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Drugs, Microbes, Host – The Elements of Chemotherapy Chapter 12 Copyright © The McGraw-Hill Companies, Inc) Permission required for reproduction or display. Learning Objectives • Identify and explain desirable characteristics of antibiotics • Identify and describe the five cellular targets of antibiotics • List and describe antibiotics targeting bacterial cell wall, plasma membrane, protein and nucleic acid synthesis, and folic acid biosynthesis. Learning Objectives • Describe how selective toxicity is achieved in targeting fungi, viruses and helminthes. Give examples of antifungal, antiviral, and antihelmintic drugs. • Describe five mechanisms of antibiotic resistance, and explain how it can be acquired by bacteria. • Describe the Kirby-Bauer and dilution assays for antibiotic sensitivity History of Antibiotics • Paul Erlich: “magic bullet”, Salvarsan. • Gerhard Domadk: Prontosil • Alexander Fleming: Penicillin Properties of Antimicrobial Agents • Synthetic antimicrobials, antibiotics, semisynthetic drugs (sources) • Desirable characteristics • Selective toxicity (target the pathogen) • Few side effects (low toxicity) • Narrow spectrum (leaves normal biota) • Localization and stability in host • Shelf life and cost Possible Adverse Reactions • Toxicity to organs • Allergies • Disruption of normal flora • Other adverse effects Therapeutic Index • What is the best drug to use? • Lowest risk of side effects versus • Highest probability of killing the pathogen • 50 µg is toxic and 5 µg is effective; T.I. = 10 • 50 µg is toxic and 1 µg is effective; T.I. = 50 • Higher T.I. are better Targets of Antibiotics Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 1. Cell wall inhibitors Block synthesis and repair Penicillins Cephalosporins Carbapenems Vancomycin Bacitracin Fosfomycin Isoniazid 2. Cell membrane Cause loss of selective permeability Polymyxins Daptomycin Ribosome Substrate Enzyme Product Site of action 50S subunit Erythromycin Clindamycin Synercid Pleuromutilins Site of action 30S subunit Aminoglycosides Gentamicin Streptomycin Tetracyclines Glycylcyclines Both 30S and 50S Blocks initiation of protein synthesis Linezolid 3. DNA/RNA Inhibit replication and transcription Inhibit gyrase(unwinding enzyme) Quinolones Inhibit RNA polymerase Rifampin 4. Protein synthesis inhibitors acting on ribosomes mRNA DNA 5. Folic acid synthesis Block pathways and inhibit metabolism Sulfonamides (sulfa drugs) Trimethoprim Folate Synthesis Inhibitors Cell Wall Inhibitors • Target peptidoglycan synthesis • Bactericidal • Active against young growing cells Inhibitors of Cell Wall Synthesis : Penicillins Nucleus • Block cross-linking of peptidoglycan R Group Betalactam Thiazolidine S CO N CH3 CH3 N Nafcillin O COOH •Beta-lactam ring •Different spectra of action •Often cause allergic reactions Inhibitors of Cell Wall Synthesis : Penicillins • Penicilinaseresistant penicillins (methicillin) • Extendedspectrum penicillins (ampicillin) • Penicillins + lactamase inhibitors (clavulanic acid) (augmentin) Inhibitors of Cell Wall Synthesis: Penicillins Figure 20.8 Inhibitors of Cell Wall Synthesis: Cephallosporins • Isolated in 1940s from the mold Cephallosporium acremonium. O C CH2 N7 S O R Group2 Cephalothin (first generation*) S or O 5 6 4 3 N1 2 COOH • -lactam ring, resistant to penicillnases. • Broad spectrum (2nd, 3rd, and 4th generations more effective against gramnegatives, 5th generations effective against MRSA) Basic Nucleus R Group1 O CH2 CH3 CH2 N OC Cefotiam (second Ngeneration) N S NH2 N CH2 CH2 N CH3 O N OH CH C COONa CH2 S Moxalactam (third N generation) N N CH3 NH2 Cefepime (fourth generation) S O N C C NH CH2 N • Less allergenic. • Administered parenterally CH3 N S N CH3 OCH3 OH N N NH2 S N N O O *New improved versions of drugs are referred to as new “generations.” Ceftobiprole (fifth generation) NH Inhibitors of Cell Wall Synthesis: Other Beta-Lactam Antibiotics • Carbapenems: powerful, but potentially very toxic. • Reserved as a last line of defense for pneumonia, septicemia, urinary tract infections • NDM-1 gene in G- bacteria causes resistance to carbapenems. Other