Antimicrobial.docx

**Antimicrobial** - Antibacterial bigger term talks about every class of bacteria. **Summary** - How does antibacterial selectivity arise? For example, is drug target specific to prokaryotic cells? - Understanding the mechanism of action helps understand how resistance might arise...

**Antimicrobial** - Antibacterial bigger term talks about every class of bacteria. **Summary** - How does antibacterial selectivity arise? For example, is drug target specific to prokaryotic cells? - Understanding the mechanism of action helps understand how resistance might arise - **Resistance can arise via** - **Drug modification** - β-Lactams, chloramphenicol, aminoglycosides, isoniazid - **Target modification** - β-Lactams, vancomycin, daptomycin, quinolones, sulphonamides, aminoglycosides, linezolid, macrolides, isoniazid - I**ncreased levels of drug target** - D-cycloserine, isoniazid - **Decreased accumulation or increased efflux** - Quinolones, chloramphenicol, aminoglycosides, macrolides **Antibacterial drug discovery** - The golden age of antibacterial drug discovery has passed, with the last novel class of clinically approved agent discovered in 2000. - Research expertise has been lost from pharmaceutical industry - Antibacterials are usually used for short periods in the treatment of acute conditions, so are less profitable than drugs used for long-term chronic conditions - Resistance will eventually arise, reducing the drug's efficacy - Newly approved antibacterials will not be used in first line treatment but will be held in reserve until older classes are ineffective **Antibacterial chemotherapy** - **Antibacterial agents** - **Bacteriostatic inhibit cell growth**, allowing the host's immune system to overcome the infection. - **Antibiotics** - 'Chemical substances produced by micro-organisms that inhibit the growth (or even destroy) other micro-organisms' - First antibiotics --- bacteria active against anthrax (Pasteur and Joubert, 1877). Procyanase from P. aeruginosa (Emmerich and Löw, 1899) - **Fungi** - Penicillium chrysogenum (penicillins) - Penicillin griseofulveum (griseofulvin) - Cephalosporium (cephalosporins) - **Bacteria** - Streptomyces resistant to (streptomycin, chloramphenicol, macrolides, tetracyclines) - Nocardia (rifamycins) **Cell walls**  **The bacterial cell (prokaryotic)** **Selective Toxicity to the Bacterial Cell** - **Differences between prokaryotic and eukaryotic cells** - Bacterial cell has a cell wall and plasma membrane (the cell wall protects the bacteria from differences in osmotic pressure and prevents swelling and bursting due to the flow of water into the cell) - Bacterial cells do not have defined nuclei. - Bacterial cells are relatively simple and do not contain organelles, e.g. mitochondria - The biochemistry of bacterial cells is very different to that of eukaryotic cells, e.g. vitamin synthesis **Inhibition of Bacterial Cell Wall Synthesis (β-Lactams, Cycloserine, Vancomycin)** **β-Lactams** - These agents interfere with cell wall synthesis in growing bacteria, resulting in a weakened cell walls, lysis and death. - Bactericidal - can affect mature cell walls as affect the balance between penicillin binding proteins (PBPs), which catalyse cell wall synthesis and murein hydrolase, which catalyses cell wall lysis - Penicillins, cephalosporins, monobactams, carbapenems all contain βlactam ring - Penicillins were discovered in 1929 by Fleming when he discovered that a mould P. notatum growing on a petri dish inhibited the growth of bacterial colonies. - **piperacillin** (and piperacillin with tazobactam) used in treatment of P. aeruginosa infections - **amoxicillin** used in treatment of community acquired pneumonia (CAP), e.g. due to Streptococcus pneumoniae - **ceftriaxone** used in treatment of N. gonorrhoea infections - **cefoxitin** used for surgical prophylaxis for some GI procedures (active against anaerobic Bacteroides fragilis) - **aztreonam** inactive against Gram positive organisms but active against Gram negative species, e.g. Haemophilus influenzae (meningitis, bacteremia, pneumonia, cellulitis) - **imipenem** active against Gram negative rods and can be used in treatment of mixed aerobic / anaerobic infections **Penicillins** - Production, isolation and demonstration of selectivity by Chain, Florey, Abraham and Heatley - Nobel prize in 1945 for Fleming, Chain and Florey - Greatest advance in production came from deep fermentation process during WWII - Variations to original method included: - Replacement of surface culture