(2/7) Lee Lecture: Chemistry and Pharmacology of Antimicrobials – Cell Wall Inhibitors Bacterial Cell Wall

- Function: The bacterial cell has extraordinarily high internal osmotic pressure, therefore the presence of the cell wall is critical for the survival of the cell. The Cell wall also functions as an anchoring point for various proteins

Gram-Staining Dichotomy - Bacterial pathogens are often classified by their cell wall structure. While the gram stain is not definitive, the

result of the test does give insight on potential susceptibility and appropriately indicated abx - Gram-Negative: Inner Membrane + Thin Peptidoglycan Cell Wall + Outer Membrane (With LPS)

o Peptidoglycan layer accounts for 90% of the cells weight, can be 20-80nm thick! - Gram-Positive: Inner Membrane + Thick Peptidoglycan Cell Wall (with WTA)

o Between the inner membrane and peptidoglycan layer is the periplasmic space Peptidoglycan Composition: There are 3 major components: 2 sugars + 1 stem peptide forming the Lipid II building block

- Sugar Residues: N-acetyl-glucosamine (NAG) and N-acetyl-muramic acid (NAM) - Stem Peptide: Consists of 5 AA, all of which are well-conserved besides the 3rd residue

o L-Ala—D-Glu--*DAP/L-Lys*--D-Ala—D-Ala - Together these form the building block: Lipid II. It is synthesized in the cytoplasm

o (1) NAG-Uridine-Diphosphate (UPPG) is converted to NAM-Ur-Diph (UPPM) o (2) UPPM in succession picks up L-Ala, D-Glu, and DAP o (3) UPPM-1,2,3 then picks up the 4th and 5th AA as a dipeptide o (4) UPPM-1,2,3,4,5 reacts with transmembrane Undecaprenol to become covalently bonded to the inner

membrane. In the process, Uridine is kicked out. The Muramic acid residue now is nucleophilic o (5) Undecaprenol-PP-M-1,2,3,4,5 attacks cytoplasmic UPPG, forming the complete Lipid II

- Lipid II is a hydrophilic compound, however is made hydrophobic upon binding with Undecaprenol, providing the capability for Lipid II to be flipped into the periplasm. It is then attached to the growing peptidoglycan polymer

- Morphology: The Peptidoglycan architecture is maintained by crosslinking. This occurs between neighboring strands through the stem peptide tails, accounting for the rigidity of the cell wall.

o à Crosslinking is catalyzed by enzyme Transpeptidase (TPase) between two terminal D-Ala residues o **The covalent intermediate of this reaction is targeted by b-lactam antibiotics o Holes: Different sized holes determine what can pass through

b-Lactam Antibiotics - Structure: Four-atom cyclic amide is the pharmacophore of all b-lactam abx. - Subclasses: Penicillins, Carbapenems, Cephalosporins, Monobactams - Mechanism of Action: A substrate mimic of D-Ala-D-Ala, TPase forms a covalent bond with the abx. This adduct

is very stable, irreversibly inactivating - Penicillin Binding Proteins (PBPs): When we discovered PCN interacted with TPase, we simultaneously found

many other enzymes with the ability to bind to PCN via Gel Electrophoresis autoradiography, such as the resistance conferring b-lactamase

Penicillins - Penicillin G: b-lactam + Thiazolidine Ring.

o Activity: Effective against Gram(+) bacteria, however acid and alkali labile. - Synthesis through 6-APA: Removal of PCN-G benzyl moiety produces 6-aminopenicillanic acid (6APA)

o 6-APA serves as the base structure for new side chains to develop new Pencillins o Improved Acid Stability: The amide bond of the b-lactam ring is unstable in acidic

conditions. Introducing an EWG side chain makes it less electronegative and less reactive decreasing the rate of acid hydrolysis. Furthermore, this makes the drugs available orally! YAY!

§ Ex: Amoxicillin (Phenol), Cloxacillin (Halogenated Phenyl). o Broader Spectrum: Hydrophobic side chains (Benzyl of PCN-G) interfer with passage

through gram(-) porins. Adding polar groups, such as –NH2 or –COOH facilitate passage across the outer membrane and increased access of b-lactams to the periplasmic space of gram-(-) bacteria

Resistance to b-Lactams - Drug Modification: b-lactamases - Target Modification: Bacterial production of different TPases “Strongest resistance mechanism” - Efflux Pumps: Usually a mutli-use efflux pump, capable of exporting the abx extracellularly

Penicillin Improvements - Addressing b-Lactamases

o Bulky Side Chains: b-lactamases hydrolyze the amide bond of the b-lactam ring. Adding bulky side chains will limit the access of the b-lactamase to the amide group.

o b-Lactamase Inhibitors: Resistance can also be overcome by administering b-lactams in combination with a b-lactamase inhibitor. Similar to b-lactams, the inhibitors also have a b-lactam ring. Their mechanism involves inactivating the enzyme without being degraded. While they do independently have antimicrobial activity, it is very weak.

