L10-12 Antimicrobials PDF - University of Sydney

COMMONWEALTH OF AUSTRALIA Copyright Regulations 1969 WARNING This material has been copied and communicated to you by or on behalf of the University of Sydney pursuant to Part VB of the Copyright Act 1968. (The Act). The material in this communication may be subject to copy...

COMMONWEALTH OF AUSTRALIA Copyright Regulations 1969 WARNING This material has been copied and communicated to you by or on behalf of the University of Sydney pursuant to Part VB of the Copyright Act 1968. (The Act). The material in this communication may be subject to copyright under the Act. Any further copying or communication of this material by you may be the subject of copyright protection under the Act. Do not remove this notice.  Acknowledgement of Country We acknowledge the traditional owners of the land on which we meet and pay our respects to their Elders, past, present and emerging, and acknowledge their ongoing cultures and connections to the lands and waters of NSW.  PHAR2921 Antimicrobials Paul Groundwater E-mail: [email protected] The University of Sydney Page 3  Australian Pharmacy Council (APC) curriculum learning domains Learning domain 1: The health care consumer The unique expertise of the pharmacist in ensuring that the consumer achieves optimal health outcomes from medicines and minimises the potential for harm. The pharmacist’s contribution to the promotion of good health and disease prevention. Disease management and care planning, including application of clinical guidelines, prescribing guidelines, medication review and new models of care. Learning domain 2: Medicines: drug action Molecular basis of drug action and the actions of drugs within living systems; molecular, cellular, biological and physical aspects. Clinical therapeutic uses of drugs and medicines in man, including contraindications for, adverse reactions to, and interactions of medicines and their relevance to treatment. Drug absorption, distribution, metabolism and excretion and influences thereon, including formulation, route of administration, dosage regimen, ageing and disease. Prospects for new approaches in therapeutics. Learning domain 3: Medicines: the drug substance Sources and purification of substances of biotechnological, chemical synthetic, immunological, mineral and plant origin used in medicine.  Antibacterial Chemotherapy Learning Outcomes  Understand the basis of antibacterial agent selectivity  Understand the different sources, mode of action, and resistance of antibacterial agents from the following classes; Inhibition of cell wall synthesis (β-lactams) Inhibition of metabolism (sulphonamides) Inhibition of nucleic acid transportation and replication (quinolones) Inhibition of protein synthesis (chloramphenicol, macrolides) Inhibition of cell membrane function (isoniazid)  Understand the role of some bacterial pathogens in infectious disease  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 – Increased levels of drug target D-cycloserine, isoniazid – Decreased accumulation or increased efflux Quinolones, chloramphenicol, aminoglycosides, macrolides “The World is facing an Antibiotic Apocalypse” Prof Dame Sally Davies (UK CMO) Tackling Drug-Resistant Infections Globally: Final Report and Recommendations. The Review on Antimicrobial Resistance, J O’Neill (chair) [https://amr-review.org/sites/default/files/160525_Final%20paper_with%20cover.pdf]  Antibacterial Drug Discovery The golden age of antibacterial drug discovery has passed, with the last novel class of clinically approved agent discovered in 2000.  