antimicrobials课件

Antimicrobials,Medicinal Chemistry Fall 2005,Antimicrobial Agents,Antibiotics natural substances produced by microorganisms Semi-synthetic antibiotics chemically modified natural products Synthetic antibiotics chemically synthesized natural substances Chemotherapeutic agents chemically synthesized agents,Microbes in History,Historical Perspective of Antibiotics,Ancient remedies and observations: 1500 BC Ancient Chinese recognized the therapeutic properties of moldy soybean curd on boils and similar infections 1871 Joseph Lister noted that urine samples contaminated with mold did not allow the growth of bacteria and tried to identify the agent antibacterial agent in the mold. 1874 William Roberts observed that cultures of the mold Penicillium glaucum did not exhibit bacterial contamination 1877 Pasteur and Joubert noted that anthrax bacilli were inhibited when the cultures were contaminated with mold 1897 Ernest Duchesne reported in his dissertation the discovery, partial refinement and successful testing of a substance with antibiotic properties Modern Era of antimicrobial therapeutics: 1928 Flemmings discovery of penicillin 1935 Domagks discovery of sulfonamides 1939 Ernst Chain, Howard Florey, Edward Abraham purified and stabilized a form of penicillin 1940s WWII Production of penicillin Isolation of Streptomycin Isolation of Chloramphenicol Isolation of Tetracycline 1950S Antibiotics in clinical usage,Antimicrobial Agents,Antibiotic therapy is prescribed for 30% of all hospitalized patients. Antibiotic therapy has grown to be one of the most misused by physicians. Widespread use has allowed for the emergence of antibiotic resistant pathogens. Selection of an antimicrobial agent is a complex procedure microbiological factors and pharmacological considerations.,Ultimate Goal: a drug that is selective against the infecting organism and has the least potential to cause harm to the host patient.,Antimicrobial Agents,Effect on microbes: Cidal (killing) effect Static (inhibitory) effect Spectrum of action Broad Spectrum effective against procaryotes which kill or inhibit a wide range of Gram+ and Gram- bacteria Narrow spectrum effective against mainly Gram+ or Gram- bacteria Limited spectrum effective against a single organism or disease,Antimicrobial Agents,Wide spectrum of activity Nontoxic to the host and without undesirable side effects Non-allergenic to the host Not eliminate the normal flora of the host Be able to reach the part of the body where the infection is occurring Inexpensive and easy to produce Chemically stable (long shelf life) Unlikely to develop microbial resistance,Characteristics of a clinically-useful antibiotic:,Your Basic Bacteria,How we think of bacteria,Antibiotic Targets,Inhibition of bacterial cell wall synthesis Interactions with the cell membrane Disruption of protein synthesis Inhibition of DNA and RNA synthesis Inhibition of cell metabolism-Folate synthesis,Antibiotic Targets,,Antimicrobial Drugs & Mode of Action,-lactams Penicillin G, Cephalothin Semisynthetic penicillin Ampicillin, Amoxycillin Glycopeptides Vancomycin Clavulanic Acid Clavamox (clavulanic acid + amoxycillin) Sulfonamides “Sulfa” drugs,Inhibit steps in cell wall (peptidoglycan) synthesis and murein assembly,“suicide” inhibitor of beta-lactamases,Inhibit cell metabolism: Folate synthesis,Antimicrobial Drugs & Modes of Action,Aminoglycosides Streptomycin Macrolides Erythromycin Tetracyclines Tetracycline Quinolones Ciprofloxacin Rifamycins Rifampicin Polypeptides Bacitracin,Inhibits translation (protein synthesis),Inhibits nucleic acid synthesis,Damages cytoplasmic membranes,Types of Antimicrobial Resistance,Intrinsic Resistance “Natural” or built in resistance based on the characteristics of a particular strain or species. Acquired Resistance Acquisition of new genetic information or mutation of the existing genome that protects the “bug” from the effects of an antibiotic.,Mechanisms of Resistance,Impermeability Some bacteria are naturally resistant to antibiotics because their cell envelope is impermeable to a particular class of antibiotics. Antibiotic Modification Enzyme inactivation Organism spontaneously produces an enzyme that degrades the antibiotic. Staphylococcus arueus produces an extracellular enzyme: -lactamase. Enzyme addition Bacteria may express enzymes that add a chemical group to the