INHIBITORS

What are some examples?

What experiments show they are SPECIFIC!!!
Do they always work?
 INHIBITORS
Used to diminish or abolish a
biological activity so that
conclusions can be drawn about
the properties of that activities or
of a process by which the activity
may play a role.
Sources of inhibitors:
Nature
Synthetic
Immunological

1. Give examples
2. Describe how work
3. Controls?
 ANTISENSE
WHAT IS?
Oligos or RNA
AAAAAAA translation

AAAAAAA No translation
antisense
SPECIFICITY!!
1. Control RNA (oligo) …. Same effect? Different effect?
2. Specific Control RNA (oligo).. Point mutant, missense,
3. Antisense from different area of RNA…. Same effect? Different
Effect?
4. Is the resulting phenotype specific for the transcript of interest or is it
generally inhibiting transcription (I.e. interferon response)
5. RESCUE
 MORPHOLINOS
WHAT IS?
Modified Oligonucleotides - either adenine,
cytosine, guanine and thymine liked to a 6
membered morpholine ring. These bases then
joined by a non-ionic phosphorodiamidate to
create the morpholino.
DESIGN: Make complementary to ATG start or 5
prime non-translated.
HOW Works:
1. Has almost as good binding affinity as native
RNA.
2. Resistant to nucleases.
3. Hinders translation.
SPECIFICITY!!
1. Control morpholino …. Same effect? Different effect?
2. Specific Control morpholino…. Point mutant, missense,
3. Do more than one morpholino give the same phenotype?
4. Antisense from different area of RNA…. Same effect? Different
Effect?
5. Is the resulting phenotype specific for the transcript of interest or is it
generally inhibiting transcription (I.e. interferon response)
6. Rescue
 RNAi
What is it??
Double stranded RNA interference (good review:
Greg Hannon, Nature, July 11 2002)
dsRNA

dsRNA DICER (RNASE III enzyme… cuts dsRNA)

RISC (nuclease…recognize and destroys target mRNAs.)
AAAAAA
21-23bp Target mRNA with
homology to dsRNA.
Shown to have multiple modes of action:
-Posttranscriptional.
-Methylation differences shown on target sites
in plants.
-Endogenous inducers of RNAi machinery
operate at level of protein synthesis (lin4)
(control expression of endogenous genes)…
SPECIFICITY!!
1. Control (luciferase, other mRNA)…. Same effect? Different effect?
2. Specific Control …. Point mutant, missense,
3. Do more than one region of the mRNA give the same phenotype?
4. Is the resulting phenotype specific for the transcript of interest or is it
generally inhibiting transcription (I.e. interferon response)… Check
multiple transcripts, Proteins, etc…
5. RESCUE
 CHEMICAL INHIBITORS
specific for particular molecule or
function
- wortmanin(fits perfectly into PI3Kinases ATP pocket)
- inhibitors of cell cycle
-cyclosporin
-aspirin/ibuprofen(many drugs used
in medicine are inhibitors!)
The How is specific for each
molecule….
Natural Occurring:
Diptheria Toxin
BoTox
Rifampicin
CREATIVE and UNIQUE
APPROACHES To
INHIBITORS
ANTIBODIES AS
INHIBITORS
MUTANTS VS INHIBITORS:
-Temporal activation.
-availability
Pharmaceutical Industry is based on inhibitors
- most that act outside cell inhibit Gcoupled
receptors
- Most that act inside of cells affect enzymes
- Hard to find inhibitors for growth factor receptors,
which could be used for anti-cancer agents.
- (Not too hard to find peptides that inhibit, but
companies don’t like because of IV administration
avenues…. Look for small organics)