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Flashcards in Antibiotics acting on Cell Wall Biosynthesis Deck (31):
1

Three general types of bacteria based on outer membrane

GP
GN
Acid Fast GP; do not stain with gram stain.
Mtg are coated with mycelia acids (C56-C90); covered in a giant layer of grease; hard to get AB into these things.
Peptidoglycan synthesis is the target for antibiotics that act on the cell wall.
Cytoplasmic membrane and on the outside of theta you he the peptidoglycan.
Outside this PG on the GN bacteria you have an outer membrane; makes more resistant.

2

GP Cell Wall

PTG is comprised of sugars cross linked via peptide bonds.
Lipoteichoic acid (LTA) is on the outside of these things.
Focus on clinically approved drugs.
PTG; layers of sugars cross linked with peptides.
2 types of sugars in PTG wall.

3

GN Cell Wall

Antibiotics acting on cell wall biosynthesis must reach the periplasm to affect Gram negative.
LPS= Lipopolysaccharide
A lot more going on
Periplasm is the PTG; sugars cross linked by peptides.
Then outer membrane, then outside of this is LPS.
LPS is something that humans detect pretty well in terms of mounting up an immune defense.
LPS; Lipid A; all these sugars that come out of the end of it; final terminus of cross linked sugars is called the O antigen.
AB that act on the cell wall for GN need to get into periplasm; need to cross through a porin; size is important; large, charged peptides that hit GN, most large molecule don't have any effect on GN because they cannot get in the space.
GP, the whole thing is on the outside.

4

PTG Biosynthesis

Starts in cytoplasm where you have to build the sugars.
Build peptide
Anchor peptide on inner side of cytoplasmic membrane.
D-Ala-D-Ala, in bacteria you see a lot of D AA (humans are L).
Assemble peptides and sugars and then somehow flip out to outside, then cross link sugars and cross link the peptides.
Build sugar backbone and cross link the peptides.
3 distinct areas where an AN can act in terms of inhibited PTG:
Starts in cytoplasm (starts with sugar synthesis) with the UDP-GlcNAc; N acetyl glucosamine.
PTG; composed of sugars, lipids, and peptides.
Cycloserines, uridylepptides inhibit things on the cytoplasm side.
Drug does not have to cross into the cell especially for GP.
Sugars in cytoplasm, pentapeptides that is attached to sugars then needs to flip across membrane to get to the outside then connect sugars and peptides and build a network on the outside of the cytoplasmic membrane that protects the bacterium and provides structural rigidity for bacterium.

5

Differences and Similarities in GP and GN

Differences: membrane permeability barriers; a lot of AB are broken down into spectrum (does it hit GN or GP or both, broad spectrum).
GP have really thick multiple layers of PG.
GN have a PG layer and also have an outer membrane.
Similarities:
How PG is biosynthesized; link sugars together than have a pentapeptide.
PG elongation by transglycosylase (TGase) action.
PG cross-linking by transpeptidase (TPase)..
Outer membrane:
Provide additional barrier that AB have to penetrate to get to PG.
GP; penicillin is small and can penetrate through porin (outside of GN).
Penetration of AB to the cytoplasmic membrane are inhibited by GN; Vancomycin (large size and cannot squeeze through porins) for GP only and penicillin for both GP and GN.

6

Peptidoglycan Assembly

Lipids, Sugars and Pentpeptides
C55 complex that tethers the sugars on the outside of the cytoplasmic membrane.
Cross link sugars then get cross linking of peptides.
1. In cytoplasm:
Sugar, lipid, lipid 1, and lipid 2 biosynthesis.
2. Translocation of lipid 2 from cytoplasm to periplasm (unknown); C55
3. In periplasm, Lipid 2 polymerization, PG cross-linking, and lipid recycle