antibiotic, inhibiting its activity. Addition of an amino, acetyl or adenosine group to aminoglycosides.,Efflux mechanisms Acquisition of an inner membrane protein which actively pumps the antibiotic out of the cell. E. coli acquires resistance to tetracyclines Alternative pathway Bacteria acquire a gene to code an alternative penicillin binding protein which is not inhibited. Alteration of the target site Point mutations, insertions or deletions alters the site of inhibition thus conferring resistance.,Mechanisms of Resistance,Transmission of Antimicrobial Resistance,Transformation bacteria takes up naked DNA and incorporate it into their genome. Conjugation Plasmids (circular portions of DNA found in the cytoplasm) are passed from one bacterium to another. Transposons Moveable genetic elements able to encode transposition. Can move between the chromosome and the plasmids and between bacteria.,Antibiotic (e.g. penicillin),Enzymes that degrade antibiotics (e.g. beta-lactamases),Plasmid with resistance genes.,Chromosome Changes to an antibiotics target,(e.g. a protein involved in cell wall synthesis prevents inhibition.),Antibiotic (e.g. streptomycin),Enzymes that alter antibiotics addition of amino, acetyl or adenosine group to aminoglycosides,Antibiotic (e.g. tetracycline, fluoroquinolone),Pumps that transport antibiotics out of the cell.,Its an uphill battle with the “bugs”,Factors That Accelerate Microbial Resistance,Inadequate levels of antibiotics at the site of infection. Duration of treatment too short Overwhelming numbers of organisms Overuse/misuse of antibiotics,Mechanisms to Reduce Antibiotic Resistance,Control, reduce or cycle usage Improve hygiene personal and in hospitals Discover or develop new antibiotics Modify existing antibiotics chemically to produce compounds inert to known mechanisms of resistance Develop inhibitors of antibiotic-modifying enzymes Define agents that would “cure” resistance plasmids,Cell Wall Structure of Gram(+) and Gram(-) Bacterium,Bacterial Cell Wall Synthesis Stage I,Formation of starting materials: takes place in the cytoplasm: N-Acetylglucosamine 1-Phosphate and uridine triphosphate (UTP) are converted to uridinediphosphoN-acetylglucosamine (UDPNAG) Condensation with elimination of pyrophosphate UDPNAG reaction with phosphoenolpyruvic acid (PEP) with transferase gives the enolic ether. Reduction of the double bond by NADPH utilizing reductase enzyme gives N-acetylmuramic acid (as the uridine derivative) Three amino acids are added to the muramyl peptide to give the tripeptide using ATP and enzymes specific for the aa. Two more aa are added D-alanine-D-alanine added after 2 D-alanines were synthesized via D-ala-Dalasynthetase. D-ala is from racemization of L-ala by racemase enzyme. UDPNAM-pentapeptide,Fosfomycin inhibits the enol-pyruvyl transferase by direct nucleophilic attack on the enzyme. Note: Mammalian enzymes are not inhibited, thus no effect on the host.,Inhibitors of Bacterial Cell Wall Synthesis Stage I,Cycloserine inhibits both alanine racemase and D-alaninyl-D-alanine synthetase Note the similarity in cycloserine and D-alanine. Cycloserine actually binds to the enzymes better than the D-alanine,Inhibitors of Bacterial Cell Wall Synthesis Stage I,Bacterial Cell Wall Synthesis Stage II,Peptidoglycan Synthesis: Reactions take place at and are catalyzed by membrane bound enzymes: The pentapeptide is linked to a phospholipid membrane- bound carrier bactoprenol (55C isoprenoid alcohol esterified with phosphoric acid). It is now anchored and the subsequent events occur in the interior of the cell membrane. A second sugar moiety is added by glycosidation and the UDP released are rephosphorylated to UTP and recycled to stage 1. 