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ANTIMICROBIAL CHEMOTHERAPY Brief historical perspective of chemotherapy Classification of antimicrobial agents with special reference to mechanism of action and chemical structures. Drugs inhibiting cell wall synthesis Drugs that Inhibit protein synthesis. Drugs that interfere with cell m...

ANTIMICROBIAL CHEMOTHERAPY Brief historical perspective of chemotherapy Classification of antimicrobial agents with special reference to mechanism of action and chemical structures. Drugs inhibiting cell wall synthesis Drugs that Inhibit protein synthesis. Drugs that interfere with cell membrane integrity. Inhibitors of RNA and DNA synthesis. Antifungal agents Antiviral agents Development of resistance to antibiotics by microorganisms: plasmid mediated and biochemical basis CHEMOTHERAPY Most people associate the term with treatments for cancer It is actually a broader term that refers to the use of chemicals or drugs to treat diseases Chemotherapy may involve drugs that target cancerous cells or tissues OR it may involve antimicrobial drugs that target infectious microorganisms Antimicrobial drugs typically work by destroying or interfering with microbial structures and enzymes thus either killing microbial cells or inhibiting their growth The first antimicrobial drugs In the early 1900s German physician and scientist Paul Ehrlich (1854–1915) set out to discover or synthesize chemical compounds capable of killing infectious microbes without harming the patient. In 1909, after screening more than 600 arsenic-containing compounds, Ehrlich’s assistant Sahachiro Hata (1873–1938) found one such “magic bullet.” Compound 606 targeted the bacterium Treponema pallidum, the causative agent of syphilis. It was found to successfully cure syphilis in rabbits and soon after was marketed under the name Salvarsan as a remedy for the disease in humans Alexander Fleming (1881–1955) In 1928, Fleming returned from holiday and examined some old plates of staphylococci in his research laboratory at St. Mary’s Hospital in London. He observed that contaminating mould growth (subsequently identified as a strain of Penicillium notatum) inhibited staphylococcal growth on one plate. Fleming is therefore credited with the discovery of penicillin, the first natural antibiotic Further experimentation showed that penicillin from the mould was antibacterial against streptococci, meningococci and Corynebacterium diphtheriae, the causative agent of diphtheria Gerhard Domagk Discovered the antibacterial activity of a synthetic dye, prontosil, that could treat streptococcal and staphylococcal infections in mice Gerhard Domagk (1895–1964) was awarded the Nobel Prize in Medicine in 1939 for his work with prontosil and sulfanilamide, the active breakdown product of prontosil in the body. Sulfanilamide, the first synthetic antimicrobial created, served as the foundation for the chemical development of a family of sulfa drugs. Selman Waksman(1888–1973) Prominent soil microbiologist at Rutgers University Led a research team that discovered several antimicrobials including actinomycin, streptomycin, and neomycin The discoveries of these antimicrobials stemmed from Waksman’s study of fungi and the Actinobacteria, including soil bacteria in the genus Streptomyces, known for their natural production of a wide variety of antimicrobials. His work earned him the Nobel Prize in Physiology and Medicine in 1952. The actinomycetes are the source of more than half of all natural antibiotics and continue to serve as an excellent reservoir for the discovery of novel antimicrobial agents. CLASSIFICATION OF ANTIMICROBIAL AGENTS Antibacterial agents can be classified based on  Type of action  Source  Spectrum of activity  Chemical structure and  Function (mode of action) CLASSIFICATION BASED ON TYPE OF ACTION Bactericidal; destroy bacteria by targeting the cell wall or cell membrane of the bacteria Bacteriostatic; slow or inhibit the growth of bacteria. the inhibition phenomenon of bacteriostatic agents involves inhibition of protein synthesis or some bacterial metabolic pathways CLASSIFICATION BASED ON SOURCE Natural: Naturally occurring mostly from fungal sources Semisynthetic: chemically altered natural product Synthetic: designed to have even greater effectiveness and less toxicity and, thus, have an advantage over the natural antibiotics that the bacteria are not exposed to the compounds until they are released CLASSIFICATION BASED ON SPECTRUM OF ACTIVITY Narrow Spectrum Broad Spectrum CLASSIFICATION BASED ON CHEMICAL STRUCTURE Different skeleton containing antibiotics display different therapeutic behaviour Similar