Inhibitors of Cell Wall Synthesis • Polypeptide antibiotics • Bacitracin: topical application against grampositives • Vancomycin: glycopeptide • Important "last line" against MRSA, methicillin resistant S. aureus (VRSA reported in July 2012) Other Inhibitors of Cell Wall Synthesis • Isoniazid – Mycolic acid formation inhibited. • This is one of the main anti-tuberculosis drugs since 1954. • Due to resistance, has to be part of multi-drug therapy. • Fosphomycin – PEP analog, blocks linking of glycan and peptide portions of peptidoglycan. • Treatment of urinary infections. • Resistance and side effects prevent wider application Protein Synthesis Aminoglycosides Inhibitors • Target 30S and 50S ribosomal subunits 50S aa aa Chloramphenicol 30S 50S aa aa Oxazolidinones • Side effect: damage to eucaryotic mitochondria. 30S mRNA mRNA is misread, protein is incorrect Formation of peptide bonds is mRNA blocked Prevent Initiation and Block ribosome assembly 30S Tetracyclines 50S tRNA is blocked, no protein is synthesized 30S 30S Erythromycin aa 50S aa mRNA Ribosome is prevented from translocating 30S 30S mRNA Inhibitors of Protein Synthesis: Aminoglycosides • Produced by Streptomyces • Binds 30S, distorts the ribosome: causes translation errors • Streptomycin: serious G- infections • Neomycin Triple antibiotic cream • Side effects: oto- and nephrotoxicity Inhibitors of Protein Synthesis Tetracyclines • Broad spectrum: tetracyclin, doxycyclin • Blocks the A site: prevents tRNA entry • Reversible reaction: bacteriostatic • Widespread resistance • Side effects Inhibitors of Protein Synthesis: Macrolide Antibiotics • Bind near the P site: Prevent translocation • Lactone ring + sugars • Bacteriostatic • Active against G+ • Erythromycin • Azithromycin and clarithromycin • Hepatotoxicity Inhibitors of Protein Synthesis: Chloramphenicol • Binds 50S subunit: Prevents peptide bond formation • Wide spectrum, cheap • Toxicity: aplastic anemias (bone marrow supression) Inhibitors of Protein Synthesis: Oxazolidinones • New class of antibiotics, developed in 2000s • Bind to 50S, prevent Nformyl-methionyl-tRNA binding to the ribosome: prevent initiation • Linezolid: used to treat MRSA and VRE: drug of “last resort”. Nucleic Acid Synthesis Inhibitors • Block: Nucleotide synthesis DNA replication RNA transcription • Chloroquine: crosslinking of double helix • Quinolones: block DNA unwinding by inhibiting helicase • Purine and pyrimidine analogs (AZT): insert into viral nucleic acid, block replication. DNA Replication: Quinolones and Fluoroquinolones • Broad spectrum, high potency, readily absorbed. • Mechanisms are conserved: lead to toxicity • Inhibit DNA gyrase: prevent DNA synthesis • Treatment of serious hospital acquired infections: urinary tract infections, pneumonia. • High risk for MRSA resistance development: not recommended for community acquired infections • Nalidixic acid used in DNA replication studies • Ciprofloxacin – used in the 2001 anthrax attack • Levofloxacin – wide spectrum, including anaerobes and anthrax. May cause damage to muscles and tendons Transcription of DNA (RNA synthesis) • Difficult target because the process is well conserved • Rifamycin, Rifampicin • Bind bacterial RNA polymerase: inhibit RNA synthesis • Tratment: tuberculosis, MRSA Antibacterial Antibiotics: Injury to the Plasma Membrane • Bind to phospholipid and lipid A, disrupt membranes • Poor selective toxicity • Must be used topically • Polymyxin B and E • Topical (kidney toxicity) • Combined with bacitracin and neomycin in over-the-counter preparation. Antibiotics and Biofilms • Highly resistant due to: • Poor penetration. • Altered gene expression pattern. • Strategies: • Treatment of plastic surfaces with antibiotic before insertion. • Daptomycin (lipopeptide), adding DNase. • Disruption of quorum sensing. Anti-Fungal Drugs • Eukaryotes: more similar to human cells • Polyenes bind OH membrane, cause loss O of selective OH permeability Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. • Target: ergosterol OH OH OH OH O OH O OH (a) • Nystatin • Amphotericin B (used to treat systemic infections) Anti-Fungal