5 glycines are added (S. aureus) in sequence to the lycine residue each carried by the specific glycyl-t-RNA. The disaccharide-decapeptide monomer unit, which upon movement through the membrane is transferred following pyrophosphatase cleavage to an acceptor not yet identified. Separation from the membrane bound anchor leaves undecaprenyl phosphate which regenerate the original phosphate alcohol ester on hydrolysis by phosphotase and repeat the cycle., binds to the membrane bound bactoprene phosphate (membrane “anchor”) thus inhibiting cleavage from the anchor to allow for transport of the monomer unit to the outside of the cell. - and of lesser significance, inhibits lycine inclusion (stage I) into the murein structure.,Inhibitors of Bacterial Cell Wall Synthesis Stage II,Bacitracin, interaction with the D-alanyl-D-alanine portion of the forming mucopeptide involving strong, but not covalent, bonding with the hydroxylated phenyl glycine residues of the antibiotic. Separation of the murein component to the outside of the membrane is thus impaired and cell wall synthesis is inhibited.,Inhibitors of Bacterial Cell Wall Synthesis Stage II,Vancomycin,Bacterial Cell Wall Synthesis Stage III,Peptidoglycan Cross-Link: outside the cell wall Polymerization of the subunits transfer of the new peptidoglycan chain from its carrier in the membrane to the cell wall. The terminal amine function of the pentaglycine side chain forms a new peptide bond at the expense of the terminal d-alanyl-D-alanine linkage of a neighboring peptidoglycan chain transpeptidation Transpeptidase enzyme cleaves the peptide bond between two D-alanine residues in the pentapeptide and become acylated via the carbonyl group of the penultimate D-alanine residue.,Bacterial Cell Wall Synthesis Stage III,Inhibitors of Bacterial Cell Wall Synthesis Stage III,Ceftriaxone,Cefalosporin C,-lactams inhibit the enzyme transpeptidase responsible for crosslinking peptidoglycans that comprise the cell wall.,-Lactam General Mode of Action,A residue of the transpeptidase opens the B-lactam ring thus acylating the enzyme. The acylated enzyme is now too sterically crowded to allow the cross-linking reaction to occur.,-Lactam Antibiotics,Penicillin,Cephalosporin,Clavulanic Acid,Thienamycin,Cell Wall Synthesis Key Antibiotic Targets,L-Ala-D-Ala,Synthetase,Racemase,Cleavage of monomer unit from cell membrane anchor to allow for transport to exterior of cell.,D-Ala,-Lacatams,Moxalactam,Thienamycin,Norcardicins,Penicillins,Cephalosporins,Absolute Requirement: -Lacatam ring Sulfur can be replaced Sulfur can be omitted Second ring is not necessary Carboxyl group can be replaced Amide side chain unnecessary,Tetrazolyl Penam,Antibiotic (e.g. penicillin),Enzymes that degrade antibiotics (e.g. beta-lactamases),Plasmid with resistance genes.,Chromosome Changes to an antibiotics target,(e.g. a protein involved in cell wall synthesis prevents inhibition.),Antibiotic (e.g. streptomycin),Enzymes that alter antibiotics addition of amino, acetyl or adenosine group to aminoglycosides,Antibiotic (e.g. tetracycline, fluoroquinolone),Pumps that transport antibiotics out of the cell.,Its an uphill battle with the “bugs”,Strategy I Decrease the penetration of the antibiotic to its target Strategy II Alter the target of the antibiotic Strategy III Inactivate the antibiotic with an enzyme,Bacteria Fight Back,Abraham and Chain (1940) During purification of penicillin they discovered bacteria that inactivated antibiotics. Enzymes penicillinases General class -Lactamases,Hydrolytic enzymes,Bacteria Fight Back,Cell Wall Structure of Gram(+) and Gram(-) Bacterium,Antibiotic Pathway,Antibiotic,Penetrate outer membrane,Arrive at cell wall and associate with penicillin binding proteins (transpeptidases),Avoid -Lactamase enzymes in the periplasmic space,Penicillins inactivate these by acylation of the active sites,“suicide substrate”,-Lactamases,Plasmid-mediated Chromosome-mediated Physical properties Substrate specificity 5. Inhibition profiles,-Lactamases cleave the N-carbonyl bond of the -Lactam thus inactivating the molecule.,Cell Wall Structure of Gram(+) and Gram(-) Bacterium,Hydrolysis by -Lactamase,-Lactamase,-Lactamase,Penicillin,Cephalosporin,Degradation of Penicillin,-Lactamase,Degradation H2O,Penicillamine,Penaldic acid,Penicilloic Acid,One Strategy to Combat Penicillinases,Penicillin G,Methicillin,Plan B: not planned,Soil samples from various parts of the world were tested:,Streptomyces olivaceus,Potent -Lactamase inhibitor,Metabolites isolated “olivanic acids”,Streptomyces clavuligerus,Potent inhibitor,“clavulanic acid”,Clauvlanic Acid Characterization,1: HPLC isolation 2: 1HNMR 3: X-ray,(z)-(2R, 5R)-3-(-hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo3.2.0heptane-2-carboxylic acid,First example of a non-traditional -Lactam from a natural source.,Bioassay,-Lactamase producing organism,Penicillin G,Sample to be screened,Organism Grows,Organism doesnt grow,Inhibition Study,Table 14.3,Irreversible Inhibitor of -Lactamases,NH2,NH2,NH,NH,NH,NH,Clavulanic