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Antibiotics Step 1: How to Kill a Bacterium. What are the bacterial weak points? Specifically, which commercial antibiotics target each of these points? Target 1: The Bacterial Cell Envelope Two types of bacteria Gram-positive: Stained dark blue by Gram -staining procedure Gram-negative: Dont take up the crystal violet stain, and take up counterstain (safranin) instead, staining pink in the Gram procedure. Structure of the bacterial cell envelope. Gram-positive. Gram-negative. Gram staining animation /courses/bio141/labmanua/lab6/imag es/gram_stain_11.swf Structure of peptidoglycan. Peptidoglycan synthesis requires cross-linking of disaccharide polymers by penicillin-binding proteins (PBPs). NAMA, N-acetyl- muramic acid; NAGA, N-acetyl-glucosamine. Antibiotics that Target the Bacterial Cell Envelope Include: The b-Lactam Antibiotics Vancomycin Daptomycin Target 2: The Bacterial Process of Protein Production An overview of the process by which proteins are produced within bacteria. Structure of the bacterial ribosome. Antibiotics that Block Bacterial Protein Production Include: Rifamycins Aminoglycosides Macrolides and Ketolides Tetracyclines and Glycylcyclines Chloramphenicol Clindamycin Streptogramins Linezolid (member of Oxazolidinone Class) Target 3: DNA and Bacterial Replication Bacterial synthesis of tetrahydrofolate. Supercoiling of the double helical structure of DNA. Twisting of DNA results in formation of supercoils. During transcription, the movement of RNA polymerase along the chromosome results in the accumulation of positive supercoils ahead of the enzyme and negative supercoils behind it. (Adapted with permission from Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. New York: Garland Science, 2002:314.) Replication of the bacterial chromosome. A consequence of the circular nature of the bacterial chromosome is that replicated chromosomes are interlinked, requiring topoisomerase for appropriate segregation. Antibiotics that Target DNA and Replication Include: Sulfa Drugs Quinolones Metronidazole Which Bacteria are Clinically Important? Gram-positive aerobic bacteria Gram-negative aerobic bacteria Anaerobic bacteria (both Gram + and -) Atypical bacteria Spirochetes Mycobacteria General Classes of Clinically Important Bacteria Include: Gram-positive Bacteria of Clinical Importance Staphylococci Staphylococcus aureus Staphylococcus epidermidis Streptococci Streptococcus pneumoniae Streptococcus pyogenes Streptococcus agalactiae Streptococcus viridans Enterococci Enterococcus faecalis Enterococcus faecium Listeria monocytogenes Bacillus anthracis Staphylococcus aureus Streptococcus viridans Gram-negative Bacteria of Clinical Importance Enterobacteriaceae Escherichia coli, Enterobacter, Klebsiella, Proteus, Salmonella, Shigella, Yersinia, etc. Pseudomonas aeruginosa Neisseria Neisseria meningitidis and Neisseria gonorrhoeae Curved Gram-negative Bacilli Campylobacter jejuni, Helicobacter pylori, and Vibrio cholerae Haemophilus Influenzae Bordetella Pertussis Moraxella Catarrhalis Acinetobacter baumannii Anaerobic Bacteria of Clinical Importance Gram-positive anaerobic bacilli Clostridium difficile Clostridium tetani Clostridium botulinum Gram-negative anaerobic bacilli Bacteroides fragilis Atypical Bacteria of Clinical Importance Include: Chlamydia Mycoplasma Legionella Brucella Francisella tularensis Rickettsia Spirochetes of Clinical Importance Include: Treponema pallidum Borrelia burgdorferi Leptospira interrogans Mycobacteria of Clinical Importance Include: Mycobacterium tuberculosis Mycobacterium avium Mycobacterium leprae Antibiotics that Target the Bacterial Cell Envelope The b-Lactam Antibiotics Mechanism of action of -lactam antibiotics. Normally, a new subunit of N- acetylmuramic