HMB304 - Lecture 2 - Antibiotic Origins & targets

what are antibiotic classes based on?

-they are based on chemical scaffolds and mode of action (MOA)

(ex. tetracyclines)

what are scaffolds?

-they are the core molecular framework that forms the structural foundation of a drug class

-use R groups to change and make new generations of a class

what is mode of action (MOA)?

-it is the specific way a drug interacts with bacterial cells to kill or stop growth

what do antibiotics in the same class share?

-they shared the same antibiotic target

what are antibiotic targets?

-they are specific molecules or processes in bacteria that antibiotics disrupt to kill or inhibit bacterial growth

what are the 2 qualities the best antibiotic targets have?

1. unique to bacteria (ex. peptidoglycan)

2. differ significantly from human counterparts (ex.ribosome) → that we won’t see toxicity

what are antibiotics designed to have?

-they are designed to have selective toxicity to target bacteria without harm to the host

how do antibiotics react with their targets?

-they react through reversible and irreversible binding (both possible)

which type of binding is the preferred method?

-irreversible is the preferred output because we want it to bind and be stuck there and not fall off

why are the ribosomes in bacteria different than the ones in antibiotics?

-bacteria have them too but they look different and have different subunits, so different binding in most cases

what are bacteriostatic drugs?

-they inhibit the growth of bacteria without directly killing them

-the number of bacteria remains constant, relying on the immune system to clear the infection (it stops growing overtime and its constant growth) → which is why it is importan to finish your antibiotic dosage given

-target cellular processes that inhibit metabolic processes needed for growth (ex. protein synthesis)

→ ex. tetracycline

what are the pros of bacteriostatic drugs?

-they preserve the microbiome which is good bacteria that is important for our body

→ our immune system knows not to come and clear our microbiome

what are the cons of bacteriostatic drugs?

-they have slower response, which makes it more likely to develop AMR if taken incorrectly and requires a functioning immune system

→ because we are waiting for our immune system to catch up

what are bactericidal drugs?

-they kill sensitive bacteria to reduce the number of live bacteria after exposure to the drug

-target cellular essential processes needed for microbial viability (ex. cell wall growth)

-the number of viable bacteria decreases rapidly

what are the pros of bactericidal drugs?

-they have rapid response (essential for severe infections), less likely to cause AMR, and are ideal for immunocompromised patients → since we aren’t relying on our immune system

what are the cons of bactericidal drugs?

-they can have potential endotoxin surge (rapid release of bacterial toxins, increased inflammation), and microbiome disruption → when a bacteria it releases its contents

-these can’t differentiate between bacteria and your microbiome

what are the 2 types of cell walls in bacteria?

1. gram-positive (+) cell wall

2. gram-negative (-) cell wall

what significantly impacts antibiotic activity?

-the bacterial cell wall structures do

what are the 2 types of spectrum of activity?

1. narrow spectrum

2. broad spectrum

what is narrow spectrum?

-its active against a limited group of bacteria (ex. Gram-positive only OR Gram-negative only)

what are the pros of narrow spectrum?

-its less likely to disrupt the microbiome, lower risk of AMR since we are impacting less bacteria

what are the cons of narrow spectrum?

-we must know the bacteria causing infection

what is broad spectrum?

-its active against a wide variety of bacteria (ex. Gram-positive AND Gram-negative) → both

what are the pros of broad spectrum?

-its treating unknown bacteria, works on multiple infections at the same time

-don’t need to get test results back beforehand

what are the cons of broad spectrum?

-its likely to disrupt the microbiome (diarrhea, rashes), higher AMR risk since more bacteria are being exposed

what are the 5 antibiotic targets?

1. cell wall growth

2. DNA/RNA synthesis

3. ribosomes

4. metabolic pathways

5. membrane integrity

what are the 3 main targets for antibiotics?

1. cell wall growth

2. DNA/RNA synthesis

3. ribosomes

what antibiotics target cell wall growth?

beta-lactams:

• penicillins

• cephalosporins

• monobactams

• carbapenems

glycopeptides:

• vancomycin

-bacitracin

what antibiotics target DNA synthesis?

fluoroquinolones:

• ciprofloxacin

• levofloxacin

• moxifloxacin

what antibiotics target RNA synthesis?

rifamycins:

• rifampin

what antibiotics target ribosomes?

