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Microbial Biotechnology (BIO-6004A) > Lecture 13: Antibiotic production > Flashcards

Lecture 13: Antibiotic production Flashcards

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1
Q

What are the two main modes of antibiotic action?

A

Bacteriostatic: inhibits bacterial growth
Bactericidal: kills bacteria

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2
Q

What is a broad-spectrum antibiotic?

A

active against a wide range of pathogenic bacteria

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3
Q

What is narrow-spectrum antibiotic?

A

Active against a specific family (or limited number) of pathogenic bacteria

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4
Q

What is minimum inhibitory concentration (MIC)?

A

Lowest concetration of a drug that will inhibit visible growth of an organism after overnight incubation

MIC <4ug/mL usually desirable

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5
Q

What is considered the golden age of antibiotic discovery?

A

1940-60

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6
Q

What are different classes of clinically used antibiotics

A
  1. Actinomycetes: Gram+, non-motile, found in soil
  2. Other bacteria
  3. Fungal
  4. Synthetic
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7
Q

What is the traditional approach to antibiotic discovery?

A
  1. Environmental (traditionally soil) sample
  2. Plated to identify bacteria and fungi
  3. Individual organisms screened for ability to prod. antibiotics
  4. fermentation and subsequent purification to isolate pure antibiotic
  5. Testing, etc
  6. New clinically useful antibiotic
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8
Q

What are important classes of natural antibiotics

A
  1. Beta-lactams: penicillins (eg. amoxicillin), cephalosporins (eg. cefacetrile)
  2. Aminoglycosides (eg. Kanamycin A)
  3. Macrolides (eg. Erythromycin)
  4. Glycopeptides (eg. vancomycin)
  5. Polymyxins (eg. colistin)
  6. Tetracyclines (eg. tetracycline)
  7. Ansamycins (eg. Rifampicin)
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9
Q

What are important classes of synthetic antibiotics

A

Generally simpler than natural compounds, as easier to produce

  1. Oxazolidinones (Linezolid)
  2. (Fluoro)quinolones (Ciprofloxacin)
  3. Azoles (metronidazole)
  4. Sulfonamides (mafenide)
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10
Q

What are the targets of antibiotics?

A
  1. Cell wall biosynthesis
    a. Beta-lactams (penicillin, cephalosporins, carbapenems, etc)
  2. Protein synthesis
    a. 50S subunit of ribsome (Macrolides, chloramphenicol, etc)
    b. 30S subunit (aminoglycosides, tetracyclines, etc
  3. Nucleic acid synthesis
    a. RNA synth (Rifampicin)
    b. DNA synth (Quinolones)
    c. DNA damage (Metronidazole, Nitrofurantoin)
  4. Folate synthesis (Sulphonamides, trimethoprim)
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11
Q

What are Beta-lactams?

A

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

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12
Q

What is carbapenase?

A

An enzyme which degrades carbapenems, a beta-lactam which is used for treatment of serious infections causes by multi-drug resistant bacteria

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13
Q

How can bacteria be antibiotic resistant?

A
  1. 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)
  2. 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
  3. TARGET MODIFICATION: modify so antibiotic can no longer bind
    a. Point mutation to target
    b. Modificaiton of target
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14
Q

How does resistance develop

A

Random mutagenesis
HGT

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15
Q

How much does it cost to take a drug to the market and how long?

A

800-1000 million
10-15 years

Discovery, preclinical trials, clinical trials, FDA

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16
Q

What are the stages of antibiotic development and clinical trials?

A
  1. Pre-clincal testing
  2. Investigational new drug application (IND)
  3. Clinical trials phase 1
  4. Clinical trials phase 2
  5. Clinical trials phase 3
  6. New drug application (NDA)
  7. Approval
17
Q

Why has antibiotic discovery slowed down?

A
  1. Drug rediscovery
  2. Lock of investment due to people thinking the problem was solved and limited financial returns
  3. Much higher regulatory hurdles
  4. Competition from highly profitable drugs for chronic diseases (e.g., high BP)
18
Q

What are the next steps for antibiotic discovery?

