Lec 5- Target discovery (2)

Question 1

Genome and proteome approaches

Answer 1

Question 2

Proteomics

Answer 2

• Proteomics: The study of the complete protein complement of a cell (or sub-fracrtion thereof e.g. isolate organelle)
• Comparison of diseased vs healthy cells, infectious vs non-infectious etc, and the identification of the proteins responsible
• More informative than the geonme (Drosophila genome 15,000 genes, human genome 22,000 genes) but how many proteins
• Relies on genome information and integration of information from a wide variety of sources

Question 3

Differential proteomics

Answer 3

• Cellular level phenotypes- is it proliferating more than usual
• Induce bad sample
• Clinical- compare healthy and diseased tissue- cancer v non-cancer
• Seperate out proteins- what is different between them
• Quantify and analyse and identify differences

Question 4

2D-page

2D gel electrophoresis

Answer 4

• Separate proteins by isoelectric point- pH at which the charge of AA is neutral- (pl) in the first dimension (Isoelectric focusing on an immobilized pH gradient)
• Then separate by size in the second (12% SDS)

Answer 5

• We can us MS to identify the proteins
• Down = glycosis= making energy
• Up= these proteins protect the cell from oxygen
• Oxidative damage to proteins is linked to neurodegeneration

Question 6

Proteomics

Answer 6

• Benzamide inhibits tumour growth
• H1299 cells treated with bengamideanalogue LAF389
• Proteins extracted and separated
• Identify the proteins that change- 14-3-3 isoforms
• Relate to biochemistry- inhibits methionine aminopeptidase
  
  • Start codon contains methionine, every protein starts with methionine and is removed once started, aminopeptidase cleaves this- by inhibiting aminopeptidase we cause the production of lots of inactive proteins= cancer cell dies
• New targets for developing assays and drugs

Question 7

Chemical proteomics

Answer 7

• Many drugs have been discovered by screening but their target is often not known
• Chemical proteomics uses the drug as a bait to capture the proteins that bind to the drug
• The target and off-target proteins can be identified
• We attach the drug to a magnetic particle to access it easily
  
  • We allow the drug to bind to any targets (enzymes, proteins) that it can
  • On removal those proteins should still be bound
  • We can then identify the protein
    
    • It could be target that causes action- on target
    • Target could cause side effect (we dont want it to bind to that protein)- off target

Question 8

Chemical proteomics: examples- trapoxin

Answer 8

Trapoxin

• A fungal product identified as an anti-cancer agent found to bind to histone deacetylase (HDAC)- packing and unpackaging DNA, control expression of DNA
• Shown to be a HDAC inhibitor- led to an understanding of epigenetics
• This led directly to the development of the clinically used HDAC inhibitors suberoylanilide hydroxamic acid (SAHA) and Romidepsin (FK228) for the treatment of lymphoma

Question 9

chemical proteomics- example thalidomide

Answer 9

• Very effective sedative and treatment for morning sickness
• Withdrawn 1961 due to teratogenicity
• In 2010 the molecular basis for this was uncovered to be cereblon which is involved in the expression of growth factors
• Development of new analogues that avoid this interaction

Question 10

Induced phenotypic approaches- Take a model organism

Answer 10

• Take a model organism
• Mutate it (chemically) and look for the disease like phenotypes
• Identify the gene that is changed by sequencing the genome
• Use RNAi to ‘silence’ genes and look for the disease like phenotypes
• NB- RNAi- small double-stranded RNA that are put into cells and produce siRNA (single-stranded) that interacts with the cellular mRNA and prevent the gene product being formed

Question 11

An example from genomics

Answer 11

• Genomics allows the study of the full gene complement of a system
• The Her2 gene (ErbB, epidermal growth factor receptor family) was identified as an oncogene in a model using chemically induced rat neuroblastoma in 1984
• Found to be overexpressed in <25% of breast carcinoma
• Kohler and Milstein had discovered the use use of Ab’s in therapy in 1975- approach used to develop Herceptin
• Trastuzumab (1988) developed as therapy, approved 2005

Question 12

Technology can deliver new knowledge and understanding

Answer 12

• The general belief is that around 3% of the human genomic DNA codes for protein products
• Evidence from total transcribed genome analysis suggested that there may be a large amount (possibly as much again) DNA being transcribed as was previously predicted
• Discovery of micro-RNA
• Is it meaningful?

Question 13

Chemical genetics

Answer 13

• Nothing new- aspirin from willow bark but not until 1970 that target identified- led to new analgesics
• Rapamycin- from Streptomyces hygroscopicus in 1970s as antifungal found to inhibit mTOR
• Current methods involve screening for a compound that gives a desired phenotype
  
  • Monastrol identified as antimitotic (from >16,000 compounds)
  • Using a screen to look at distribution of H-Ras and Raf1 >73,000 compounds screened and one compound identified- MCP1

Question 14

Systems biology and systems medicine

Answer 14

• While looking at individual targets allows us to develop specific pharmaceuticals, it is common for resistance to develop or of-target effects to become apparent after time
• System biology and systems medicine aim at look at a whole biological system, and to develop functional models that allow us to predict the effects of chanhing one component e.g. targeting one protein
• Also aim to develop systems wide screens for identifying biomarker patterns that can be used in diagnosis and monitoring of treatment

Question 15

Drug fail

Answer 15

• Very expensive to fail in phase I and II clinical trials- one of the reasons why drugs are so expensive
• We can use these approaches to target discovery we can screen for much more effective drugs, as well as potentially identify why it fails to make the whole process more efficient