30S subunit:

• aminoglycosides

• tetracyclines

50S subunit

• macrolides

• lincosamides

• chloramphenicol

• oxazolidinones

what is central dogma?

-it is the flow of genetic information within a biological system

what are the general 3 steps of central dogma?

1. DNA polymerase facilitates DNA replication (DNA → DNA)

2. RNA polymerase facilitates transcription (DNA → mRNA

3. Ribosomes facilitate translation (RNA → Protein)

what are supercoils?

-these are extra twists in bacterial chromosomes that allow for compaction

-aka a coiled-coil

what does positive supercoiling do?

-its right-handed and overwinds dsDNA in the same direction as the double helix

(ds = double strand)

what is DNA helicase

-it introduces positive supercoils into the chromosome when unwinding DNA (right-handed)

-helps to neutralize the coil

-unwinds the DNA

what is DNA gyrase?

-it relaxes DNA during replication by introducing negative supercoils (left-handed) so it doesn’t turn into a ball and get helicase stuck

how do helicase and gyrase work together?

-they coil in opposite directions in order to create a linear piece of DNA nicely

what is the process of DNA gyrase forming dsDNA breaks?

1. gyrase (has 4 domains) binds dsDNA

2. gyrase forms dsDNA break & passes DNA segment through → its 4 domains remain bound to each DNA segment

3. gyrase ligates loose ends together & unbinds DNA

(ss= single stranded)

what are fluoroquinolone antibiotics?

-they are broad-spectrum, bactericidal antibiotics

-fully synthetic

• used/effective for urinary tract infections (UTIs) and pneumonia

-causes inhibition of DNA gyrase, inhibiting DNA replication & leading to cell death (inhibits DNA gyrase)

(ex. ciprofloxacin)

what is the process of fluoroquinolones inhibiting DNA gyrase?

1. gyrase binds dsDNA

2. gyrase forms ds break & fluoroquinolones bind gyrase → prevents religasation from happening

3. gyrase is trapped by fluoroquinolones

→ its permanent ds breaks create a physical barrier to movement of the replication fork, DNAP/RNAP, and DNA helicase → cell growth is inhibited (cell stress) → cell death

*this happens at multiple sights throughout a cell

what is RNA polymerase (RNAP)?

-it binds to DNA and transcribes the DNA strand

what are rifamycin antibiotics?

-these are broad-spectrum, bactericidal antibiotics

-natural products and semi-synthetic

-capable of penetrating eukaryotic cells → good for targeting pathogens that hide in our cells

-effective for tuberculosis, leprosy (mycobacterial infections)

-it inhibits RNAP

what is the process of rifamycins inhibiting RNAP?

1. it binds to RNAP and physically blocks mRNA transcript elongation

2. it inhibits transcription and prevents the synthesis of essential proteins

**can only bind to unoccupied RNAP (goes for every RNAP in every cell)

what are the 2 subunits prokaryotic ribosomes contain?

1. 30S small ribosomal subunit

2. 50S large ribosomal subunit (70S ribosome)

what is the process of translation in prokaryotes?

1. transfer RNAs (tRNAs) deliver amino acids to matching codons in the mRNA template in the A-site

2. peptide bonds are formed between the aa in the A-site and growing polypeptide in the P-site

3. the ribosome advances one codon, shifting tRNA positions and releasing empty tRNAs from the E-site

what are the 3 sites of a prokaryotic ribosome?

1. A → aminoacyl site (acceptor)

2. P → peptidyl site (grows polypeptide)

3. E → exit site

what are 3 examples of protein synthesis inhibitors to remember?

1. tetracyclines

2. clindamycin

3. macrolides

what are the 3 ribosomal targets?

