Antibiotics and Chemotherapy  

Prepared by :

Microbiology and Immunology Department

Objectives of the Lecture

1. Classification of Antibiotics: 2. Classification according to the spectrum: 3. Classification according to the mode of action:

§  Inhibition of cell wall synthesis. §  Disruption of cell membrane. §  Inhibition of protein synthesis. §  Inhibition of nucleic acid synthesis.

4.   Antifungal drugs: 5.   Antituberculous drugs and its treatment strategy: 6.   Antiviral drugs:

Cell Membrane

The boundary layer between the cell’s cytoplasm and the cell’s external environment (Cytoplasmic membrane, Plasma membrane)

-  40% phospholipid and 60% proteins. -  The phospholipids are arranged as a parallel layer known as the phospholipid bilayer. -  Each phospholipid molecule consists of a polar head formed of a hydrophilic (water soluble) phosphate group with glycerol and a non polar hydrophobic (water insoluble) tail composed of fatty acids.

3. Classification according to the mode of action: II-Disruption of cell membrane

-Bacterial membranes do not contain sterols (different from Fungi and mammalian cell membranes). -Gram negative have an additional lipid bi-layer which acts as a barrier to harmful substances entering the cell (Antibiotics). Membrane active agents act by destroying the integrity of the membrane causing:

- leakage of the cytoplasmic content Or - Impairment of metabolic functions

-Most membrane active agents (antibiotics that attack the plasma membrane) act by this way. -Some agents have poor selectivity (can act on mammalian cell membranes) and consequently cannot be used systemically. They are used as antiseptics or disinfectants e.g: Alcohols, Quaternary ammonium compounds and biguanides.

Characters of Bacterial Membranes

A-Polymyxin B and Polymyxin E (colistin): -They have detergent-like properties, thus they bind tightly to negatively charged phosphate groups on LPS in the outer membrane of Gram-negative bacteria destroying the permeability barrier. B- Polyenes: Amphotericin B and nystatin: -Are antifungal agents, have strong affinity towards sterols, particularly ergosterol, however, and low selectivity to cholesterol (found in membrane of human cells). -The hydrophobic polyene region binds to the hydrophobic sterol ring system within fungal membranes causing leakage of cytoplasmic constituents.

C- Imidazoles, triazoles and Naftidine: -All act by inhibiting the biosynthesis of the sterol components of the fungal membrane causing rapid defects in fungal membrane integrity.

The Ribosome

-Responsible for protein biosynthesis -Present in the cytoplasm - Composed of RNA (rRNA) and proteins. - Bacterial ribosome: 70S composed of: -Small subunit: 30S(16S rRNA)+20 proteins -Large subunit: 50S(23S rRNA+5SrRNA)+30proteins

Eukaryotic (human) ribosome is 80S (60S+40S)

THE RIBOSOME IS A GOOD TARGET FOR ANTIBIOTIC

30S 50S

70S

There are 4 phases/stages of translation in the bacterial ribosome

A) Initiation: The mRNA binds to the 30S ribosomal subunit to form a complex to which first the initiator tRNA and then the 50S ribosomal subunit bind. tRNA carries the initiator amino acid (N- formylmethionine). Initiator tRNA occupies the P-site.

III-Inhibition of protein synthesis

P site (pepetidyl )

A site (Aminoacyl)

b) Elongation: Repeated cycles of aminoacyl tRNA binding, addition of the amino acid to the nascent peptide chain (peptide bond formation) and release of the deacylated tRNA. c) Translocation: Movement of the ribosome along the mRNA to allow the addition of more amino acids (carried on tRNA). d) Termination: Termination of the protein synthesis, release of the newly synthesized peptide and dissociation of the ribosome into its subunits.