Reasons 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 bactericidal kill bacterial cells  Antibiotics ‘chemical substances produced by micro-organisms that inhibit the growth (or even destroy) other micro-organisms’ (S.A. Waksman, 1942 — Nobel Prize, 1952) 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 (streptomycin, chloramphenicol, macrolides, tetracyclines) Nocardia (rifamycins)  Cell walls Gram positive Gram negative Mycobacteria Bacillus anthracis Escherichia coli Mycobacterium tuberculosis Clostridium difficile Pseudomonas aeruginosa Mycobacterium leprae Staphylococcus aureus Neisseria gonorrhoeae Mycobacterium avium complex Streptococcus pneumoniae Treponema pallidum Chlamydia trachomatis  The Bacterial Cell (Prokaryotic) β-LACTAMS VANCOMYCIN QUINOLONES CHLORAMPHENICOL TETRACYCLINES MACROLIDES SULPHONAMIDES ISONIAZID  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 wall, 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 β- lactam 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.  β-Lactams 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 O thiazolidine ring H H 4 R 6 5 S N R R Me 3 H 7 N 1 2 Me O S β-lactam ring H COOH RCOOH H H 4 no substituted acetic acid in 6 5 S fermentation medium H2N R R Me penicillin acylase from E. coli 3 P. chrysogenum 7 1 Penicillin G N 2 Me O S H COOH 6-aminopenicillanic acid  Penicillins R1 R2 name (trade name) comments PhCH2 Na Penicillin G (original US) requires addition of corn steep liquor (BenPen) MeCH2CH=CHCH2 Na Penicillin F (original UK) PhOCH2 Na Penicillin V increased acid stability (Stabillin) OMe resistant to Staphylococcal penicillinase Methicillin Na (Celbenin) OMe Cl O Na Flucloxacillin increased acid stability (Flopen, Staphylex, Flubiclox) (oral absorption) -lactamase resistant H H F N Me β R1 S O N Me H N R Na Ampicillin broad spectrum, including Me (Ampicyn, Austrapen) Gram negative H NH2 O H COOR2 Me Me O Pivampicillin increased acid stability C Me H2 (Pondocillin, Miraxid) (oral absorption) O HO R Na Amoxycillin increased acid stability (Alphamox, Amoxil) (oral absorption) H NH2 R Et N used in combination with Na Piperacillin tazobactam (tazocin or zosyn) H HN N O (Piril) to treat P. aeruginosa infections O O  Other β-Lactams O H H R1 N S Cephalosporins first isolated H N from Cephalosporium in 1948 R2 O COOH by Brotzu S Cephalosporins, e.g. Ceftazidime (Fortum) Active against Salmonella H2N typhi (typoid fever) R1 = N Me Me R2 = H2C N 7-ACA nucleus consists of N O COOH dihydrothiazine ring fused to β- lactam ring OH Latest cephalosporins belong O NH2 Me H N to the 3rd/4th generations and R N H S have a broad spectrum of H N N activity O SO3H O COOH Cephalosporinase activity increasing (ESBL and AmpC) Monobactam (Aztreonam) Carbapenem (Imipenem) S H2N R= N Me Me N O COOH  Penicillins — acid sensitivity and oral administration  The β-lactam ring is sensitive to hydrolysis (acid or enzyme catalysed) H H H H H R1 N S N S Me R1 Me O N Me O HN Me O O H COOR2 H COOR2 H other inactive products  Sensitivity to acid can be overcome by; the introduction of an electron-withdrawing group group on the amide side-chain as in Penicillin V the introduction of a bulky group on the amide side-chain (steric hindrance) as in Ampicillin or Amoxicillin  Penicillins — acid sensitivity and oral administration  The β-lactam ring is sensitive to hydrolysis (acid or enzyme catalysed) H H H H H R1 N S N S Me R1 Me O N Me O HN Me O O H COOR2 H COOR2 H other inactive products  Sensitivity to acid can be overcome by; the introduction of an electron-withdrawing group group on the amide side-chain as in Penicillin V the introduction of a bulky group on the amide side-chain (steric hindrance) as in Ampicillin or Amoxicillin  Penicillins — acid sensitivity and oral administration H NH2 H H H H H Ph N N S S Me O Me O N O N Me Me O O COOH H COOH H Penicillin V Ampicillin Heterocycles, such as the isoxazole ring in Flucloxacillin, also have an electron-withdrawing effect and so increase acid stability  Penicillins — oral administration H2N H H H H  Some penicillins are zwitterionic N S Me (doubly charged) and are poorly O N Me H absorbed through the gut wall O O O H C(Me)3 (Ampicillin, Amoxicillin) O O  These penicillins disrupt the gut Pivampicillin ENZ-OH flora and may cause diarrhoea non-specific esterase -H 2C=O  Absorption can be enhanced (after absorption) through the preparation of prodrug esters H2N H H H H N S Me O N Me O H OH O Ampicillin  β-Lactamases  Over 5,000 different β-lactamases  β-Lactamases are present in the H H periplasmic space and cleave the R N S R N S β-lactam ring, leading to the O O N N inactivation of these antibiotics O EserO O CH2  Different β-lactamases have E-ser-OH COOH OAc COOH different substrates, e.g. AmpC [cephalosporins, Nukaga et al., J. Biol. Chem., 2004, 279, 9344-9352] and some, e.g. extended spectrum β- lactamases (ESBL) hydrolyse even third generation cephalosporins but not carbapenems [see Tumbarello et al., J. Antimicrob. Chemother., 2004, 53, 277]  Carbapenemases!  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. [TR Walsh, Intn. J. Antimicrob Agents, 2010, 36S3, S8]  β-Lactamase Inhibitors (BLIs)  Clavulanic acid (from Streptomyces clavuligerus) weak antibiotic action but suicide inhbitor 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 O O H H O H O N O OH S S N Me N N N Me N Me O O O H OH H OH H OH O O O Clavulanic acid Sulbactam Tazobactam O OH O O OH O O H Ser70 -CO2 O O N O HN O N O O H O Ser70 Ser70 OH Ser70 COOH COOH i ii iii H Acyl-enzyme intermediates  Non β-Lactam BLIs Inhibitors O H 2N O OH H H 2N S N B N O N H N O N N O OSO3H O OSO3 COOH Avibactam Relebactam Vaborbactam  Zavicefta – avibactam and ceftazidime  Imipenem/ cilastatin / relebactam (cilastatin inhibits dehydropeptidase which degrades imipenem)  Vabomere — vaborbactam and meropenem  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 [Understanding Antibiotic Resistance, H. Wickens and P. Wade, Pharm. J., 2005, 274, 501 Antibiotic Resistance — the Problem Intensifies, S.B. Levy, Adv. Drug Deliv. Rev., 2005, 57, 1446] 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 CH2OH CH2OH  Peptidoglycan consists of parallel O O H OH H OH sugar backbones composed of H H OH H O H alternating NAG and NAM H H OH OH  Peptide chains are attached to the H HN CH3 H 3C H HN CH3 NAM through the carboxylic acid COOH H residue O O  Peptide chains are then linked N -Acetylglucosamine (NAG) N -Acetylmuramic acid (NAM) together to give extra strength to the cell wall through crosslink formation, catalysed by peptidoglycan NHCOCH3 transpeptidase HOH2C O O HO  Crosslinking of peptide chains H O NHCOCH3 HOH2C O inhibited by the β-lactams H3C