Antimicrobials.ppt

Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,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,Date,Event,300Bc,Aristotle,Greek philosopher and scientist,studied and wrote about living organisms.,1675,Antony van Leeuwenhoek discovered bacteria.,1796,Edward Jenner laid the foundation for developing vaccines.,1848,Ignc Flp Semmelweis discovered simple handwashing could prevent passage of infection from one patient to another.,1857,Louis Pasteur introduced the germ theory of disease.,1867,Joseph Lister showed evidence that microbes caused disease and pioneered the use of antiseptics during surgery to kill germs.,1876,Robert Koch,by studying anthrax,showed the role of bacteria in disease.,1928,Alexander Fleming is credited with discovering penicillin.,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,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,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,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.,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.,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,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,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 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,H,2,O,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:,1,HNMR,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,NH,2,NH,2,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,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(CH,3,),2,5 Active,5a lose H Inactive,Carbon#Modification Effect,6loss of OH or CH,3,Active&more stable,7Cl,Br,NO,3,N(CH,3,),2,Active,8 -,9 Cl and CH,3,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,N-methyl,Glucosamine,Streptose,Streptodine,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