by deep fermentation process (100,000 litre vessels) - Replacement of P. notatum by P. chrysogenum - Addition of corn steep liquor --- gave rise to Penicillin G instead of F (due to production of phenylacetic acid and incorporation into structure) - Only mono-substituted acetic acids incorporated by moulds, so range of possible penicillins produced this way is small **Penicillins**  - Very difficult to synthesise - β-lactam ring must intact if it destroyed no effect - β-lactam is sensitive to acid. - semi-synthesis **Penicillins** - highlight flucloxacillin and amoxycillin - Flucloxacillin is acid stable due electron withdrawing. - Amoxycillin is acid stable bulkier group of R1 **Other β-Lactams**  **Carbapenemase** - Spectacular increase in carbapenem resistance genes (especially KPC, OXA and metallo-β-lactamase) - NDM-1 metallo-β-lactamase (blaNDM-1 gene) characterized in 2008 and NDM-1 positive isolates now found throughout the World - blaNDM-1 gene also carries resistance to macrolides, aminoglycosides, rifampicin, sulfamethoxazole, tigecycline, and aztreonam - **So what treatment is effective? Colistin (a polymyxin) and rifampicin in combination.** **β-Lactamase Inhibitors (BLIs)** - Clavulanic acid (from Streptomyces clavuligerus) weak antibiotic action but suicide inhibitor of β-lactamases - Coamoxiclav --- amoxicillin and clavulanic acid - Unasyn --- ampicillin and sulbactam - Tazocin or Zosyn --- piperacillin and tazobactam - These combinations **are not** active against carbapenemases - **Non β-Lactam BLIs Inhibitors**  - Zavicefta -- avibactam and ceftazidime (activity against ESBL and KPC) - Imipenem/ cilastatin / relebactam (cilastatin inhibits dehydropeptidase which degrades imipenem) (activity against KPC and AmpC) - Vabomere --- vaborbactam and meropenem (activity against KPC) - These combinations are active against carbapenemases - Currently no approved BLIs which inhibit metallo-β-lactamases such as NDM-1 **Antibiotic resistance** - **Microbiological** - **Intrinsic resistance** is the natural resistance an organism has to an antibiotic, e.g. P. aeruginosa has efflux pumps for the β-lactams - **Acquired resistance** due to a chance mutation in genetic material or the acquisition of resistance genes via a plasmid - **Clinical** - the failure to achieve an antimicrobial concentration which inhibits the growth of an organism **Penicillins Disrupt peptidoglycan formation** - Prokaryotic cell wall composed of peptidoglycan (polymer consisting of sugar and peptide units) - **Gram positive bacteria** (stained by crystal violet-iodine complex) are surrounded by a cytoplasmic membrane and cell wall containing peptidoglycan linked to teichoic acids (polyhydroxylated phosphate polymers) - **Gram negative bacteria** have a thinner cell wall (peptidoglycan and associated proteins) surrounded by outer membrane of lipid, lipopolysaccharide and protein - Complex cell wall helps protect against influx of water due to higher salt concentration within cell - β-Lactams interfere with peptidoglycan formation through their interaction with the penicillin binding proteins (PBPs) - PBPs classified by size (PBP1 biggest *etc*.) and are essential in the final stages of peptidoglycan synthesis and activities include D-alanine carboxypeptidase, removal of D-ala from peptidoglycan precursor, **peptidoglycan transpeptidase** and peptidoglycan endopeptidase **Peptidoglycan** - Peptidoglycan consists of parallel sugar backbones composed of alternating NAG and NAM. - Peptide chains are attached to the NAM through the carboxylic acid residue - Peptide chains are then linked together to give extra strength to the cell wall through crosslink formation, catalysed by peptidoglycan transpeptidase - Crosslinking of peptide chains inhibited by the β-lactams - A diagram of chemical formulas Description automatically generated with medium confidence **Peptidoglycan formation**  - Do not memorise amino acid sequence but D-alanine at the end is important. **Penicillins -- mode of action** A diagram of a chemical reaction Description automatically generated - β-Lactams bind covalently to active site of enzyme, preventing access of peptidoglycan fragments and attack of hydroxy group on D-Ala residue **Other Agents Which Target Cell Wall synthesis** - D-Cycloserine (DCS) and **Vancomycin** both target cell wall synthesis - Peptidoglycan biosynthesis consists of a number of steps and these agents target different parts of the process; - Cycloserine (Seromycin) is an inhibitor of two key bacterial enzymes --- alanine