CONH-aa n  Peptidoglycan Formation (NAM-NAG)n L-Ala D-Glu L-LysGlyGlyGlyGlyGlyNH2 CH3 D-Ala HOOC N O H D-Ala (NAM-NAG)n (NAM-NAG)n L-Ala L-Ala D-Glu D-Glu ENZYME OH L-LysGlyGlyGlyGlyGlyNH2 -D-Ala L-LysGlyGlyGlyGlyGlyNH2 CH3 D-Ala D-Ala HOOC N O ENZYME O O H D-Ala H H (NAM-NAG)n L-Ala D-Glu (NAM-NAG)n L-Ala L-LysGlyGlyGlyGlyGlyNH2 D-Ala D-Glu L-LysGlyGlyGlyGlyGlyNH O CH3 D-Ala HOOC N O H peptidoglycan D-Ala  Penicillins — Mode of Action H H R N S R N S Me Me O N O HN Me ENZYMEO Me O O COOH COOH ENZYME OH H 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 OH NH2 OH Me Me O HO O CH2OH O O Cl O O H O HO OH Me Me O H Cl H O H2N H H O H O N N N N N H H H H NH HN CO2H O H2NOC O NHMe O D-Cycloserine (DCS) Vancomycin OH HO OH  Formation of Cell Wall Crosslinks (NAM-NAG)n L-Ala D-Glu L-LysGlyGlyGlyGlyGlyNH2 CH 3 D-Ala HOOC N O H D-Ala (NAM-NAG)n (NAM-NAG)n L-Ala L-Ala D-Glu D-Glu ENZYME OH L-LysGlyGlyGlyGlyGlyNH 2 -D-Ala L-LysGlyGlyGlyGlyGlyNH 2 CH 3 D-Ala D-Ala HOOC N O ENZYME O O H D-Ala H H (NAM-NAG)n L-Ala (NAM-NAG)n D-Glu L-LysGlyGlyGlyGlyGlyNH 2 L-Ala D-Glu D-Ala L-LysGlyGlyGlyGlyGlyNH O CH 3 D-Ala HOOC N O H peptidoglycan D-Ala  Cycloserine  D-Cycloserine (DCS) is produced by Streptomyces garyphalus and Streptomyces lavendulae and can be used in treatment of tuberculosis  D-Ala-D-Ala required for synthesis of bacterial cell wall but this is the unnatural enantiomer of Ala  Bacteria must produce D-Ala from natural L-Ala, using alanine racemase (ALR) — produces a racemic mixture (50:50) from either alanine enantiomer  Rare example of the destruction of chirality by an enzymatic process  ALR uses pyridoxal 5′-phosphate (PLP) as co-factor to cause racemisation via an imine (Schiff’s base), thus increasing the acidity of the α-hydrogen of the alanine  DCS inhibits alanine racemase, thus preventing the formation of the D-Ala required for crosslink formation  DCS also inhibits D-Ala-D-Ala ligase (Ddl) the enzyme responsible for the coupling of the two D-Ala residues to give the D-Ala-D-Ala dipeptide which is added to the carboxy terminal L-Lys to form the pentapeptide [M. Sugiyama et al., J. Biol. Chem., 2004, 279, 46143]  Alanine Racemase (ALR) R CO2H N H Me Schiff's base R CO2 RCHO CO2 R CO2H H 3N N H N H Me H Me H Me L-Ala planar carbanion B (allows delocalisation) H R R N CO2H N CO2H [RCHO = PLP] H H H Me H Me hydrolysis H3N CO2 H3N CO2 H Me H Me L-Ala D-Ala  Alanine Racemase (ALR) R CO2H N H Me Schiff's base R CO2 RCHO CO2 R CO2H H 3N N H N H Me H Me H Me L-Ala planar carbanion B (allows delocalisation) H R R N CO2H N CO2H [RCHO = PLP] H H H Me H Me hydrolysis H3N CO2 H3N CO2 H Me H Me L-Ala D-Ala  Alanine Racemase (ALR) R CO2H N H Me Schiff's base R CO2 RCHO CO2 R CO2H H 3N N H N H Me H Me H Me L-Ala planar carbanion B (allows delocalisation) H R R N CO2H N CO2H [RCHO = PLP] H H H Me H Me hydrolysis H3N CO2 H3N CO2 H Me H Me L-Ala D-Ala  Alanine Racemase (ALR) R CO2H N H Me Schiff's base R CO2 RCHO CO2 R CO2H H3N N H N H Me H Me H Me L-Ala planar carbanion B (allows delocalisation) H R R N CO2H N CO2H [RCHO = PLP] H H H Me H Me hydrolysis H 3N CO2 H 3N CO2 H Me H Me L-Ala D-Ala  D-Ala-D-Ala Ligase Me H O H3N Me H Me H O Me H ATP ADP D-Ala-D-Ala H O O O N CO2 ligase H3N H3N P O H 3N ligase O O O O Me H D-Ala-D-Ala Me H Me H ENZYME-OH....L-Lys O CO2....L-Lys O N N ENZYME H H O Me H O acyl-enzyme intermediate  Cycloserine H O H2N NH O D-Cycloserine (DCS)  Orally available antibiotic  Broad