1. Abiola Muhammad, ADEOSUN Department of Biochemistry, Lead City University, Ibadan 2. What are Antibiotics? Antibiotics: chemical substances produced by microorganisms that inhibits the growth or kills other microorganisms Antimicrobial agents: chemical substances from a biological source or produced by chemical synthesis that kills or inhibits the growth of microorganisms. 3. Sources of Antibiotics Natural : mainly fungal sources (e.g Benzylpenicillin and Gentamycin) Semi synthetic: chemically altered natural compound (e.g Ampicillin & Amikacin). Synthetic: chemically designed in the lab (e.g Moxifloxacin and Norfloxacin). 4. What are the Roles of Antibiotics? Bacteriostatic effect: To inhibit multiplication @ [drug] = MIC minimal inhibitory concentration. Bactericidal effect: To kill (destroy) the bacteria population @ [drug] = MBC minimal bactericidal concentration. There is a much closer relationship between the MIC and MBC values for bactericidal drugs than for bacteriostatic drugs. 5. Principle of Selective toxicity: What is an ideal Antibacterial? Selective target target unique Bactericidal kills Narrow spectrum does not kill normal flora High therapeutic index ratio of toxic level to therapeutic level Few adverse reactions toxicity, allergy Various routes of administration IV, IM, oral Good absorption Good distribution to site of infection Emergence of resistance is slow 6. Antibiotics classification Antibiotics are usually classified based on their structure , Function and/or spectrum of activity 1. Structure - molecular structure. -Lactams - Beta-lactam ring Aminoglycosides - vary only by side chains attached to basic structure 2. Function - how the drug works, its mode of action. 5 functional groups These are all components or functions necessary for bacterial growth Targets for antibiotics: Inhibitors of cell wall synthesis Inhibitors of protein synthesis Inhibitors of membrane function Anti-metabolites Inhibitors of nucleic acid synthesis 3. Spectrum of Activity: Narrow spectrum Broad Spectrum In these discussions, we will primarily use the functional classification, but will point out where 7. 1. Inhibitors of Cell Wall Synthesis: Beta-lactams: Penicillins Cephalosporins Monobactams Carbapenems Glycopeptides Fosfomycins 8. Inhibitors of Cell Wall Synthesis: The Penicillins1928 - Alexander Fleming Bread mold (Penicillin notatum) growing on petri dish 1939 - Florey, Chain, and Associates Began work on isolating and synthesizing large amounts of Penicillin. 1944 - Used in WWII to treat infections Late 1940s - available for general use in US Penicillins as well as cephalosporins are called beta-lactam antibiotics and are characterized by three fundamental structural requirements: the fused beta-lactam structure (shown in the blue and red rings, a free carboxyl acid group (shown in red bottom right), one or more substituted amino acid side chains (shown in black). The lactam structure can also be viewed as the covalent bonding of pieces of two amino acids - cysteine (blue) and valine (red). The beta-lactam nucleus itself is the chief structural requirement for biological activity; metabolic transformation or chemical alteration of this portion of the molecule causes loss of all significant antibacterial activity Figure 1: beta lactam structure 9. Figure 2: The beta lactam ring 10. Mechanism of Actions of Beta lactams All penicillin derivatives produce their bacteriocidal effects by inhibition of bacterial cell wall synthesis. Specifically, the cross linking of peptides on the mucosaccharide chains is prevented. If cell walls are improperly made cell walls allow water to flow into the cell causing it to burst. 