Bacterial screens in vitro Flashcards
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)
Pyranzizoic acid does not bind the mycogacterial proteins
It has multiple proteins with low affinity because its an analogue of nicotic acid
- So you can’t find one target
Gene library
If knock out is impossible.
- Add extra copies of genes in several bacteria
- Every bacteria has part of DNA with extra gene copies
- Which bacteria has decreased susceptibility»_space; Identify this region in genome.
And isolate resistant mutants with point mutations.
- Sequence genomes
- Look for common features of mutants.
Arabinogalactam
Important to keep outer membrane
Genetic approach pyrazinamide
- Found 80-90% colonies mutation in pncA < = nicotine amidase:
Required for modification from pyrazinamide to active form pyrazinoic acid. - 10-20% resistant colonies
Various mutations
Probably affecting regulation of pncA.
Because these were senistive for pyrazinoic acid.