structural units have similar pattern of toxicity and effectiveness  β‐lactams  β‐lactam/β‐lactamase inhibitor combinations  Aminoglycosides  Macrolides  Quinolones and fluoroquinolones CLASSIFICATION BASED ON FUNCTION Function means how a drug works or what is its mode of action Major processes or functions, which are responsible for bacterial growth, are cell wall synthesis, cell membrane function, protein synthesis, nucleic acid synthesis All such processes are targets for antibiotics; therefore, antibacterials, which interfere or disturb these processes in different ways can be subdivided into  Cell wall synthesis inhibitors  Inhibitors of membrane function,  Inhibitors of protein synthesis,  Inhibitors of metabolic pathways and  Inhibitors of nucleic acid synthesis. DRUGS THAT INHIBIT BACTERIAL CELL WALL SYNTHESIS Bacteria are classified as Gram positive or Gram negative on the basis of staining characteristics Gram positive bacterial cell wall is much thicker than Gram negative bacterial cell wall. It contains peptidoglycan and teichoic acid and it may or may not be surrounded by protein or polysaccharide envelope Gram negative bacterial cell wall contains peptidoglycan, an extra lipid layer outside made up of lipopolysaccharide, lipoprotein, phospholipid and protein. The cell wall is confined in the periplasmic space between 2 lipid layers The cell wall provides shape and rigidity and also protects the bacteria from changes in osmotic pressure  The rigid cell wall possessed by most bacteria is lacking in the host cells. This is the prime target for agents that exhibit selective toxicity Inhibitors of bacterial cell wall synthesis act on the formation of peptidoglycan layer Bacteria that lack peptidoglycan such as mycoplasma are resistant to such agents PEPTIDOGLYCAN Peptidoglycan also known as murein is a combination of glycans (sugars) and peptide cross links Glycans: N-acetyl glucosamine (NAG) N-Acetyl Muramic acid (NAM) Gram positive bacteria have about 50-100 glycan layers Gram negative bacteria have about 1-2 glycan layers BACTERIAL CELL WALL SYNTHESIS Cell wall synthesis proceeds in 3 stages  Cytoplasmic stage  Synthesis of precursors (NAG & NAM): Inhibited by Cycloserine &Fosfomycin  Membrane stage (Elongation and transfer)  Transfer of the precursors from cytosol to membrane and incorporation into the growing peptidoglycan. Inhibited by Bacitracin and Vancomycin  Extracellular stage (Cross linking)  Cross linking of linear chains of peptidoglycans by membrane bound transpeptidases: Inhibited by β-lactams Stage 1: Synthesis of precursors Stage 2: Elongation and Transfer The two sugars NAG and NAM are put outside the bacterial cell A protein critical for this stage is bactoprenol (lipoprotein) An enzyme peptidoglycan synthase takes the bactoprenol complex and attaches the 2 sugars to the growing peptidoglycan Recycling of bactoprenol is very important because the bacteria has limited supply of bactoprenol so if it doesn’t get recycled, there will be a shortage of bactoprenol An enzyme called phosphatase recycles bactoprenol back to its original state which removes the phosphate that was left when the sugar complex was removed from it  Bacitracin inhibits the process of dephosphorylation of bactoprenol leading to insufficient bactoprenol for attachment of the NAMpp+NAG complex  Vancomycin attaches itself to the last 2 amino acids(D-alanine) thereby inhibiting the flipping of the bactoprenol NAMpp+NAG complex Stage 3: Cross linking of polymers In stage 2, the glycans are put together in polymeric form, there is no attachment between one strand of sugar and the other strand Neighbouring peptidoglycan chains are cross linked through their peptide side chain The reaction is catalysed by transpeptidase which is a type of Penicillin Binding Protein Β-lactam antibiotics inhibit transpeptidase MICROBIAL SOURCES OF ANTIBIOTICS β-lactams This group of compounds includes Penicillins, Cephalosporins, Monobactams, and Carbapenems Characterized by the presence of a β-lactam ring found within the central structure of the drug molecule. The β-lactams block the crosslinking of peptide chains during the biosynthesis of new peptidoglycan in the bacterial cell wall. They act by inhibiting transpeptidases by acting as alternative substrates They mimic the D-alanyl-D-alanine residues and react covalently with the transpeptidase The cells become deformed in shape and eventually burst through the combined action of a weakened cell wall, high internal osmotic pressure and uncontrolled activity of autolytic enzymes in the cell wall Penicillins Adding an amino group (−NH2) to penicillin G created the aminopenicillins (i.e., ampicillin