1. block the amino-acyl tRNA binding between P & A sites (this inhibits peptide bonds formation from happening) in 50S subunit → ex. chloramphenicol

2. block polypeptide exit tunnel (E-site) in 50S subunit → ex. macrolides

3. block amino-acyl tRNA from binding to the A site in 30S subunit → ex. tetracyclines

4. bind to the A-site and cause misreading of mRNA (creating faulty proteins) in 30S subunit → ex. aminoglycosides (doesn’t stop translation but makes it awful)

**helps overcome resistance since we have many options

what is a gram-positive (+) cell wall?

-predominantly peptidoglycan (up to 90%)

-porous

-most substances pass through (including antibiotics + nutrients and things it needs to survive)

-also have teichoic acid

-has N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM)

what is teichoic acid?

-it is embedded between PG (peptidoglycan) layers and creates a negative charge

-its important for bacteria and maintaining pressure inside the cell

-has 2 types

what are the 2 types of teichoic acid?

1. wall teichoic acid

2. lipoteichoic acid

what is wall teichoic acid?

-its covalently linked to PG (peptidoglycan)

what is lipoteichoic acid?

-its connected to cell membrane with a lipid anchor

what is a gram-negative (-) cell wall?

-only 5-10% peptidoglycan

-has outer membrane, lipopolysaccharide, porins (that live in the outer membrane), and inner membrane

-harder to get into

what is the outer membrane?

-it is composed of a lipid bilayer (similar to the inner membrane)

what is lipopolysaccharide (LPS)

-it faces into the environment

-similar to teichoic acids in gram-positive

• contributes to a negative charge

• stabilizes membrane

• protection from antibiotics (removes ease in which drugs can enter the cell)

what are porins?

-they live in the outer membrane

-transmembrane proteins that transport molecules

-can be selective against antibiotics which is in favour of our bacteria

what is peptidoglycan (PG)?

-provides structure and prevents osmotic lysis (cell burst from water entering the cell)

-composed of 2 glucose derivatives that form a disaccharide (NAM and NAG), in alternating chains

what does NAG stand for?

-it stands for N-acetylglucosamine

-aka NAG or GlcNAc (prof uses this more)

what does NAM stand for?

-it stands for N-acetylmuramic acid

-aka NAM or MurNAc (prof uses this more)

what is osmotic lysis?

-it is when the cell bursts from water entering the cell

how are the chains linked in PG

-they are linked by peptide cross-bridges of varying composition (2-5 amino acids long)

-differs based on bacteria

-forms 4 → 3 cross-links

why does PG use D-amino acids?

-it does this to protected from proteases and degradation

-it helps keep the cell wall safe

what are the L-amino acids for in PG?

-that are used to allow our proteins to be detected by the ribosme

what is the tetrapeptide cross-linking made out of in the PG?

-it is made out of AAs

where does PG biosynthesis start? as what?

-it begins in the cytoplasm, as Lipid II (a precursor formation)

what is the process of Lipid II biosynthesis?

1. starts in the cytoplasm as UDP-MurNAc pentapeptide

2. then UDP-MurNAc pentapeptide + C55 undecaprenyl phosphate chain (lipid tail) (in the inner membrane) interact with enzyme MraY to create Lipid I

3. then Lipid I interacts with enzyme MurG + UDP-GlcNAc to create Lipid II

4. since the lipid tail is anchoring the entire Lipid II in the inner membrane, it needs to get out of the cell

5. so Lipid II interacts with the enzyme flippase to get it out of the cytoplasm and into the periplasm (the lipid tail, since hydrophobic, stays in the inner membrane but rest of lipid II is on the periplasm side)

6. then once lipid 2 is in the periplasm it interacts with penicillin binding proteins (PBPs) creating peptidoglycan

what do the 2 phosphates in UDP-MurNAc pentapeptide do?

-they help the reaction (between C55-P + UDP-MurNAc) happen and reacts with the phosphate group on C55-P

what does the enzyme MraY do?

-it is responsible for reacting to the lipid tail (C55-P) and helps with the release of UDP and lose on phosphate, so that the lipid tail and (now) lipid I can stick together

what does flippase do in lipid II biosynthesis?

-it translocates lipid II to the outerface of the inner membrane

what is UDP?

