INHIBITORS by alicejenny

VIEWS: 9 PAGES: 16

									        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)

								
To top