1-Aminoglycosides and aminocyclitol antibiotics

They bind to the 30S subunits leading to: -Interfere with the initiation step and assembly of the 70S ribosome -Disturb the shape of the A-site and interfere with the positioning of the anti-codons of the amionacyl tRNA. -This leads to misreading of the mRNA -This results in the formation of toxic , nonfunctional proteins which has a lethal effect. - These antibiotics require the organism to be growing aerobically, consequently, they do not act on anaerobic organisms. - Cannot be taken by mouth, nephrotoxic, neurotoxic “8th” nerve (ototoxicity).

2-Tetracyclines

They bind to the 30S subunits leading to: - They interfere with the binding of the aminoacyl tRNA at the A-site, consequently, blocking the protein synthesis. - Tetracylines are transported inside the cell as a Mg complex by active transport (against conc gradient) achieving 50 fold increase in conc inside than outside. - Mammalian cells do not take up tetracyclines. - Tetracyclines are bacteriostatic than bactericidal, consequently, they should not be taken with β-lactams. - Permanent yellow staining of teeth limits its use for children

3-Chlorarmphenicol, Lincomycin and Clindamycin : They bind to the 50S subunits : They inhibit the peptidyl transferase reaction consequently, they prevent elongation.

4-Macrolides and azalides They bind to the 50S subunits : Macrolides (e.g erythromycin): block translocation -It is a valuable alternative to penicillin in penicillin-hypersensitive patients.

Azalides (e.g azithromycin): have a similar action to the macrolides with improved intracellular penetration.

5- Fusidic acid They bind to the 50S subunits : - Fusidic acid block the elongation factors which supply energy needed during the different stages of translation. - It is particularly good for eradicating staphylococcal infections of bones and joints. Used in combination with cloxacillin or flucloxacillin to prevent development of resistance. - Very popular in creams and ointments for topical use. 6-Oxazolidinones (linezolid)

- A new class of synthetic antibiotics first introduced in 1987. - Inhibit the initiation step (formation of 70S from 30 and 50S). - For serious Gram positive infections like MRSA and VRE in hospital acquired infections. - Methicillin-resistant Staphylococcus aureus and vancomycin- resistant Enterococcus are two of the most important antibiotic-resistant organisms that cause nosocomial infections.

IV-Inhibition of nucleic acid synthesis

1-Quinolones: - Selectively inhibit DNA gyrase (topoisomerase II) ( not found in mammalian cells). (The enzyme responsible for uncoiling the supercoiled DNA, before replication). - Effective against Ps. aeruginoea and used for treatment of urinary tract infections

DNA is supercoiled

2-Nitroimidazoles (metronidazole) and nitrofurans (nitrofurantoin):

-They cause DNA breakage by a direct chemical action rather than by inhibition of a gyrase. -Metronidazole is active only against anaerobic bacteria and protozoa. The nitro group of metronidazole is activated by the low redox potential within cells, this activated metronidazole attacks the DNA.

C=N O

OH

+ - R1

R2

3-Rifampicin:

-It acts upon the σ (sigma) subunit of RNA polymerase (transcription). It inhibits the initiation stage of transcription in which the first

nucleotide is incorporated in the RNA chain.

The most potent antituberculous agent available, it penetrates well into cerebrospinal fluid (CPS).

4-(5-Fluorocytosine):

- Antifungal agent which inhibits DNA synthesis.

-  It is converted inside fungi to 5-flurouracil (by a deaminase) which acts as an inhibitor for thymidylate synthetase which adds methyl group to uracil ring (which gives thymine) thus inhibiting DNA synthesis. - 5-flourocytosine is an important antifungal agent in treatment of life-threatening infections.

V-Folate antagonists (Antimetabolites)

Petridine PABA

DHF

THF

Folic acid

Important cofactor in the synthesis of adenine, guanine, thyamine and cytosine (DNA nucleotides bases).

Dihydropteroic acid

Dihydrofolate synthetase

Petridine synthetase

q Metabolite: Any substance synthesised or modified by a metabolic process, often understood to mean an intermediate or final product in the breakdown from a parent molecule.

q Antimetabolite: a substance bearing a close structural resemblance

to one required for normal physiological functioning, and exerting its effect by interfering with the utilization of the essential metabolite.