acid (NAMA) and N-acetylglucosamine (NAGA) disaccharide with an attached peptide side chain is linked to an existing peptidoglycan polymer. This may occur by covalent attachment of a glycine () bridge from one peptide side chain to another through the enzymatic action of a penicillin-binding protein (PBP). In the presence of a -lactam antibiotic, this process is disrupted. The -lactam antibiotic binds the PBP and prevents it from cross-linking the glycine bridge to the peptide side chain, thus blocking incorporation of the disaccharide subunit into the existing peptidoglycan polymer. Mechanism of penicillin-binding protein (PBP) inhibition by -lactam antibiotics. PBPs recognize and catalyze the peptide bond between two alanine subunits of the peptidoglycan peptide side chain. The -lactam ring mimics this peptide bond. Thus, the PBPs attempt to catalyze the -lactam ring, resulting in inactivation of the PBPs. Six Ps by which the action of - lactams may be blocked: (1) penetration, (2) porins, (3) pumps, (4) penicillinases (- lactamases), (5) penicillin-binding proteins (PBPs), and (6) peptidoglycan. Category Parenteral Agents Oral Agents Natural Penicillins Penicillin G Penicillin V Antistaphylococcal penicillins Nafcillin, oxacillin Dicloxacillin Aminopenicillins Ampicillin Amoxicillin and Ampicillin Aminopenicillin + b- lactamase inhibitor Ampicillin-sulbactam Amoxicillin-clavulanate Extended-spectrum penicillin Piperacillin, ticaricillin Carbenicillin Extended-spectrum penicillin + b-lactamase inhibitor Piperacillin-tazobactam, ticaricillin-clavulanate The Penicillins INTRODUCTION Antibacterial agents which inhibit bacterial cell wall synthesis Discovered by Fleming from a fungal colony (1928) Shown to be non toxic and antibacterial Isolated and purified by Florey and Chain (1938) First successful clinical trial (1941) Produced by large scale fermentation (1944) Structure established by X-Ray crystallography (1945) Full synthesis developed by Sheehan (1957) Isolation of 6-APA by Beecham (1958-60) - development of semi-synthetic penicillins Discovery of clavulanic acid and b-lactamase inhibitors /microbelibrary/files/ccImages/Artic leimages/Spencer/spencer_cellwall.html Acyl side chain 6-Aminopenicillanic acid (6-APA) STRUCTURE Side chain varies depending on carboxylic acid present in fermentation medium b-Lactam ring Thiazolidine ring present in corn steep liquor Penicillin G Benzyl penicillin (Pen G) R = Phenoxymethyl penicillin (Pen V) R = Penicillin V (first orally active penicillin) Shape of Penicillin G Folded envelope shape Properties of Penicillin G Active vs. Gram +ve bacilli and some Gram -ve cocci Non toxic Limited range of activity Not orally active - must be injected Sensitive to b-lactamases (enzymes which hydrolyse the b-lactam ring) Some patients are allergic Inactive vs. Staphylococci Drug Development Aims To increase chemical stability for oral administration To increase resistance to b-lactamases To increase the range of activity SAR Conclusions Amide and carboxylic acid are involved in binding Carboxylic acid binds as the carboxylate ion Mechanism of action involves the b-lactam ring Activity related to b-lactam ring strain (subject to stability factors) Bicyclic system increases b-lactam ring strain Not much variation in structure is possible Variations are limited to the side chain (R) Penicillins inhibit a bacterial enzyme called the transpeptidase enzyme which is involved in the synthesis of the bacterial cell wall The b-lactam ring is involved in the mechanism of inhibition Penicillin becomes covalently linked to the enzymes active site leading to irreversible inhibition Covalent bond formed to transpeptidase enzyme Irreversible inhibition Mechanism of action L-Ala D-Glu L-Lys L-Ala D-Glu L-Lys L-Ala D-Glu L-Lys L-Ala D-Glu