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Medical Microbiology > 16. The leaky antibacterial discovery pipeline > Flashcards

16. The leaky antibacterial discovery pipeline Flashcards

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1
Q

What is the AMR problem in the rich world?

A

AMR in hospital infections

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2
Q

What are ESKAPE pathogens?

A

Pathogens that have significant resistance to treatment that are critical.

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3
Q

What are the ESKAPE pathogens?

A
  1. Glycopeptide-resistant enterococci
  2. MRSA
  3. ESBL/carbapenemase-producing Enterobacteriaceae like E. coli, K. pneumoniae and other enterobacterales.
  4. Pan-resistant non-fermentors like P. aeruginosa and A. baumanii
  5. Neisseria gonorrhoeae
  6. Mycobacterium tuberculosis
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4
Q

When were most antibiotic classes discovered?

A
  1. In the golden age
  2. 1940s-1962
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5
Q

What was the antibiotic discovery void?

A
  1. Between the discovery of quinolones in 1962 and Oxazolidinones in 2000.
  2. There were no new antibiotic classes, just variations of existing classes like carbapenems.
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6
Q

Is the antibiotic discovery void still going on?

A
  1. no
  2. But very few new classes of antibiotics have discovered since
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7
Q

What currently dominates antibiotic discovery?

A
  1. ß-lactam and ß-lactamase inhibitors
  2. Not novel antibiotics
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8
Q

What ESKAPE pathogens do we have successful new treatments for?

A

Carbapenemase producing enterobacterales

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9
Q

What ESKAPE pathogens are more challenging to develop successful new treatments for?

A
  1. Acinetobacter
  2. Pseudomonas
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10
Q

Why did antibiotic development stall?

A
  1. Perception
  2. Profitability compared to other drugs
  3. Regulations
  4. It is difficult
  5. Gram-negatives are hard to develop drugs for.
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11
Q

Stalling antibiotic development: Perception

A
  1. There was a view that we had solved the problem by the end of the 60s.
  2. It was thought we had all the tools we needed to treat infections and didn’t need anymore.
  3. Also the view that Infectious diseases were a thing of the past.
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12
Q

Stalling antibiotic development: Profitability

A
  1. It is hard for companies to make a profit.
  2. This is because most infections remain susceptible to existing antibiotics.
  3. Patients with resistant infections take a new drug for a short time and then move on to the next treatment or die.
  4. This leads to reduced sales compared to drugs for chronic conditions it is a lot more difficult to make money.
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13
Q

Stalling antibiotic development: Regulations

A
  1. These are important but they push the costs of developing drugs up.
  2. To use the same drug for a different infection caused by the same pathogens requires separate clinical trials.
  3. Eg different clinical trials for respiratory infections, UTIs etc
  4. This cost lots of time and money.
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14
Q

Why is resistance in gram-positives less of a worry?

A

They are easier to develop new treatments for.

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15
Q

What is a problem with current antibiotic targets?

A

They target only a small range of processes in the bacteria cell.

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16
Q

What are the main current targets for antibiotics?

A
  1. Protein synthesis
  2. Peptidoglycan synthesis
  3. Bacterial membrane stability
  4. Nucleic acid metabolism or manipulation like transcription.
  5. General metabolism like the synthesis of nucleic acids.
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17
Q

How were new antibiotic targets identified?

A
  1. Using the large amounts of bacterial genomic data that started increasing in the 90s
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18
Q

What is phenotype based drug discovey?

A
  1. This is what was used during the golden age.
  2. Assay candidates for antimicrobial activity by challenging them with a pathogen.
  3. Then, observing its activity and zone of inhibition.
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19
Q

What is the disadvantage of phenotype based drug discovery?

A
  1. The mechanisms of killing the pathogen could be undesirable.
  2. This means that they could target things that would also kill eukaryotic cells and therefore would not be good drugs.
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20
Q

What is target-based drug discovery?

A
  1. It is a method where you identify a target and then find a molecule/drug that inhibits it.
  2. This means you have a clearer idea of the mechanism of killing and use a target that won’t affect human cells.
  3. You screen molecules against the target.
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21
Q

What genes can be used as targets in target-based drug discovery?

