1、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 procedureGram-negative: Dont take up
2、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,http:/student.ccbcmd.edu/courses/bio141/labmanua/lab6/images/gram_stain_11.swf,Structure of pept
3、idoglycan. 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
4、 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 C
5、hloramphenicol 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 move
6、ment 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 o
7、f 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 a
8、re 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 Staphylococcu
9、s 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
10、 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
11、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,Atyp
12、ical Bacteria of Clinical Importance Include:,Chlamydia Mycoplasma Legionella Brucella Francisella tularensis Rickettsia,Spirochetes of Clinical Importance Include:,Treponema pallidumBorrelia burgdorferiLeptospira interrogans,Mycobacteria of Clinical Importance Include:,Mycobacterium tuberculosis My
13、cobacterium 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 li
14、nked 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 antibioti
15、c 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 cataly
16、ze 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: penetration, porins,
17、 pumps, penicillinases (-lactamases), penicillin-binding proteins (PBPs), and peptidoglycan.,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 b
18、y 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
19、of clavulanic acid and b-lactamase inhibitors,http:/www.microbelibrary.org/microbelibrary/files/ccImages/Articleimages/Spencer/spencer_cellwall.html,STRUCTURE,Side chain varies depending on carboxylic acid present in fermentation medium,Shape of Penicillin G,Folded envelope shape,Properties of Penic
20、illin 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
21、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 s
22、train (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 cel
23、l 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,Mechanism of action - bacterial cell wall s
24、ynthesis,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
25、 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-
26、 Pencillin mimics D-Ala-D-Ala.,Mechanism of action - bacterial cell wall synthesis,Alternative theory- Penicillin mimics D-Ala-D-Ala.,Mechanism of action - bacterial cell wall synthesis,Penicillin can be seen to mimic acyl-D-Ala-D-Ala,Mechanism of action - bacterial cell wall synthesis,Penicillin An
27、alogues - 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 slow2) By total synthesis only 1% overall yield (impractical)3) By semi-synthetic procedures Use a naturally occurring structure as the
28、 starting material for analogue synthesis,Penicillin Analogues - Preparation,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 r
29、eactive,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 ringit has a limited range of activit
30、y,Problem 1 - Acid Sensitivity,Reasons for sensitivity,1) Ring Strain,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,
31、Reasons for sensitivity,X,Problem 1 - Acid Sensitivity,3) Acyl Side Chain - neighbouring group participation in the hydrolysis mechanism,Reasons for sensitivity,Problem 1 - Acid Sensitivity,Conclusions,The b-lactam ring is essential for activity and must be retained Therefore, cannot tackle factors
32、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,Penicillin V (orally active),Problem 1 - Acid Sensitivity,Examples,Very successful semi-synthetic penicillinse.g. ampicillin, oxacillin,Better
33、 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),Problem 2 - Sensitivity to b-Lactamases,Notes on b-Lactamases,Enzymes that inactiva
34、te 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
35、 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,Problem 2 - Sensitivity to b-Lactamases,Strategy,Block access of penicillin to active site of enzyme by
36、 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),Problem 2 - Sensitivity to b-Lactamases,Examples - Methicillin (Beecham - 1960),Methoxy groups block acc
37、ess 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 Po
38、or 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 infl
39、uences 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 O
40、xacillin (parenteral) as well as Dicloxacillin (oral),Problem 3 - Range of Activity,Factors Cell wall may have a coat preventing access to the cell Excess transpeptidase enzyme may be present Resistant transpeptidase enzyme (modified structure) Presence of b-lactamases Transfer of b-lactamases betwe
41、en strains 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 Acti
42、vity,R= hydrophobic results in high activity vs. Gram + bacteria and poor activity vs. Gram - bacteria Increasing hydrophobicity has little effect on Gram + activity but lowers Gram - activity Increasing hydrophilic character has little effect on Gram + activity but increases Gram - activity Hydroph
43、ilic 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 pe
44、nicillin,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
45、 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 reducer (lansoprazo
46、le, or Prevacid) is sometimes added.,Helicobacter pylori,Helicobacter pylori is linked to stomach inflammation, which may also result in gastric ulcers and stomach cancer,In the early 20th century, ulcers were believed caused by stressIn 1982, Robin Warren and Barry Marshall, two Australian physicia
47、ns, suggested link between H. pylori and ulcersMedical community was slow to accept (first abstract describing such results was rejected for a poster),In 2005, the two researchers, Barry Marshall and J. Robin Warren, received the Nobel Prize in medicine for their discovery of the bacterium Helicobac
48、ter pylori and its role in gastritis and peptic ulcer disease,Problem 3 - Range of Activity,Prodrugs of Ampicillin (Leo Pharmaceuticals - 1969),Properties Increased cell membrane permeability Polar carboxylic acid group is masked by the ester Ester is metabolised in the body by esterases to give the
49、 free drug,Problem 3 - Range of Activity,Mechanism,Ester is less shielded by penicillin nucleus Hydrolysed product is chemically unstable and degrades Methyl ester of ampicillin is not hydrolysed in the body - bulky penicillin nucleus acts as a steric shield,The aminopenicillins include Ampicillin (parenteral) as well as Amoxicillin and Ampicillin (both oral),
