Transcript Inhibitors
Methods and Logic 2007 Inhibitors Examples How they work Specificity Definition Used to diminish or abolish a biological activity so that conclusions can be drawn about the properties/or role of that activity. Types of inhibitors “Pharmacological” (natural or synthetic chemicals) “Genetics” (RNA interference) “Immunological” (neutralizing antibodies) Chemical Inhibitors Natural -Aspirin: (willow bark) COX2 -Wortmannin: (Fungus) (PI3K) -Cyclosporin: (Fungus) calcineurin (immunosuppressant) -Diphtheria Toxin (bacteria) inhibits elongation factor (protein synthesis -Botulinum Toxin (BoTox) (bacteria) (SNAP25) peripheral nerve endings Chemical Inhibitors Designed Kinase inhibitors -Gleevec (Abl, c-kit, PDGFR) cancer -Roscovitine (Cdk) polycystic kidney disease -SB203580 (p38MAPK) -LY294002 (PI3K) -PD 098059 (ERK) Phosphatase inhibitors Methylase inhibitors Acetylase inhibitors Deacetylase inhibitors Specificity IC 50 for other related molecules Use lowest dose possible Verify it does not affect other pathways Use two independent non-related types of inhibitors (not always possible) Antisense (single-stranded) DNA mRNA Protein Antisense oligonucleotide RNAse H RNAi (double stranded) Morpholinos (zebrafish) Modified oligonucleotides (25bp) Adenine, Cytosine, Guanine and Thymine with a morpholine ring instead of deoxyribose ring. Bases are linked by phosphorodiamidate instead of phosphate to create the morpholino. Complementary to ATG start, 5’UTR or splice junction Prevents translation or splicing RNAi-specificity 1. Blast RNAi sequence + Verify that control RNA/proteins are not affected 2. Use more than one siRNA against the same gene : do you get the same phenotype? 3. Control siRNA with mismatches 4. Is the resulting phenotype specific for the transcript of interest or is it generally inhibiting a cellular response. • Rescue with a form of cDNA that has silent mutations that make it resistant to RNAi Neutralizing antibodies Inhibitors versus genetics -Temporal action (FAST!) -Reversibility -Availability -Can be used in different species -Can be used as a drug in human Use in medicine Cancer: Chemicals: Gleevec (Abl, c-kit), BAY 439006 (B-Raf) neutralizing antibodies: Avastin (VEGF), Herceptin (HER2) Viral infections (HIV): RNAi