Exam 1- Cushman Lec 1- Bacterial Cell wall + Beta Lactams

Question 1

How does the structure of the cell wall in Gram (+) bacteria differ from Gram (-) bacteria?

Answer 1

Gram (+):
-Drugs penetrate the outer layers of cell wall effectively
-Bacterial membrane is the main barrier
-B-lactamases excreted through cell wall to external environment
-B lactamases produced in larger quantities
-Thick peptidoglycan layer
-Have 1 membrane
-Peptidoglycan residue is replaced by L-lysine residue (COOH is replaced with H)
-Peptidoglycan is cross-linked by a bridge between the L-Lys strand and the terminal D-Ala of a second molecule

Gram (-):
-Outer membrane excludes drugs, preventing penetration
-Porins in outer membrane allow some drugs to pass
-B-lactamases are confined to periplasmic space
-Thin peptidoglycan layer
-Have 2 membranes (inner + outer) with periplasmic space between -have a more complex wall that is lipoidal
-Peptidoglycan contains meso-diaminopimelic acid residue (DAP)
-Peptidoglycan is cross-linked by a bridge between DAP residue of one strand and the terminal D-Ala of another

Question 2

What is the role of beta lactamases?

Answer 2

Hydrolyze beta lactam and inactivate the drugs

Question 3

Which enzyme is responsible for cross-linking peptidoglycan strands?

Answer 3

Transpeptidase

Question 4

How does transpeptidase cross-link peptidoglycan strands?

Answer 4

-Covalent bond

-Creates a Gly bridge between:
Gram (-): DAP residue of one strand and D-Ala of another
Gram (+): L-Lys strand of one and terminal D-Ala of another

-Transpeptidase active site has a serine residue (CH2OH)

Question 5

Why is cross-linking of peptidoglycan strands important?

Answer 5

Confers strength to the bacterial cell wall

-cell wall will lyse and the bacteria will die without it

Question 6

What well-known antibiotic is a beta-lactamase?

Answer 6

Penicillin

Question 7

What is the mechanism of action of beta-lactam antibiotics?

Answer 7

Inhibit the transpeptidases that cross-link the peptidoglycan strands in the bacterial cell wall together

Question 8

How do beta-lactam antibiotics inhibit peptidglycan?

Answer 8

They acylate the transpeptidase Ser residue in the enzyme active site to form a stable product

-this inactivates the transpeptidase and inhibits cross-linking, causing a defective bacterial cell wall

-cell wall is now subject to osmotic stress and the bacterial cell lyses

(see next notecard)

Question 9

How does penicillin fool the transpeptidase into believing it is a peptidoglycan?

Answer 9

-Causes the transpeptidase to think it is a D-Ala-D-Ala residue at the end of a peptidoglycan
*peptidoglycan attacks the double bonded O just like it does in peptidoglycan (carbonyl)

-Forms a tetrahedral intermediate
*electrons move from H to O, forming another double bonded O (carbonyl)
**this breaks the four-membered ring

Question 10

Why are penicillins/ beta-lactams reactive?

Answer 10

They have a four-membered ring
*this causes a lot of ring string and therefore, reactivity

Question 11

What are the mechanisms of resistance that can occur against beta-lactams?

Answer 11

Decreased cellular uptake of the drug

Mutation of the penicillin-binding proteins that decreases their affinity for penicillins

Presence of an efflux pump that pumps antibiotics out of the cell

Induction/elaboration of bacterial B-lactamases that catalyze the hydrolysis of the B-lactam moiety
**note that this resistance mechanism is basically the same thing as the moa mechanism. Carbonyl is attacked, forms intermediate, ring structure breaks
–creates a regenerated B-lactamase and unstable hydrolyzed penicillin

Question 12

What percent of the US population is allergic to B-lactams?

Answer 12

6-8%

Question 13

What causes the allergenicity seen with B-lactams?

Answer 13

The drug acts as a hapten

-acylates host proteins, raising antibodies, and resulting in an allergic reaction

Question 14

If a person is allergic to one penicillin can they be given another?

