Lesson 2: Selectivity And Mechanism Of Action Of Antibiotics PDF

Lesson 2: Selectivity and mechanism of action of antibiotics Prepared by Dr/ Essam KOTB Associate Professor of Microbiology, IAU University [email protected]...

Lesson 2: Selectivity and mechanism of action of antibiotics Prepared by Dr/ Essam KOTB Associate Professor of Microbiology, IAU University [email protected] +2/01121343400 +966/0563533550  Selective toxicity of antibiotics Selective toxicity is the ability of the drug to target the disease-causing bacteria, while causing no or minimal harm to the patient. This is due to the differences between pathogenic bacteria and human or animal cells in both structure and functions. For centuries, mercury was used to treat syphilis. But it was extremely toxic, it makes hair and teeth fall out and destroys the nervous system thus, killing the patients (it has no selective toxicity). Ehrlich used dyes but soon, people went die crazy (also due to the unselectivity).  Ehrlich then tested hundreds of arsenic derivatives against rabbits infected with syphilis. Some compounds killed both bacteria and rabbits. Some killed neither. Finally, he found that compound 606 (later named salvarsan), killed the bacteria and left rabbits unharmed. This was the first magic bullet that humans had been hoping for. At that time, it was the world's most popular drug for syphilis. Ehrlich’s work on syphilis proved that ‘magic bullets’ are there, just screen for them. With this he established the principle “drugs should demonstrate selective toxicity”.  Sulfonamides were then appeared (1900's) as the second synthetic magic bullets. This was initiated by prontosil red which is a sulphonamide dye that was very effective against streptococcal infections. Penicillin was then discovered (1928) as third magic bullet and the first natural antibiotic.  Mode of action of antibiotics Inhibition of one of the following: 1. Cell wall synthesis 2. Protein synthesis 3. Nucleic acid synthesis 4. Formation of metabolites (antimetabolites) A small number of antibiotics target the bacterial cell membrane. Such antibiotics are often highly toxic to the host. Can you suggest a reason for this? – Because the membrane of animal and human cells has a very similar structure to that of bacteria. – The potential for such antibiotics to adversely affect eukaryotic cells is therefore greater and these antibiotics generally demonstrate poor selective toxicity. Figure 2 Main antibiotic modes of action. 1. Inhibit cell wall synthesis Cross-linking between peptidoglycan chains forms a strong, mesh-like structure that gives the cell wall rigidity and protects the underlying cell membrane from osmotic damage when water moving into the cell by osmosis. Examples of cell wall synthesis inhibitors are the ß- lactam antibiotics. These include penicillins, cephalosporins, carbapenems, and monobactams. Figure 4 Core ring structures of β-lactam antibiotics. The β-lactam ring is shaded red in each case.  All ß-lactam antibiotics contain a core chemical structure called a ß-lactam ring (Figure 4) which determines the mode of action of this class. Monobactams are monocyclic and bacterially-produced β- lactam antibiotics. The β-lactam ring is not fused to another ring, in contrast to most other β-lactams. Monobactams are effective only against aerobic Gram-negative bacteria (e.g., Neisseria, Pseudomonas.  The β-lactam antibiotics target the bacterial cell wall by inhibiting the enzymes responsible for cross-linking adjacent molecules in the peptidoglycan layer. Figure 8 Peptidoglycan molecules consist of a backbone of carbohydrate units with sets of amino-acid residues attached (yellow). They are cross-linked by bridges (red), providing structure and strength.  The ß-lactam antibiotics bind to these enzymes, which are known as penicillin-binding proteins (PBPs) at the -NH2 terminal preventing them from forming cross-links. Figure 9 Reaction of penicillin with a PBP.  The ß-lactam ring contains 3C and 1N atoms with 90 degrees bond angle which encourage it to react effectively. The -NH2 side chain of the PBP reacts with the ß- lactam ring to form a new side chain or what is called new ring strain. This reaction opens the ß-lactam ring and effectively deactivates the enzyme (PBP) to bind to its normal substrate during the peptidoglycan cross-linking process.  