spectrum  Used in combination therapies for the treatment of TB  Serious side-effects, including depression and convulsions  Side-effects due to binding as an agonist to neuronal N-methyl-D-aspartate (NMDA) receptors  Inhibits enzymes which metabolize and synthesize neurotransmitter GABA  Resistance due to over-production of D-Ala racemase  Vancomycin (Vancocin)  Vancomycin is a glycopeptide antibiotic produced by Streptomyces orientalis which is the last resort in treatment of MRSA  Not absorbed orally so usually given i.v. and can be toxic to ears and kidneys  Oral vancomycin used in treatment of Clostridioides difficile-associated disease OH NH2 OH Me Me O HO O CH2OH O O Cl O O HO OH Me Me O H Cl H O O H H H O N N N N N H H H H HN CO2H O H2NOC O NHMe Vancomycin OH HO OH  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) O N O F N O O Me N H  Vancomycin (Vancocin)  Vancomycin is hydrophilic and forms hydrogen bonds to the terminal D-Ala-D-Ala 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-Lactate ligase which synthesises an ester (D-Ala-D-Lac) 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 micoorganisms (J.R. Knox et al., Structure, 2000, 8, 463)  Treatment for VRE infections can involve linezolid  Vancomycin Resistance Me H O CO2 D-Ala-D-Lac H 3N ligase Me H Me H O Me H ATP ADP O D-Ala-D-Lac O O H 3N H 3N P O ligase O O O Me H D-Ala-D-Ala H N CO2 ligase H3N O Me H D-Ala-D-Ala Me H Me H ENZYME-OH....L-Lys O CO2....L-Lys O N N ENZYME H H O Me H O acyl-enzyme intermediate Formation of Cell Wall Crosslinks NAM NAM H2N-L-Ala-D-Glu-L-Lys L-Ala D-Ala-D-Ala L-Ala NAM D-Glu D-Glu HO2CGlyGlyGlyGlyGlyNH2 L-LysGlyGlyGlyGlyGlyNH2 L-LysGlyGlyGlyGlyGlyNH2 CO2H D-Ala D-Ala NAM-NAG CYTOPLASM L-Ala D-Glu L-LysGlyGlyGlyGlyGlyNH2 D-Ala D-Ala CARRIER CARRIER NAM-NAG LIPID LIPID L-Ala D-Glu CELL MEMBRANE L-LysGlyGlyGlyGlyGlyNH2 D-Ala D-Ala GROWING CELL WALL CELL WALL  Formation of Cell Wall Crosslinks (NAM-NAG)n L-Ala D-Glu L-LysGlyGlyGlyGlyGlyNH2 CH 3 D-Ala HOOC N O H D-Ala (NAM-NAG)n (NAM-NAG)n L-Ala L-Ala D-Glu D-Glu ENZYME OH L-LysGlyGlyGlyGlyGlyNH 2 -D-Ala L-LysGlyGlyGlyGlyGlyNH 2 CH 3 D-Ala D-Ala HOOC N O ENZYME O O H D-Ala H H (NAM-NAG)n L-Ala (NAM-NAG)n D-Glu L-LysGlyGlyGlyGlyGlyNH 2 L-Ala D-Glu D-Ala L-LysGlyGlyGlyGlyGlyNH O CH 3 D-Ala HOOC N O H peptidoglycan D-Ala  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  Daptomycin (Lipopeptide) 10 of the 13 amino acids form a depsipeptide (a peptide in which at least 1 amide [CONH] bond has been replaced by an ester [COO] link) ring, with the other 3 attached to this ring though a threonine residue  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 causes 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 [V. Laganas, J. Alder and J. A. Silverman, Antimicrob. Agents Chemother., 2003, 47, 2682 – 2684; L. Robbel and M. A. Marahiel, J. Biol. Chem., 2010, 285, 27501 – 27508]  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 walls and daptomycin resistance is due to its inability to diffuse through these thicker cell walls to 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 Paenibacillus sp. LC231 strain with daptomycin resistance found in Lechuguilla Cave, that has been isolated from the surface for over 4 Million years [Pawlowski, Nature Commun, 2016, 7, 13803]  The Bacterial Cell (Prokaryotic) β-LACTAMS VANCOMYCIN QUINOLONES CHLORAMPHENICOL TETRACYCLINES MACROLIDES SULPHONAMIDES ISONIAZID  Quinolone