Drugs Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. • Azoles N • Inhibit ergosterol synthesis N • Griseofulvin • effective against ringworm • inhibits microtubules • prevents cell division C Cl (b) Antiparasitic Chemotherapy • Quinolones (anti-malaria): disrupt different life stages of plasmodium. • Metronidazole: anti-protozoan drug (Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis) • Mebendazole and albendazole: broad spectrum anti-helmintic drugs, block glucose utilization • Pyrantel: paralyzes muscles of intestinal roundworms. Anti-Virals: Inhibitors of Virus Entry • Few antivirals • Toxicity problems • Amantadine, Relenza and Tamiflu • Inhibits the entry of Influenza A virus • Fuzeon • Blocks binding of HIV to the GP-41 receptor • Drug resistance. Antiviral Drugs: Inhibition of Nucleic Acid Synthesis • Acyclovir • Disrupts Herpesviruses replication • Purine analog • Ribavirin • Blocks RNA synthesis (RSV and hemorrhagic fever) •Anti HIV agents: Reverse transcriptase inhibitors AZT Antiviral Drugs: Nucleoside and Nucleotide Analogs Antiviral Drugs: Nucleoside and Nucleotide Analogs Anti-Viral Drugs: Inhibition of Viral Assembly/Release/Spread • Protease inhibitors • Indinavir, saquinavir • Used in combination with reverse transcriptase inhibitors • Interferons prevent spread of viruses to new cells • Glycoprotein produced by immune cells • Viral hepatitis Mechanism of Antibiotic Resistance 1. Drug inactivation S R 1 O N S R Penicillinase COOH O C OH Active penicillin N H CH3 CH3 COOH Inactive penicillin 1. Inactivation of a drug like penicillin by penicillinase, an enzyme that cleaves a portion of the molecule and renders it inactive. 2. Decreased permeability Drug Normal receptor 2 Different receptor 3. Activation of drug pumps Drug Inactive drug pump 3 Active drug pump 4. Change in drug binding site 2. The receptor that transports the drug is altered, so that the drug cannot enter the cell. 3. Specialized membrane proteins are activated and continually pump the drug out of the cell. 4. Binding site on target (ribosome) is altered so drug has no effect. 4 5. Use of alternate metabolic pathway Drug acts 5 A B C X C1 D Product D1 5. The drug has blocked the usual metabolic pathway (green), so the microbe circumvents it by using an alternate, unblocked pathway that achieves the required outcome (pink). Mechanisms of Acquiring Antibiotic Resistance Genes Transfer of Resistance • Resistance (R) factors (plasmids): transferred by conjugation, transformation or transduction • Transpozons: duplicated and inserted from one plasmid to another or from a plasmid to a chromosome Preventing Drug Resistance • Limit drug use - less selective pressure • Proper drug use - viruses are not affected, use full dose to ensure elimination of pathogens • Narrow range antibiotics - kill only the targeted microbes; less likely complications • Multiple drug treatments - drugs can work synergistically; much less likely to get drug resistance. Diffusion Assays Kirby-Bauer Disc Diffusion Test* • Disk Diffusion Assay Oxytetracycli ne 30g (R<17 mm;S 22mm) Enrofloxacin 5 g (R < 17 mm;S 22 mm) • Kirby-Bauer 0 mm ENR 1 5 2 3 Gentamicin 10 g (R < 17 mm; S 21 mm) 4 S OT R 30 • Standardized conditions CTX 30 GN I 10 AMP I 10 S R • Zones of inhibition C 30 Cefotaxime 30 g Ampicillin10g Chloramphenicol 30 g (R < 14 mm; S 23 mm) (R<14mm;S22mm) (R < 21 mm; S 21 mm) • Larger zone indicates more susceptible = Zone of Inhibition • Smaller zone indicates more resistant Disc Diffusion Test (schematic). Example and evaluation of a sensitivity test, agar diffusion method. R = resistant, I = intermediate, S = sensitive = Region of bacterial growth ENR 5 = Antibiotic carrier (disc) (b) E-Test Strips • Drug gradient used • Can determine MIC • Read where the zone touches the strip • MIC: Minimal inhibitory concentration. • MBC: Minimal bactericidal concentration. Tube Dilution Assay Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Same inoculum size of test bacteria added Control • Drug diluted in series 0 • Inoculate and incubate Negative control (a) • Look for growth (MIC) (b) 0.2 0.4 0.8 1.6 3.2 6.4 12.8 m g/ml Increasing concentration of drug Growth No growth