Structural Biology and Antimicrobial Design

Question 1

What is an antimicrobial?

Answer 1

Broad term that covers Antibacterial, antiviral, antifungal and antiparasitic agents.

Question 2

What are antibiotics?

Answer 2

A natural chemical compound produced by a micro-organism that inhibits (static) or kills (cidal) other micro­‐organsisms at low concentrations.

(technically this definition doesn’t include synthetic agents. Nowadays, most antibiotics are synthetic.)

Question 3

How was penicillin discovered?

Answer 3

Sir Alexander Fleming.
Found penicillium colony growth on plates of staphylococcal colonies- the area around penicillium was clear of staph.
Collaborative effort with Ernst Boris Chain and Sir Howard Walter Florey. Nobel Prize for Medicine 1945.

Question 4

What did Dorothy Hodgkin do in 1945?

Answer 4

X-ray crystallography pioneer.
Determined the position of every atom of penicillin in 3D space.
Showed structure of penicillin with beta lactam ring.

Question 5

What are the mechanisms of action exploiting selective toxicity?

Answer 5

Cell wall synthesis.
Protein synthesis.
Nucleic acid synthesis.
Folate synthesis.
Disruption of cytoplasmic membrane.

These are processes that also happen in human cells- risk of toxicity of drugs are not specific.

Penicillins, cephalosporins, glycopeptides, carbapenems and monobactams all target features that are unique to bactera- not toxic to humans.

Question 6

Why are sugars in bacterial periplasm good antibiotic targets?

Answer 6

They are unique to bacteria, so the antibiotic target doesn’t interfere with eukaryotic cells eg. peptidoglycan.
Increased specificity.

Question 7

Describe peptidoglycan.

Answer 7

NAG and NAM units- both are derived from glucose.
Glycosidic units are linked by transglycosidases (beta 1,4 linkages).
Spaced out by pentapeptide bridges.
Transpeptidases (penicillin binding proteins) catalyse the crosslinking of D-alanine‐D­‐alanine NAM pentapeptides.
Can cross link sugars with peptides, which protects bacteria from mechanical stress.

Question 8

How is cross linking prevented?

Answer 8

The beta lactam ring- 4 membered ring made of 3 carbons, 1 nitrogen and a double bonded oxygen- substrate mimetic.
Transpeptidases (PBPs) “mistakenly” use the beta lactam as a “building block”.
Acylation of the enzyme prevents further catalysis.
Cell wall synthesis ceases followed by cell lysis.

eg. penecillin and D-Ala-D-Ala have similar structures. Penicillin is the beta lactam what modifies the active site of transpeptidases. The only structural difference in D-Ala-D-Ala from penicillin is a more stable N link in penicillin.

Question 9

What are some general mechanisms of resistance?

Answer 9

Enzyme production.
Alteration of membrane permeability- this is altered to block drugs, as drugs need to enter cells to work.
Alteration of target site- drug can’t bind, but the enzyme still works.
Efflux pumps.
Altered metabolic pahways (salvage).
Sharing of DNA.

Question 10

Describe some beta lactams.

Answer 10

Penicillins- many kinds, all built of the beta lactam structure with a 5 membered ring.
Cephalosporins- have a 6 membered ring.
Carbapenems- different R groups on the 5 membered ring.
Monobactams- can recognise the beta lactam ring.

Question 11

How are antibiotics used globally?

Answer 11

Used in human medicine, crops and animals- bacteria can gain resistance through the use of antibiotics on crops and animals, as well as humans.

Prophylaxis- use of antibiotics as a preventative measure.
pre-emptive/empiric- if patient has an infection but not known what is it, patient is given lots of antibiotic to try and clear infection.
Targeted- specific use.

Question 12

What are some specific fight-back strategies?

Answer 12

Production of beta-lactamases (most common and important mechanism for Gram
negatives).

Changes in the active site of PBPs resulting in lower affinity for beta‐lactams (e.g. production of PBP2a conferring methicillin resistance in Staphylococcus aureus).

Reduced expression of outer membrane proteins
(e.g. Carbapenem resistance in Enterobacter spp., Klebsiella pneumoniae).

Efflux pumps (e.g. Pseudomonas aeruginosa).

Question 13

Why are beta lactamases a severe threat?

Answer 13

Beta lactamases hydrolyse the b-lactam ring of penicillins.
Penicillins are unable to bind to their substrates.
The ring is the active part of the molecule- if this is modified, it is inactive against transpeptidases- not recognised at all.

Discover/design beta- lactam antibiotics that evade enzymatic inactivation.
Discover/design beta-lactamase inhibitors so that the partner antibiotic can still bind to the substrate.

There are families of beta lactamases that are all slightly different- a whole arsenal of anti-antibiotic warfare.

