08/01/2018 WEEK WEEK WEEK 27 27 27 Overview – Slide Set 3 The Bacterial Cell ( Prokaryotic ) SULPHONAMIDES OSPAP Programme • Agents targeting the cytoplasm TRIMETHOPRIM – [Sulfonamides] NITROFURANTOIN – Trimethoprim – Nitrofurantoin • Agents targeting the ribosomes MPHM14 Antibacterial – Chloramphenicol – Linezolid Chemotherapy – Tetracyclines – Macrolides – Aminoglycosides • Agents targeting the nuclear material – Rifamycins – Fluoroquinolones • Updates and new developments • Clinical applications http://whyfiles.org/126dna_forensic/images/dna.gif Slide 1 of 75 OSPAPMPHM14 Antineoplastic Chemotherapy Slide 2 of 75 OSPAPMPHM14 Antineoplastic Chemotherapy Slide 3 of 75 OSPAPMPHM14 Antineoplastic Chemotherapy WEEK WEEK WEEK 27 27 27 Agents acting in the cytoplasm Sulfonamide Antibacterials Folic Acid Synthesis • Many common biochemical processes in cytoplasm of all cells • Sulfonamides were the first synthetic antibacterials used clinically • Rely on differences between prokaryotic and eukaryotic cells for • Bacteriostatic action selectivity • – Agents targeting metabolic processes only found in bacteria provide Sulfonamides are structural mimetics of para -aminobenzoic acid selective antibacterials (PABA, essential metabolite) and compete for a key enzyme in the PABA • Bacteria synthesise several vitamins, e.g. folic acid, not synthesized synthesis of folic acid in mammalian cells • Folic acid is an essential metabolite for mammals (cannot be – These processes are good targets for antibacterial agents synthesised by mammalian cells), so this interference in folic acid • The sulfonamides are antifolates (like methotrexate) and interfere synthesis is the basis of the selectivity of the sulfonamides with the bacterial biosynthesis of folic acid • Sulfonamides are extremely rarely used now due to extensive • Trimethoprim is also an antifolate, targeting a different part of the resistance in both G+ve and G-ve bacteria Folic acid Dihydropteroic acid folic acid biosynthetic pathway – Alterations to the enzyme target, dihydropteroate synthase (DHPS) Sulfonamides resemble PABA and • Nitrofurantoin acts on several processes in the cytoplasm, some – Mutations to gene for DHPS resulting in poor sulfonamide binding similar to mammalian processes, but… are mistakenly used instead, • Only sulfamethoxazole (as Co-trimoxazole) and sulfadiazine in BNF preventing synthesis of folic acid – Is prodrug and only activated efficiently in bacterial cells Slide 4 of 75 OSPAPMPHM14 Antineoplastic Chemotherapy Slide 5 of 75 OSPAPMPHM14 Antineoplastic Chemotherapy Slide 6 of 75 OSPAPMPHM14 Antineoplastic Chemotherapy 1 08/01/2018 WEEK WEEK WEEK 27 27 27 NH2 Co-trimoxazole (Septrin) UTIs OMe Trimethoprim N • Although sulfonamides rarely used now, co-trimoxazole is sometimes H2N N OMe still useful for urinary tract infections OMe – Susceptibility should be established 1 st [culture / MS methods] • Trimethoprim is an antifolate agent through dihydrofolate reductase • Also useful for: (DHFR) inhibition; bacteriostatic – Pneumonia due to Pneumocystis jirovecii (fungus, was called Pneumocystis carinii ) – • Used for UTIs (good renal excretion as unchanged drug), acute and Although reduction of DHF to THF also occurs Toxoplasmosis and nocardiasis in mammalian cells, trimethoprim has about • chronic bronchitis (well distributed), pneumocystis pneumonia 40,000x greater affinity for the bacterial Co-trimoxazole associated with rare, serious ADRs, especially in elderly enzyme than the mammalian version – hence patients • Can be used as monotherapy or in combination (synergistic) with the selectivity – Was replaced by amoxicillin or cephalexin, but less commonly used now due to sulfamethoxazole (Co-trimoxazole) in ratio 5 : 1 resistance (amoxicillin) and increased risk of C. diff infection after use (cephalexin) – Similar pharmacokinetics gives best combination • Nitrofurantoin more commonly used now • Co-trimoxazole acts on two enzymes in the same biosynthetic – (Relatively) broad spectrum, bactericidal agent, particularly against E. coli , most sequence (sequential blocking ) Enterococci , Klebsiella sp., Staphylococci and Streptococci • Doses of both drugs are lower than would be required if either used (But poor activity vs Proteus sp. and inactive vs Pseudomonas sp.) alone so side-effects (and possibly resistance) can be minimised – Rapid renal elimination after oral admin: low serum, high urinary concentration Slide 7 of 75 OSPAPMPHM14 Antineoplastic Chemotherapy Slide 8 of 75 OSPAPMPHM14 Antineoplastic Chemotherapy Slide 9 of 75 OSPAPMPHM14 Antineoplastic Chemotherapy WEEK WEEK WEEK 27 27 The Bacterial Cell ( Prokaryotic ) 27 Agents targeting protein synthesis via ribosome Nitrofurantoin MoA • Activated by several bacterial enzymes