A

Genes that are essential for growth

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22
Q

How can you use transposons to identify drug targets in bacteria?

A
  1. Create a library of bacteria with transposons inserted into lots of different genes.
  2. If the bacteria cannot grow then you know the gene is essential.
  3. Not very efficient and based on chance.
  4. Identify the gene after its been knocked out.
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23
Q

How can you use inducible plasmids to identify drug targets in bacteria?

A
  1. Make a panel of inducible plasmids with RNase to knock out specific genes.
  2. Create a library of plasmids that target a massive range of genes.
  3. As you induce the expression of the plasmid transcription is blocked.
  4. Obverse the viability of the cell and if the bacteria dies the gene was essential.
24
Q

What other gene targets were identified using genomic approaches?

A
  1. Screened H. influenzae, E.coli and S. pneumoniae.
  2. Identified lots of biochemical pathways the contain essential genes.
  3. Produced known targets like cell wall biosynthesis.
  4. New targets like amino acid metabolism and lipid metabolism.
25
Q

what is PDF?

A
  1. Peptide deformylase
  2. A newly identified target for antibiotics.
  3. Lots of companies started developing antibiotics with this target.
26
Q

What does PDF do?

A
  1. Like eukaryotes protein synthesis starts with methionine.
  2. Unlike eukaryotes, a formyl group is added to the amino group of the methionine before translation.
  3. The formyl group needs to be removed from Met before the protein can mature.
  4. PDF does this and was flagged as essential in many species.
27
Q

What does the structure of PDF tell us?

A
  1. The structure shows PDF is highly conserved across gram positive and gram negative bacteria.
  2. An antibiotic targeting PDF could treat lots of pathogens.
28
Q

Why were PDF inhibitors considered to be a good idea?

A
  1. It was effective at killing multiple bacterial species.
  2. It was safe in humans in phase 1 trials.
  3. It was thought there was no homologue in humans but that’s not the case but it doesn’t contribute to toxicity
29
Q

What problems with PDF inhibitors were shown in gram negative bacteria during clinical trials?

A

There was significantly increased efflux to remove the drug before it could act on its target.

30
Q

What problems with PDF inhibitors were shown in S. aureus during clinical trials?

A
  1. Massive amounts of resistance was seen.
  2. Slight resistance was expected but it was expected to be target site modification.
  3. However these mutants contained a mutation in the FMT gene that bypassed the formylation pathway.
  4. This meant the PDF gene became non-essential and PDF was no longer a good target for an antibiotic as it didn’t kill the bacteria.
  5. This killed PDF inhibitors as broad spectrum antibiotics.
31
Q

What is leucine tRNA synthetase?

A

Attaches leucine to the tRNA that is then recruited to the ribosome.

32
Q

Why were inhibitors of leucine tRNA synthetase looked as a good target for antibiotics?

A
  1. It stops the attachment of leucine to the tRNA.
  2. This target has not been used before and any tRNA synthetase has not been explored before as systemic agents.
  3. The inhibitor uses a boron atom that mimics lots of tetrahedral chemistry in bacterial enzymatic reactions with a novel mechanism.
33
Q

How well did leucine tRNA synthetase inhibitors work?

A
  1. They worked well against lots of high-priority strains like carbapenem-resistant Klebsiella.
  2. They had activity comparable to carbapenems.
  3. They are safe and tolerated well in phase 1 trials.
34
Q

What happened with leucine tRNA synthetase inhibitors in phase 2 trials?

A
  1. They were tested on 14 patients with complicated UTIs.
  2. Very quickly, three patients developed a massive increase in MIC and became resistant to the inhibitor.
  3. These were not developed further.
35
Q

What do phase 2 trials test?

A

The drugs’ ability to treat infection

36
Q

How was resistance developed to leucine tRNA synthetase inhibitors in phase 2 trials?

A
  1. The causative E coli developed rapid mutations in the tRNA synthetase.
  2. This was not seen in in vitro test but developed rapid target site modifications in humans.
  3. It was hard to guess this would happen.
37
Q

What are 2 key problems with target based drug discovery?

