C.%20elegans

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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

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1. Brief Overview of C. elegans
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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, gt99.9 Sequenced (6
chromosomes) 19 000 Genes ( 5000 essential
genes)
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The C. elegans Life Cycle
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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.
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2. C. elegans as Model in Biomedical Research
Validated C. elegans disease models

• CNS
• Depression, Psychosis
• Parkinsons, Alzheimers
• Pain

• Metabolic
• Type II diabetes
• Obesity

• Other
• Cardiovascular (arrhythmia)
• Oncology
• Muscle disease

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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
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Conserved pathways
Example serotonergic synapse
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3. C. elegans in the Drug Development Line.
Versus cells and other model organisms
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Moving directly to genes and compounds effective
in human disease and agricultural pest control
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• Uses C. elegans to
• Identify/Validate targets.
• Assay development and screening of compounds.
• Other Companies
• - Exelixis Pharmaceuticals
• - Axys Pharmaceuticals
• - Cambria Biosciences
• - Hoffmann-La Roche

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

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Pharyngeal Pumping Depends on Voltage-Gated
Channels
Mutants in Critical Voltage-Gated Channels Can
Be Rescued with Corresponding Human Channel
Humanization
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In vivo Screening for Ion Channel Modulators
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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
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Humanized Worms to Screen for Inhibitors of a
Human Voltage-Gated Channel
Screen for worms that dont load with dyes using
worm sorter. (Amount of dye in gut correlates
with drinking rate)
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Cell Sorting of Worms (Furlong et al (2001)
Nature Biotech. 19153-156)
Can also be used to measure fluorescence in
individual worms. Can be automated to sample
96-well plates.
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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.

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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?

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Prozac What Genes Mediate its Side-Effects?
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High Concentrations of Prozac Cause Acute
Response in C. elegans
Control
1mg/ml Prozac, 20min
- Nose hypercontraction - Paralysis - Sudden
egg-laying
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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 4143-152
1. Nose-contraction in response to Prozac
doesnt 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.
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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.
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Nose Resistant to Fluoxetine mutants (15 mutants
found, affecting 7 genes)
Note none have obvious neuromuscular defects
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Cloning C. elegans Mutants

1. Map the gene genetically.
2. Align genetic and physical maps.
3. Clone by rescue using candidate DNA region.

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NRF-6 and NDG-4 form a Novel Transmembrane Family
(GM06434 is from Drosophila)
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Hydrophobicity Profiles
Dendogram
Note worms have lots of members in this new
protein family
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Blast Search No Vertebrate Homolog of Nrf-6
There seems to be no vertebrate homolog...
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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).