to reactive nitrosyl radical • Reacts with cytoplasmic proteins and other macromolecules • Inhibits: protein synthesis, aerobic (carbohydrate) energy metabolism, DNA synthesis, RNA synthesis, and cell wall synthesis • Resistance rare, even although introduced into clinic in 1952; resistance may be due to non-specific activity – resistance would require concurrent changes to many enzymes, proteins and pathways = unlikely • Although broad spectrum, rapid and efficient absorption from GI tract reduces risk to GI bacteria (low risk of C. diff resulting) Nitrosyl radical CHLORAMPHENICOL TETRACYCLINES MACROLIDES AMINOGLYCOSIDES LINEZOLID http://whyfiles.org/126dna_forensic/images/dna.gif Slide 10 of 75 OSPAPMPHM14 Antineoplastic Chemotherapy Slide 11 of 75 OSPAPMPHM14 Antineoplastic Chemotherapy Slide 12 of 75 OSPAPMPHM14 Antineoplastic Chemotherapy 2 08/01/2018 WEEK WEEK WEEK 27 27 27 Transcription and translation Transcription and translation - revision Chloramphenicol (Chloromycetin, Cm) TRANSCRIPTION Transcription and • Genetic code on DNA template transcribed into messenger RNA by RNA polymerase • Originally obtained from Streptomyces venezuelae , translation describe the • In bacteria, mRNA moves to ribosomes [ cf in eukaryotes, mRNA exits nucleus into cytoplasm] now prepared synthetically • processes by which the TRANSLATION Bacteriostatic with broad spectrum of activity; only R,R -isomer is active genetic code on DNA is • Ribosomes have two subunits: 30S and 50S in bacteria [70S] (40S and 60S in eukaryotic cells [80S]) • Highly lipophilic and penetrates most tissues converted into proteins • mRNA binds to small subunit of ribosome, followed by initiator: tRNA-methionine (crosses blood-brain barrier) In bacteria, these Replication • Large ribosome subunit binds to small subunit. Large ribosome subunit has two binding sites, P • Active against Neisseria meningitidis , Streptococcus processes all take place in Translation and A in the peptidyl transferase centre (PTC) pneumoniae and Haemophilus influenzae (causes of meningitis) the cytoplasm, making an • DNA Transfer RNA (tRNA) – amino acid Transfer RNAs carry amino acids to the ribosome site where mRNA binds (charged tRNA) • Can be used in treatment of meningitis in patients with β-lactam allergies and easy target for • Only tRNA with codon of complementary sequence to that on mRNA can bind to ribosome drug of choice against typhoid fever antibacterial agents Transcription Codon = 3 nucleotides (triplet), which codes for a specific amino acid • But…. severe toxicity possible, so only given systemically in life-threatening Selectivity is possible, as • Ribosome moves along mRNA from 5′ to 3’: once peptide bond formed, non-acylated tRNA leaves infections: benefit must outweigh risks Messenger RNA (mRNA) RIBOSOME (rRNA) bacterial ribosomes are P site and peptide-tRNA moves from the A to the P site • Side-effects after systemic admin include aplastic anaemia (bone marrow cannot different to mammalian A new charged tRNA-amino acid (as specified by the mRNA codon) enters A site replenish blood cells) – unpredictable and may occur weeks after treatment ribosomes • Peptide chain grows as amino acids added until stop codon reached, then leaves ribosome through ceases Protein http://highered.mcgraw- protein exit tunnel • Commonly used topically in treatment of bacterial conjunctivitis hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120077/micro06.swf::Protein%20Synthesis Slide 13 of 75 OSPAPMPHM14 Antineoplastic Chemotherapy Slide 14 of 75 OSPAPMPHM14 Antineoplastic Chemotherapy Slide 15 of 75 OSPAPMPHM14 Antineoplastic Chemotherapy WEEK WEEK WEEK 27 27 27 Chloramphenicol: MoA & resistance Resistance to chloramphenicol Linezolid (Zyvox ®) • Resistance to chloramphenicol in Pseudomonas aeruginosa is due to efflux pumps (via both chromosomal and plasmid transmission) • First member of oxazolidinone agents • Chloramphenicol binds to large ribosome subunit (50S) at the peptidyl • An RNA methyltransferase (cfr ) methylates a key part of the 23S subunit – Only example currently licensed transferase centre A site, preventing binding of the next charged tRNA near to ‘fenicol’ binding site and blocks the binding • New synthetic class of antibacterials • www.youtube.com/watch?v=0VINqUF-r5I • Resistance also arises due to chloramphenicol acetyltransferases (CAT) • • Discovered in 1990s, approved in 2000 Binding involves 4 H-bonds from chloramphenicol to the ribosome and which acetylate chloramphenicol so that it no longer binds to the PTC A 2+ • Active against G+ve bacteria, such as: coordination to a Mg ion in the catalytic site site. CAT genes are both plasmid (e.g. CatC in S. aureus ) and – MRSA • Selectivity arises due to differences between the conformations