11. Bacteria Cell Wall Synthesis The cell walls of bacteria are essential for their normal growth and development. The peptidoglycan (which provide rigid mechanical stability) is composed of glycan chains, which are linear strands of two alternating amino sugars (N- acetylglucosamine and N-acetylmuramic acid) that are cross-linked by peptide chains. (NAG-NAM). In gram-positive microorganisms, the cell wall is 50 to 100 molecules thick, but it is only 1 or 2 molecules thick in gram-negative bacteria 12. Bacteria Cell Wall Synthesis (cont) The biosynthesis of the peptidoglycan involves about 30 bacterial enzymes and may be considered in three stages. The first stage is precursor formation in the cytoplasm. The product, uridine diphosphate (UDP)-acetylmuramyl-pentapeptide, called a "Park nucleotide. The last reaction in the synthesis of this compound is the addition of a dipeptide, D- alanyl-D-alanine. The second stage, UDP-acetylmuramyl-pentapeptide and UDP-acetylglucosamine are linked to form a long polymer. The third and final stage involves the completion of the cross-link. This is accomplished by a transpeptidation reaction that occurs outside the cell membrane. The transpeptidase itself is membrane bound. The terminal glycine residue of the pentaglycine bridge is linked to the fourth residue of the pentapeptide (D-alanine), releasing the fifth residue (also D-alanine). Penicillin binds at the active site of the transpeptidase enzyme that cross-links the peptidoglycan strands. It does this by mimicking the D-alanyl-D-alanine residues that would normally bind to this site. Penicillin irreversibly inhibits the enzyme transpeptidase by reacting with a serine residue in the transpeptidase. This reaction is irreversible and so the growth of the bacterial cell wall is inhibited. 13. Bacteria Cell Wall Synthesis (cont): The PBPs and Binding of Penicillins Related targets of penicillins and cephalosporins collectively termed penicillin- binding proteins (PBPs) PBPs functions are diverse: catalyze the transpeptidase reaction, maintam shape, forms septums during division, Inhibit autolytic enzymes. Binding to PBPs results in: Inhibition of transpeptidase: transpeptidase catalyzes the cross-linking of the pentaglycine bridge with the fourth residue (D-Ala) of the pentapeptide. The fifth reside (also D-Ala) is released during this reaction. Spheroblasts are formed. Structural irregularities: binding to PBPs may result in abnormal elongation, abnormal shape, cell wall defects. 14. Figure 3. The transpeptidase reaction in Staphylococcus aureus that is inhibited by penicillins and cephalosporins. 15. Figure 4. Comparison of the structure and composition of gram-positive and gram-negative cell walls. 16. Other Inhibitors of Cell Wall Synthesis: Glycopeptide Include two compounds with similar structures; Vancomycin and Teicoplanin Teicoplanin not FDA approved in the U.S. Both are of high molecular weight (1500-2000 daltons) Glycopeptides have a complex chemical structure Inhibit cell wall synthesis at a site different than the beta-lactams All are bactericidal All used for Gram-positive infections. (No Gram- negative activity) Pharmaceutical research and development has been very active in this area recently resulting in new antimicrobials and classification In Gram-Positives: The drugs enter without any problem because peptidoglycan does not act as a barrier for the diffusion of these molecules. In Gram-Negatives: Glycopeptides are of high molecular weight (1500-2000 daltons), stopping them from passing through the porins of gram-negative bacteria (i.e., glycopeptides have no activity against Gram-negatives). Vancomyci n 17. Other Inhibitors of Cell Wall Synthesis: Glycopeptide MOA Glycopeptides inhibit the final cell wall stage of the peptidoglycan synthesis process The pocket-shaped glycopeptide binds the D-ala-D-ala terminal of the basic sub-unit theoretically waiting to be incorporated into the growing peptidoglycan Because it is so bulky, the glycopeptide inhibits the action of the glycosyltransferases and transpeptidases (which act as a kind of cement) - blocks pentaglycine from joining molecules, thereby blocking peptidoglycan growth. Glycopeptides are bactericidal, but slow-acting 18. Other Inhibitors of Cell Wall Synthesis: Fosfomycins Spectrum of Action Fosfomycin: Acts to inhibit cell wall synthesis at a stage earlier than the penicillins or cephalosporins. FDA approved 1996. It is a broad spectrum agent Mode of Action: Inhibits the first step of the peptidoglycan synthesis process (Actual step of inhibition is not completely understood) 19. 