Answer 14

NO

cross-sensitivity is common, so if a person has an allergy to one B-lactamase, they are likely to have an allergy to another

Question 15

What tests are available to test for B-lactam allergy?

Answer 15

Topical flare and wheal tests

Question 16

Under which conditions do penicillins degrade?

Answer 16

Acidic and Basic

Question 17

How does pencillin degrade under acidic conditions?

Answer 17

-The side chain has echimeric assistance (participates in mechanism of reaction to assist hydrolysis)

-Side chain carbonyl attacks the main carbonyl to form a five-membered ring

*Penicillin breaks down to Penicilloic acid

*Note that the nucleophilicity of the R on the side chain determines the reactivity. If electron attracting (electronegative) the O is less nucleophilic and penicillin is more stable. If electron-donating, the O is more nucleophilic and less stable (cannot be given po)

-Creates a penillic acid

-Also can create a penicillenic acid

Question 18

How does penicillin degrade under basic conditions?

Answer 18

OH- group attacks main carbonyl and creates Penicilloic acid

Question 19

True or False: Penicillin hydrolysis products have antibiotic activity

Answer 19

False
-they have to antibiotic activity

Question 20

True or False: Hydrolysis of the B-lactam is irreversible

Answer 20

True
-once the 4-membered ring is opened, it cannot close again

Question 21

True or False: Electronegative substituents on the side chain carbonyl reduce the nucleophilicity of the side chain and makes the B-lactam more stable

Answer 21

True

-the penicillin is stabilized against hydrolysis under acidic conditions
-the first step in the hydrolysis reaction is decelerated

Question 22

What element present in the side chain will make it more electronegative, and therefore stabilize the penicillin?

Answer 22

O

-Penicillin V is more stable than Penicillin G because V has an extra oxygen in its side chain which is electronegative and decreases the nucleophilicity of the main carbonyl

Question 23

What pH is best for penicillin storage?

Answer 23

6-6.8

Question 24

What molecules can catalyze penicillin degradation reactions, and should therefore be kept away from penicillin solutions?

Answer 24

Heavy metal ions

Question 25

How does lipophilicity of the penicillin side chain affect protein binding?

Answer 25

More lipophilic side chains result in more protein binding

Question 26

How does protein binding affect the action of B-lactams?

Answer 26

Protein binding reduces bioavailability -reduces the effective concentration of the free drug

Question 27

Penicillins are excreted by which routes?

Answer 27

Renal Biliary

Question 28

For penicillins excreted by the kidneys, what percent of excretion is glomerular vs tubular?

Answer 28

Glomerular filtration: 10% Tubular secretion: 90%

Question 29

How is penicillin excretion affected by kidney disease/failure?

Answer 29

Penicillin half-life increases

Question 30

What are the 2 mechanisms of tubular secretion?

Answer 30

One for anions and one for cations *Note that penicillins are anions

Question 31

How does probenecid affect penicillin excretion?

Answer 31

Penicillins are anionic and so is probenecid -these two drugs compete with each other for secretion mechanisms -therefore, when probenecid is administered with penicillin it increases the half-life of penicillin

Question 32

What are the 5 nomenclatures to know for B-Lactam ring systems?

Answer 32

These are added to the four-membered ring of penicillins: Penam (5-membered ring with S and no double bond) Penem (5-membered ring with S + double bond) Carbapenem (5-membered ring with no S but has a double bond) Cephem (6-membered ring with S + double bond) Monobactam (no extra ring, just NH)

Question 33

What are the B-lactam sensitive penicillins?

Answer 33

Benzylpenicillin (Penicillin G) Phenoxymethyl Penicillin (Penicillin V)

Question 34

What points are important to know about Benzylpenicillin (Penicillin G)?

Answer 34

Works on: Gram + cocci B-lactam sensitivity: Yes Admin: Parenteral Precautions: In individuals with history of allergies/asthma

Question 35

What is the main difference between Penicillin G and V?

Answer 35

V is more stable in acid (can give po) -due to extra oxygen on side chain (more electrophilic)

Question 36

What are the B-lactamase-resistant parenteral penicillins?

