Antimicrobial Agents

1. Antimicrobial Agents • Most other drugs to be covered affect body systems to cure imbalance or malfunction • E.g. reduce inflammation, relieve pain, etc. • Antimicrobials used to inhibit or destroy living organisms which are “uninvited guests” • Drugs must harm invader without harming host • Notion of selective toxicity • Lectures will focus more on interactions between drug and microbe than drug and host.

2. Selective toxicity • The more distantly related the invader, the more targets available for the drug to hit • The less likelihood of direct toxic effects. • Prokaryotes biochemically least similar • Fungi and Protozoa are eukaryotes, so more closely related to humans. • Helminths (worms) also animals • Viruses use our own cell machinery • Cancer cells ARE our cells.

3. History of Antimicrobial Therapy • 1909 Paul Ehrlich • Differential staining of tissue, bacteria • Search for magic bullet that would attack bacterial structures, not ours. • Developed salvarsan, arsenic derivative used against syphilis. http://www.chemheritage.org/EducationalServices/pharm/antibiot/activity/stain/salvarsa.gif

4. Timeline • 1929 Penicillin discovered by Alexander Fleming • Messy lab, cool damp weather, luck • 1940 Florey and Chain mass produce penicillin for war time use, becomes available to the public. • 1935 Sulfa drugs discovered • 1943 Streptomycin discovered • Western civilization fundamentally changed

5. Historical distinctions • Antibiotics: substances produced by organisms that have inhibitory effects on other organisms. • Penicillin, streptomycin • Synthetic drugs: produced in a lab. • Salvarsan, sulfa drugs • Nowadays, most antimicrobials are semi-synthetic • Chemically modified versions of natural products • Distinction between “antibiotics” and “synthetic drugs” slowly being abandoned.

6. Selective toxicity means safer for host • Antibiotics generally have a low MIC • Minimum inhibitory concentration • Effective at lower doses • Good therapeutic index • Safer; larger quantity must be administered before harmful side effects occur. e.g. Ti = LD50 / ED50 Where LD = lethal dose ED = effective dose

7. Where do antibiotics come from? • Soil dwelling organisms • Several species of fungi including Penicillium and Cephalosporium • E.g. penicillin, cephalosporin • Species of actinomycetes, Gram positive filamentous bacteria • Many from species of Streptomyces • Also from Bacillus, Gram positive spore formers • A few from myxobacteria, Gram negative bacteria • New sources explored: plants, herps, fish

8. Antibiotics are common in nature • Many are discovered every year, but not all are useful • May belong to previously recognized family, so not really new • Toxicity to host makes them unusable • They may have poor chemical properties • Insoluble, unstable, rapidly metabolized • No longer effective against resistant organisms

9. What and why • Antibiotics are secondary metabolites • Substances not essential for the growth of the organism • Typically produced at onset of stationary phase • When growth slows down • Production inhibited by presence of nutrients • Why do microbes make them? • No one is sure • Habitat guarding: prevents outsiders from establishing themselves when residents inhibited

10. Mostly antibacterial compounds • Most infections the result of bacteria or viruses • Except in Tropical medicine, fungal, protozoal, and helminth infections of humans less common. • Viral infections generally untreatable with antimicrobial drugs • Our focus is primarily on antibacterial drugs • Taxonomy of living things: review • 3 Domains: 2 of Prokaryotes and 1 Eukaryotes • One prokaryotic Domain, Archaea, not involved in disease.

11. Bacteriostatic vs. Bactericidal • Antibiotics differ by mode of action • Bacteriostatic compounds inhibit the growth of bacteria • Holds invaders in check; host immune system does the killing • Bactericidal compounds directly kill the bacteria • Location and severity of infection affect choice of antibiotic • E.g. CNS infection calls for bactericidal treatment.

12. Antibacterial agents • There are 5 principle targets for antimicrobial agents to work against bacterial cells • Inhibition of cell wall synthesis • Inhibition of protein synthesis • Attack on cell membranes • Disruption of nucleic acid synthesis • Interference with metabolism

13. Review and Overview of Bacterial Targets • Bacterial cell walls • Except for Mycoplasma and relatives, all bacteria of the Domain Eubacteria possess peptidoglycan • Peptidoglycan provides shape and structural support to bacterial cells • Bacterial cytoplasm is generally hypertonic compared to their environment • Net flow of water: into cell • Wall under high osmotic pressure

14. Cell walls continued • Chemical structure of peptidoglycan contributes to its function • Polysaccharide chains composed of 2 alternating sugars, N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) • Cross-linked in 3 dimensions with amino acid chains • A seamless, bag-like molecule which resists osmotic pressure • A breach in peptidoglycan endangers the bacterium

15. Glycan chains cross-linked with amino acids • G- and G+ vary w/ DAP vs. lysine and at the interbridge. • Note the presence of unusual “D” amino acids. • Peptides attached to NAM.

16. Peptidoglycan is a 3D molecule Cross links are both horizontal and vertical between glycan chains stacked atop one another. http://www.sp.uconn.edu/~terry/images/other/peptidoglycan.gif; http://www.alps.com.tw/cht/img/anti-allergy_002.jpg

17. There is no molecule similar to peptidoglycan in humans, making drugs that target cell wall synthesis very selective in their toxicity against bacteria.