racemase (ALR) and D-Ala-D-Ala ligase (Ddl) - **Vancomycin (Vancocin)** is a glycopeptide antibiotic and prevents the release of the disaccharide from its lipid carrier **Vancomycin (Vancocin)** - Vancomycin is a glycopeptide antibiotic and is the last resort in treatment of MRSA. - Not absorbed orally so usually given i.v and can be toxic to ears and kidney. - Oral vancomycin used in treatment of Clostridioides difficile -- associated disease. - Vancomycin is produced to stop bacterial cell wall formation. (to stop synthesis of peptidoglycan)  **MRSA** - Methicillin Resistant *Staphylococcus Aureus* - *S. aureus* causes skin infections (boils *etc*.) as well as toxic shock syndrome (TSS), pneumonia, septicaemia and meningitis - Penicillin-resistant strains known since 1960s - Epidemic MRSA (EMRSA) strains emerged in the 1990s - Resistance due to a plasmid (*blaZ*) mediated β-lactamase **AND** a chromosomal (*mecA*) gene which was acquired from an unknown bacterium, which codes for an altered penicillin binding protein (PBP-2a) - PBP-2a has decreased affinity for binding β-lactams - *mecA* gene also confers resistance to many other antibiotics, *e.g.* ciprofloxacin, erythromycin and trimethoprim-sulfamethoxazole - Only available oral antibiotic for treatment of MRSA infections is Linezolid (*Zyvox*) - A structure of a chemical formula Description automatically generated **Vancomycin** - Vancomycin is hydrophilic and forms hydrogen bonds to the terminal D-Ala-Dala sequence -- preventing crosslink formation and blocking the release of the disaccharide from the carrier lipid. - Resistance to vancomycin occurs through alteration in the ligase activity. - The Vancomycin resistant Enterococci (VRE) Van A phenotype produces **a D-Ala-D-lactase ligase which synthesis an ester (D-Ala-D-Lae)** rather than an amide (D-Ala-D-Ala) - D-Ala-D-lactate sequence has 1000-fold reduction in affinity for vancomycin but can still be added to L-Lys **and** act as precursor for crosslink formation - Such altered ligases are also produced by vancomycin producing microorganisms - Treatment for VRE infections can involve linezolid **Vancomycin Resistance**  - 특정 장알균들이 반코마이신 저항성이 생기는 이유는 D-Ala-D-Ala가 D-Ala-D-lactate로 바뀌기 때문이다. 서열이 D-Ala-D-lactate로 바뀌면 반코마이신이 펩티도클리칸 전구체에 결합을 못하게 되고, 따라서 세균의 세포벽 형성을 억제할 수 없게 된다. **Formation of cell wall crosslinks** A diagram of a cell membrane Description automatically generated **Daptomycin (Lipopeptide)** - Cubicin (daptomycin for injection) was approved by the FDA in 2003 for the treatment of complicated skin and skin structure infections (SSSI) caused by MRSA - In 2006, the FDA approved a new indication the treatment of *S. aureus* bacteraemia due to MRSA, including right-sided endocarditis - Binds strongly to pulmonary surfactant, so cannot be used in the treatment of pneumonia - Originally isolated by Eli Lilly and Co. from a strain of *Streptomyces roseosporus* from a soil sample from Mount Ararat in Turkey  - Lipid like tail structure is important. **Daptomycin (Lipopeptide)** - Calcium ions are essential for the rapid bactericidal activity against Gram positive bacteria. - Mode of action believed to involve the insertion of daptomycin into the lipid bilayer, facilitated by the lipid tail, promoting weak hydrophobic interactions with the phospholipid bilayer. - Interaction of daptomycin, calcium and phosphatidyl glycerol promotes mild disturbances in the lipid membrane and cause content leakage. **Mechanism of Action of Daptomycin** - Interaction with the cytoplasmic membrane alters permeability - Daptomycin oligomerisation (which is promoted by binding to Ca2+) creates a large pore in the membrane, allowing potassium efflux, membrane depolarization, and eventually cell death - A diagram of a cell structure Description automatically generated **Resistance to daptomycin** - Point mutations in mprF and yycG genes; mprF enzyme influences the nature of the phospholipid content (it catalyses the addition of lysine to membrane phosphatidylglycerol) - Point mutations in yycG gene, which is believed to be involved in cell permeability - Cases of S. aureus which are not susceptible to daptomycin are often associated with vancomycin-unresponsive strains- vancomycin resistant (VRSA) or vancomycin intermediate S. aureus (VISA) have thickened cell wall and daptomycin resistance is due to its inability to diffuse through these thicker cell walls so its site of action at the lipid membrane. - Daptomycin resistance is still rare and, as there are established daptomycin surveillance