Introduction to Antibiotics

Professor Syed Ziaur Rahman

Department of Pharmacology

Jawaharlal Nehru Medical College, AMU, Aligarh

Email: rahmansz@yahoo.com

Content in this presentation

• Definition and concept of AB activity

• Possible mechanism of AB actions

• Classification of AB on the basis of MoA and Spectrum

Definitions

• Antibiotics are the substances produced by microorganisms, having the property of inhibiting the growth of or destroying other microorganisms in high dilution (very low concentrations) - Waksman.

• Antibiotics with Antibacterial, Antifungal and Antitumor actions

Evolution of Chemotherapy

• Pre Ehrlich Era before 1891 • The period of Paul Ehrlich • Post Ehrlich Era after 1935

• Some historical Terms during Pre Paul Ehrlich Era

• Works of Pasteur, Joubart, Babes

• Some historical Terms during Paul Ehrlich Era • Heptaphore, Toxophore • Magic Bullet – Methylene Blue, Arsephenamine (Salvarsan),

• Some historical Terms after 1935 • Pontosil (converted to Sulfonilamide • Sulfonilamide (Gelmo in 1908) • Penicillin (Fleming, Chain & Florey)

Medical Museum of Ibn Sina Academy, Aligarh

Salvarsan in a vial

Mechanism of Action of ABs

• To understand the mechanism of action of antibiotics, one should know the basic difference between

• Prokaryotic and Eukaryotic cells • Gram+ and Gram- Bacteria

Difference between Prokaryotic and Eukaryotic

AB acts at 30S & 50S ribosome.

ABs doesn’t acts at 40S & 60S ribosome. Adapted Picture Courtesy

Difference between Gram- and Gram+ Bacteria

Picture Courtesy

Bacterial cell wall

• Peptidoglycan (murein) a polymer consisting of sugars and amino acids that forms mesh-like layer outside plasma membrane forming the cell wall.

• Sugar component consists of alternating residues of β-(1,4) linked N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM).

• Attached to N-acetylmuramic acid is a peptide chain of three to five amino acids (Pentapeptide).

• Peptide chain can be cross-linked to peptide chain of another strand forming 3D mesh-like layer.

• Peptidoglycan serves a structural role in the bacterial cell wall, giving structural strength, as well as counteracting the osmotic pressure of the cytoplasm.

• Peptidoglycan is also involved in binary fission during bacterial cell reproduction.

Biosynthesis of Bacterial Wall

• Cell wall synthesis starts by conversion of UDP-N acetyl glucosamine (UDP-G) to UDP-N-acetylmuramic acid (UDP-M) in the presence of enzyme enolpyruvate transferase

• UDP-M acquires the pentapeptide • Alanine racemase and Alanine-alanine ligase helps in the formation of pentapeptide unit.

• UDP is then removed from UDP-M pentapeptide by bactoprenol membrane lipid carrier and N acetyl glucosamine is added to it which is carried by UDP-G

• These all reactions occur in cytoplasm. The resulting molecule formed is transported across plasma membrane by bactoprenol.

• Elongation of the peptidoglycan channel occurs with the help of enzyme transglycosylase.

• Strength to the peptidoglycan chain is provided by cross linking of elongation chains with the help of transpeptidase.

Inhibition of Biosynthesis of Cell Wall

• Enolpyruvate transferase is inhibited by Fosfomycin. • Transpeptidase is inhibited by β-lactam antibiotics

• Penicillin, Cephalosporins, Cephamycins, Monobactams, Carbapenems.

• Dephosphorylation of bactoprenol is inhibited by Bacitracin.

• Alanine racemase and alanine ligase is inhibited by Cycloserine.

• Transglycosylase is inhibited by Vancomycin.

•Arabinogalactan synthesis inhibitors:

•Ethambutol inhibits arabinogalactan synthesis and thus incorporation of mycolic acid in the cell wall of mycobacteria.

Inhibiting the synthesis of Cell Wall of Mycobacterium

Drugs inhibiting translation

(Inhibition of Ribosomal Protein Synthesis)

Protein synthesis in Bacteria

Accomplished with the use of 70S ribosome, mRNA and tRNA

Steps taken place in protein synthesis

• 70S ribosome consists of two subunits 30S and 50S. • m-RNA transcribed by DNA becomes attached to 30S. • 50 S then binds to 30S to form 70S.