Antibacterials  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  Quinolone Antibacterials O O CO2H F CO2H Me N N N N Et HN Nalidixic acid Ciprofloxacin (Cipro, Ciproxin) (1st gen) active against Gram -ve (2nd gen) active against P. aeruginosa O O F CO2H F CO2H H N N N N N O Me F F Me H 2N H H F Levofloxacin (Cravit, Levaquin) Trovafloxacin (Trovan) (3rd gen) used in treatment of community- (4th gen) improved Gram +ve activity acquired pneumonia  Quinolone Antibacterials  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 chain. 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 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 (J. Ruiz, J. Antimicrob. Chemother., 2003, 51, 1109)  Quinolone Antibacterials  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) or 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 the quinolones [J. Ruiz, J. Antimicrob. Chemother., 2003, 51, 1109]  Bacillus anthracis  Causative agent of anthrax  B. anthracis is a Gram positive spore forming rod-shaped bacterium  Commonly found in soil of grazing areas (only 3 cases of anthrax in NSW in ~40 years)  Also used as biological warfare agent (Sep/Nov 2001 spores sent in US mail, led to 22 cases of anthrax and 5 deaths)  Patients with cutaneous anthrax can be completely cure; intestinal and inhalational (pulmonary) anthrax have very high mortality rates  Limited vaccine availability in Australia  Treatment usually involves ciprofloxacin or doxycycline Socrative Room 145682  The Bacterial Cell (Prokaryotic) β-LACTAMS VANCOMYCIN QUINOLONES CHLORAMPHENICOL TETRACYCLINES MACROLIDES SULPHONAMIDES ISONIAZID 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 Antibacterials  Sulphonamides were the first specific and synthetic antibacterials  Discovered in 1932 when a red dyestuff (Prontosil), manufactured at the Bayer Corporation (IG Farben), was found to prevent the multiplication of bacteria in animal experiments (but not in in vitro tests)  Gerhard Domagk who discovered the antibacterial effect of the sulphonamides was awarded the Nobel Prize in Medicine in 1939 (http://nobelprize.org/nobel_prizes/medicine/laureates/1939/domagk-lecture.pdf)  Deaths of more than 100 patients in 1937 who used Elixir Sulfanilamide was a result of a soluble form (using toxic diethylene glycol as the solvent) and led to increased regulation of drugs by the US Food and Drug Administration  Sulphonamide (M&B 693) used to treat Winston Churchill, who had contracted pneumonia at a meeting to discuss D-Day landings held in 1943 in North Africa. Prontosil is an azo dye and the sulphonamide group was originally introduced to help increase the binding of the dye to wool  Sulphonamide Antibacterials N SO2NH2 azo reductase H2N N H 2N NH2 + H 2N SO2NH2 NH2 NH2 Prontosil Sulphanilamide  Prontosil is inactive in vitro as it is a prodrug which requires activation via an azo reductase in vivo. Using prontosil Domagk was able to control streptococcal infections in mice and staphylococcal infections in rabbits  Sulphanilamide is produced in vivo and was the first synthetic antibacterial agent  Daniele Bovet and co-workers at Institute Pasteur (Paris) discovered the in vivo metabolism to sulphanilamide  Sulphonamide Antibacterials  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 (-SO2NR2) 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 H H N SO2N R R'  Folic Acid Synthesis OH 6-Hydroxymethylpterin OH N pyrophosphokinase (PPPK) N