INHIBITORS

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INHIBITORS

• What are some examples?
• What experiments show they are SPECIFIC!!!
• Do they always work?

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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.
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Sources of inhibitorsNatureSyntheticImmunologi
cal1. Give examples2. Describe how work3.
Controls?
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ANTISENSE
WHAT IS?

• Oligos or RNA

AAAAAAA
translation
AAAAAAA
No translation
antisense
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• 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

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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.

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• 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

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RNAi

• What is it??
• Double stranded RNA interference (good review
  Greg Hannon, Nature, July 11 2002)

DICER (RNASE III enzyme cuts dsRNA)
RISC (nucleaserecognize and destroys target
mRNAs.)
AAAAAA
21-23bp
Target mRNA with homology to dsRNA.
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• 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)

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• 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

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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.
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• Natural Occurring
• Diptheria Toxin
• BoTox
• Rifampicin

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CREATIVE and UNIQUE APPROACHES To INHIBITORS
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• ANTIBODIES AS INHIBITORS

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• MUTANTS VS INHIBITORS
• -Temporal activation.
• -availability

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• 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 dont like because of IV administration
  avenues. Look for small organics)