Question 14

When developing future beta- lactamase inhibitors, what is needed t make them successful?

Answer 14

High affinity for the active site of targets.
Mimic the natural substrate.
Stabilise interactions in the active site.
Rapid cell penetration.
An appropriate beta- lactam partner.
Sufficient serum/periplasm concentrations.

Question 15

What is the problem with the world view on fungal infections?

Answer 15

The effect of fungal infections on human health are often overlooked.
Difficult to ascertain epidemiological data due to misdiagnosis and lack of mycological surveillence.

Question 16

What is an iatrogenic disease?

Describe the effects of Aspergillus fumigatus on human health.

Answer 16

Iatrogenic disease is one that is caused by medical treatment.

Aspergillus fumigatus is a fungus. Spores are released from conidia and are everywhere. If we are healthy, the immune system prevents fungi growing in the lung. Infections can occur if the patient is severely immunosuppressed.
Aspergullis can disseminate (spread) from site of infection to other parts of the body.
Mortality rates of invasive aspergillosis is up to 90% depending on the site of infection (eg. the brain is difficult to treat).

Question 17

How are antifungals use clinically for invasive disease?

Answer 17

Echinocandins eg. caspofungin- affect glucan synthesis.
Polyenes eg. liposomal amphoterican B- affect phospholipid bilayer of fungal cell membrane.
Azoles eg. voriconazole- affects ergosterol.

(These three classes of drugs are becoming ineffective, toxic and resistance is emerging. We need better and more rapid diagnostic tests, and safer and more effective drugs).

Question 18

Describe the fungal cell wall.

Answer 18

These are unique to fungi- not part of human cells, and contain different sugars from bacteria.
Contain galactomannan- highly variable- helps fungus to evade the immune response (first bit seen by immune system).
Made up of chains of glucan and chitin.
Chitin- monpolymer of N-acetylglucosamine (one of the peptidoglycan components).
Membrane conatins chitin synthases and glucan synthases.

Question 19

Describe high throughput screening.

Answer 19

Require purified protein and an optimised enzyme assay.
Screen large libraries e.g. 80,000 compounds for activity against a protein target.
Follow‐up structure-based activity relationships.
Chemist input to optimise and select
classes of compounds that show the most promise for being drugs.

Question 20

What is the rule of 5?

Answer 20

Properties for oral drugs to optimise absorption and penetartion.
All commonly used drugs have these in common.
<5.
All of these make up physiochemical properties.

Question 21

Why use the rule of three?

Answer 21

< 3 hydrogen bond acceptors.
< 3 hydrogen bond donors.
MW < 300Da.
ClogP < 3.
All of these make up physiochemical properties.

Want to find smaller molecules that have a great degree of inhibition.

Question 22

What is fragment screening?

Answer 22

Require purified protein only- don’t need an enzyme assay.
Early triage of targets.
Screen small libraries e.g. 1000 fragments.
Explore more “chemical space” by combining molecular building blocks.
Measures binding NOT enzyme inhibition.
Requires extensive multi‐disciplinary follow-­‐up.
Usually involves elaboration of a “scaffold” fragment.

Question 23

What is ligand efficiency?

Answer 23

Efficiency of ligand to bind into the site of its receptor. Several smaller fragments have a better ligand efficiency that one big molecule.
Nanomolar binding- greater degree of inhibition with a smaller number of atoms.

Question 24

In the search for new antifungals, how can essential genes be targeted?

Answer 24

Human homologues.
Betetr to look at inhibiting enzymes that make the precursor to chitin (UDP-GlcNAc)- fungus needs chitin synthase enzymes to grow, but difficult to inhibit the entire family.
Searching for small molecules selective for A.fumigatus that reproduce the genetic phenotype.

Question 25

How does bio-layer interferometry work?

Answer 25

Purify protein of interest. Immobilise on the end of a BLI tip. Dip the tip in a solution with different compounds and determine what binds.
Target is bound to sensor which sends out a wavelength readout.

Question 26

What does Xray crystallography do?

Answer 26

Confirms binding.
Provide a 3D structure of protein and fragment.
“Complexes” shows the molecular mechanism of binding and are crucial for generating structure activity relationships (SAR).

Question 27

How does fragment elaboration work?

Answer 27

Binding site comprises of three binding pockets.
Screening locates molecular fragments that bind to one, two or all three pockets.
A lead compound is designed by organising all three fragments around a core template.
Growing out of a single fragment.

Question 28

What are some hurdles in drug discovery?

Answer 28

Need to know if drug inhibits or not.
Does the target have a human analogue that means it may be non-specific and toxic to human cells.
Try to reduce attrition- the failure rate of compounds.