N OH N OPP H2N N N 2ATP 2ADP H2N N N H H NH2 Dihydropteroate synthetase (DHPS) HO2C O H CO2H CO2H OH N CH2CH2CO2H OH H N N N N L-Glu N N H H H 2N N N H 2N N N H H Dihydrofolic acid  Folic Acid Synthesis OH 6-Hydroxymethylpterin OH N pyrophosphokinase (PPPK) N N OH N OPP H2N N N 2ATP 2ADP H2N N N H H NH2 Dihydropteroate synthetase (DHPS) HO2C O H CO2H CO2H OH N CH2CH2CO2H OH H N N N N L-Glu N N H H H 2N N N H 2N N N H H Dihydrofolic acid  Folic Acid Synthesis OH 6-Hydroxymethylpterin OH N pyrophosphokinase (PPPK) N N OH N OPP H2N N N 2ATP 2ADP H2N N N H H NH2 Dihydropteroate synthetase (DHPS) HO2C O H CO2H CO2H OH N CH2CH2CO2H OH H N N N N L-Glu N N H H H 2N N N H 2N N N H H Dihydrofolic acid  Folic Acid Synthesis OH 6-Hydroxymethylpterin OH N pyrophosphokinase (PPPK) N N OH N OPP H2N N N 2ATP 2ADP H2N N N H H NH2 Dihydropteroate synthetase (DHPS) HO2C O H CO2H CO2H OH N CH2CH2CO2H OH H N N N N L-Glu N N H H H 2N N N H 2N N N H H Dihydrofolic acid  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 H H H-bond H H H H N N N 670 pm π−π stacking 690 pm C S S ionic bond O O O O O O NHR NR  Prevention of Folic Acid Synthesis OH 6-Hydroxymethylpterin OH N pyrophosphokinase (PPPK) N N OH N OPP H 2N N N 2ATP 2ADP H 2N N N H H NH2 Dihydropteroate synthetase H2NO2S SO2NH2 OH N L-Glu x N N H H 2N N N H  Prevention of Folic Acid Synthesis OH 6-Hydroxymethylpterin OH N pyrophosphokinase (PPPK) N N OH N OPP H 2N N N 2ATP 2ADP H 2N N N H H NH2 Dihydropteroate synthetase H2NO2S SO2NH2 OH N L-Glu x N N H H 2N N N H  Prevention of Folic Acid Synthesis OH 6-Hydroxymethylpterin OH N pyrophosphokinase (PPPK) N N OH N OPP H2N N N 2ATP 2ADP H 2N N N H H NH2 Dihydropteroate synthetase H2NO2S SO2NH2 OH N L-Glu x N N H H2N N N H  Sulphonamides N Sulphapyridine (M&B 693) HN Used in treatment of pneumonia. 2-Pyridyl more active H 2N S O than other isomers O N Sulphathiazole HN S Higher therapeutic index but poorly ionised and so H2N S O poorly soluble (pKa ~10) (metabolites can crystallise O and block kidney tubules) N Sulphadiazine Increased acidity of NH proton due to electron HN N withdrawing pyrimidine so lower pKa (6.5) and mostly H2N S O ionised in blood (pH 7.4). Meningococcal meningitis O N Sulphamethoxypyridazine (Sulfametopyrazine) HN N Half-life of 37 hours (one administration per day). UTI, H2N S O OMe chronic bronchitis O N O Sulphamethoxazole Me Used in combination with trimethoprim (another HN antifolate) in Co-trimoxazole to treat UTI, gonorrhoea H2N S O and pneumocystis carinii (jiroveci) pneumonia in HIV O patients  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 (O. Sköld, Drug Resistance Updates, 2000, 3, 155)  Trimethoprim NH2 OMe N H2N N OMe OMe  Trimethoprim (Monotrim, Proloprim) is an antifolate agent which is a dihydrofolate reductase (DHFR) inhibitor  Used in combination (synergistic) with sulphamethoxazole (Co-trimoxazole, Resprim, Bactrim)  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  Co-trimoxazole NH2 CO2H OH OH N HO2C N N OPP N N H Dihydropteroate H 2N N N synthetase (DHPS) H2N N N L-Glu O H CO2H Sulphamethoxazole OH N CH2CH2CO2H H N N N H H 2N N N FOLIC ACID NADPH O H O H CO2H NADP CO2H OH N CH2CH2CO2H OH N CH2CH2CO2H H H H N NADP N N N NADPH N N H H Dihydrof olate reductase H2N N N (DHFR) H2N N N H H TETRAHYDROFOLIC ACID DIHYDROFOLIC ACID Trimethoprim  The Bacterial Cell (Prokaryotic) β-LACTAMS VANCOMYCIN QUINOLONES CHLORAMPHENICOL TETRACYCLINES MACROLIDES