Metabolite and Antimetabolite

Folate antagonists 1. Sulphonamides:

-Structural analogues of PABA. -They competitively inhibit the incorporation of PABA into dihydropteroic acid and there is some evidence for their incorporation into false folate analogues which inhibit subsequent metabolism.

2. Dihydrofolate reductase (DHFR) Inhibitors

• Trimethoprim: is a selective inhibitor of bacterial DHFR. The bacterial enzyme is several thousand times more sensitive than the mammalian enzyme. • Pyrimethamine: is a selective inhibitor of plasmodial DHFR. Both are structural analogues of the dihydropteroic acid portion of the DHF substrate. • Proguanil: it is metabolized in the liver to cycloguanil, an active selective inhibitor of plasmodial DHFR.

Cotrimazole: sulphamethoxazole+trimethoprim Fansidar: sulphadoxine + pyrimethamine

• Methotrexate: is a potent DHFR inhibitor, but it has no selectivity towards microbial DHFR and cannot therefore be used to treat infections; however, it is widely used as an anticancer agent.

• Trimetrexate: A derivative of methotrexate which is used for treatment of Pneumocystis carinii infections in AIDS patients.

Sulphonamides types and combinations 1-  Rapidly absorbed and rapidly excreted e.g. Sulfadiazine and Sulfaisoxazole. These are still commonly used for treating acute urinary tract infections. 2-  Rapidly absorbed , slowly excreted e.g. Sulfamethoxazole. It is used in combination with trimethoprim (co-

trimoxazole) to prevent emergence of resistant strains 3-Non-absorbable e.g. Succinyl- and phthalylsulfathiazole. These have been extensively used together with antibiotics, for pre­operative suppression of bowel flora, may be used for bowel infections. 4-  Sulfonamides for special uses e.g. Sulfacetamide It is still widely used in the form of eye drops and eye ointment.

4. Antifungal agents 1- Topical: a- Polyenes: Nystatin and Natamycin are used for candidiasis. b-Imidazoles: Clotrimazole, Miconazole and Econazole are useful for tinea and candidiasis. 2- Systemic: a-Amphotericin B: used For invasive yeast. b-Flucytosine: used for invasive yeasts. c-ketoconazole (orally) and Miconazole (intravenously) for histoplasmosis, chronic and invasive candidiasis and dermatophytes. d-Griseofulvin: Accumulates in skin inhibiting dermatophytes. Used for tinea of the scalp and nails.

5. Antituberculous drugs 1- First-line drugs: Are used for in the beginning of the treatment, all are given orally. a-Rifampicin b-Isoniazid c-Ethambutol 2- Secondary drugs: Are less frequently used because of their side effects. a-Streptomycin: b-Pyrazinamide: c-Thiacetazone

TB treatment strategy Standard therapy for active TB consists of a six-month regimen:

-Two months with Rifater (Isoniazid, rifampin and pyrazinamide). -Four months with Rifamate (Isoniazide and rifampin) and

ethambutol (Myambutol) or Streptomycin.

-Treatment takes that long because the disease organisms grow very slowly and, unfortunately, also die very slowly.

- Multiple drugs are used to reduce the likelihood of resistant organisms emerging.

1-Non selective antiviral agents: Examples a-Amantadine: It inhibits viral uncoating. It is used for prophylaxis against infection with influenza A virus. b-Iodoxuridine: It inhibits nucleic acid synthesis. It is restricted to topical treatment of herpes-infected eyes. 2-Selective antiviral agents: Examples a-  Phosphonoacetic acid: It inhibits DNA synthesis of herpes viruses. b- Interferons: -Low molecular weight proteins, produced by virus-infected cells. - Act indirectly by inducing the infected cell to form a second protein that inhibits the transcription of viral mRNA.

6. Antiviral drugs