-it stands for uridine diphosphate and helps facilitate the reactions in lipid II biosynthesis

what does penicillin binding proteins (PBP) do in lipid II biosynthesis?

-it helps stick together bits to make peptidoglycan

what does the lipid tail do?

-it keeps lipid I and II anchored /attached to the inner membrane

what is the final precursor to PG?

-lipid II is the final precursor to this

what is lipid II ready to do once its made?

-it is the final precursor to PG, so it is ready to be incorporated by penicillin-binding proteins (PBPs)

what does PBP domains with transglycosylase activity do?

-they incorporate the disaccharide core of Lipid II into the growing glycan strand

• transglycosylase → aka glycosyltransferase

what does PBP domains with transpeptidase domain do?

-they catalyze the formation of 4 → 3 cross-links between peptide strands

→ 4th residue of one peptide chain is linked to the 3rd residue of another

what are the 2 domains of a PBP?

1. transpeptidase

2. glycosyltransferase

• 1 protein with 2 domains

what does transpeptidase do in PG biosynthesis?

-it helps incorporate peptides into the peptide strand

what does glycosyltransferase do in PG biosynthesis?

-it helps stick together our disaccharide component

why do we need to know how PGs and lipid II are made?

-we need to know how these are made so that we know how to stop them

what is the process of peptidoglycan (PG) biosynthesis?

1. lipid II + polysaccharide bone interact with transglycosylase (PBP), which sticks the 2 sugars together

2. we lose lipid tail (undecaprenyl pyrophosphate) because we do not need it anymore

3. now that the 2 sugars are stuck together, with the backbone alternating NAM-NAG-NAM-NAG… it interacts with D₂D-transpeptidase (PBP)

4. this causes it to lose 1 D-Ala, the 5th AA from one of the chains causing the 4→3 cross-link to occur

what are the 2 examples of inhibitors of the cell wall growth highlighted in class?

1. β-lactam antibiotics

2. glycopeptide antibiotics

what are β-lactam antibiotics?

-they are broad-spectrum, bactericidal natural products

-extremely common (2/3 of prescribed antibiotics on the market)

-they are the death star of antibiotics

-they were the first ones we discovered

-they use structural mimicry to bind to the active site of PBP transpeptidases, inhibiting PG cross-linking essential for cell wall synthesis

(ex. penicillin → D-Ala-D-Ala motif)

what are glycopeptide antibiotics?

-they are narrow-spectrum, bactericidal natural products (Gram +)

-they bind to D-ala-D-ala motif of peptidoglycan precursors, preventing PG incorporation

-they are used as a drug of last resort

-the resistance of these in the clinic is a growing concern

why are glycopeptide antibiotics a last resort?

-we don’t like to use it unless we have to, which protects its efficacy meaning the less we use the less resistance we will see of it out there

what is the process of action for glycopeptide antibiotics?

1. glycopeptide (such as vancomycin) binds to D-Ala-D-Ala

2. which then inhibits PG synthesis

3. causing bacterial growth inhibition and disruption of cell wall

how does β-lactam inhibit cell wall growth?

-it binds to the active site of PBP transpeptidase to prevent 4→3 cross-linking

how does glycopeptide inhibit cell wall growth?

(ex. vancomycin)

-it binds to terminal D-Ala-D-Ala to prevent PG incorporation

→ physically blocks the step that incorporates sugars

what does incomplete PG biosynthesis do?

-it compromises the integrity of the cell envelope, leading to osmotic stress and bursting of the cell

how old are antibiotics?

-they are older than dinosaurs

how did antibiotics come to exist?

-It all began in the dirt with antibiotic-producing bacteria

→ bacteria produced them

why were antibiotics made by bacteria?

-they weren’t made by bacteria for us to harness and utilize, they are made by bacteria for their own competitive advantage, we just figured out how to make it something that works for us

what is petrichor?

-it is the earthy smell after rain

what is the earthy smell after rain due to?

-it is due to the compound geosmin

what is geosmin?

-a compound that makes the earthy smell after rain through petrichor

what is geosmin produced by?

-it is produced by a phyla of soil bacteria called Actinobacteria/Actinobacteriota