As the bacterial cell grows, the inhibitory reaction of ß-lactams has 2 effects: 1. Prevent cell division due to the none formation of transverse septa therefore growing in form of long strings. 2. Cell bursting as a result of osmotic damage due to the weakness and unrepair of the existing cell wall. Figure 3 Lysis of a bacterium with a defective cell wall. Part (a) is a schematic diagram showing the sequence of events that lead to the osmotic lysis of a bacterium. Initially the cell wall and the cell membrane beneath it are intact. As water enters by osmosis, the cell wall becomes defective. Eventually the cell contents and surrounding membrane expand through the defective cell wall, the membrane then ruptures and the cell contents spill out; that is, the cell lyses. In the light micrograph in part (b), the intact near-spherical cell appears orange–yellow on the black background; while the lysed cell has collapsed and lost most of its contents and so has a shrivelled shape and appears mostly black.  Glycopeptide antibiotics are peptides that are covalently attached to carbohydrate moieties (glycans). Best example is vancomycin which is originally produced by Streptococcus orientalis. They are especially effective against gram positive bacteria such as methicillin resistant Staphylococcus aureus (MRSA). They inhibit bacterial growth by interfering with cell wall biosynthesis. This occurs through binding to and sequestering lipid II. This prevents the recycling of bactoprenol phosphate, the lipid transporter that is shared by peptidoglycan and wall teichoic acid biosyntheses Therefore, inhibit glycosyl-transferase linking of OH group of lipid II with the sugar moiety after transport to the cell wall (glycosylation). Resistance to glycopeptides occurs through the reprogramming of peptidoglycan formation, in which a d-lactate unit replaces the terminal d-alanine in lipid II, a substitution sufficient to prevent glycopeptide binding in position.  Antibiotics targeting bacterial cell membrane For therapeutic use, due to side effects on host cell membrane, only daptomycin and polymyxins are approved. 1. Daptomycin cause calcium-dependent membrane depolarization, which results in the cessation of macromolecular synthesis and disruption of the cellular membrane in G+ve bacteria. 2. Polymyxins bind to lipid A of the lipopolysaccharide in the bacterial membrane, resulting in membrane disintegration in G- ve bacteria. 2. Inhibit protein synthesis Prokaryotic (70S) and eukaryotic (80S) ribosomes differ structurally and functionally. Therefore, 70S ribosomes are an easy antibiotic targets in the bacterial pathogen which are not present in the host cells. Table 1 Examples of antibiotics inhibiting protein synthesis. Ribosomal Outcome Antibiotic Structure Example target class drug Small (30S) Errors give Aminoglycos All contain Streptomycin subunit rise to faulty ides amino sugar proteins that rings (red) disrupt the cell membrane Tetracyclines also inhibit 30S Table 1 Examples of antibiotics inhibiting protein synthesis. Ribosomal Outcome Antibiotic Structure Example target class drug Large (50S) First steps of Oxazolidine All contain Linezolid subunit protein synthesis s oxazolidone (initiation) are ring (red) impaired thus bacteria cannot grow and divide Macrolides also inhibit 50S  3. Inhibit nucleic acid synthesis Differences between enzymes that carry out the synthesis of nucleic acids in eukaryotic and prokaryotic cells allow antibiotics to target them in bacterial pathogens easily, for example: 1. Fluoroquinolones (= ciprofloxacin) inhibit DNA replication by inhibition of bacterial DNA unwindases, so that the DNA can't make a copy of itself. Figure 6 The fluoroquinolone (ciprofloxacin) all contain the chemical structure highlighted in red. DNA gyrase or helicase is used instead of unwindase 1. Rifamycins – inhibit RNA synthesis by inhibiting RNA polymerase. Therefore, ultimately stop new proteins being made. 4. Inhibit metabolite formation These antibiotics are called antimetabolites. Which are structural analogues to natural metabolic intermediates. For example, trimethoprim inhibits the synthesis of folic acid vitamin (vit B9) which bacteria must make themselves. Trimethoprim