医学课件抗生素

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. CategoryParenteral AgentsOral Agents Natural PenicillinsPenicillin GPenicillin V Antistaphylococcal penicillins Nafcillin, oxacillinDicloxacillin AminopenicillinsAmpicillinAmoxicillin and Ampicillin Aminopenicillin + b- lactamase inhibitor Ampicillin-sulbactamAmoxicillin-clavulanate Extended-spectrum penicillin Piperacillin, ticaricillinCarbenicillin Extended-spectrum penicillin + b-lactamase inhibitor Piperacillin-tazobactam, ticaricillin-clavulanate The Penicillins INTRODUCTIONINTRODUCTION Antibacterial agents which inhibit bacterial cell wall synthesisAntibacterial agents which inhibit bacterial cell wall synthesis Discovered by Fleming from a fungal colony (1928) Discovered by Fleming from a fungal colony (1928) Shown to be non toxic and antibacterialShown to be non toxic and antibacterial Isolated and purified by Florey and Chain (1938)Isolated and purified by Florey and Chain (1938) First successful clinical trial (1941) First successful clinical trial (1941) Produced by large scale fermentation (1944)Produced by large scale fermentation (1944) Structure established by X-Ray crystallography (1945) Structure established by X-Ray crystallography (1945) Full synthesis developed by Sheehan (1957)Full synthesis developed by Sheehan (1957) Isolation of 6-APA by Isolation of 6-APA by Beecham Beecham (1958-60) (1958-60) - development of semi-synthetic penicillins- development of semi-synthetic penicillins Discovery of clavulanic acid and Discovery of clavulanic acid and b b-lactamase inhibitors-lactamase inhibitors /microbelibrary/files/ccImages/Artic leimages/Spencer/spencer_cellwall.html Acyl side Acyl side chainchain 6-Aminopenicillanic acid6-Aminopenicillanic acid (6-APA)(6-APA) STRUCTURESTRUCTURE Side chain varies depending on carboxylic acid present in fermentation mediumSide chain varies depending on carboxylic acid present in fermentation medium b b-Lactam-Lactam ringring ThiazolidineThiazolidine ringring present in corn steep liquor Penicillin G Benzyl penicillin (Pen G)Benzyl penicillin (Pen G) R =R = Phenoxymethyl penicillin (Pen V)Phenoxymethyl penicillin (Pen V) R =R = Penicillin V (first orally active penicillin) Shape of Penicillin GShape of Penicillin G Folded envelope shape Folded envelope shape Properties of Penicillin GProperties of Penicillin G Active vs. Gram +ve bacilli and some Gram -ve cocciActive vs. Gram +ve bacilli and some Gram -ve cocci Non toxicNon toxic Limited range of activityLimited range of activity Not orally active - must be injectedNot orally active - must be injected Sensitive to Sensitive to b b-lactamases -lactamases (enzymes which hydrolyse the (enzymes which hydrolyse the b b-lactam ring)-lactam ring) Some patients are allergic Some patients are allergic Inactive vs. Inactive vs. StaphylococciStaphylococci Drug DevelopmentDrug Development AimsAims To increase chemical stability for oral administrationTo increase chemical stability for oral administration To increase resistance to To increase resistance to b b-lactamases-lactamases To increase the range of activityTo increase the range of activity SARSAR ConclusionsConclusions Amide and carboxylic acid are involved in bindingAmide and carboxylic acid are involved in binding Carboxylic acid binds as the carboxylate ionCarboxylic acid binds as the carboxylate ion Mechanism of action involves theMechanism of action involves the b b-lactam ring-lactam ring Activity related to Activity related to b b-lactam ring strain -lactam ring strain (subject to stability factors)(subject to stability factors) Bicyclic system increases Bicyclic system increases b b-lactam ring strain-lactam ring strain Not much variation in structure is possibleNot much variation in structure is possible Variations are limited to the side chain (R)Variations are limited to the side chain (R) Penicillins Penicillins inhibit inhibit a a bacterial bacterial enzyme enzyme called called the the transpeptidase transpeptidase enzyme enzyme which which is is involved