Antimicrobial Drug Discovery PDF

Microbiology 302 ANTIMICROBIAL DRUG DISOCVERY Dr Tarryn Swart Biosciences Building, Room 417 (4th Floor) Course Outline (12 Lectures + 6 practical’s): 1. Introduction to the discovery of penicillin and...

Microbiology 302 ANTIMICROBIAL DRUG DISOCVERY Dr Tarryn Swart Biosciences Building, Room 417 (4th Floor) Course Outline (12 Lectures + 6 practical’s): 1. Introduction to the discovery of penicillin and the golden age of antibiotic discovery 2. Classes of antibiotics, biological activity and mechanism of action 3. Mechanisms of resistance 4. Antimicrobial Resistance (AMR), ESPAKE pathogens, and the search for new antimicrobials  ANTIMICROBIAL DRUG DISCOVERY 1. The discovery of penicillin and the Golden Age of antibiotic discovery  Before the discovery of penicillin 1. Def antimicrobial – substance that kills microorganisms (antibiotics, antivirals, antifungals, antiparasitics). 2. Fleming and other researchers were interested in the antimicrobial properties of lysozyme. 3. Lysozyme is present in saliva and has antimicrobial activity especially against Gram +ve bacteria. 4. Component of the human immune system - low toxicity. 5. Hydrolyzing 1,4-beta-linkages between N- acetylmuramic acid and N-acetylglucosamine in Alexander Fleming Laboratory Museum, St Mary’s Hospital, London, UK the cell wall. 6. Causes the cells to burst/lyse and die. 7. Studying the gram +ve pathogenic bacterium Staphylococcus aureus.  1. A fungus caused lysis of adjacent The discovery of penicillin Staphylococcus colonies on an agar plate 2. Not all fungal species were antibacterial 3. Antimicrobial activity retained in broth in which the Penicillium fungus had been grown 4. Inhibited the growth of different gram –ve and gram –ve bacteria 5. Penicillin was heat stable (56-80°C) and water soluble 6. Addition of glucose to fungal culture delayed the production of inhibitory activity 7. Slow rate of kill of Staphylococcus 8. Concentrations of penicillin require to kill bacteria did not affect mice cytotoxicity, low MIC 9. Penicillin may be useful to treat bacterial infections Development of penicillin as an antibiotic - the problem of supply 1. Little interest in Fleming’s work after publication in 1929. 2. Fleming's attempts to purify penicillin were unsuccessful so he lost interest. “the production of penicillin for therapeutic purposes…almost impossible” Harold Raistrick. 3. 1937: Howard Florey and Ernst Chain further research enabled the testing and production of the drug – ‘Penicillin Project’ at Oxford University 4. Could only produce a few mgs of penicillin per week by fermentation. Inefficient production but enough for mouse trials in 1939 (8 mice infected with Streptococcus, 4 injected with penicillin survived 5. First human trial in 1941 – treatment was successful, but the patient died because they ran out of penicillin. 6. Started re-purifying penicillin from patient urine 7. 1941: US Dept of Agriculture labs optimize fermentation medium by adding corn- steep liquor – 10X increase in penicillin production. 8. Very few Penicillium isolates (1/1000) produce penicillin. 9. USDA screened 1000s of Penicillium cultures and found one strain isolated from rotting melon that produced 6x more penicillin  By 1943: there were 20 US companies producing penicillin with sufficient stocks for the US and its allies Penicillin available to the public for medical use –the first antibiotic –in 1946 “The discovery of penicillin changed the course of history. Penicillin saved thousands of wounded soldiers and civilians during the biggest of the wars, and its discovery laid the foundations of the antibiotic era and subsequent development of other more potent antibiotics.” Development of penicillin G and other B-lactam antibiotics 1. Fleming, Florey and Chain awarded the Nobel Prize in 1945 for ”the discovery of penicillin and its curative effect in various infectious diseases”. 2. Hodgkin and Low solved the structure by X-ray crystallography analysis in 1945 showing the Penicillin G presence of the 4-membrane beta lactam ring. 3. Discovery of new natural penicillins (e.g cephalosporins) Cephalosporin C and synthesis of new derivatives. The emergence of penicillin-resistant bacterial pathogens 1. Four penicillin-resistant S. aureus strains isolated from hospital patients treated with penicillin in 1942. 2. Late 1960s: more than 80% of both community and hospital-acquired strains of S. aureus are penicillin resistant. 