Inhibitor screening using infection models in vivo Flashcards
Drug discovery process
5 steps
Normally
- Discovery & Screening:
HTS & Target validation.
Target screen and in vitro screen pathogens. - Lead optimization:
Combinational chemistry/ Structure based design.
Pre-clinical in vivo screen. - ADMET: Adsorption, Distribution, Metabolism, Excretion, Toxicity studies
- Clinical trials
- NDA approved
In vitro screen is in Drug discovery and pre-clinical.
For in vivo studies: Model should look like:
Able to screen libraries
- High specimen number = good sample for examination
- Small animals
- Easy read out (colourisation etc)
Danio reria as a model:
10 characteristics
- Embyro develop externally
- Small animal
- Vertebrate
- Cheap maintenance
- Production of large clutches of eggs
- Genetic screening possible
- Embryo models = translucent
- Follow development in vivo
- Development in 24 hours
- Fish embryos pose minor ethical problems
Example real time analysis
- GFP expressed on T-cells
> Screen for compounds that destroy T-cells
> Look what happens to T-cells
Forward chemical screen for leukemia modified trangenic line
- Compound LDK block formation of T-cells > Block leukemia > New medication?
- Next level look in mice
- You can do this also for infection diseases
Host pathogen interaction: Zebrafish for mycobacterial infection
- In blood vessel put fluorescent bacteria
- Real time analysis of the respons to infection (real time dosis response curve)
- Use zebrafish to create mycobacterium environment
- M. marinum is closely related to M. tuberculosis. (causes tuberculosis like disease in cold blood animals)
M. marinum: Grows rapid and has optimal temperature of 28-32.
Tuberculosis: Proces
4 stages
- Persist and replicate in MQ
- Granuloma formation: Environment completely different from other bacteria.
- Change metabolism
- Dormancy
Granuloma:
2 characteristics
- Bacteria in MQ
- Surrounded by B-/T-cells
- Use zebrafish to create same environment
Need new Tuberculose drugs
4 reasons
- MDR en XDR en TDR resistant strains of tuberculosis
- Treatment during HIV co-infections
- Useless prophylaxis for contact persons, but epidemic setting in Africa etc.
- Need to shortening treatment length
Known drugs for tuberculosis:
- Isoniazid:: Test method: Infect embryo’s for 28 hours > Start treatment 1 day after infection. (Start later, drugs must be better)
- Rifampin
- Ethambutol
- Pyrazinamide
Don’t start infection to early
- Bacteria will overgrow the fish
- It’s to small to inject
- Doens’t have immune system
Compound screen during one week
Day -1: Male and femlae fish kept seperate overnight
Day 0: Male and female fish are put together»_space; Eggs
Day 0: Injection with M.marinum 1-4 cell stage
Day 1: Eggs are transferred in 12 wellsh plates
Day 2: Compounds are diluted to 10 uM in 96 wellsh plate
Day 6: Analysis
Advantages in vivo screen
- Identify host targets (immune modulation)
- Test for toxicity and killing bacteria in same time
- Identify (bacterial) targets essential for charged metabolism
- Discard compounds that only work in vitro
- Identify compounds that only work in vivo
- Forword / reverse pharmacology
- Screen combinations of compounds (synergistic effect)
- Screen target in vivo
- Test developmental defects induced by compounds
- Test for immune modulating compounds
- Follow infection in real time (test concentration dosis)
- Test duration of treatment
- Try to reduce the amount of compounds that end up in clinical trials that will fail at the end (To safe costs)
Disadvantages in vivo screen
- Problems optimization (unknown target) >> So no HTS - Low blood volume - Difficult to assess PK/ PD - With fluorescent find false positives, because difficult to see
PBTZ
Modified benzothiazinones second generation:
- No developmental defects and worked very well in combi with other known drugs
- Normal BTZ: abnomal structures in embryo