Answer 36

Methicillin Nafcillin

Question 37

What causes methicillin to be resistant to B-lactamase?

Answer 37

Steric hinderance -unable to have nucleophilic attack by the enzyme on the B-lactam carbonyl

Question 38

What are important points to know about methicillin?

Answer 38

-Unstable to acid in the stomach, must be administered by injection -Discontinued due to resistance

Question 39

Why is methicillin so unstable in stomach acid?

Answer 39

The oxygen on the side chain, instead of reducing electronegativity of the main carbonyl, increases it -oxygen in the side chain moves electrons to the carbonyl and makes it more electronegative -this is due to the resonance effect

Question 39

What is the most important bug to remember that is resistant to methicillin?

Answer 39

MRSA (methicillin-resistant staphylococcus aureus) -because of mutation in transpeptidase

Question 40

What gene codes for the mutated penicillin-binding protein (transpeptidase) in MRSA?

Answer 40

mecA (methicillin resistance gene)

Question 41

What penicillin binding protein is coded for by mecA?

Answer 41

PBP2A *this is the protein found in MRSA *it can cross-link peptidoglycan but does not react with methicillin and many other B-lactams

Question 42

What are the important points to remember about Nafcillin?

Answer 42

-Not sensitive to beta lactamase -Slightly more stable than methicillin in acid

Question 43

What are the B-lactamase-Resistant oral penicillins?

Answer 43

Oxacillin Cloxacillin Dicloxacillin

Question 44

Why are oxacillin, cloxacillin, and dicloxacillin B-lactamase resistant?

Answer 44

They all have an isoxazole structure and large 6-membered ring that sterically hinders the carbonyl

Question 45

Which B-lactamase resistant penicillin is the only one still used orally?

Answer 45

Dicloxacillin

Question 46

What are the B-Lactamase-Sensitive, Broad-Spectrum, Oral Penicillins?

Answer 46

Ampicillin Amoxicillin

Question 47

What organisms are sensitive to ampicillin?

Answer 47

Gram- (Salmonella, Shegella, Proteus mirabilis, Escherichia coli, Haemophilis influenzae, Neisseria gonorrhea)

Question 48

How is ampicillin able to be transported into Gram - bacteria?

Answer 48

The porin channels are hydrophilic and transport ionic compounds -ampicillin has a charged amino group at physiological pH, allowing it to be transported through porins

Question 49

Why is ampicillin stable in acid?

Answer 49

The amino group is protonated in the stomach -the charged nitrogen group becomes more electron-attracting -this decreases nucleophilicity of the carbonyl and it does not participate in ring-opening

Question 50

What points are important to remember about amoxicillin?

Answer 50

-Analog of ampicillin ---only difference is the addition of a hydroxyl group to the aromatic ring **Better oral absorption than ampicillin

Question 51

What are the B-lactamase inhibitors?

Answer 51

Clavulanic Acid Sulbactam Tazobactam Avibactam

Question 52

When are B-lactamase inhibitors used?

Answer 52

In combination with B-lactamase-sensitive penicillins -enhance the activity of these penicillins against resistant strains

Question 53

What is the MOA of the B-lactamase inhibitors?

Answer 53

Acylate the serine hydroxyl group in the active site of the B-lactamase

Question 54

What is the brand name of Potassium Clavulanate + Amoxicillin?

Answer 54

Augmentin

Question 55

What is the brand name of Sulbactam + Ampicillin?

Answer 55

Unasyn

Question 56

What is the brand name of Sulbactam + Pipericillin?

Answer 56

Zosyn

Question 57

What is the bran name of Avibactam + Ceftazidime?

Answer 57

Avycaz

Question 58

What is the B-Lactam-Sensitive, Broad-Spectrum, Parenteral Penicillin?

Answer 58

Piperacillin

Question 59

What bacteria is piperacillin active against?

Answer 59

Gram - Gram +

Question 60

Why does piperacillin have enhanced potency over the B-lactam-sensitive oral penicillins?

Answer 60

The added side chain fragment on piperacillin resembles a longer section of the peptidoglycan chain than ampicillin