2. Inhibitors of Protein Synthesis Aminoglycosides -(Bactericidal) : Gentamicin, Tobramycin, Amikacin MLSK (Macrolides, Lincosamides, Streptogramins, Ketolides) (Bacteriostatic) Erythromycin, Clindamycin, Quinupristin-Dalfopristin (Synercid), Clarithromycin, Azithromycin, Telithromycin Tetracyclines (Bacteriostatic) Tetracycline, Doxycycline, Minocycline Glycylcyclines - Tigecycline Phenocols (Bacteriostatic), Chloramphenicol Oxazolidinones Linezolid (Bactericidal for Streptococci; Bacteriostatic for Enterococcus and Staphylococci) Ansamycins - Rifampin (Bacteriostatic or Bactericidal depending on organism and concentration) 20. Inhibitors of Protein Synthesis These classes interferes with ribosomes Most are bacteriostatic Resistance to tetracycline and Macrolide is common. 21. Overview of Protein Synthesis Figure 5: Overview of protein synthesis 22. Tetracyclines Analogs; Doxycycline Minocycline, and Tigecycline Enter microorganisms in part by passive diffussion, and in part by active transport Binds to 30S subunits & blocks the binding of amino acyl tRNA to the acceptor site on the mRNA-ribosome complex. Active against; many gram+ve & gram ve, rickettsiae, chlamydiae, mycoplasmas. 23. Macrolides Characterized by Macrocyclic lactone rings + deoxy sugars Prototype: Erythromycin (from streptomyces erythreus) Semisynthetic derivatives: clarithramycin , ketolides and azithromycin Inhibit 50S ribosomal RNA near peptidyl transferase centre, thereby preventing peptide chain elongation by blocking of polypeptide exit tunnel. As a result,m pepidyl tRNA is dissociated from the ribosome Active against: pneumococci, streptococci, staphylococci, H. Pyroli, Ricketssia spp, chlamydia spp Hemophilus influenza and Campylobacter are less susceptible Resistances: Usually plasmic encoded, reduced permeability of membrane, active efflux, or by production of esterases (by enterobaceriaceae) that hydrolyzes macrolides Action of clindamycin and streptogramins is related to that of erythromycin 24. Chloramphenicol Chloramphenicol binds reversibly to the 50S subunit of the bacterial ribosome and inhibit peptide bond formation Bacteriostatic broad-spectrum antibiotic that is active against both aerobic and anaerobic gram +ve & gram - ve organisms. It is active also against Rickettsiae but not Chlamydiae. Clinically significant resistance is due to production of chloramphenicol acetyltransferase, a plasmid-encoded enzyme that inactivates the drug. 25. MOA of MLSK Translation: 1.Initiation 2. Elongation 3Termination Figure 6: MOA OF MLSK (Macrolides, Lincosamides, Streptogramins, Ketolides) 26. 3. Inhibitors of Membrane Function Lipopeptides Polymyxins (A,B,C,D, and E) Polymyxin B and E can be used therapeutically Polymyxin B derived from Bacillus polymyxa var. aerosporus Polymyxin E derived from Bacillus polymyxa var. colistinus = Colistin. Colistin exsists as two forms: Colistin sulfate intestinal infections, topical, powders, media Colistimethate sodium most active, effective form Cyclic Lipopeptides All Bactericidal 27. Polymyxin Mode of Action Target =Membrane phospholipids (lipopolysaccharides (LPS) and lipoproteins) 1. Outer and Cytoplasmic Membrane Effect: Polymyxins are positively charged molecules (cationic) which are attracted to the negatively charged bacteria. The negative charge of bacteria is due to LPS in the outer membrane and the peptidoglycan (notably the teichoic acid). The antibiotic binds to the cell membrane, alters its structure and makes it more permeable. This disrupts osmotic balance causing leakage of cellular molecules, inhibition of respiration and increased water uptake leading to cell death. The antibiotic acts much like a cationic detergent and effects all membranes similarly. Toxic side effects are common. Little or no effect on Gram-positives since the cell wall is too thick to permit access to the membrane. Gram-positives are naturally resistant. 