and amoxicillin) that have increased spectrum of activity against gram negative pathogens. Furthermore, the addition of a hydroxyl group (−OH) to amoxicillin increased acid stability, which allows for improved oral absorption. Methicillin is a semisynthetic penicillin that was developed to address the spread of enzymes (penicillinases) that were inactivating the other penicillins. Changing the R group of penicillin G to the more bulky dimethoxyphenyl group provided protection of the β-lactam ring from enzymatic destruction by penicillinases, giving us the first penicillinase-resistant penicillin. Penicillins There are four types of penicillins: The natural penicillins are based on the original penicillin-G structure. Natural penicillins are active against gram-positive streptococci, staphylococci, and some gram-negative bacteria such as meningococcus. Penicillinase-resistant penicillins or beta lactamase resistant penicillins, anti staphylococcal penicillins (e.g. methicillin, oxacillin, cloxacillin) are active against beta-lactamase producing bacteria, that inactivates most penicillin antibiotics. Aminopenicillins such as ampicillin and amoxicillin are effective against a wider range of bacteria and have a better oral absorption. Extended-spectrum penicillins or ureidopenicillins/antipseudomonal penicillins (e.g. mezlocillin, piperacillin, ticarcillin). Cephalosporins The β-lactam ring of cephalosporins is fused to a six-member ring, rather than the five-member ring found in penicillins. This chemical difference provides cephalosporins with an increased resistance to enzymatic inactivation by β-lactamases. The drug cephalosporin C was originally isolated from the fungus Cephalosporium acremonium in the 1950s and has a similar spectrum of activity to that of penicillin against gram-positive bacteria but is active against more gram-negative bacteria than penicillin. Another important structural difference is that cephalosporin C possesses two R groups, compared with just one R group for penicillin, and this provides for greater diversity in chemical alterations and development of semisynthetic cephalosporins. Cephalosporins The family of semisynthetic cephalosporins is much larger than the Penicillins These drugs have been classified into generations based primarily on their spectrum of activity, increasing in spectrum from the narrow-spectrum, first-generation cephalosporins to the broad- spectrum, fourth-generation cephalosporins. A new fifth-generation cephalosporin has been developed that is active against methicillin-resistant Staphylococcus aureus (MRSA). Cephalosporins Used to treat pneumonia, tonsillitis, staph infections, bronchitis, otitis media, various types of skin infections, gonorrhoea, urinary tract infections Cephalosporin antibiotics are also commonly used for surgical prophylaxis. Cephalexin can also be used to treat bone infections. The first generation Cephalosporins Have quite similar spectrums of activity. They have excellent coverage against most gram-positive pathogens but variable to poor coverage against most gram negative pathogens. The first generation includes: cephalothin cefazolin cephapirin cephradine cephalexin cefadroxil The second generation Cephalosporins Have expanded gram negative spectrum in addition to the gram positive spectrum of the first generation cephalosporins. Cefoxitin and cefotetan have good activity against Bacteroides fragilis. Enough variation exists between the second generation cephalosporins in regard to their spectrums of activity against most species of gram negative bacteria, that susceptibility testing is generally required to determine sensitivity. The second generation includes: cefaclor cefamandole cefonicid ceforanide cefuroxime The third generation Cephalosporins Have much expanded gram negative activity. However, some members of this group have decreased activity against gram -positive organisms. They have the advantage of convenient dosage regimen, but they are expensive. The third generation includes: cefcapene cefdaloxime cefditoren cefetamet cefixime ceftazidime cefodizime cefoperazone cefotaxime cefpimizole cefpodoxime ceftibuten ceftriaxone The fourth generation cephalosporins Extended-spectrum agents with similar activity against gram- positive organisms as first-generation cephalosporins. They also have a greater resistance to beta-lactamases. Many fourth generation cephalosporins can cross blood brain barrier and are effective in meningitis. The fourth generation includes: cefclidine cefepime cefluprenam cefozopran cefpirome cefquinome Fifth Generation Cephalosporin Ceftaroline; active against MRSA Retains the activity of older