A
  1. Resistance and it developing in new ways.
  2. Problems with the target like different isoforms.
38
Q

Why was fatty acid biosynthesis selected as a viable new antibiotic target?

A
  1. The enzymes that carry it out are different enough from the human ones.
  2. Isoniazid is a treatment for TB that targets fatty acid biosynthesis
39
Q

What is an enyolACP reductase?

A
  1. An enzyme in fatty acid biosynthesis.
  2. Isoforms include FabK, FabL and the main one, FabI.
40
Q

What is triclosan?

A
  1. an antimicrobial compound that targets FabI.
  2. Not a drug.
  3. Often used in coatings of household goods.
41
Q

What is the problem with targeting EnoylACP reductases?

A
  1. There are many isoforms that exist in different bacteria.
  2. If you increase the activity against one isoform, you usually decrease activity against the other.
42
Q

What EnoylACP reductases does S. aureus use?

A

FabI

43
Q

What EnoylACP reductases does S. pneumoniae use?

A

FabK

44
Q

What EnoylACP reductases does E. faecalis and P. aeruginosa use?

A

FabI and FabK

45
Q

What EnoylACP reductases does B. subtilis use?

A

FabI and FabL

46
Q

What does an antibiotic targeting EnoylACP reductases need to do?

A

Have antibiotic activity against the main isoforms FabI and FabK

47
Q

What did testing FAB inhibitors show?

A
  1. They were tested against S. aureus, H. influenzae, E. coli and S. pneumoniae.
  2. FAB inhibitors were very potent against S. aureus even compared to ciprofloxacin.
  3. Against gram negative like E. coli they worked in vitro but not in vivo. This shows problems with getting the FAB inhibitors into the gram negative cell.
  4. There is a trade off in activity. If you improve the activity against FabI you reduce activity against FabK.
48
Q

How much time and effort is needed to develop 1 potential antibiotic?

A
  1. 100,000s man hours and billions of dollars
  2. Essentiality screening and identifying targets.
  3. Testing up to 500,000 compounds from synthetic libraries.
  4. 16 hits
  5. 5 leads
  6. 1 active target
  7. This target has since been abandoned
49
Q

What are the Lipinski rules?

A
  1. A set of rules made up that defines the chemical properties that are associated with drug activity in small molecules.
  2. No more the 5 H-bond donors
  3. Molecular weight under 500
  4. Log P under 5 (measure of hydrophobicity)
  5. No more than 10 H-bond acceptors.
50
Q

What did the Lipinski rules influence?

A
  1. What molecules went into chemical libraries that were screened for antibacterial activity.
  2. This limits the scope of molecules that were screened for antibacterial activity and we might not be looking in the right place.
51
Q

What can failure of a drug be due to?

A
  1. Problems with target
  2. Problems with the drug
52
Q

What problems with the drug can prevent it from being developed further?

A

Its chemistry and chemical properties.

53
Q

Are the Lipinski rules generally accurate?

A
  1. Yes
  2. They have been good for drugs historically.
  3. They are not as good for antibiotics
54
Q

Why are the Lipinski rule not good for antibiotics?

A
  1. They are acting on bacterial cells not human cells so they need different properties.
55
Q

How do antibiotics break the Lipinski rules?

A
  1. They tend to be larger/ heavier.
  2. They tend to be more hydrophilic.
56
Q

What are the lessons learned from target based antibiotic discovery?

A
  1. Finding new targets is not difficult but finding a good one is.
  2. Essentiality is the start of target validation.
  3. Resistance can arise in unexpected ways.
  4. Efficacy against multiple targets is hard.
  5. Antibiotic chemistry is different from other drugs.
  6. Getting drugs into gram-negatives is hard.
57
Q

What is target based whole cell screening?

A
  1. Comparing the activity of an agent in a whole cell to a cell with a mutation that makes the target conditionally essential.
  2. This happens when you knock out specific genes and then make other downstream genes essential as they are the only mechanisms left.
  3. This helps you identify the mechanism the antibiotic uses to kill the bacteria.
  4. It is phenotypic screening in a defined mutation background.

Medical Microbiology (16 decks)

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