programs around the World which monitor its in vitro activity. The bacterial cell (prokaryotic)  **Quinolone Antibacterials** - **Synthetic antibacterial. (synthetic agents)** - Nalidixic acid was discovered in 1962 during the synthesis and purification of chloroquine (anti-malarial). Nalidixic acid and other first-generation quinolones have weak anti-bacterial (bactericidal) activity - First generation quinolones only used to treat urinary tract infections - All quinolones well absorbed orally and usually highly serum-protein bound, giving long half-lives. Used in high doses due to protein binding and weak activity - Side-effects include GI disturbance, rashes, prolongation of the QT interval, fatigue, dizziness, visual disturbances, convulsions (particularly if used concomitantly with NSAIDs), and spontaneous tendon ruptures - Later generations have broader spectrum, mostly due to the introduction of a fluorine at the 6-position - Now have 2nd, 3rd and 4th generation quinolones A group of chemical formulas Description automatically generated - Red part structure important for activity. **Quinolone Antibacterial** - Bactericidal - Inhibit bacterial DNA gyrase and topoisomerase IV. - The right-handed helical nature of DNA means that positive supercoils (knots) form ahead of replication sites when DNA strands act as templates for new strands. - In order for DNA replication to proceed these supercoils must be removed by the gyrase or topoisomerase IV relaxing the DNA chian. By catalysing the formation of negative supercoils, these enzymes remove the positive supercoils and give a tension free DNA double helix. - DNA gyrase and topoisomerase IV relax bacterial DNA by cutting one of the strands, passing the other strands, passing the other strand through the cut and then resealing the cut. - Quinolones bind to these enzymes, preventing them from relaxing the DNA helix and so preventing replication. - Quinolones target the DNA gyrase in Gram negative bacteria and the topoisomerase IV in Gram positive. - Mammalian cells do not have DNA gyrase or topoisomerase IV (they do have topoisomerases I and II but quinolones do not bind to these enzymes) hence these agents have some selectivity - Inhibition of DNA gyrase and topoisomerase IV leads to cell death, especially if cell is also dealing with the other toxic effects of quinolones at the same time. - Resistance to the quinolones arises through two major mechanisms; Alterations in the target enzymes - Decreased accumulation of the quinolones in cells due to the impermeability of the membrane or the over-expression of efflux pumps - Alterations to the DNA gyrase occur *via* mutations in the quinolone- resistance determining region (QRDR) of the *gyrA* gene which encodes the two A subunits of the tetrameric enzyme (*gyrB* encodes the two B subunits) - Similar mutations have been described in topoisomerase IV which decrease quinolone binding **Resistance to Quinolone Antibacterials** - Decreased uptake or increased efflux has also been found for the quinolones. - Quinolones cross the outer membrane via specific porins (all quinolones) of diffusion through the phospholipid bilayer (hydrophobic quinolones only). - Porins are protein channels which allow passive diffusion of a specific agent across the cell membrane. - The outer membrane of P. aeruginosa has very low permeability to small hydrophobic molecules giving this bacterium intrinsic resistance to the quinolones. - E. coli has three main porins and a decrease in the level of one of these (OmpF) is associated with an increase in resistance to the quinolones. - P. aeruginosa (Gram negative) and S. aureus (Gram positive) exhibit well characterised efflux pumps for quinolones. **Agents which act on bacterial metabolic processes** - Agents which target bacterial metabolic processes will be selective antibacterials if they target a process which is specific to the bacteria. - Bacteria synthesise a number of vitamins, e.g. folic acid, and these processes can be targeted by antibacterial agents. - The sulphonamides are antifolates (like methotrexate) and interfere with the bacterial biosynthesis of folic acid. **Sulphonamide Antibacterial** - Gerhard Domagk discovered the antibacterial effect by chance. -  - Produced in vivo first synthetic antibacterial agent - A black hexagon with black text Description automatically generated- generalised structure - When para-aminobenzoic acid (PABA) added to bacterial culture at same time as sulphonamide, drug had little or no effect. - Sulphonamides (sulpha