3. Second-generation, semisynthetic methicillin introduced in the 1960s to combat penicillin resistance, but within 20 years MRSA = >29% of hospital infections in the USA. 4. The development of resistance goes hand in hand with the introduction of new generations of penicillin into clinical practice 5. Antimicrobial Resistance is declared a Global Health Problem in 1981 (‘Statement regarding Worldwide Antibiotic Misuse”). The race to overcome penicillin-resistance  ANTIMICROBIAL DRUG DISCOVERY 2. Classes of antibiotics, biological activity and mechanism of action Classes of antibiotics and their mechanism of action The majority of clinically important antibiotics are natural products 1. In the context of drug discovery, natural products (NPs) are small molecules or peptides produced by living cells, mostly microorganisms, and usually as secondary metabolites. 2. Features of antimicrobial NPs: Chemical diversity Targeted for biological function in living cells They have evolved to be active in the cellular environment (solubility etc.) They are often extremely potent with low MICs Often produced as chemical defense against other, pathogenic or competitive microorganisms and viral pathogens The majority of clinically important antibiotics are natural products Bacteriostatic: restricting growth & reproduction, does not kill. When removed, bacteria will grow again. Bactericidal: causing bacterial cell death. Summary of the mechanism of action of clinically important antibiotics 1 1. Antibiotics targeting the cell wall The bacterial cell wall Gram negative Gram positive  The bacterial cell wall The importance of Peptidoglycan 1. Essential component of the bacterial cell envelope, maintains cell shape and prevents bursting due to turgor pressure. 2. Glycan chains crosslinked by short peptides, forming a net-like structure around the cytoplasmic membrane 3. Peptidoglycan structure varies in different bacteria modified to resist lysozyme activity 4. Peptidoglycan precursors are synthesized in the cytoplasm and transported across the membrane into the periplasmic space. 5. Comprises a backbone of alternating units of N-acetylglucosamine(GlcNAc) and N- acetylmuramic acid, with the N-acetylmuramic acid residues cross-linked to peptides, catalysed by transpeptidases.  Antibiotics targeting the cell wall β-Lactams e.g. penicillin G, methicillin, amoxicillin, cephalosporin All contain a β-lactam ring Mechanism of action is interference with the synthesis of peptidoglycan Mostly used against gram +ve bacteria. Mechanism of action Lactam antibiotics interrupt bacterial cell-wall formation as a result of covalent binding to essential penicillin-binding proteins (PBPs) PBPs are enzymes involved in the terminal steps of peptidoglycan cross-linking in both Gram-negative and Gram-positive bacteria. Every bacterial species has its own distinctive set of PBPs that can range from three to eight enzymes Summary of the mechanism of action of clinically important antibiotics 1 2  2. Membrane Disrupting Antibiotics Selective permeability of membranes essential for bacterial survival. Requires ATP dependent K+ gradient across the membrane. Targets of antimicrobials are membrane disruption and the metabolic steps of fatty acid synthesis and membrane phospholipids. A) Polymyxin B, a bactericidal antibiotic, is one of very few drugs for treating Gram -ve bacteria e.g. Pseudomonas aeruginosa. Detergent-like peptides having lipophilic and hydrophilic groups disrupts membrane integrity. Interacts electrostatically with the bacterial outer Membrane. Mg2+ and Ca2+ ions that stabilize the membrane structure are displaced leading to the insertion of the polymyxin molecule into the membrane. This process destabilizes the membrane integrity. disintegration of the bacterial membrane leading to cell lysis and death  2. Membrane Disrupting Antibiotics B) Valinomycin, is an ionophore, disrupts cellular membrane potential and inhibits oxidative phosphorylation by forming pores in cellular membrane. (Oxidative phosphorylation is a cellular process that harnesses the reduction of oxygen to generate high-energy phosphate bonds in the form of ATP) C) Daptomycin: widely used in bloodstream, wound, and soft skin infections caused by β-lactam especially vancomycin-resistant Staphylococcus aureus. Binds and inserts into the cell membrane in a phosphatidylglycerol-dependent fashion It aggregates in the membrane It alters the curvature of the membrane by depolarization, K+ ions are released from cytoplasm to extracellular matrix easily. Summary of the mechanism of action of clinically important antibiotics 1 2 3  3. Antibiotics that target nucleic acid synthesis Topoisomerases are involved in the control of topological transitions of DNA supercoiling Essential enzymes for DNA replication and mRNA transcription –unwinding and rewinding of DNA DNA gyrase catalyses the ATP-dependent negative