Acid,Other clavams,Proposed Biosynthetic Pathway,Liras & Rodriquez-Garcia, 2000,oxidative deamination,Synthesis of ()Methyl clavulanate,See scheme 14.3 for synthesis,Bacterial Protein Synthesis,http:/www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/protsyn/translation/translation.html,Binding of aminoacyl-t RNA to acceptor site Peptidyl transfer from the peptidyl t RNA to the newly bound aminoacyl t RNA on the acceptor site Translocation of the synthesized peptidyl t RNA from the acceptor site to the donor site,Inhibition Sites of Bacterial Protein Synthesis,Antibiotics that Inhibit Protein Synthesis,Aminoacyl - tRNA formation use of “imposter amino acids”,N-ethylglycine,Inhibitors of initiation complex formation and tRNA-ribosome interactions,Tetracyclines & Aminoglycosides,Antibiotics that Inhibit Protein Synthesis,Chloramphenicol,Inhibitors of peptide bond formation & translocation,Erythromycin A,Antibiotics that Inhibit Protein Synthesis,Tetracyclines,Discovered in 1947 Bacteriostatic (almost always) Enter via porins (G-) and by their lipophilicity in (G+). Low toxicity, broad spectrum for both Gram- and Gram+ bacteria Selectivity results from transfer into bacterial cells but not mammalian cells Primary binding site is 30s ribosomal subunit. Prevents the attachment of amino acyl-tRNA to the ribosome and protein synthesis is stopped Resistance associated with ability of compound to permeate membranes and alteration of the target of the antibiotic by the microbe,Tetracyclines “SAR”,Carbon # Modification Effect 1 any No activity 2 Slight activity 3 any No activity 4 must have -N(CH3)2 5 Active 5a lose H Inactive,Carbon # Modification Effect 6 loss of OH or CH3 Active & more stable 7 Cl, Br, NO3, N(CH3)2 Active 8 - - 9 Cl and CH3 Less active 10 Cannot change 11 Cannot change Loss of activity 11a Cannot change 12 Cannot change 12a Change OH stereochemistry Decreases activity or remove,Amphoteric Compound,Strong Chromophore pKa = 7.2 7.8,Strong Chromophore pKa = 2.8 3.3,pKa = 9.1-9.7,Cannot be modified,Zwitterion exists at ph = 4 7 (ph of duodenum),Degradation/Instability of Tetracyclines,Aminoglycosides,represents some of the oldest antibiotics bactericiocidal works against Gm+ and Gm- bacteria binds to the S12 protein on the 30s ribosome to block normal activation of the initiation complex can alter membrane permeability increase membrane leakage can alter elongation of the peptide chain,Streptomycin,Macrolide Antibiotics,active against Gm+ bacteria can be bacteriostatic or bacteriocidal depending upon the organism binds with high affinity to bacterial 50s ribosomes and interacts with the 23s ribosomal RNA protein synthesis is inhibited by the blockage of chain elongation,Erythromycin A,Mode of Action of Erythromycin A,Penetrates the periplasmic area by diffusing through porin lined aqueous channels. Enters bacterial cycoplasm by using energy dependent electron transport associated with oxidative phosphorylation. Drug interacts with ribosomes to prevent protein synthesis Drug binds the ribosome and causes a conformational change Ribosomes break down and associate with mRNA causing inhibition of normal synthesis,active against Gm+ and Gm bacteriostatic mode of action involves reversible binding to the 50s ribosomal subunit binds aminoacyl-tRNA and prevents translocation of the peptide chain,Chloramphenicol,For DNA replication to occur, the two strands must be separated. Separation results in excessive positive supercoiling (overwinding).,Bacterial DNA Synthesis,Bacterial DNA Synthesis,To avoid this, DNA gyrase is responsible for continually introducing negative supercoils into DNA. Both strands of the DNA are cut during this process thus allowing passage of a portion of the DNA through the break which is then resealed.,Synthetic Antibiotics - Quinolones,Inhibit DNA synthesis Interfere with the activity of DNA gyrase Bacteriocidal Newer agents are called fluoroquinolones Broad spectrum of activity,Quinoline,Mode of Action,Quinolones selectively inhibit bacterial DNA synthesis. Target DNA-gyrase (topoisomerase II enzyme found in procaryotic cells) 4-Quinolones inhibit ATP-dependent DNA supercoiling by binding to “subunit A” of DNA-gyrase.,Alteration of the target of the antibiotic in G+ resistance is due to a change in the DNA gyrase (mutation) Decrease penetration of the antibiotic in G- resistanc