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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.

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inhibitors

INHIBITORS

What are some examples?

What experiments show they are SPECIFIC!!!

Do they always work?

slide2

INHIBITORSUsed 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
Sources of inhibitors:NatureSyntheticImmunological1. Give examples2. Describe how work3. Controls?
antisense
ANTISENSE

WHAT IS?

Oligos or RNA

AAAAAAA

translation

AAAAAAA

No translation

antisense

slide5
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
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:

  • Has almost as good binding affinity as native RNA.
  • Resistant to nucleases.
  • Hinders translation.
slide7
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

slide8

dsRNA

dsRNA

RNAi

What is it??

Double stranded RNA interference (good review: Greg Hannon, Nature, July 11 2002)

DICER (RNASE III enzyme… cuts dsRNA)

RISC (nuclease…recognize and destroys target mRNAs.)

AAAAAA

21-23bp

Target mRNA with homology to dsRNA.

slide9
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)…

slide10
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

slide11

CHEMICAL INHIBITORSspecific 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….

slide12
Natural Occurring:

Diptheria Toxin

BoTox

Rifampicin

slide15
MUTANTS VS INHIBITORS:

-Temporal activation.

-availability

slide16
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)