super-coiling of double-stranded supercoiled DNA ahead of the RNA polymerase/Replisome complexes Topoisomerase IV regulates positive supercoiling A) Quinolones and fluorouinones (e.g. ciprofloxacin) Inhibit DNA gyrase by interfering with the DNA cleavage/resealing function of the enzyme. Broad-spectrum antibiotics, used for urinary tract infections, hospital-acquired infections with suspected resistance to older classes of antibiotics. Widespread use for veterinary purposes - rapid development of resistance. B) Aminocoumarins (e.g. novobiocin) bind to the βsubunit of DNA gyrase, competing for the binding of ATP Inhibition of ATP hydrolysis -gyrase is unable to hydrolyse the DNA C) Anthracyclines DNA intercalators that bind to the major groove of the DNA and inhibit topoisomerase activity Also used as anticancer drugs. Side effects are a problem  3. Antibiotics that target nucleic acid synthesis D) Ansamycins (rifamycins) e.g. rifampicin Bacterial secondary metabolites (NPs) Effective against Gram +ve and some Gram -ve bacteria. Bactericidal, inhibiting transcription by binding to the active site of bacterial RNA polymerase, RpoB Rifamycins are used to treat TB and leprosy, also anti-viral activity Problem with spontaneous resistance Antibiotics that target DNA synthesis and replication https://www.youtube.com/watch?v=jmWuju1S9_E  Antibiotics targeting DNA synthesis and replication https://www.biomol.com/resources/biomol-blog/how- do-antibiotics-affect-nucleic-acid-synthesis Summary of the mechanism of action of clinically important antibiotics 1 2 3 4  4. Antibiotics that target metabolic pathways Targets enzymes that catalyse biological metabolic reactions e.g. purine biosynthesis where the products are essential for cell viability. Antibiotics most often resemble the enzyme substrate and block the reaction – competitive inhibition Inhibition of folate biosynthesis - folate is an essential vitamin required for nucleotide biosynthesis Para-aminobenzoic acid (PABA) is a substrate for folic acid synthesis that is a coenzyme in the reactions of purines, pyrimidine, and amino acids synthesis. Sulfonamides (sulfamethoxazole) are synthetic broad-spectrum antibiotics acting against both Gram +ve and Gram-ve bacteria. Inhibits conversion of PABA to dihydropteroate – problems with allergies – sulphur. Dihydropyrimidines (trimethoprim) inhibits dihydrofolate reductase - converts dihydrofolate to tetrahydrofolate, inhibits purines and thymidylate synthesis Trimethoprim used in combination with sulfamethoxazole Antibiotics that target metabolic pathways Metabolic pathway in bacteria Summary of the mechanism of action of clinically important antibiotics 1 5 2 3 4  5. Antibiotics targeting protein synthesis Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues – Bacteria cannot survive without proteins. Three phases of protein synthesis: 1. Initiation: ribosome is assembled in the correct place on an mRNA (A-site: aminoacyl) 2. Elongation: correct amino acid is brought to the ribosome, joined to the polypeptide chain and the entire assembly moves one position along the mRNA (P-site: peptidyl) 3. Termination: stop codon is reached, newly synthesized polypeptide is released (E-site: exit)  Antibiotics targeting protein synthesis A) Aminoglycosides e.g. streptomycin, kanamycin Inhibit protein synthesis, broad spectrum of activity that cause translation errors, leading to cell death. Only effective against certain Gram -ve bacteria and some Gram +ve bacteria, low levels of resistance. Streptomycin was the first drug effective against TB, but issues with toxicity makes present day use is limited. Not absorbed during digestion, so only injected. B) Tetracyclines Broad-spectrum antibiotics, active against both Gram +ve and Gram-ve bacteria. Inhibit protein synthesis by mimicking tRNA, inhibiting growth. Use is decreasing due to increasing resistance, but still used for acne, urinary tract, and respiratory tract infections, as well as chlamydia infections. C) Macrolides e.g. erythromycin Mainly effective against Gram +ve bacteria e.g. Legionella, Mycoplasma, and Rickettsia infections. Inhibits translation by binding to 23S rRNA, blocking the E site resulting in the premature release of an incomplete polypeptide. D) Amphenicols e.g. chloramphenicols Binds to the 50 S subunit of the 70 S ribosome and inhibiting the action of peptidyl transferase, thus preventing peptide bond formation. Also prevents binding of aminoacyl tRNA to the peptidyl transferase active site Antibiotics targeting protein synthesis ANTIMICROBIAL DRUG DISCOVERY 3. Mechanisms of resistance APPROVAL FOR CLINICAL USE EMERGENCE OF RESISTANCE What is antibiotic resistance The bacterium continues to grow and multiply in the presence of MIC's of the antibiotic. Require higher doses for effective control of the resistant bacterium. Mechanisms of resistance 1. Antibiotic modification: Addition of acetyl, phosphate, or adenyl groups to aminoglycosides by N-acetyl transferases (AAC), O-phosphotransferases (APH), and O-adenyltransferases (ANT). Other examples include chloramphenicol acetyl transferases (CAT) and bleomycin N-acetyltransferases (BlmB). 