is a structural analogue of dihydrofolic acid (an intermediate compound in the folic acid pathway). Trimethoprim out-competes dihydrofolic acid to react with a specific bacterial enzyme (dihydrofolate reductase) in the pathway, thereby interrupting folic acid synthesis and inhibiting bacterial growth (Figure 7). Therefore, these antibiotics are competitive enzyme inhibitors. Figure 7a The underlying competitive inhibition mechanism. Figure 7b The folic acid pathway. Trimethoprim (I) prevents the enzyme (E) dihydrofolate reductase reacting with the intermediate compound dihydrofolic acid (S), thereby blocking the pathway at the point shown.  Sulfonamides (sulfa drugs) are also antimetabolites. – They are competitive inhibitors of dihydropteroate synthetase in the folic a pathway because of their structural similarity with para-amino benzoic acid (PABA). – Therefore, they are bacteriostatic same as trimethoprim. Inhibiting the bacterial growth. – For this sometimes mixed with trimethoprim. – The antimetabolites are safe for human because human are in contrast with bacteria, acquire folic acid through diet. – The effective group of Sulfonamides is sulfonamide group (SO2NH2).  1-Broad-spectrum versus narrow-spectrum antibiotics Factors determining the spectrum of activity: 1. Ability to penetrate the bacterial cell. – In G+ve bacteria, the cell wall has a thick peptidoglycan layer which is relatively porous, giving greater access to antibiotics, allowing them to more easily penetrate the cell and/or interact with the peptidoglycan itself in case of ß-lactams. – In G-ve bacteria, peptidoglycan layer is greatly reduced and is protected by a second outer membrane which is an effective barrier, regulating the passage of antibiotics and other large molecules into the cell. Therefore, generally Gram-negative are resistant to antibiotics. – In other words:  Figure 10 Arrangement of the cell wall in (a) Gram-positive and (b) Gram-negative bacteria. 1. The widespread of the target among bacteria. 2. Bacterial resistance to the antibiotic. – Not all Gram-positive and Gram-negative bacteria are affected by a single antibiotic because of the specificity of the antibiotic/bacterial target interaction, whether the bacterial species be penetrated, has the target and whether the bacteria are resistant to the antibiotic. 2-Bactericidal versus bacteriostatic antibiotics The activity of most antibiotics depends on the dose (concentration). While some classes have consistent antibacterial effects such as: ß-lactams which are always bactericidal. Antimetabolites are bacteriostatic. 1. What happens to bacteria when a bacteriostatic antibiotic (antibiotic A) introduced during the exponential phase of growth? Answer: What will happen to bacterial growth if antibiotic A is removed from the culture at the point indicated on the graph? Answer: 1. What happens to bacteria when a bactericidal antibiotic (antibiotic B) introduced during the exponential phase of growth? Answer: What will happen to bacterial growth if antibiotic B is removed from the culture? Answer: So, during antibiotic treatment we can conclude: Bactericidal antibiotics kill pathogenic bacteria during the exponential phase of growth and relief the infection directly. Bacteriostatic antibiotics stop pathogenic bacteria during the exponential phase of growth and the cells remain viable. – This gives time for the host’s immune system to be activated and target the bacterial pathogen and relief the infection indirectly. Summary This lesson introduced some of the basic biology and chemistry that underpins antibiotic activity. You looked at the main modes of antibiotic action and learned why these drugs demonstrate selective toxicity. You should now be able to: Recognise different types of commonly used antibiotics Recall the characteristic features of bacterial and human or animal cells Explain why antibiotics have selective toxicity Demonstrate how commonly used antibiotics affect bacterial growth Summarise the main mechanisms by which antibiotics stop infections from spreading and kill bacteria. Having explored different types of antibiotic in some detail, you should now be well prepared to move on to Lesson 3 which discusses antibiotic resistance mechanisms. Thanks for your attention Questions?