drugs) are bacteriostatic and antifolates. - Bacteria require PABA (essential metabolite) for the synthesis of folic acid and lack the protein for folate uptake. - Folic acid is an essential metabolite for mammals (cannot be synthesised by mammalian cells) so this interference in folic acid synthesis is the basis of the selectivity of the sulphonamides. - Many thousands of analogues tested and sulphonamide group (-SO~2~NR~2~) and a free amino group at the para position were found to be essential. - Only variations in R and R' lead to active sulphonamides. - Variation in R leads to inactive prodrugs which can be hydrolysed to the active amino group in vivo. - Variations in R' (especially heterocyclic substituents) are responsible for the many sulphonamides used clinically. **Prevention of Folic acid synthesis** - Sulphanilamide has similar molecular dimensions and properties to PABA so is mistaken for it by *dihydropteroate synthetase* - Sulphonamides bind to the PABA binding domain of the active site of *dihydropteroate synthetase and* some are incorporated into macromolecule - Once sulphonamide is incorporated into macromolecule further reaction with L-glutamic acid will not produce folic acid - Incorporation is reversible since the addition of excess PABA results in the formation of folic acid -  A diagram of a chemical structure Description automatically generated **Bacterial resistance to sulphonamides** - Chromosomal resistance through mutations in the dihydropteroate synthetase (folP) gene in E. coli, S. aureus, P. jiroveci (fungal pneumonia infection), Campylobacter jejuni (gastroenteritis), Neisseria meningitidis (bacterial meningitis) leads to alterations in the sulphonamide binding site of the DHPS) - Sulphonamide resistance in Gram-negative bacteria is plasmid-borne. **Trimethoprim** A structure of a chemical formula Description automatically generated - Trimethoprim (Monotrim, Proloprim) is an antifolate agent which is a dihydrofolate reductase (DHFR) inhibitor. - Used in combination (synergistic) with sulphamethoxazole (Co-trimoxazole, Resprim, Bacterium. - Co-trimoxazole acts on two enzymes in the same biosynthetic sequence (**sequential blocking**) - Doses of both drugs are lower than would be required if either used alone so side-effects (and possibly resistance) can be minimised  Agents which target protein synthesis - Agents which target the ribosome and so inhibit protein synthesis include: - A diagram of different chemical formulas Description automatically generated with medium confidence **Transcription and translation of DNA**  ***TRANSCRIPTION*** - DNA → messenger RNA - Messenger RNA leaves nucleus for cytoplasm ***TRANSLATION*** - Messenger RNA binds to ribosome (giant ribonucleoprotein) - Ribosome has two subunits (30S and 50S in bacteria \[70S\], 40S and 60S in eukaryotic cells \[80S\]) - Ribosome small subunit attaches to mRNA - Initiator tRNA-methionine binds to site - Large ribosome subunit binds to small subunit. Large ribosome subunit has two binding sites, P and A in the peptidyl transferase centre (PTC) - **Transfer RNAs carry amino acids to the ribosome site where mRNA binds (charged tRNA). tRNA has 3 nucleotides (triplet) --- codes for a specific amino acid --- and binds to the complementary sequence on the mRNA** - Ribosome moves along mRNA from 5′ to 3 ′ since once the peptide bond has formed the non-acylated tRNA leaves the P site and the peptide-tRNA moves from the A to the P site. A new tRNA-aa (as specified by the mRNA codon) enters the A site - Peptide chain grows as amino acids added until stop codon reached, then leaves ribosome through protein exit tunnel **Chloramphenicol (Chloromycetin, Cm)** - Originally obtained from Streptomyces venezuelae, now prepared synthetically. - Bacteriostatic with broad spectrum of activity and only R, R -- isomer is active. - Highly lipophilic and penetrates most tissues (crosses blood-brain barrier) - Active against Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae (causes meningitis) - Used in treatment of meningitis in patients with (β- lactam allergies and drug of choice against typhoid fever - Severe toxicity so not given systemically but used in treatment of bacterial conjunctivitis - Side-effects include aplastic anaemia (bone marrow cannot replenish blood cells) -- unpredictable and may occur weeks after treatment ceases - A structure of a chemical formula Description automatically generated - Chloramphenicol binds to large ribosome subunit (50S) at the peptidyl transferase centre A site, preventing