Bacterial screens in vitro Flashcards
(28 cards)
In vitro/ vivo screen phases
(From drug) - Potential antimycobacterial molecules in compound libaries >> (In vitro activity) Lead identification, MIC/MBC determination, Cytotoxicity >> (To target) - Target identification and validation - Preclinical development - Lead optimization, medicinal chemistry - In vivo studies
Target based drug design phases
From target:
- Identification and validation of a potential drug target
To enzyme inhibition:
- Enzyme expression and purification
- Enzyme based screening
- 3d structur determination
- Fragment based screening
To drug:
- Validation against M.tuberculosis and MIC/ MBC determination
- Preclinical development: Lead optimization, Medicinal chemistry
- In vivo studies
Improve screen of Wakksman
When known in vitro activity > Look for the target. In stead of looking for the target first
Target dogma: Target based discovery
5 dogmas
- Target should be known
- Target should be essential
- Target should be specific for bacteria
> There are several antibiotics that work on bacteria but are not specific for the bacteria - Target should be present in most bacteria
- No multiple targets
Compound dogma: Target based discovery
2 dogmas
- Should follow lipinski rules: Compounds should be in that quarter (smaller than 500 molecular weight)
- Should be from synthetic library
Unconventional tuberculoses treatment
4 reasons
- TB drugs specific for mycobacteria: Isoniazid, Bedaquiline, Ethambutol, Pyrazinamide, Ethionamide, PAS
- TB drugs, target unknown:
Pyrazinamide, PAS, Isoniazid - Multiple targets:
Isoniazid, Probably pyrazinamide - Do not obey lipinski rules:
Molecular mass should be less than 500
Ideal drug should be:
- Prodrug: Prodrug moves in bacterie > After metabolism it becomes active
- Multiple targets
- Bactericidal
Optimised form of Waksman’s screenings platform
- Compound library is optimised and use different compound libraries
- Screen is optimised
- Grow conditions
Screen is optimized:
5 ways
- More easy to see things
- Identifying suboptimal compounds
- Identify specific compounds
- Quickly avoid known (groups of antibiotics)
- Test on persistent forms
Grow Conditions optimised:
2 ways
- Grow medium important for bacteria, to grow and express virulence factors
- Mimic in vivo growth conditions in host:
Iron limitations/ nitrogen, phosphate source/ Carbon source
Compound library optimised:
Use different compounds libraries:
- Synthetic compounds
- Natural compounds (Fungi, Plant extracts, Deep sea organisms, Atinobacteria)
Actinobacteria and related species
- Best antibiotic producers: Are gram positive:
- Huge structure, uses lots of nutrients, uses itself as nutrion source. Also other bacteria use their structure as nutrion source.
- So actinobacteria, secrete antibiotics against other bacteria
- 1/50 produced by actinobacteria are usefull drugs
- But low hanging fruits are already found»_space; Find bacteria like actinobacteria that can be used for screen.
Screen optimized:
- More easy to see things
- Add growth factors (Resazurin is blue, turns pink when growth)
- Add other indicators (GFP, Luciferase (sense metabole actives that admit light))
See dead of life.
Screen optimized:
- Identifying suboptimal compounds or specific compounds (you won’t find with general screen)
- Stress signal indicators
- Specific pathway indicators (Cell enveloppe biogenesis, DNA gyrase, Compounds or specific compounds)
- Mix sensitive and resistent ones on a plate and add compounds
- Change permeability of gram negative bacteria (knock out efflux/ more membrane porins)
Screen optimized:
- Quickly avoid known (groups of antibiotics)
Add resistance mutations / genes to test strain.
- Example E.coli screening strain has incorperated resistance to 15 antibiotics
Screen optimized:
- Test on persistent forms
- Phenotype form > Persistent form is genetically sensitive to a drug but survives.
- Test these after washing antibiotics away
- Biphasic killing
- Only thing that will work is very long treatment
Biphasic killing Tuberculosis
One bacteria population but two form of killing:
- Sensitive bacteria will be killed immediately
- Population that persist for a long time. This one grows slower and antibiotics only kill very active ones.
Advantages Natural compound screens
- Identify prodrugs
- Identify compounds that can penetrate the micro organism
- Unexpected targets
- Possibly multiple targets
Disadvantages Natural compound screen
- Purification and characterization of compound from complex mixture is difficult
- Problem for optimization because of unknown target
- Problems with nuisance/overlast compounds (detergent, antiseptics)
Solution for Problem for optimization because of unknown target
Sequence before and after resistance and look wich part of genome changes
Solution for problem : Purification and characterization of compound from complex mixture is difficult
- Purification by HPLC, analysis with mass spec/NMR
Look for target after in vitro screen:
Benzothiazones
5 phases
Screened for mycobacterial growth inhibitors.
- Genetic approach: Couldn’t do knock out. So added gene library.
- Mining literature/ Homology search»_space; Enzyme they found was important for making arabinogalactam.
- Biochemical approach: Looked for epimerization (forming arabinogalactam)
With benzothiazone: Epimerization wasn’t possible. - Structural approach
Yes it binds with high affinity to DprE - Did it also work in vivo?
Yes: TB inhibitor.
Look for target after in vitro screen:
Pyrazinamide:
6 characteristics
- First lines drug TB
- Crucial for sterilizing treatment
- Found in vivo screening : Realy speeded up killing bacteria
- Only in vivo active & no real target found yet
- Hardly active in culture (depends on pH and type of media)
- Pyrazinamide acid is activated drug
- Nicotinic acid (vit B3) analogue
Why don’t find mutation in target gene in pyrazinamide?
In genetic approach:
- Mutations in target gene are lethal
- Pyranizoic aid has multiple targets
- Pyranizoic acid has no protein target (but lowering pH for example)
