1. INHIBITORS What are some examples? What experiments show they are SPECIFIC!!! Do they always work?

2. 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.

3. Sources of inhibitors: Nature Synthetic Immunological 1. Give examples 2. Describe how work 3. Controls?

4. ANTISENSE Oligos or RNA AAAAAAAtranslation AAAAAAA antisense No translation WHAT IS?

5. 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

6. 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.

7. 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

8. RNAi What is it?? Double stranded RNA interference (good review: Greg Hannon, Nature, July 11 2002) dsRNA DICER (RNASE III enzyme… cuts dsRNA) 21-23bp RISC (nuclease…recognize and destroys target mRNAs.) AAAAAA Target mRNA with homology to dsRNA.

9. 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)…

10. 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

11. 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….

12. Natural Occurring: Diptheria Toxin BoTox Rifampicin

13. CREATIVE and UNIQUE APPROACHES To INHIBITORS

14. ANTIBODIES AS INHIBITORS

15. MUTANTS VS INHIBITORS: -Temporal activation. -availability

16. 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)