§ Inhibitors = Clavulanic acid, Sulbactam, Tazobactam § Fan-favorite: Augmentin (Amoxicillin + Clavulanic Acid)

Cephalosporins Structure: While Penicillins have 1 side chain, Cephalosporins have 2. These side chains, R1 and R2 modulate cephalosporin activity. There are dozens of combinations. The central component is the dihydrothiazine ring. Activity: Depending on the generation of development, the spectrum of activity may vary

- 1st Generation: Gram(+). This includes cefazolin and cephalexin (Keflex) - 2nd Generation: Gram(+) and a little Gram(-), such as H. Influenzae. This includes Cephaclor - 3rd Generation: Gram(-) > Gram(+). This includes Cefixime - Addressing b-Lactamases

o Bulky Side Chains: Similar to Penicillins, addition of bulky side chains can protect the abx o 7-a-Methoxy: b-lactamases can inactivate cephalosporins. The addition of a 7-a-methoxy group can

increase resistance against the b-lactamases Carbapenems

Structure: A Penicillin/Cephalosporin combo. Sulfur removed from ring. Prototype = Thienamycin Activity: Broad Spectrum Abx! Both Gram(+) and Gram(-). It is naturally resistant to the extended spectrum b-lactamases (ESBL), and in addition can target another cell-wall biosynthetic enzyme, L-D-Transferase (LdT).

- Self-Infliction: At high concentrations, the amine group can attack the b-lactam Amide bond. This drug can therefore be very unstable during the manufacturing process. By modifying the Amine sidechain to an Imine group, this problem can be solved. See Imipenem.

- Addressing Resistance o Dehydropeptidase-I (DHP-I): Found in the renal brush border can hydrolyze Imipenem

§ DHP-I Inhibitor: Usage of Cilastatin is common to improve Imipenem bioavailability Monobactams Indication: Gram(-) bacteria for individuals allergic to PCN Non-b-Lactam Cell Wall Inhibitors

- Fosfomycin (Monurol) o Indication: Uncomplicated UTI. Active against carbapenem-resistant Klebsiella pneumoniae

§ It is like our gram(-) last resort for resistant shit - Bacitracin

o This is a polypeptide antibiotic produced by Bacillus subtilis. It inhibits cell-wall biosynthesis by preventing dephosphorylation of the lipid carrier of peptidoglycan precursors

o Toxicity: Interferes with sterol biosynthesis, preventing addition of cholesterol to human cell membranes. As a result, it is exclusively used in topical formulations

- Vancomycin (Vancocin) o This tricyclic glycopeptide has a MW 1500Da, preventing its passage across bacterial outer membranes.

Therefore, it is only active against gram(+) bacteria. It is bactericidal against most susceptible bacteria § MoA: Binds very tightly to D-Ala-D-Ala reisudes at the end of Lipid II. \ the peptide bond is

inaccessible to TPase, preventing crosslinking of the peptidoglycan cell wall and inducing lysis. o The future? This used to be the abx we could rely upon, especially for treating MRSA and cephalosporin-

resistant pathogens. Nowadays, the outlook is quite bleak. o Vancomycin intermediate susceptible S aureus (VISA Cells): Have abnormal peptidoglycan cell walls,

thicker and less crosslinked. They act as a vancomycin sponge. Resistant, but they grow slowly. - Televancin (Vibativ)

o Semi-synthetic derivative of Vancomycin with an additional hydrophobic side chain and polar phosphate group. It can bind to and increase the permeability of the cell membrane, x10 bactericidal activity

(2/9) Lee Lecture: Topoisomerase Inhibitors: Quinolones DNA Gyrase (Topo II): This bacterial enzyme is essential for maintaining bacterial life, as it can relax positively supercoiled DNA and entangle negative supercoils. Its activity is constitutive, either for transcription or DNA replication. DNA gyrase is composed of two critical subunits:

- Subunit A: DNA cleavage and DNA re-ligation - Subunit B: ATPase providing the energy for the reaction

Rise of the Quinolones - Nalidixic Acid was the first clinically useful quinolone. Though it was broad spectrum (-,+), it was weakly active - Target: Gram(-) DNA-Gyrase, a topo of transcription and replication

o Gram(+) Topoisomerase IV, a topo for chromosomal segregation during replication

- Gyrase MoA: Quinolones binds to the complex of DNA and DNA Gyrase, forming a Ternary Complex. When bound, DNA Gyrase is still capable of cleaving DNA, however the quinolone prevents re-ligation of the DNA strands, leading to DNA fragmentation and cell death. \, consider these bad boys bactericidal. Dr. Lee kind-of said they are specific to the A-subunit

- Selectivity: Since human Topoisomerases are dimers of single subunits, they are non-homologous to the bacterial DNA Gyrase target (2 unique subunits – dimerized) and therefore quinolones are selective.