A
  1. Synthetic antibiotics
  2. Semi-synthetic antibiotics
  3. Natural products
  4. Alternatives
    a. vaccines, probiotics, antibody-drug conjugates
19
Q

What is an example of semi-synthesis being a reliable route for new compounds?

A

Semi-synthetic beta lactams

Penicillin (1st gen) -> Amoxicillin (2nd gen) -> Piperacillin (3rd gen)

16 penicillin semi-synthetic derivitives today

20
Q

How are natural product antibiotics synthesised?

A

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

21
Q

How can we activate these (potential) antibiotic pathways in bacteria and fungi?

A

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

22
Q

How do we hunt for antibiotics in new environments?

A

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

23
Q

How can we culture the uncultureable?

A

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

24
Q

What are Trojan Horse Antibiotics?

A

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

25
Q

How can we revive old antibiotics?

A

Antibiotics overlooked before may be relooked at as more and more become resisitable

E.g., those that are narrow spectrum or dont produce large yields

Example: Bicyclomycin
produced by streptomyces strains. Discovered 1972. Active towards multi-drug resistant gram-negative bacteria. Inhibits RNA transcription termination factor Rho.

26
Q

What are common features of non-ribosomal peptides?

A
  1. Peptide backbone
  2. Non-proteinogenic amino acids
  3. Oxidative crosslinks
  4. D-amino acids
  5. Cyclisation
  6. acylation and glycosylation
27
Q

What is ‘penicillin non-ribosomal peptide synthase’ (NRPS) also called?

A

ACV synthase

28
Q

What is ACV synthase?

A
  1. massive multidomain protein
  2. each module incorporates one amino acid

composed of modules containing domains:
‘A’ Domain = adenylation domain
‘T’ Domain = thiolation domain
‘C’ Domain = condensation domain
‘PCP’ Domain = Peptidyl carrier protein
‘E’ Domain = Epimerisation domain
T & PCP domain for substrate tethering

29
Q

How do A and PCP domains interact in ACV synthase?

A

‘Aminoacyl thioester formation’

30
Q

How do C domains interact within ACV synthase?

A

Amide bond formation

31
Q

How is D-valine residues incorporated in ACV synthase?

A
  1. Activation and condensation of L-valine
    a. D-valine cant be incorporated
    b. instead A and C domains select for L-valine
  2. Epimerisation (E) domain
    a. L and D isomers in equilibrium but the thioesterase (TE) only
    hydrolyses the peptide with D-valine. This provides high
    stereochemical purity of the final tripeptide
32
Q

What does each domain of the ACV synthase do?

A

A: specificity for loading correct amino acid onto adjacent PCP
C: catalyse the peptide bond forming step. Substrate specificity for
the correct enantiomers
E: catalyse epimerisation of peptidyl-thioester
TE: catalyses the off-loading hydrolysis

33
Q

How does ACV get converted to mature penicillin?

A

L,L,D-ACV -(oxidation)-> Isopenicillin N -(Epimerase)-> Penicillin N -(Amidase)-> 6-aminopenicillanic acid (6-APA) -> chemical or enzymatic modifcation to penicillin

34
Q

What are glycopeptides?

A
  1. natural products produced by actinomycete bacteria
  2. used against multi-drug resistant Gram+ bacterial infections like
    MRSA
  3. Two origninal compounds: Vancomycin, Teicoplanin
35
Q

What is the mechanism of action for glycopeptides

A
  1. Forms 5 H bonds with D-Ala-D-Ala termini of bacterial
    peptidoglycan
  2. Very specific and very strong 3D interaction that inhibits both
    transglycosylation and transpeptidation
  3. Osmotic pressure kills the cell in absence of well-structured
    peptidoglycan
36
Q

How do bacteria become resistant to glycopeptide?

A

VanA seven-gene transposon - upregulated when Glycopeptide detected

37
Q

What bacterial organisms/strains are Glycopeptide resistant

A
  1. Originally detected in eterococci, but vancomycin-resistant Staphylococcus aureus (VRSA) was subsequently detected (MIC >32 ug/mL)
  2. MRSA - heteroresistant to vancomycin (hVISA)
38
Q

How do we overcome Glycopeptide resistance?

A

Synthetically redesigned Glycopeptide antibiotic

Microbial Biotechnology (BIO-6004A) (17 decks)

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