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
Benzothiazinones in vivo ontdekking
- Go for mutated bacteria that show less infection. These mutated ones had DprE2 mutant
» - Compound with target DprE
- Add compound»_space; Lesser bacteria present
DprE function:
- Involved in biosynthesis of D-arabinose
- Essential for transformation to DPA
- No DprE: Whole outer membrane isn’t formed
Pre- discovery: normally
Based on their disease focus, companise and scientist work to understand disease
Drug discovery: normally
Researchers select a target , such as a gene or protein then search for a molecule or compound that may act on the target to alter the disease
Pre clinical testing normally
Early safety and efficacy tests are undertaken in computational models , cells and animals
Clinical trial phase one normally
The candidate medicine tested on healthy volunteers.
Clinical trial phase two normally
Researches evaluate the candidate medicines effecicacy in patients with disease
phase 3 clinical trial normally
Study in 1000 to 5000 patients to see safety , efficacy , overall benefit-risk relation of medicine
Licensing approval normally
Information and results from all studies combined and submitted to regulatory agencies.
Forward pharmacology
- Compound discovered
- Assay for biological activity
- Determine mechanism
Process:
- Cell or physiologically directed
- Unbiased as to the compounds mechanism of action
- Must determine the mechanism of action often using in vitro methods
Reverse pharmacology
- Isolate a therapeutic target
- Identify a compound that affects the target
- Modify drug to maximize effects
- Demonstrate the desired biological function in vivo
Process:
- Moleculary target directed
- Compound has demonstrated in vitro activity
- Must demonstrate in vivo activity
- Must demonstrate the compound acts by the proposed mechanism of action