Fluoroquinolones - The second generation quinolone drugs are lead the by the prototype Norfloxacin, still used in developing

countries. They are much more potent with an even more broad spectrum of action - Levofloxacin and Ciprofloxacin accumulate to high levels in the bloodstream and can be used for the tx of STDs,

lower respiratory tract and skin infections. - Low levels of toxicity: These 2nd generation abx exhibit low toxicity, and can be used long-term for extensive

therapy, such as the 6 month tx for anthrax infection. Resistance to Quinolones: Same as usual. Efflux pumps, target modification, and ‘target protect’ – which is weak.

- Most prevalent are mutations in DNA gyrase, especially the A subunit. - Moxifloxacin: 3rd generation fluoroquinolone, shows some good activity against Cipro-resistant bacteria.

Novobiocin: Coumarin-containing antibiotic - Target: DNA Gyrase B subunit. (I’m having trouble deciphering, but I think he means to say quinolones only act on A. but idk. Whatevs.) - Clinical Use: It is frequently used along with quinolones due to synergistic activity.

Cationic Licopeptides – “The most important class of Abx… for now…” – Dr. Lee ••Daptomycin

- Target: Gram (+) Cocci Cell Membranes (like S. aureus, MRSA, MSSA) - MoA: While the MoA is largely unknown, some evidence suggests that

Daptomycin’s lipophilic tail gets inserted in the membrane, meanwhile the positively charged peptide side chains (coordinated with Ca2+) interact with the negatively charged lipids of the bacterial cell membrane. Hence cationic

o Bacterial Surface is negatively charged: Composed of Wall teichoic acids (WTA) and Lipoteichoic acids (LTA)

- Bacterial Resistance: o Target Modification (-Ala): The negatively charged phosphate groups of LTA can be neutralized by

adding Ala side-chains. The N-terminal free amine of the Ala side chain is positively charged and will negate the electrostatic interactions, conferring resistance to daptomycin

o Target Modification (-Lys): The phosphatidylglycerol’s of the bacterial cell membrane can also modified via Lysinylation. Thus, the lysine additions are positive, effectively inverting the membrane’s charge.

••Polymixin E (Colistin) - Target: Gram (-) multidrug-resistant P. aeruginosa and A. baumanii - MoA: Similar to daptomycin, polymyxin interacts with the outer membrane, eventually

causing repulsion via accumulative electrostatic interactions, forming a pore, leading to lysis and cell death. Boom.

- Clinical Use: Traditionally topical preparations, however have recently been approved for urinary and blood infx. - Bacterial Resistance

Daptomycin (Cubicin)

o Target Modification (LPS): Addition of aminoarabinose or ethanolamine to LPS will neutralize their phosphate-based negative charges, making electrostatic interactions weak, conferring resistance to polymyxin

o Target Modification (Phosphate group blockade): Mobile Colistin Resistance (MCR1) is an extrachromosomal enzyme based on a plasmid that can confer low level resistance by placing an ethanolamine moiety on the phosphate groups of LPS. (mentioned below in the cationic antimicrobial peptides)

••Selectivity of Cationic peptides - The human cell membrane primarily has hydrophobic interactions, it does not have the electrostatic interactions

found on bacterial cell membranes. Great. So mammals have neutral membranes. ••The Big Fear

- Cationic Licopeptides, such as Daptomycin and Polymyxin, when used in excess, can produce bacterial resistance as we have seen. This however poses a great challenge to the natural abilities of the human immune system.