28. 4. Antimetabolites Folate Pathway Inhibitors: Sulfonamides, Trimethoprim/Sulfamethoxazole The drug resembles a microbial substrate and competes with that substrate for the limited microbial enzyme The drug ties up the enzyme and blocks a step in metabolism Figure 7: Competitive Antagonism 29. Synthesis of Tetrahydrofolic Acid Humans do not synthesize folic acid. Good selective target Sulfonamides (sulfadiazine, sulfamethoxazole, sulfadoxine) Bacteriostatic Introduced in 1930s first effective systemic antimicrobial agent Used for treatment of acute, uncomplicated UTIs Trimethoprim/Sulfamethoxaz ole TMP/SXT is bactericidal Broad spectrum Synergistic action Figure 8: synthesis of THF 30. Anti-metabolite (conts) The combination SXT (thrimethoprim-sulfamethoxazole) is synergistic and the association provides a bactericidal effect Natural Resistance Enterococcus low level and poorly expressed S. pneumoniae Ps. aeruginosa (impermeability) 31. 5. Inhibitors of Nucleic Acid Synthesis (Qunolones & Furanes) Quinolones: Humans do synthesize DNA - shared process with bacteria Do tend to see some side effects with Quinolones Some drugs withdrawn from market quickly All are bactericidal 32. Quinolones Mode of Action Small and hydrophilic, quinolones have no problem crossing the outer membrane. They easily diffuse through the peptidoglycan and the cytoplasmic membrane and rapidly reach their target. Target = Topoisomerases (DNA-gyrase) Rapid bactericidal activity 33. Quinolones inhibit DNA synthesis Mode of Action A typical E. colis chromosome is 1400 microns long (1 micron in diameter when supercoiled), enough to fit in the E. colis bacterial cells which is 2-3 microns long The bacterial chromosome consists of a single circle of DNA DNA is double-stranded forming a left-handed double helix All topoisomerases ( which are involved in DNA replication, transcription and recombination) can relax DNA but only gyrase which carry out DNA supercoiling. The main quinolone target is the DNA gyrase which is responsible for cutting one of the chromosomal DNA strands at the beginning of the supercoiling process. The nick is only introduced temporarily and later the two ends are joined back together (i.e., repaired). The quinolone molecule forms a stable complex with DNA gyrase thereby inhibiting its activity and preventing the repair of DNA cuts 34. Resistance to Quinolones Natural Resistance Gram Positives 1st generation quinolones S.pneumoniae decreased activity to Ofloxacin and Ciprofloxacin Ps. aeruginosa decreased activity to Norfloxacin and Ofloxacin 35. Furanes Nitrofurantoin Mode of Action: The drug works by damaging bacterial DNA. In the bacterial cell, nitrofurantoin is reduced by flavoproteins (nitrofuran reductase). These reduced products are are highly active and attack ribosomal proteins, DNA, respiration, pyruvate metabolism and other macromolecule within the cell. It is not known which of the actions of nitrofurantoin is primarily responsible for its bactericidal acitivity. Natural Resistance: Pseudomonas and most Proteus spp. are naturally resistant. 36. Questions? 37. Bibliography Katzung, B.G. Basic and Clinical Pharmacology, 12th Edition, Chapters 44 and 45, p774-783. D Avis, B. D., Chen, L. L. & Tai, P. C. Misread protein creates membrane channels: an essential step in the bactericidal action of aminoglycosides. Proc. Natl Acad. Sci. USA 83, 61646168 (1986). Wise, E. M. Jr & Park, J. T. Penicillin: its basic site of action as an inhibitor of a peptide cross-linking reaction in cell wall mucopeptide synthesis. Proc. Natl Acad. Sci. USA 54, 7581 (1965). Davis, B. D. Mechanism of bactericidal action of aminoglycosides. Microbiol. Rev. 51, 341350 (1987).