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

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4a. Voltage-gated channel(in vivo screening)

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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

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• 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).