do Random Mutagenesis
Identify and molecularly clone
mutant resistant to compound
Identify human homologue
High Concentrations of Prozac Cause
Acute Response in C. elegans
Control
1mg/ml Prozac, 20min
- ”Nose” hypercontraction
- Paralysis
- Sudden egg-laying
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.
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.
Nose Resistant to Fluoxetine mutants
(15 mutants found, affecting 7 genes)
Note: none have obvious neuromuscular defects
Cloning C. elegans Mutants
3.
1. Map the gene genetically.
2. Align genetic and physical maps.
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).