Lecture 13: Antibiotic production Flashcards
What are the two main modes of antibiotic action?
Bacteriostatic: inhibits bacterial growth
Bactericidal: kills bacteria
What is a broad-spectrum antibiotic?
active against a wide range of pathogenic bacteria
What is narrow-spectrum antibiotic?
Active against a specific family (or limited number) of pathogenic bacteria
What is minimum inhibitory concentration (MIC)?
Lowest concetration of a drug that will inhibit visible growth of an organism after overnight incubation
MIC <4ug/mL usually desirable
What is considered the golden age of antibiotic discovery?
1940-60
What are different classes of clinically used antibiotics
- Actinomycetes: Gram+, non-motile, found in soil
- Other bacteria
- Fungal
- Synthetic
What is the traditional approach to antibiotic discovery?
- Environmental (traditionally soil) sample
- Plated to identify bacteria and fungi
- Individual organisms screened for ability to prod. antibiotics
- fermentation and subsequent purification to isolate pure antibiotic
- Testing, etc
- New clinically useful antibiotic
What are important classes of natural antibiotics
- Beta-lactams: penicillins (eg. amoxicillin), cephalosporins (eg. cefacetrile)
- Aminoglycosides (eg. Kanamycin A)
- Macrolides (eg. Erythromycin)
- Glycopeptides (eg. vancomycin)
- Polymyxins (eg. colistin)
- Tetracyclines (eg. tetracycline)
- Ansamycins (eg. Rifampicin)
What are important classes of synthetic antibiotics
Generally simpler than natural compounds, as easier to produce
- Oxazolidinones (Linezolid)
- (Fluoro)quinolones (Ciprofloxacin)
- Azoles (metronidazole)
- Sulfonamides (mafenide)
What are the targets of antibiotics?
- Cell wall biosynthesis
a. Beta-lactams (penicillin, cephalosporins, carbapenems, etc) - Protein synthesis
a. 50S subunit of ribsome (Macrolides, chloramphenicol, etc)
b. 30S subunit (aminoglycosides, tetracyclines, etc - Nucleic acid synthesis
a. RNA synth (Rifampicin)
b. DNA synth (Quinolones)
c. DNA damage (Metronidazole, Nitrofurantoin) - Folate synthesis (Sulphonamides, trimethoprim)
What are Beta-lactams?
E.g., Penicillin G
1. most widely used class of antibiotic
2. Broad spectrum B-lactam antibiotics active against both gram+ and gram- bacteria
TARGET: peptidoglycan
Mechanism of action (MOA):
1. penicillin binding protein (PBP) is transpeptidase, that catalyses peptidoglycan cross-linking
2. Nromal crosslinking catalyses by the PBP
3. Inhibition of penicillins
What is carbapenase?
An enzyme which degrades carbapenems, a beta-lactam which is used for treatment of serious infections causes by multi-drug resistant bacteria
How can bacteria be antibiotic resistant?
- DESTRUCTION: enzymatically degrade or modify antibiotic
a. e.g., B-lactamases degrade B-lactams by hydrolytically breaking the
B-lactam ring in penicillin and other B-lactams
b. e.g., enzymatic modification of aminoglycosides (N-
acetyltransferases; AAC, O-adenyltransferases; ANT, O-
phosphotransferases; APH) - EXCLUSION: prevent the antibiotic from entering the cell, or remove it
from the cell before it can do damage
a. e.g., the tetracycline TetA efflux pump - TARGET MODIFICATION: modify so antibiotic can no longer bind
a. Point mutation to target
b. Modificaiton of target
How does resistance develop
Random mutagenesis
HGT
How much does it cost to take a drug to the market and how long?
800-1000 million
10-15 years
Discovery, preclinical trials, clinical trials, FDA
What are the stages of antibiotic development and clinical trials?
- Pre-clincal testing
- Investigational new drug application (IND)
- Clinical trials phase 1
- Clinical trials phase 2
- Clinical trials phase 3
- New drug application (NDA)
- Approval
Why has antibiotic discovery slowed down?
- Drug rediscovery
- Lock of investment due to people thinking the problem was solved and limited financial returns
- Much higher regulatory hurdles
- Competition from highly profitable drugs for chronic diseases (e.g., high BP)
What are the next steps for antibiotic discovery?
- Synthetic antibiotics
- Semi-synthetic antibiotics
- Natural products
- Alternatives
a. vaccines, probiotics, antibody-drug conjugates
What is an example of semi-synthesis being a reliable route for new compounds?
Semi-synthetic beta lactams
Penicillin (1st gen) -> Amoxicillin (2nd gen) -> Piperacillin (3rd gen)
16 penicillin semi-synthetic derivitives today
How are natural product antibiotics synthesised?
Dedicated biosynthetic pathways exist in bacteria and fungi for production of antibiotics.
Genes for these pathways (which there are many pathways) are almost always clustered in bacteria and fungi
e.g., teicoplanin gene cluster
How can we activate these (potential) antibiotic pathways in bacteria and fungi?
Genetic manipulation (e.g., crispr cas 9 or other)
Cloning into a heterologous host
Classic methods: screening media, random mutagenesis, co-culturing with other organisms
How do we hunt for antibiotics in new environments?
less than 1% of all microorganisms have been cultured in the lab.
Most exist in complex and competitive communities
e.g., Burkholderia cepacia (opportunistic human athogen) -> Enacyloxin lla
How can we culture the uncultureable?
Use a reaction vessel embeded in the soil to grow microorganisms that cant grow in lab.
Once colony isolated, can often grow in the lab
What are Trojan Horse Antibiotics?
Molecules featuring an antibiotic conjugated to a chemical features that enables cellular import
often conjugated to siderophores
Microcin E492 natural example
Particularly promising for gram- bacterial infections where outer membrane prevents many durgs entering