7

PG Assembly: Cytoplasm

Whole process starts with the uridine diphosphate sugar: N-acetyl glucosamine.
2 enzymes:
MurA, PEP (phosphoenol pyruvate); catalyzes the esterification of OH group, replaces phosphate group that is on the PEP; have acid attached at that OH.
MurB, NADPH (proton transfer); catalyzes reduction of double bond, go to methyl group.
Side chain attached to the sugar comes from PEP and is canalized by 2 enzymes.
Acid side chain; provides tether
Need to attach N-terminus of AA to this sugar backbone.
FOSFOMYCIN; used for UTI; inhibits this first step, MURA enzyme; structural mimic for PEP; gets in enzyme and epoxide gets opened up and inhibits the enzyme.
Once we have acid side chain, we have UDP-MurNAc
Now have enzymes that will work at Carbox. acid and attach pentapeptide at that position:
Attach alanine
All enzyme used ATP to do this.
L-alanine; form amide bond
Then D-glutamic acid (first one in side chain).
Then DAP; diamminopamenic acid; 1 amino groups, used for forming amid bone between glutamic acid and the other is free.
Have second amino group on the end.
Then attach D-Ala D-Ala; attached as the dipeptide; MurF with the ATP binds DalaDala.
This is now the UDP-MurNAc Pentapeptide (lipid-free).

8

Peptidoglycan Assembly

Attach the lipid
Bacteria needs to prepare itself to work near the membrane and flip everything on the outside of cytoplasmic membrane.
C55 undecaprenyl (building blocks to make cholesterol); nice lipid interface for this molecule.
Undecaprenyl hangs out in membrane; MRAY catalyzes reaction; take monophosphate on end of C55 and nucleophilic attack; leaving group is uridine with one phosphate. UMP leaves, MRAY acts as a transferase to form lipid 1.
Are inhibitors of this: tunicamycin (not clinically approved); acts on MRAY to inhibit lipid 1 synthesis.
No clinically used AB to inhibit this enzyme.
Lipid 1 (1 sugar) and lipid 2 (2 sugars) difference is the number of sugars.
Now form the disaccharide
UDP-GlcNAc; same sugar MurG catalyzes reaction where health attack of the OH on this sugar (muramic acid) to replace the O-UDP (leaves); catalyzes this disaccharide; MurG is a transglycosylase; transferees sugar across; have disacchrade (lipid2) and have the pentapeptide.
Lipoglycopeptide ramoplanin has been suggested to inhibit Lipid 2 synthesis (acts on MurG-remains controversy); does this really inhibit MurG?
The MRAY inhibitors are not used clinically, and ramoplanin is in developmentto treat C diff (2,3,4 years will see in clinic).
Now we have lipid 2 and is anchored into the membrane
Start MRAY catalyzes transfer of sugar and pentapeptide to C55 lipid;; anchors into cytoplasmic membrane
MURG catalyzes formation; tranglycosylase, transfers sugar, now have disaccharide, in cytoplasm, have disaccharide, pentapeptide attached to C55 lipid via diphosphate.
Now have to translocate where whole thing needs to flip across membrane; mechanism is unknown; represents future drug target.
On exterior of cytoplasmic membrane we have lipid 2.
First step when on external space in periplasm is cross linking of sugars.
Footings that represent sugars, walls are going up based o pentapeptides, builds up distance away from surface.
Enzyme is a transglycosylase similar to MurG, adds lipid to growing chain of disaccharide, continue building disaccharide base, polymerize sugar with pentapeptide sitting out.
MOENOMYCIN AND CLOROBIPHENUL-VANCOMYCIN inhibit this transglycosylase step.
Once this formed have ability to cross link peptides.
Inner strand cross linked also; forms layers of sugars and more peptides; complicated.
C55 group has a diphosphate on the end; phosphatase that plucks off 1 of these groups and recycles this to the undecaprenyl monophosphate; this is required; recycles undecaprenyl system.
BACITRACIN inhibits this enzyme PPiase, works great to basically shut down process of PG biosynthesis.
Common in triple antibiotic topical ointments.
Monophsohate is flipped back into cytoplasm and is ready to undergo attachment of sugar via MRAY.
Transpeptidase cross links peptide strands; a lot of these; while family of these that vary across different bacteria; PENICILLINS AND CEPHALOSPORINS act on this transpeptidase and block transpeptidation step; all groups of AB act on outside of membrane; not requirement for these to penetrate; GN need to penetrate into periplasm where these enzymes are present do not have to penetrate cytoplasmic membrane.

9

PG Assembly (Periplasm)

Only a few compounds have been found to inhibit the transglycosylation step of PG assembly; such as moenomycins (not clinically used agent) and chlorbiphenyl-vancomycin.
Hydrolyzing ATP the phosphate bonds are easily hydrolyzed; so phosphate makes good LG and transglycosylase catalyzes attack of OH On this long chain of sugars to replace that phosphate group; bind is formed, now C55 diphosphate is now untethered and have long chain of saccharide units.
Some bifunctional transpeptidase transglycsylases; whether inhibit one or the other can be blurry.