involved in in the the synthesis synthesis of of the the bacterial bacterial cell cell wallwall The The b b-lactam ring is involved in the mechanism of inhibition-lactam ring is involved in the mechanism of inhibition Penicillin Penicillin becomes becomes covalently covalently linked linked to to the the enzymes enzymes active active site site leading to irreversible inhibitionleading to irreversible inhibition Covalent bond formed Covalent bond formed to transpeptidase enzymeto transpeptidase enzyme Irreversible inhibitionIrreversible inhibition Mechanism of actionMechanism 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 synthesisMechanism of action - bacterial cell wall synthesis NAMNAMNAMNAGNAG NAMNAMNAMNAGNAG NAMNAMNAMNAGNAG Bond formation inhibited by penicillin Cross linkingCross linking Mechanism of action - bacterial cell wall synthesisMechanism of action - bacterial cell wall synthesis Penicillin inhibits final crosslinking stage of cell wall synthesisPenicillin inhibits final crosslinking stage of cell wall synthesis It It reacts reacts with with the the transpeptidase transpeptidase enzyme enzyme to to form form an an irreversible covalent bondirreversible covalent bond Inhibition of transpeptidase leads to a weakened cell wall Inhibition of transpeptidase leads to a weakened cell wall Cells swell due to water entering the cell, then burst (lysis)Cells swell due to water entering the cell, then burst (lysis) Penicillin Penicillin possibly possibly acts acts as as an an analogue analogue of of the the L-Ala-L-Ala-g g-D-Glu -D-Glu portion portion of of the the pentapeptide pentapeptide chain. chain. However, However, the the carboxylate carboxylate group group that that is is essential essential to to penicillin penicillin activity activity is is not not present present in in this portionthis portion Mechanism of action - bacterial cell wall synthesisMechanism of action - bacterial cell wall synthesis Alternative theoryAlternative theory- Pencillin mimics D-Ala-D-Ala.- Pencillin mimics D-Ala-D-Ala. Normal MechanismNormal Mechanism Mechanism of action - bacterial cell wall synthesisMechanism of action - bacterial cell wall synthesis Alternative theoryAlternative theory- Penicillin mimics D-Ala-D-Ala.- Penicillin mimics D-Ala-D-Ala. Mechanism inhibited by penicillinMechanism inhibited by penicillin Mechanism of action - bacterial cell wall synthesisMechanism of action - bacterial cell wall synthesis Penicillin can be seen to mimic acyl-D-Ala-D-AlaPenicillin can be seen to mimic acyl-D-Ala-D-Ala PenicillinPenicillinAcyl-D-Ala-D-AlaAcyl-D-Ala-D-Ala Mechanism of action - bacterial cell wall synthesisMechanism of action - bacterial cell wall synthesis Penicillin Analogues - PreparationPenicillin Analogues - Preparation 1) By fermentation1) By fermentation vary the carboxylic acid in the fermentation medium vary the carboxylic acid in the fermentation medium limited to unbranched acids at the limited to unbranched acids at the a a-position i.e. RCH-position i.e. RCH 2 2 COCO 2 2 HH tedious and slowtedious and slow 2) By total synthesis2) By total synthesis only 1% overall yield (impractical)only 1% overall yield (impractical) 3) By semi-synthetic procedures3) By semi-synthetic procedures Use Use a a naturally naturally occurring occurring structure structure as as the the starting starting material material for for analogue synthesisanalogue synthesis Penicillin Analogues - PreparationPenicillin Analogues - Preparation Fermentation Penicillin acylasePenicillin acylase or chemical hydrolysisor chemical hydrolysis Semi-synthetic penicillinsSemi-synthetic penicillins Penicillin Analogues - PreparationPenicillin Analogues - Preparation ProblemProblem - - How How does does one one hydrolyse hydrolyse the the side side chain chain by by chemical chemical means in presence of a labilemeans in presence of a labile b b-lactam ring?