2. Antibiotic degradation: β-lactam antibiotics (e.g. penicillins) are degraded by β- lactamases, that hydrolyze the antibiotic. 3. Antibiotic efflux: ATP-dependent antibiotic efflux pumps remove the antibiotic from the cell often resulting in multidrug resistance. Mechanisms of resistance 4. Target modification: Target modification includes various target alterations, e.g. 23S rRNA or 16S rRNA methylation, alterations in the peptidoglycan precursors (e.g. glycopeptides), or synthesis of alternate low-affinity targets (PBPs, penicillin binding proteins) that reduce or completely block antibiotic from associating with the target. 5. Sequestration: Expression of proteins that can associate with the antibiotic and block them from reaching their targets. 6. Target bypass: Generation of additional antibiotic targets or subunits that are not susceptible to binding of the antibiotic. Or upregulating the expression of the target so that the antibiotic is diluted out. 7. Impermeability: The antibiotic cannot pass across the membrane of Gram –ve vs Gram +ve bacteria – bacteria alters the cellular permeability Mechanisms of antibiotic resistance (G) Impermeability  ANTIMICROBIAL DRUG DISCOVERY 4. Antimicrobial Resistance (AMR), ESPAKE pathogens, and the search for new antimicrobials The ESKAPE Pathogens Methods of horizontal gene transfer Horizontal gene transfer contributes to the spread of 1. In conjugation, DNA is antibiotic resistance through the exchange of genetic transferred between bacteria through material across genera, which increases the potential for a tube between cells. After the donor a harmful, antibiotic resistant bacteria to develop. cell pulls itself close to the recipient using a structure called a pilus, DNA is transferred between cells. In most cases, this DNA is in the form of a plasmid. 2. In transduction, DNA is accidentally moved from one bacterium to another by a virus.  Methods of horizontal gene transfer 3. Transposable elements are chunks of DNA that "jump" from one place to another. They can move bacterial genes that give bacteria antibiotic resistance or make them disease-causing. 4. In transformation, a bacterium takes up a piece of DNA floating in its environment. Imagine that a harmless bacterium takes up DNA for a toxin gene from a pathogenic species of bacterium. If the receiving cell incorporates the new DNA into its own chromosome (homologous recombination), it too may become pathogenic. SELF-RESISTANCE MECHANISMS IN PRODUCER ORGANISMS Transfer of resistance genes to clinical isolates could occur by a variety of routes (shown by arrows), each using horizontal gene transfer mechanisms potentially involving plasmids, integrons (genetic elements with a site-specific recombination system able to integrate, express and exchange gene cassettes), or transposons. Direct transfer of resistance determinants from producers in the soil to clinical strains is possible but unlikely. More likely first movement from the producer soil bacteria to non-producer soil bacteria (for example Mycobacterium species), followed by transfer to clinical pathogens through several carriers. Or a more important route, could involve direct transfer from environmental bacteria (found in bodies of water, aquaculture, livestock animals, wildlife, and plants) to clinical isolates, implying greater probability of these pathways for dissemination of resistance genes to clinical strain. MGE-mediated (i.e. phage, plasmids, and integrons) dissemination of AMR across different microbial reservoirs MGE(mobile genetic elements). An estimated 1.2 million people died in 2019 from antibiotic-resistant bacterial infections In 2019, an estimated 1.2 million people died from antibiotic-resistant bacterial infections – more deaths than HIV/AIDS or malaria - and that antimicrobial-resistant infections played a role in 4.95 million deaths..  Surveys of antimicrobial resistance in animals -2021 Number of resistance rates grouped by year and animal Resistance rates grouped by pathogen and animal species. species. Action against antimicrobial resistance requires a ONE HEALTH APPROACH https://iris.who.int/bitstream/handle/10665/376479/WHO-EURO-2024- 9510-49282-73655-eng.pdf?sequence=1