Methods and Logic 2007
Inhibitors
Examples
How they work
Specificity
Definition
Used to diminish or abolish
a biological activity so that
conclusions can be drawn
about the properties/or role
of that activity.
Types of inhibitors
“Pharmacological”
(natural or synthetic chemicals)
“Genetics”
(RNA interference)
“Immunological”
(neutralizing antibodies)
Chemical Inhibitors
Natural
-Aspirin: (willow bark) COX2
-Wortmannin: (Fungus) (PI3K)
-Cyclosporin: (Fungus) calcineurin
(immunosuppressant)
-Diphtheria Toxin (bacteria) inhibits
elongation factor (protein synthesis
-Botulinum Toxin (BoTox) (bacteria)
(SNAP25) peripheral nerve endings
Chemical Inhibitors
Designed
Kinase inhibitors
-Gleevec (Abl, c-kit, PDGFR) cancer
-Roscovitine (Cdk) polycystic kidney disease
-SB203580 (p38MAPK)
-LY294002 (PI3K)
-PD 098059 (ERK)
Phosphatase inhibitors
Methylase inhibitors
Acetylase inhibitors
Deacetylase inhibitors
Specificity
IC 50 for other related molecules
Use lowest dose possible
Verify it does not affect other pathways
Use two independent non-related types of
inhibitors (not always possible)
Antisense (single-stranded)
DNA
mRNA
Protein
Antisense
oligonucleotide
RNAse H
RNAi (double stranded)
Morpholinos (zebrafish)
Modified oligonucleotides
(25bp)
Adenine, Cytosine, Guanine
and Thymine with a
morpholine ring instead of
deoxyribose ring. Bases are
linked by phosphorodiamidate
instead of phosphate to create
the morpholino.
Complementary to ATG start, 5’UTR or splice junction
Prevents translation or splicing
RNAi-specificity
1. Blast RNAi sequence + Verify that control
RNA/proteins are not affected
2. Use more than one siRNA against the same gene :
do you get the same phenotype?
3. Control siRNA with mismatches
4. Is the resulting phenotype specific for the
transcript of interest or is it generally inhibiting a
cellular response.
•
Rescue with a form of cDNA that has silent
mutations that make it resistant to RNAi
Neutralizing
antibodies
Inhibitors versus genetics
-Temporal action (FAST!)
-Reversibility
-Availability
-Can be used in different species
-Can be used as a drug in human
Use in medicine
Cancer:
Chemicals: Gleevec (Abl, c-kit), BAY 439006 (B-Raf)
neutralizing antibodies: Avastin
(VEGF), Herceptin (HER2)
Viral infections (HIV): RNAi