- Cationic Antimicrobial Peptides: As an immunological mechanism, the tissues and organs of our body produces cascades of antimicrobial peptides that are positively charged. Neutrophils and platelets are the front-line. If antibiotic resistance induced by antibiotic therapies forms, it can be a major threat to the population

o Examples of host defense peptides: Cathelicidin, a-defensin - Thus, certain resistance mechanisms for either polymyxin or daptomycin

may also confer resistance to our cationic antimicrobial peptides of the innate immune system. ---------> He said these peptides would not be tested upon. However, I would know neutrophils + platelets (2/12) Lee Lecture: Antibiotics and Human (gut) microbiota Antibiotic-Induced Dysbiosis

- General: It is easy to define and recognize dysbiosis, being an imbalance in the gut microbiota. However, it is impossible to define a healthy microbiota as each individual has their own unique microflora. The overuse/misuse of abx may lead to the development of many disorders associated with alteration of gut microbiota

o Dysbiotic Phenotype: Allergy, behavior syndrome, cancer, CVD, infection, inflammation, obesity, T2DM - RoA: IV Antibiotics: Small but substantial percentages of IV-administered abx are excreted into the bile - Abx Specificity: Individual abx exhibit a general tendency for how they affect the abundance/diversity of the gut

microbiota. Depending on which abx is taken, some bacterial species may increase or decrease o Taxonomical Trends: The effect of an abx on microbiota is best stratified by phylum, largely due to the

inability to regrow individual species in the lab - Gene Expression: Abx can alter bacterial gene expression in gut microbiota by directly sensing abx.. Upon

recognition of an antibiotic, a dedicated sensor protein produces a signal that activates a Transcription Regulator (TR) to alter gene expression. Gene expression may be upregulation, or downregulation, depending on the mechanism producing resistance.

- Sub-MIC: Bacteria may also indirectly sense abx, secondary to abx dosed at sub-MIC. Many bacteria have Global Regulators (GR) that are activated when movement or growth is inhibited. They upregulate the production of virulence factors promoting bacterial survival. Hence Abx @ sub-MIC induce virulence gene expression

o Example given: Sub-MIC b-lactam therapy targeting PBP1 transpeptidase will arrest septum formation, allowing for the upregulation of virulence factors

- Overall: The combination of direct and indirect effects of abx on gut microbiota lead to altered gut diversity, abundance, and metabolic profiles that ultimately effects the host physiology

-Cidal and –Static: Conditionally binary - We often consider the macrolide Azithromycin to be bacteriostatic, and macrolide Telithromycin

to be bactericidal. However the –cidality of an antibiotic is unique to each bacteria. For example, when Azithromycin used to treat P. aeruginosa at 4xMIC, it is bactericidal

- MBC: Minimum Bactericidal Concentration – For Azithromycin and P. aeruginosa, 4xMIC Susceptibility: Conditionally Variable

- In similar fashion, abx susceptibility of a gut bacterium can be very different, especially when comparing mono-cultures and mixed cultures.

o Ex: Collective Abx resistance: S. aureus expresses CAT, which breaks down Chloramphenicol, of which S. pneumoniae is susceptible to. So, S. aureus protects S. pnuemoniae from chloramphenicol tx. This is an example of a resistance mechanism that should be considered when determining susceptibility

- The Susceptibility Breakpoint: The antibiotic concentration determined by the FDA and CDC to be safe for therapy based on the risk versus benefit. Factors associated with these guidelines include MIC and the Bug.

o If MIC < Breakpoint, Bug is susceptible. If MIC > Breakpoint, Resistance. The effects of temporal alteration of the intestinal microbiome

- Immune System Development: Passage of the bacterial metabolites from the mother to offspring is responsible for the functional development of the innate immune system. Transfer of the microbiota may occur via the placenta or milk.

- Critical Development Windows: Antibiotic use during critical development windows in childhood and adulthood may lead to serious health consequences.

o Jess T et al (2014)- mother mouse exposed to PCN became fat. Passage of her microbiota to gnotobiotic mice induced obesity. Holy cow

- Abx at birth: Increased risk of infection, asthma, allergies, and T1DM. Abx tx will cause a significant loss in microbial diversity and enrichment for the resistsnce genes.

- Abx during infancy: During the first months of life, abx tx can greatly increase the risk of childhood obesity. Continued tx by age 1 can increase the risk of T2DM.

- Adulthood: Effects of abx during critical development windows can predispose future susceptibility to C. diff C. diff - CDI

- Promotion of Hospital-Acquired C. difficile infection: Cephalosporins, Clindamycin, Fluoroquinolones - Clostridium scindens, a normal bacteria of the gut microbiota is responsible for converting primary bile acid

(cholic acid) to secondary bile acid (deoxycholic acid). Deoxycholic acid prevents the growth of C. diff - If treated with these abx, susceptible C. scindens will be eradicated, exposing the host to greater susceptibility to

C. diffi infection. Best of luck – please let me know if you find any errors/mistakes in my logic. I’ll be trying to make a practice exam