18. Gram positive & Gram Negative • Gram positive bacteria have a thick cell wall • Peptidoglycan directly accessible from environment • Gram negative bacteria have a different wall • Thin layer of peptidoglycan • Surrounded by an outer membrane composed of lipopolysaccharide, phospholipids, and proteins • OM is a barrier to diffusion of molecules including many antibiotics • Some hydrophobic antibiotics may diffuse in. • Porins allow passage of only some antibiotics

19. Gram negative cell structure

20. Ribosomes: site of protein synthesis • Prokaryotic ribosomes are 70S; • Large subunit: 50 S • 33 polypeptides, 5S RNA, 23 S RNA • Small subunit: 30 S • 21 polypeptides, 16S RNA • Eukaryotic are 80S Large subunit: 60 S • 50 polypeptides, 5S, 5.8S, and 28S RNA • Small subunit: 40S • 33 polypeptides, 18S RNA

21. Differences in structure between prokaryotic and eukaryotic ribosomes make antibiotics that target protein synthesis fairly selectively toxic against bacteria. Drug entry into host cells and into host mitochondria (w/ prokaryotic ribosomes) apparently not a major problem.

22. Interference with nucleic acid synthesis • Bacterial DNA is negatively supercoiled • Supercoiling is maintained by gyrase, a type II topoisomerase. • Inhibition of gyrase and type IV topoisomerase interferes with DNA replication, causes cell death • Eukaryotic topoisomerases differ in structure

23. Cell membranes as targets • Bacterial cell membranes are essentially the same in structure as those of eukaryotes • Antibiotics also affect Gram neg. cell walls, ie. Outer membrane together with cell membrane • Anti-membrane drugs are less selectively toxic than other antibiotics, i.e, worse therapeutic index • Many antifungal drugs make use of cell membrane differences.

24. Other Targets • Metabolic inhibitors • Mostly target the folic acid synthesis pathway • Many bacteria can and do synthesize a large proportion of needed cofactors • Humans require folic acid in the diet (a “vitamin”), thus folic acid synthesizing enzymes are not an available target in humans • Selectively toxic

25. Drug selection • Identity of infectious agent • Many infections viral, antibiotic administration is a dangerous waste. • Identification of bacterium provides rational basis for choice • Administration depends on severity of situation • Choice of drug influenced by location of infection • Certain bacteria are more likely at certain anatomical sites, directing “blind” choice • Not all drugs reach compartments equally

26. Spectrum • When specific testing is not done or delayed, antibiotic with a broad spectrum is administered • Broad spectrum antibiotics can penetrate Gram – outer membranes, resist inactivation, etc. • Shotgun: better chance of inhibiting pathogen • Downsides • Normal microbiota found in ecological balance • Death of normal microbiota results in overgrowth of native, resistant bacteria (endogenous infection; “superinfection”) or allows invasion by outside opportunists.

27. Drug administration • Antibiotics administered oral, i.v., i.m. • Same caveats apply, i.e. acid instability, delayed absorption with food for oral • i.v. gives higher, quicker concentrations, reaches more compartments with sufficient dose quickly

28. Problem sites • Endocarditis: fibrin from inflammation • CSF because of blood-brain barrier • Osteomyelitis • Artificial joints, valves • biofilms make it difficult for drug to reach target • Abscesses: inflammatory barrier restricts access; also bacteria stop growing • Intracellular infections • Penetration into host cells, not just microbe

29. Host factors and toxicity • Antibiotics have been so successful because of their selective toxicity • But “any substance that has a biological effect will have a biological side effect” • Allergies and intolerance • Immediate and delayed type hypersensitivities when drug acts as a hapten • Intolerance: e.g. erythromycin makes me puke. • Age: renal function, development, type of pathogen

30. Host factors and toxicity-2 • Renal • Many antibiotics cleared through renal action, so renal function affects dose, choice. • Liver: Good hepatic function needed for metabolism of some antibiotics • Pregnancy • Beware of developmental effects, teratogenesis • Host defenses • Influences choice of bactericidal vs. bacteriostatic • Genetic background, metabolic factors

31. Combination therapy • Some valuable reasons why combination therapy is used • Synergistic effects between two drugs • Polymicrobial infections, e.g. abdominal injuries • Avoid or circumvent microbial resistance

32. Antibiotic resistance • Inherent: Outer membrane of Gram negative bacteria, wall-less bacteria. • Mutations: change in transport protein, ribosome, enzyme, etc. Normally harmful mutations are selected FOR in the presence of antibiotic. • Plasmids: through conjugation, genetic information allowing cell to overcome drug. http://www.mun.ca/biochem/courses/3107/ images/Stryer/Stryer_F32-13.jpg

33. Mechanisms of drug resistance • Alteration of target: active site of enzyme changes, ribosome changes. • Alteration of membrane permeability: transport protein changes, drug no longer enters; drug that does enter is actively pumped out. • Enzymatic destruction of drug: penicillinases (beta lactamases) • “End around” inhibitor: bacteria learns to use new metabolic pathway, drug no longer effective.