10

PG Assmebly-Tpase

The beta lactam antibiotics: the most celebrated antibiotics that kill bacteria by blocking the PG transpeptidases (TPase).
Penicillins and Cephalosporins
Tpases are essentially serine active site Tpases.
Serine in active site that comes in and attacks the end of peptide; end have 2 D alanines that kick off one of the D alanine; activates as an ester; now DAP, free amine group (not important to know which amino acid is the free one because some bacteria use ornithine or lysine), all use something with amen group; Tpase catalyzes reaction, activates and catalyzes the attack of N, release from enzyme and have DAP, D-alanine isopeptide bond cross link.
Serine based active site enzyme.
Penicilins and cephalosporins come in ant act as peptide mimic and get into active site and instead of attacking, opens up an activated ring that is present on drugs, act as suicide substrate for these Tpases.
Resistance mechanisms; inactivating the warhead on these molecules.
Cidal AB.
Have to be actively growing for drugs to work; needs be doing maintenance on PG for these to work.
During process of Tpases breaks down; PG is a structural support; Osmotic pressure with bacterial cell; contained by PG; if PG weakens, bacteria explodes because osmotic pressure overcomes ability of PG to hold it together.
Contents leak out.

11

Penicillin Binding Proteins

PBPs
Tapes are all variants of active site "Ser" hydrolyses.
The beta lactam ABs act as suicide substrates for PG Tpases, and autoradiographs of 14C-penicilloyl-proteisn (PBPs) of E coli and other pathogens separated on SDS-page.
PBPs refer to proteins that can be labeled by penicillin, which include Tgase, Tpase, as well as many other enzymes.
THERE ARE BIFUNCTIONAL TRANSPEPTIDASES/TRANSGLYCOSYLASES; distinguish what you're inhibiting with AB is more complicated (vancomycin is where this becomes a little compicated).
Beta lactam strain amide bond ring; Tpase; attack carbonyl and opens it up, stall at this position, now enzyme is dead and cannot continue cross linking peptides.
Next step; attack with water, the enzyme would be free again and the ABs wouldn't work.
Major form of resistance; beta lactamases; active site serine, do this thing but the attack of water and releases inactivated drug.
PBP: something that can be radio labeled with radioactive penicillin.

12

Beta Lactam Antibiotics

1. Penicillins; 5 membered ring with S at the top; expand ring and go to 6, then get cephalosporins.
2. Cephalosporins
3. Carbapenems; Carbon instead of S
4. Monobactams; one ring
5. Clavams; calvulani acid; not used as AB; juice substrates for beta lactamases; used in combination with other beta lactams.

13

Five generations of Penicillins

On the basis of narrow vs broad spectrum activities and whether there is antipseudomonal activity.
Bacteriacidal.
First choice, don't have to penetrate and very few side effects because no human targets.
Hypersensitivty reactions are the most common side effects.
Largely based on evolution of core in terms of.....

14

1. Narrow Spectrum Penicillinase-Sensitive

PenG: Poor Acid stability
PenV: Good Acid Stability
Beta lactase sensitive

15

2. Narrow Spectrum
Penicillinase (another term for beta lactamase, active site serine that can hydrolyze beta lactam warhead and inactivate the drug). Resistant

Methicillin
Oxacillin
Beta lactamase resistant
Penicillinase Resistant due to bulky side chains.

16

3. Broad spectrum Aminopenicillins

Ampicillin
Amoxicillin
Orally active, penicillinase sensitive; active again H. influenza, E coli.

17

4. Broad Spectrum Antipseudomonal

Carbenicillin
Ticarcillin
Improvement of outer membrane penetration because of side chains.
IV administration, active against P. aeruginosa.

18

5. Extended Spectrum

Piperacillin
Active against P.aeruginosa.
Increased activity against Enterobacteriacae (IV).

19

1st to 4th generation Cephalosporins

Side chain modifications have led to differential penetration through the porin-GN active.
Varied antibacterial and pharmacokinetic properties.
Hypersensitive reaction are the most common side effects.

20

1. First generation cephalosporins

Cephalothin
Cephazolin
Narrow spectrum
Best for GP

21

2. Second generation cephalosporins

Cefamandole
Cefuroxime
Cefoxitin
Expanded Spectrum
Good for GP
Better for GN; targeting GN organisms.