L-Lys L-Ala D-Glu L-Lys L-Ala D-Glu L-Lys L-Ala D-Glu L-Lys L-Ala D-Glu L-Lys L-Ala D-Glu L-Lys Mechanism of action - bacterial cell wall synthesis NAM NAM NAMNAGNAG NAM NAM NAMNAGNAG NAM NAM NAMNAGNAG Bond formation inhibited by penicillin Cross linking Mechanism of action - bacterial cell wall synthesis Penicillin inhibits final crosslinking stage of cell wall synthesis It reacts with the transpeptidase enzyme to form an irreversible covalent bond Inhibition of transpeptidase leads to a weakened cell wall Cells swell due to water entering the cell, then burst (lysis) Penicillin possibly acts as an analogue of the L-Ala-g-D-Glu portion of the pentapeptide chain. However, the carboxylate group that is essential to penicillin activity is not present in this portion Mechanism of action - bacterial cell wall synthesis Alternative theory- Pencillin mimics D-Ala-D-Ala. Normal Mechanism Mechanism of action - bacterial cell wall synthesis Alternative theory- Penicillin mimics D-Ala-D-Ala. Mechanism inhibited by penicillin Mechanism of action - bacterial cell wall synthesis Penicillin can be seen to mimic acyl-D-Ala-D-Ala Penicillin Acyl-D-Ala-D-Ala Mechanism of action - bacterial cell wall synthesis Penicillin Analogues - Preparation 1) By fermentation vary the carboxylic acid in the fermentation medium limited to unbranched acids at the a-position i.e. RCH2CO2H tedious and slow 2) By total synthesis only 1% overall yield (impractical) 3) By semi-synthetic procedures Use a naturally occurring structure as the starting material for analogue synthesis Penicillin Analogues - Preparation Fermentation Penicillin acylase or chemical hydrolysis Semi-synthetic penicillins Penicillin Analogues - Preparation Problem - How does one hydrolyse the side chain by chemical means in presence of a labile b-lactam ring? Answer - Activate the side chain first to make it more reactive Note - Reaction with PCl5 requires involvement of nitrogens lone pair of electrons. Not possible for the b-lactam nitrogen. Problems with Penicillin G It is sensitive to stomach acids It is sensitive to b-lactamases - enzymes which hydrolyse the b- lactam ring it has a limited range of activity Problem 1 - Acid Sensitivity Reasons for sensitivity 1) Ring Strain Relieves ring strain Acid or enzyme Problem 1 - Acid Sensitivity 2) Reactive b-lactam carbonyl group Does not behave like a tertiary amide Interaction of nitrogens lone pair with the carbonyl group is not possible Results in a reactive carbonyl group Tertiary amide Unreactive b-Lactam Folded ring system Impossibly strained Reasons for sensitivity X Problem 1 - Acid Sensitivity 3) Acyl Side Chain - neighbouring group participation in the hydrolysis mechanism Further reactions Reasons for sensitivity Problem 1 - Acid Sensitivity Conclusions The b-lactam ring is essential for activity and must be retained Therefore, cannot tackle factors 1 and 2 Can only tackle factor 3 Strategy Vary the acyl side group (R) to make it electron withdrawing to decrease the nucleophilicity of the carbonyl oxygen Decreases nucleophilicity Penicillin V (orally active) Problem 1 - Acid Sensitivity Examples electronegative oxygen Very successful semi- synthetic penicillins e.g. ampicillin, oxacillin Better acid stability and orally active But sensitive to b-lactamases Slightly less active than Penicillin G Allergy problems with some patients Natural penicillins include Penicillin G (parenteral) and Penicillin V (oral) Gram-positive bacteria Streptococcus pyogenes, Viridans group streptococci, Some Streptococcus pneumoniae, Some Enterococci, Listeria monocytogenes Gram-negative bacterai Neisseria meningitidis, Some Haemophilus influenzae Anaerobic bacteria Clostridia spp. (except C. difficile), Antinomyces israelii Spirochetes Treponema pallidum Leptospira spp. Problem 2 - Sensitivity to b-Lactamases Notes on b-Lactamases Enzymes that inactivate penicillins by opening b-lactam rings Allow bacteria to be resistant to penicillin Transferable between bacterial strains (i.e. bacteria can acquire resistance) Important w.r.t. Staphylococcus aureus infections in hospitals 80% Staph. infections in hospitals were resistant to penicillin and other antibacterial agents by 1960 Mechanism of action for lactamases is identical to the mechanism of inhibition for the target enzyme But product is removed efficiently from the lactamase active site b-Lactamase Bulky group Problem 2 - Sensitivity to b-Lactamases Strategy Enzyme Block access of penicillin to active site of enzyme by introducing bulky groups to the side chain to act as steric shields Size of shield is crucial to inhibit reaction of penicillins with b- lactamases but not with the target enzyme (transpeptidase) ortho groups important Problem 2 - Sensitivity to b-Lactamases Examples - Methicillin (Beecham - 1960) Methoxy groups block access to b-lactamases but not to transpeptidases Active against some penicillin G resistant strains (e.g. Staphylococcus) Acid sensitive (no e-withdrawing group) and must be injected Lower activity w.r.t. Pen G vs. Pen G sensitive bacteria (reduced access to transpeptidase) Poorer range of activity Poor activity vs. some streptococci Inactive vs. Gram - bacteria Problem 2 - Sensitivity to b-Lactamases Examples - Oxacillin Orally active and acid resistant Resistant to b-lactamases Active vs. Staphylococcus aureus Less active than other penicillins Inactive vs. Gram - bacteria Nature of R & R influences absorption and plasma protein binding Cloxacillin better absorbed than oxacillin Flucloxacillin less bound to plasma protein, leading to higher levels of free drug Oxacillin R = R = H Cloxacillin R = Cl, R = H Flucloxacillin R = Cl, R = F Antistaphylococcal Penicillins include Nafcillin and Oxacillin (parenteral) as well as Dicloxacillin (oral) Gram-positive bacteria Some Staphylococcus aureus, Some Staphylococcus epidermidis Problem 3 - Range of Activity Factors 1. Cell wall may have a coat preventing access to the cell 2. Excess transpeptidase enzyme may be present 3. Resistant transpeptidase enzyme (modified structure) 4. Presence of b-lactamases 5. Transfer of b-lactamases between strains 6. Efflux mechanisms Strategy The number of factors involved make a single strategy impossible Use trial and error by varying R groups on the side chain Successful in producing broad spectrum antibiotics Results demonstrate general rules for broad spectrum activity. Problem 3 - Range of Activity 1. R= hydrophobic results in high activity vs. Gram + bacteria and poor activity vs. Gram - bacteria 2. Increasing hydrophobicity has little effect on Gram + activity but lowers Gram - activity 3. Increasing hydrophilic character has little effect on Gram + activity but increases Gram - activity 4. Hydrophilic groups at the a-position (e.g. NH2, OH, CO2H) increase activity vs Gram - bacteria Results of varying R in Pen G Problem 3 - Range of Activity Examples of Aminopenicillins include: Class 1 - NH2 at the a-position Ampicillin and Amoxicillin (Beecham, 1964) Ampicillin (Penbritin) 2nd most used penicillin Amoxicillin (Amoxil) Problem 3 - Range of Activity Active vs Gram + bacteria and Gram - bacteria which do not produce b-lactamases Acid resistant and orally active Non toxic Sensitive to b-lactamases Increased polarity due to extra amino group Poor absorption through the gut wall Disruption of gut flora leading to diarrhea Inactive vs. Pseudomonas aeruginosa Examples of Aminopenicillins Include: Properties Amoxicillin is sometimes used together with clarithromycin (Biaxin) to treat stomach ulcers caused by Helicobacter pylori, a Gram - bacteria Also, a stomach acid re