• 50S subunit contains two sites A site (acceptor) and P site (peptidyl).

• 70S unit moves along m-RNA so that successive codons of m-RNA pass from A-site to P-site. This stage is called “Initiation Complex”.

• Codons means triplet of 3 nucleotides carrying the codes for a specific Aa needed for protein synthesis

• t-RNA with its existing nascent Aa chain (A1-A3) is already attached at P-site of the complex by complementary codon : anticodon pairing.

• The incoming t-RNA carries another anticodon Aa residue (A4) to be added to the growing peptide chain.

• Peptide bond forms between the peptide chain and newly attached Aa with the help of enzyme peptidyl transferase.

• The incoming aminoacyl t-RNA with a new Aa (A4) binds to the A-site by complementary base pairing and added to the growing peptide chain.

• The peptide chain (A1-A3) on the t-RNA attached to the p-site is then transferred to the t-RNA linked to A-site. This process is called transpeptidation. \

• The t-RNA at the P-site has lost its peptide and transferred it to aminoacyl t-RNA of the A-site which now consists of 4 Aa (A1-A4) peptide chain

• This t-RNA which has lost its peptide chain is then ejected out from the P-site, while the t-RNA at the A-site (with 4 Aa) is translocated to the P-site. This process is called translocation.

• The free A-site is now ready to receive a new t-RNA, with new Aa (A5) and relevant anticodon attached to it. This whole process is repeated.

• All of these steps keep on repeating till there is a termination codon on the mRNA. At this point protein synthesis stops.

• Usually several ribosomes ie polysomes simultaneously translate on a single m-RNA template.

Steps taken place in protein synthesis

Aminoglycosides

•Binds to 30S subunits. •Freezing of initiation complex

•Interference with polysomes formation •Accumulation of Non-functional ribosomes (monosomes)

•Misreading of mRNA code

Tetracycline and Glycylcyclines

Inhibit 30S ribosomes to prevent attachment of aminoacyl-tRNA attachment to A site of 50S.

Chloramphenicol

•Binds to 50S ribosome •Prevents binding of aminoacyl t-RNA to A-site of 50S

•Inhibits peptidyl transferase that results in inhibition of peptide formation

•Inhibits transfer of peptide chain from P to A site (Transpeptidation).

Macrolides, Lincosamides & Streptogramins

•Binds reversibly to the P-site of 50S ribosomes • Inhibits “Translocation” of growing peptide chain from A-site to P-site.

Linezolid

Binds to 50S ribosome near the interface with 30S and thus inhibits “initiation complex”.

Streptogramins and Aminoglycosides

All drugs inhibiting protein synthesis are bacteriostatic except Streptogramins and Aminoglycosides.

Aminoglycosides and Tetracycliens

•Act at 30S ribosome •Remember AT 30 years

Streptogramins, Erythromycin, Lincosamide, Linezolid, Chloramphenicol

•Act at 50S ribosome. •S – Streptogramins •E – Erythromycin •L – Lincosamide •L – Linezolid •Coffee – Chloramphenicol

•Sell Coffee at 50 rupees.

Drugs affecting cell membrane

Drugs affecting cell membrane

•Act by disruption of cell membrane osmotic integrity by displacing Ca2+ and Mg2+ from membrane lipid phosphate

•Leakage of intacellular ions and molecules from the cells.

Drugs affecting cell membrane

• Polypeptide antibiotics: polymixin B, colistin and tyrothricin. • Bacitractin is also a polypeptide but acts by inhibiting cell wall

synthesis. • Polyene antibiotics: Amphotericin B, Nystatin, Hamycin,

Natamycin • Bind to ergosterol present in fungal cell membrane and alter

permeability by forming pores through which K+, Na+, H+, Mg2+ and other macromolecules leak out

• Azoles (Ketoconazole, Itraconazole, Voriconazole, Fluconazole) • Inhibit ergosterol biosynthesis

Suppression of DNA Synthesis

• Nucleic acid synthesis can be inhibited by five different mechanisms:

1. By inhibiting the synthesis of folates, purines and pyruvate

2. By altering the base pairing properties of the template

3. By inhibiting either DNA or RNA polymerase

4. By inhibiting DNA gyrase

5. By directly damaging DNA and its functioning

1. Inhibiting the synthesis of Folates

• Drugs affecting intermediary metabolism. • Important metabolic step amenable to inhibition by the

drugs is FA synthesis. • Drugs inhibiting the FA synthesis are folic acid synthase

(dihydropteroate synthase) results in formation of FA by incorporation of PABA.