SULPHONAMIDES ISONIAZID  Agents which target protein synthesis Agents which target the ribosome and so inhibit protein synthesis include; HO H X R1 R2 H R3 H NMe2 H H CH2OH OH R R HN H NH2 O2N Me OH O OH O OH O O Chloramphenicol Tetracyclines O Me Me HO OH OH NMe2 Me Me Me HO O Me Et O O O O OMe Me Me OH Erythromycin O Me Transcription and translation Replication Translation DNA Transfer RNA (tRNA) – amino acid Transcription Messenger RNA (mRNA) RIBOSOME Protein  Transcription and translation 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 Protein Synthesis at the Ribosome Protein Synthesis at the Ribosome  tRNA-amino acid complex (charged tRNA) O 3' NH3 A O C H R C A 5' C G C G 3 nucleotides complement those on mRNA (C-G, A-U) [http://www3.interscience.wiley.com:8100/legacy/college/boyer/0471661791/structure/tRNA/trna_intro.htm]  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) HO H  Active against Neisseria meningitidis, Streptococcus CH2OH pneumoniae and Haemophilus influenzae (causes of meningitis) HN H O2N  Used in treatment of meningitis in patients with β- CHCl2 lactam allergies and drug of choice against typhoid O fever  Severe toxicity so not given systemically but used in treatment of bacterial conjuctivitis  Side-effects include aplastic anaemia (bone marrow cannot replenish blood cells) – unpredictable and may occur weeks after treatment ceases  Neisseria meningitidis Diplococcal Gram negative organism Cause of meningococcal disease (meningitis and sepsis) Meningitis – inflammation of the membranes protecting the brain and spinal cord (meninges) Symptoms – headache, inability to tolerate light, fever, rash, confusion, death Meningitis progresses rapidly so requires rapid diagnosis (microscopic examination of CSF) and treatment  Chloramphenicol (Chloromycetin, Cm)  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 PTC  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. (S. Schwartz et al., FEMS Microbiology Rev., 2004, 29, 519) HO H CH2F O HN H S Me CHCl2 O O  Chloramphenicol (Chloromycetin, Cm)  Originally obtained from Streptomyces venezuelae, now prepared synthetically  Bacteriostatic with broad spectrum of activity and HO H only R,R-isomer is active CH2OH  Highly lipophilic and penetrates most tissues (crosses HN H blood-brain barrier) O 2N  Active against Neisseria meningitidis, Streptococcus O CHCl2 pneumoniae and Haemophilus influenzae (causes of meningitis) CAT  Used in treatment of meningitis in patients with β- lactam allergies and drug of choice against typhoid HO H O fever O CH3  Severe toxicity so not given systemically but used in HN H treatment of bacterial conjuctivitis O 2N  Side-effects include aplastic anaemia (bone marrow O CHCl2 cannot replenish blood cells) – unpredictable and may occur weeks after treatment ceases  Aminoglycosides  Streptomycin isolated from Streptomyces griseus by Selman Waksman and first used clinically in treatment of tuberculosis in 1944 (Nobel prize 1952).  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. OH OH O H 2N O H 2N Me O Me HO O HN HO H2N HO OH O NH O NH OH 2 OH 2 Me O NH2 O NH2 H 2N H2N Gentamicin Tobramycin H2N NH HN OH H N NH2 O HO OH O NH CHO Me OH O Streptomycin HO O HO NHMe HO  Concentration- versus time-dependent antibacterial activity Time-dependent  Providing the concentration remains above the minimum inhibitory concentration (MIC) it 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