generation cephalosporins having broad spectrum activity against gram negative bacteria  The carbapenems and monobactams also have a β-lactam ring as part of their core structure They inhibit the transpeptidase activity of penicillin-binding proteins. The only monobactam used clinically is aztreonam. It is a narrow-spectrum antibacterial with activity only against gram-negative bacteria. In contrast, the carbapenem family includes a variety of semisynthetic drugs (imipenem, meropenem, and doripenem) that provide very broad-spectrum activity against gram-positive and gram-negative bacterial pathogens. β-Lactamase Inhibitors Bind to β-lactamases and inactivate them Commercially available inhibitors include clavulanic acid, sulbactam and tazobactam They have little direct antimicrobial activity however when combined with an antibiotic extend the antibiotics spectrum of activity and increase stability against β-lactamases Glycopeptides-Vancomycin Discovered in the 1950s as a natural antibiotic from the actinomycete Amycolatopsis orientalis. Similar to the β-lactams, vancomycin inhibits cell wall biosynthesis and is bactericidal. However, in contrast to the β-lactams, the structure of vancomycin does not directly inactivate penicillin-binding proteins. Rather, vancomycin is a very large, complex molecule that binds to the end of the peptide chain of cell wall precursors, creating a structural blockage that prevents the cell wall subunits from being incorporated into the growing (NAM-NAG) backbone of the peptidoglycan structure Vancomycin is bactericidal against gram-positive bacterial pathogens, but it is not active against gram-negative bacteria because of its inability to penetrate the protective outer membrane. 
INHIBITORS OF PROTEIN SYNTHESIS (TRANSLATION) Proteins are made up of individual units of amino acids Instruction for making proteins are found in the DNA STEP 1 DNA instructions are transcribed into RNA which is a section of DNA Transcription is a way of taking information from DNA and making RNA, The RNA made is called mRNA (messenger RNA) Why is the DNA sending the mRNA and not going by itself?  DNA cannot leave the nucleus  Too much information is contained in the DNA  STEP 2 The mRNA then leaves the nucleus and travels to the ribosome in the cytoplasm. This mRNA carries all the codons which will be translated into proteins Each codon produces one amino acid STEP 3 The ribosome “reads” the mRNA in 3 letter sequences called codons A U G C C A G A C C U U A G U Codon STEP 4 Ribosome will match a tRNA molecule to one codon at a time tRNA (amino acid carrier) has a 3 letter sequence called an anticodon; each tRNA has an anticodon that corresponds to the amino acid it carries  STEP 5 Addition of amino acids continues until the entire mRNA is translated Protein synthesis requires the input of mRNA template, ribosomes, tRNAs and various enzymatic factors Prokaryotes have 70s ribosomes, the small subunit is 30s and the large subunit is 50s SITE AT WHICH ANTIBIOTICS ACT 30s ribosomal unit  Aminoglycosides  Tetracyclines 50s ribosomal unit  Macrolides  Lincosamides  Chloramphenicol  Oxazolidinones Protein Synthesis Inhibitors That Bind the 30s Subunit  AMINOGLYCOSIDES Consist of at least two amino sugars linked by glycosidic bonds to an aminocyclitol ring Prevent proof reading that happens during the protein synthesis. Large, highly polar bactericidal drugs that bind to the 30S subunit of bacterial ribosomes, impairing the proofreading ability of the ribosomal complex. This impairment causes mismatches between codons and anticodons, resulting in the production of proteins with incorrect amino acids and shortened proteins that insert into the cytoplasmic membrane. Disruption of the cytoplasmic membrane by the faulty proteins kills the bacterial cells. The aminoglycosides, which include drugs such as streptomycin, gentamicin, neomycin, and kanamycin, are potent broad-spectrum antibacterials. TETRACYCLINES In contrast to aminoglycosides, these drugs are bacteriostatic Inhibit protein synthesis by binding to the 30s ribosomal subunit thereby preventing the binding of aminoacyl tRNA to the mRNA ribosome complex at the acceptor site Naturally occurring tetracyclines produced by various strains of Streptomyces were first discovered in the 1940s, and several semisynthetic tetracyclines, including doxycycline and tigecycline have also been produced. Although the tetracyclines are broad spectrum in their coverage of bacterial pathogens, side effects that can limit their use include phototoxicity, permanent discoloration of developing teeth, and liver toxicity with high doses or in patients with kidney impairment. 