• Sulfonamides, Paraaminosalicyclic acid (PAS) and Dapsone are structural analogues of PABA. • These act as competitive inhibitors of FA synthase.

•Dihydrofolate reductase (DHFRase) inhibitors: • DHFRase is enzyme responsible for conversion of

DHFA to THFA (active form required for transfer for one carbon units).

•Drugs inhibiting the enzyme are Trimethoprim, Pyrimethamine, Proguanil and Methotrexate.

1. Inhibiting the synthesis of Folates

•PABA + glutamate + pteridine – DHFA – THFA – DNA

•Cotrimoxazole causes sequential blockage •Bacteriocidal

1. Inhibiting the synthesis of Folates

Chemotherpy Drugs

• FA Analogues: Methotrexate, Pemetrexed, Pralatrexate

• Pyrimidine Analogues: Cytarabine, 5 FU and Floxuridine, Capecitabine, Gemcitabine

• Purine Analogues: 6-MP, Thioguanine (6-TG), Fludarabine, Cladribine, Pentostatin

1. Inhibiting synthesis of Folates, Purines and Pyruvate

2. By altering the base pairing properties of the template

• Topically applied antiseptics such as Acrifavine • These agents intercalate (get inserted) in the DNA to inhibit its

synthesis

• Acrifavine by intercalating doubles the distance between adjacent base pairs • Causes deletion of a base, or an insertion of an extra base • Causes mispairing between the two bases

3. By inhibiting either DNA or RNA polymerase

•Many anticancer drugs inhibit RNA polymerase • Prevent transcription and consequentially protein synthesis

•Rifampicin inhibits transcription by inhibiting DNA dependent RNA polymerase. • It does not bind to mammalian RNA polymerase

3. By inhibiting either DNA or RNA polymerase

4. By inhibiting DNA Gyrase

•DNA replication occurs on the straight strands of DNA and in this process positive supercoils introduced.

•DNA Gyrase nicks the double stranded DNA, introduces negative supercoils and then reseals the nicked ends. • This prevents excessive positive supercoiling. • In gram negative bacteria this function is carried out by

enzyme Topoisomerase II. • In gram positive bacteria this function is carried out by

enzyme Topoisomerase IV but in slightly different manner.

•Quinolones (Nalidixic Acid and Fluroquinolones) and Novobiocin •Blocks block cutting and resealing activity of DNA gyrase

•Block the decatenating (delinking) action of topoisomerase IV

4. By inhibiting DNA Gyrase

5. By directly damaging DNA and its functioning

•Many anticancer drugs do make covalent bonding with the bases of DNA & prevent replication

•Metronidazole generates reactive 5’-nitro radicals in anaerobic conditions • Results in DNA helix destabilization and strand

breakage.

•Nitrofurantoin also considered to be acting by the destruction of DNA.

Nucleotide/nucleoside analogues:

•Drugs that are structurally similar to nucleosides (nitrogen base + sugar) or nucleotides (nitrogen base + sugar + phosphate) get incorporated into DNA or RNA. • Results in the formation of faulty nucleic acids that may

be non-functional or unstable which degrades easily.

• Idoxuridine, Acyclovir (NRTI), etc are analogues of nucleosides/nucleotides.

AB Spectrum

• Narrow spectrum e.g. ATT, Procain Penicillin G or Cloxacillin – gm + bacteria

• Broad spectrum – gm + bacteria, gm - bacteria, spirochetes, Chlamydia and Rickettsia, etc.

• Extended spectrum – gm + bacteria and gm - bacteria (Amoxycillin and Ampicillin)

Next classes

• In the next class, we will cover • Principles of Dosing & • AB Resistance

If any query, please email or call me!

Thank you…

The notes were prepared by the author after consulting books by HL Sharma/KK Sharma and R.S. Satoskar/Nirmala Rege