binding of the next charged tRNA - Selectivity arises due to differences between the conformations of bacterial and eukaryotic PRC) - Resistance to chloramphenicol in Pseudomonas aeruginosa is due to efflux pumps (*e.g. cm1A7, P. aeruginosa*, chromosome and *cm1A6, P. aeruginosa*, plasmid) - **Resistance also arises due to chloramphenicol acetyltransferases (CAT)** which acetylate chloramphenicol so that it no longer binds to the PTC A site. CAT genes are both plasmid (*e.g. CatC*, *S. aureus*) and chromsome- derived (*e.g. CatB3*, *Salmonella typhimurium*) - Florfenicol does not contain 3-OH so is not acetylated. Cm-resistant strains in which resistance is due to CATs **are** susceptible to florfenicol. -  Aminoglycosides - Streptomycin isolated from Streptomyces griseus by Selman Waksman and first used clinically in treatment of tuberculosis in 1944. - Aminoglycosides are bactericidal in a concentration -- dependent manner. - Used in treatment of Gram positive and negative (and mycobacterial) infections. - Gentamicin is the aminoglycoside of choice for most nosocomial Gram-negative infections. - A diagram of a molecule Description automatically generated with medium confidence **Concentration -- versus time-dependent antibacterial activity** - **Time -- dependent** - Providing the concentration remains above the minimum inhibitory concentration (MIC) is the time (duration) that bacteria are in contact with the antibacterial agent which is important. - β-Lactams are time-dependent bactericidal agents. They should remain in contact with the PBPs for sufficient time to interfere with cell wall synthesis. - Short dosing intervals will ensure that the concentration remains above the MIC. - Macrolides are also time dependent. - **Concentration dependent** - The absolute concentration of a concentration-dependent antibacterial agent is the most important factor. The best responses occur when the concentrations are \> 10 × the MIC at the site of infection. - Concentration-dependent antibacterial agents can exhibit delayed bacterial regrowth; the suppression of growth after a brief exposure to the drug is known as the Post Antibiotic Effect (PAE). - Aminoglycosides are concentration-dependent antibacterials, as are the fluoroquinolones. **Aminoglycoside mechanism of action** - Bind to the 30S subunit of prokaryotic ribosome at the A site. - The tRNA which is complementary to the mRNA (cognate tRNA) is selected on the basis of two key sets of interactions (process is known as decoding); - The tRNA anticodon must match the mRNA codon. - Only when the cognate tRNA binds to the A site, conformational changes allow interactions between the codon and anticodon double helices. - A 1492 and A1493 normally in interior of rRNA double helix but flip out of the double helix and bind to tRNA residues of the codon-anticodon triplet. - Non-cognate tRNA does not cause this flipping out. - Aminoglycosides bind to RNA as a result of interactions with phosphate backbone and hydrogen bonding (for example to A1408), and cause adenines to flip out, mimicking the conformation induced by the cognate tRNA. - Cause incorporation of incorrect amino acids and misreading of mRNA. -  **Aminoglycoside selectivity and resistance** - **Selectivity** - Highly ionised and cannot permeate the phospholipid bilayer or mammalian cell membrane - Can be taken up, even by Gram negative bacteria. - **Resistance** - Resistance due to decreased uptake or increased efflux. - Mutations of ribosomal (A1408 to G1408) - Methylation of the ribosome (on A1408), decreasing the binding affinity of the aminoglycosides. - Production of aminoglycoside-modifying enzymes (for example, aminoglycoside N-acetyltransferases (AAT)) **Linezolid (Oxazolidinones)** - Only active against Gram positive bacteria - FDA approved for treatment of vancomycin resistant E. faecium (VRE) infections, pneumonia (both community-acquired \[CAP\] and nosocomial) and complicated skin and skin structure infections \[cSSSI\] - Orally available alternative to vancomycin for treatment of MRSA. - Reserve antibacterial agent. - (S)-enantiomer is active A black and white diagram of a molecule Description automatically generated - Binds to the PTC A site, disturbing the binding of the next charged tRNA - Binds to a pocket surrounded by G2061, A2451, C2452, A2503, U2504, G2505, U2506, and U2585 - Binding induces the movement of these bases, preventing charged tRNA from binding. - No effect on mammalian cytoplasmic protein synthesis but inhibits mitochondrial protein synthesis (linked to myelosuppression on extended use) **Linezolid resistance** - Gram negative intrinsically resistant due ti efficient bacterial efflux pumps (RND superfamily) - Resistance in Gram positive (e.g. MRSA) due to decreased linezolid uptake, and a methyltransferase (encoded by cfr gene) which methylates the 23S rRNA subunit. - Mutations in the bases surrounding the 8 base binding sites of the drug are also responsible for resistance. **Macrolides (Erythromycin, E-mycin)** - Macrolide denotes a large lactone (ester) ring and these antibiotics also contain 2 sugar units -- a desosamine and a cladinose - Erythromycin \[R=H\] (14-membered ring) first isolated from Saccharopolyspora erythraea in 1952 by Eli Lilly - Erythromycin (Ilosone, Erythromid) has a broad antibacterial spectrum which is similar to that of the penicillins and so is an alternative for penicillin-allergic patients - Eythromycin is mildly basic (pKa 8) due to aminosugar group and may be administered as the HCl salt - Pro-drugs esters, e.g. Erythromycin ethyl succinate \[R=CO(CH2)2CO2Et\] Erythroped) are used to mask taste of bitter drug) **Erythromycin** - Macrolides block exit of nascent protein tunnel by binding to a high affinity site, leading to the arrest of protein elongation and the dissociation of shortened peptidyl-tRNAs from the ribosome. - Main component of binding pocket is nucleotide 2058. In bacteria this is adenine (A) and macrolides bind strongly to this nucleotide. In eukaryotic cells this nucleotide is guanine (G) and is too bulky to allow favourable interactions with the 14-membered macrolides. - Desosamine sugar (formation of 3 hydrogen bonds between 2′-OH and A2058 and A2509, NMe2 and A2505), ring hydroxyls (hydrogen bonds between the 6, 11 and 12-OH and nucleotides) and lactone (hydrophobic interactions) play key role in macrolide binding to this site **Resistance to Erythromycin** - Resistance arises due to modifications to the ribosome - Inducible or constitutive erm (erythromycin ribosome methylase) gene gives rise to resistance in Streptococci \[Inducible -- only expressed in presence of antibiotic; constitutive -- expressed without any regulation\] - In Streptococcus pneumoniae, ribosomal methylase dimethylates a single site, A2058 (on N-6), resulting in a decreased binding affinity for erythromycin due to the increased size of this nucleotide. This can only take place during ribosome assembly (very narrow time window) as A2058 buried deep within ribosome **Tuberculosis (TB) consumption** - TB mostly caused by mycobacterium tuberculosis infection of pulmonary alveoli - 'Short course' treatment regimen involves isoniazid + ethambutol + pyrazinamide + rifampicin for 2 months then rifampicin and isoniazid for 4 months - Directly observed therapy (DOT) can be used to improve patient compliance - Treatment of MDR-TB requires more toxic, less effective 2nd line drugs and 3 × as lengthy treatment which is 100 × more expensive **Mycobacteria** - Mycobacteria are **aerobic organisms**, characterised by **thicker cell walls than Gram positive and negative organisms** - The cell wall / plasma membrane is hydrophobic / waxy and is rich in very long chain fatty acids, known as mycolic acids - The cell wall is resistant to agents which interfere with cell wall synthesis, e.g. β-lactams and mycobacterial infections are difficult to treat ― primarily due to the nature of the cell wall **Isoniazid (isonicotinoylhydrazine, INH)** - Synthetic agent - Activity against TB discovered in 1952 - Prodrug, activated by mycobacterial catalase-peroxidase, KatG - Catalase-peroxidases (CPs) protect bacteria from hydroperoxides, and hydroxy radicals present in aerobic conditions and - KatG activates INH to give the is nicotinoyl anion or radical, which complexes with NADP to give INH-NADP adduct - - INH-NADP adduct inhibits InhA, an NADPH-dependent enoyl-acyl carrier protein reductase, by preventing access of natural substrate - InhA is part of the mycobacterial type II fatty acid synthase system (FASII) which elongates fatty acid precursors giving the long-chained mycolic acids -  **Resistance to INH** - Mycobacterium tuberculosis resistance to isoniazid is linked to; - Mutations / deletions in the KatG gene regulatory region, resulting in a loss of catalase-peroxidase activity (and no INH activation) - Overexpression of the InhA gene (so insufficient INH-NADP complex for inhibition) - Increased N-acetyltransferase activity (N-acetylation reduces activity of INH) A close-up of a document Description automatically generated