Protein Synthesis Inhibitors That Bind the 50s Subunit

Macrolides MACROLIDES Macrolide antibacterial drugs have a large, complex ring structure and are part of a larger class of naturally produced secondary metabolites called polyketides Macrolides are broad-spectrum, bacteriostatic drugs that blocks translocation which is the process by which the ribosome moves along the mRNA by one codon after the peptidyl transferase reaction has joined the peptide chain to the aminoacyl tRNA in the A site. This results in release of incomplete polypeptides from the ribosome The first macrolide was erythromycin. It was isolated in 1952 from Streptomyces erythreus and prevents translocation. LINCOSAMIDES Include the naturally produced lincomycin and semisynthetic clindamycin Although structurally distinct from macrolides, lincosamides are similar in their mode of action to the macrolides through binding to the 50S ribosomal subunit and block the elongation of the peptide chain by inhibiting peptidyl transferase Lincosamides are particularly active against streptococcal and staphylococcal infections. Lincosamides are bacteriostatic or bactericidal depending on the concentration Lincomycin has been superceded by clindamycin which exhibits improved antibacterial activity MECHANISM OF ACTION Binds to the 50s ribosomal subunit in the region of the A site The normal binding of the aminocyl-tRNA in the A site is affected in such a way that the peptidyl transferase cannot form a new peptide bond with the growing peptide chain on the tRNA in the P site The altered orientation of this region of the aminoacyl-tRNA in the A site is sufficient to prevent peptide bond formation OXAZOLIDINONES The oxazolidinones, including linezolid, are a new broad-spectrum class of synthetic protein synthesis inhibitors that bind to the 50S ribosomal subunit of both gram-positive and gram-negative bacteria. However, their mechanism of action seems somewhat different from that of the other 50S subunit-binding protein synthesis inhibitors already discussed. They bind to the 50s ribososmal subunit preventing the formation of tertiary N-formylmethionine tRNA(f-methionine-tRNA)-70s ribosomal initaition complex that initiates protein synthesis  INHIBITORS OF MEMBRANE FUNCTION POLYMYXINS The polymyxins are natural polypeptide antibiotics that were first discovered in 1947 as products of Bacillus polymyxa; only polymyxin B and polymyxin E (colistin) have been used clinically. They are lipophilic with detergent-like properties and interact with the lipopolysaccharide component of the outer membrane of gram-negative bacteria, ultimately disrupting both their outer and inner membranes and killing the bacterial cells. Polymyxin E is used in the treatment of serious gram negative bacterial infections particularly those caused by Pseudomonas aeruginosa DAPTOMYCIN The antibacterial daptomycin is a cyclic lipopeptide produced by Streptomyces roseosporus It seems to work like the polymyxins, inserting in the bacterial cell membrane and disrupting it. However, in contrast to polymyxin B and colistin, which target only gram-negative bacteria, daptomycin specifically targets gram-positive bacteria. It is typically administered intravenously and seems to be well tolerated, showing reversible toxicity in skeletal muscles.