22

3. Third generation Cephalosporins

Cefotaxime
Ceftriaxone
Ceftazilime
Broad spectrum
Optimized for both GP and GN.

23

4. Fourth generation

Cefipime
Broad spectrum for both GP and GN
beta lactamase (penicillinase) resistant; inactivates warheads.

24

5th Generation cephalosporins

Ceftaroline; approved in the US
Ceftobiprole; approved in Canada and Europe
Unique aspects; broad spectrum GP and GN.
Both bind to PBP-2a (enzyme), a PBP responsible for primary resistance of MRSA.
Methicillin; o methoxy side chains; bully side chains result in molecule being resistance to hydrolysis by beta lactase or penicillinase.
MRSA not resistant to methicillin because of a beta lactamase; resistant because uses a different PBP that has low affinity for all penicillins and cephalosproins presented previously.
Have ability to inhibit PBP-2a (bifunctional tranpeptidase/transglycosylase).
These 2 can inhibit PBP2a; active against MRSA!!!!!
Highly modified

25

Structure-activity relationships for ceftaroline

Thiazole ring; anti MRSA activity
Thought to help with affinity for PBP-2a.
Phosphono group that gets hydrolzyed as a prodrug.
Has charged group that helps with solubilizing drug.
Another group that gives it beta lactamase resistance as well.

26

Glycopeptide AB

Glycopeptide AB like vancomycin and teicoplanin act by forming complex with the uncross-linked peptide strands hence blocking the transpeptidation step.
Hypersensitvity, auditory impairment, ototoxicity, and nephrotoxicity are common side effects (follow blood level drug concentration); more than beta lactams.
Very large complicated structures.
Cannot penetrate porins in GN, only used in GP.
Peptide backbone
Don't look like average peptide, functional groups look like tyrosine, some are modified with additional O, C, chlorine, highly modified peptide backbone.
Glyco-sugar attached

27

Glycopeptide mechanism of action

Binding affinity to DalaDala and block cross linking; some block transglycosylation steps as well, so big; other ways that newer ones interact with membrane.
Glycopeptides cannot penetrate the pores of the GN outer membrane; restricted to treating life threatening GP pathogens such as staphylococcal, streptococcal, and enterococcal infections.
Things resistant to beta lactam AB.

28

Five Types of Glycopeptides

Type 1: vancomycin
Type 2: beta-avoparcin
Type 3: ristocetin A
Type 4: Teicoplanin; lipid chain up on sugar
Type 5: Complestatin; more simple and a lot go halogenation.
Transition form type 1 to type 2 has to do with what the AA is; go from leucine to tyrosine; cross linking into type 3
Not orally bioavailable so IV or IM injection.

29

Recent Approvals: Complicated skin and skin-structure infections (cSSSI)

Televancin; MRSA
Dalbavancin; half life of 2 weeks, single dose and patients leave the hospital; MRSA; decreases costs.
Different than vancomycin
Chloro-biphenyl vanco; difference in these in if they can inhibit Transglycosylase step; MRSA
Resistance phenotypes for vanco type AB is complicated.

30

Bacitracin Mode of Action

Unique antibiotic
Bacitracin inhibits the PPiase by forming a complex with the C55 lipid phosphate.
See in topical ointments; triple AB.
C55 bisphosphate needs to be recycled; first step needs, extra P needs to be removed; happens via PPiase that cleaves off (phosphatase); does this by coordinating with Mg coordinating with bisphosphate end group and binds substrate and blocks action of this enzyme.

31

Summary

Inhibition of precursor synthesis (in cytoplasm).
Inhibition of PG assembly (in periplasm).
Inhibition of peptidoglycan cross-linking (in periplasm and cell wall).
Bactericidal but inactive against resting cells.
Major classes of antibiotics:
1. beta lectams: classified based on their spectrum and sensitivity to beta lactamases:
-Penicillins; 5 membered ring (5 generations)
-Cephalosporins 6 membered ring (5 generations)
Other beta lactams (carbapenems, monolactams, clavams).
2. Glycopeptides:
-Vancomycin, Teicoplanin, chlorobiphenyl-vancomycin.
Functionally not a big different, but difference in resistant phenotypes and what they treat.
3. Others:
-Fosfomycin, tunicamycin, lipsidomycin, mureidomycin, moenomycin, bacitracin.