-lactam ring? AnswerAnswer - Activate the side chain first to make it more reactive - Activate the side chain first to make it more reactive NoteNote - - Reaction Reaction with with PClPCl 5 5 requires requires involvement involvement of of nitrogens nitrogens lone pair of electrons. Not possible for the lone pair of electrons. Not possible for the b b-lactam nitrogen.-lactam nitrogen. Problems with Penicillin GProblems with Penicillin G It is sensitive to stomach acids It is sensitive to stomach acids It is sensitive to It is sensitive to b b-lactamases - -lactamases - enzymes which hydrolyse the enzymes which hydrolyse the b b- - lactam ringlactam ring it has a limited range of activity it has a limited range of activity Problem 1 - Acid SensitivityProblem 1 - Acid Sensitivity Reasons for sensitivityReasons for sensitivity 1) Ring Strain1) Ring Strain Relieves ring strainRelieves ring strain Acid or enzyme Problem 1 - Acid SensitivityProblem 1 - Acid Sensitivity 2) Reactive 2) Reactive b b-lactam carbonyl group-lactam carbonyl group Does not behave like a tertiary amideDoes not behave like a tertiary amide Interaction of nitrogens lone pair with the carbonyl group is not possible Interaction of nitrogens lone pair with the carbonyl group is not possible Results in a reactive carbonyl groupResults in a reactive carbonyl group Tertiary amideTertiary amide Unreactive b b-Lactam-Lactam Folded ring system Impossibly strained Reasons for sensitivityReasons for sensitivity X Problem 1 - Acid SensitivityProblem 1 - Acid Sensitivity 3) Acyl Side Chain3) Acyl Side Chain - neighbouring group participation in the hydrolysis mechanism- neighbouring group participation in the hydrolysis mechanism Further reactions Reasons for sensitivityReasons for sensitivity Problem 1 - Acid SensitivityProblem 1 - Acid Sensitivity ConclusionsConclusions The The b b-lactam -lactam ring ring is is essential essential for for activity activity and and must must be be retainedretained Therefore, cannot tackle factors 1 and 2Therefore, cannot tackle factors 1 and 2 Can only tackle factor 3Can only tackle factor 3 StrategyStrategy Vary Vary the the acyl acyl side side group group (R) (R) to to make make it it electron electron withdrawing withdrawing to to decrease the nucleophilicity of the carbonyl oxygendecrease the nucleophilicity of the carbonyl oxygen DecreasesDecreases nucleophilicitynucleophilicity Penicillin VPenicillin V (orally active)(orally active) Problem 1 - Acid SensitivityProblem 1 - Acid Sensitivity ExamplesExamples electronegativeelectronegative oxygenoxygen Very Very successful successful semi-semi- synthetic penicillinssynthetic penicillins e.g. ampicillin, oxacilline.g. ampicillin, oxacillin Better acid stability and orally activeBetter acid stability and orally active But sensitive to But sensitive to b b-lactamases-lactamases Slightly less active than Penicillin GSlightly less active than Penicillin G Allergy problems with some patientsAllergy 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 SpirochetesTreponema pallidum Leptospira spp. Problem 2 - Sensitivity to Problem 2 - Sensitivity to b b-Lactamases-Lactamases Notes on Notes on b b-Lactamases-Lactamases Enzymes that inactivate penicillins by opening Enzymes that inactivate penicillins by opening b b-lactam rings-lactam rings Allow bacteria to be resistant to penicillinAllow bacteria to be resistant to penicillin Transferable Transferable between between bacterial bacterial strains strains (i.e. (i.e. bacteria bacteria can can acquire resistance)acquire resistance) Important w.r.t. Important w.r.t. Staphylococcus aureusStaphylococcus aureus infections in hospitals infections in hospitals 80% 80% StaphStaph. . infections infections in in hospitals hospitals were were resistant resistant to to penicillin penicillin and other antibacterial agents by 1960and other antibacterial agents by 1960 Mechanism Mechanism of of action action for for lactamases lactamases is is identical identical to to the the mechanism of inhibition for the target enzymemechanism of inhibition for the target enzyme But But product product is is removed removed efficiently efficiently from from the the lacta