Modes of Action of Antimicrobials, Biology tutorial

Introduction:

Different antimicrobial agents act by interfering with synthesis of biological molecules necessary for life (nucleic acids), folate synthesis, synthesis of microbial cell wall, integrity of plasma membrane and ribosomal function.

Antibacterial Agents:

Inhibitors of cell wall synthesis: Cell wall synthesis inhibitors are bactericidal and cell wall synthesis can be reserved by ß-lactams like Penicillins and Cephalosporins, that inhibit peptidoglycan polymerization and transpeptidation reactionwith concomitant release of C-terminal D-alanine. They act as artificial substrate for D-alanyl-D-alanyltranspeptidases. Cycloserine and Vancomycin binds to precursors of peptidoglycan layer in bacterial cell walls resulting in inhibition of synthesis of peptidoglycan. Cycloserine, resembles alanine and inhibits addition of alanine in peptide chain whereas Vancomycin combines with cell wall substrates, inhibits transglycosidase enzyme and prevents peptidoglycan chain elongation.

Inhibitors of protein synthesis: Different steps in protein synthesis are vulnerable to inhibition by different groups of antibiotics. Inhibitors may inhibit protein synthesis in prokaryotes, eukaryotes or both like aminoglycosides enduringly bind to numerous sites at 30S and 50S subunits and freeze 30S initiation complex (30S-mRNA-tRNA), so initiation can no longer take place. They slow down protein synthesis which was already started and impair translational accuracy leading to misreading of mRNA sequence and/or premature termination of protein synthesis like Streptomycin interferes with formation of 30S initiation complex.

Inhibitors of nucleic acid synthesis and cell division: Rifampicin blocks RNA synthesis and functions by binding to DNA-dependent RNA polymerase thus inhibiting initiation of RNA synthesis while quinolones bind to A subunit of DNA gyrase and inhibits bacterial DNA gyrase (topoisomerase II and IV). It crosses outer membrane of gram negative bacteria via porins and prevents super coiling of DNA. This inhibits DNA synthesis and thus disrupting spatial arrangement of DNA.

Inhibitors of other metabolic processes: The sulphonamides are analogues of para-aminobenzoic acid and competitively inhibit the formation of dihydropteric acid by dihydropteroate synthase during which para-aminobenzoic acid is incorporated into the synthesis of folic acid while Trimethoprim binds to dihydrofolatereductase and inhibits the formation of tetrahydrofolic acid thereby inhibiting the synthesis of folic acid. These are called antimetabolite antimicrobials. Polymyxinsbind with anionic lipopolysaccharide molecules by displacing calcium and magnesium from outer cell membrane of gram negative bacteria. This results in leakage of cell contents and cell lysis.

Antifungal Agents:

Various classes of antifungal agents target plasma membrane, sterol biosynthesis, DNA biosynthesis and β glucan biosynthesis. Antifungals can be categorized in three classes based on mode of action.

Inhibitors of fungal cell membranes: Antifungal activities of Azoles, polyenes, and allylamine/thiocarbamates, can be attributed to skill to inhibit synthesis of ergosterol or their direct interaction with ergosterol. Antifungal agents can inhibit synthesis of ergosterol or form ergosterol binding compounds. They block synthesis of ergosterol by co-catalyzing cytochrome P-450-dependent 14 α -demethylation of lanosterol causing inhibition of C14 α -demethylase. This leads to depletion of ergosterol and accumulation of sterol precursors in membranes of fungi exposed to imidazoles, comprising 14-methylatedsterols, resulting in formation of the plasma membrane with modified structure (holes in cell membrane) and function.

Inhibitors of DNA synthesis: Flucytosine a cytosine analogue inhibits synthesis of macromolecule thymidylate. It can act directly on fungal organisms by aggressively inhibiting uptake of nucleic acids and not directly by intracellular metabolism to 5-fluorouracil. Antifungal activity of 5- Fluorouracil is exerted through its conversion in numerous active metabolites like 5-fluorodeoxyuridine monophosphate (DNA synthesis inhibitor) and fluorouridine triphosphate (RNA synthesis inhibitor), which inhibits protein synthesis by being falsely incorporated into fungal RNA or inhibits thymidylate synthase and interfers with the biosynthesis of fungal DNA. This leads to unbalanced cell growth and lysis.

Inhibitors of fungal cell wall: Echinocandins are cyclic hexapeptides linked to the long chain fatty acid like Caspofungin. They are noncompetitive inhibitors of biosynthesis of 1, 3- β- glucansynthetase and prevent more than 90 % of glucose integration in glucan. This results in disruption of fungal cell wall and subsequent death. Griseofulvin is concentrated by dermatophytes by the energy-dependent process. It interacts with and disrupts mitotic spindle formation in dividing cells by interacting with polymerized microtubules in susceptible fungi.

Antiviral Agents:

Antiviral agents act directly on viruses to prevent them from multiplying therefore able to hinder a virus-specific function or interfere with cellular function so that virus cannot reproduce. They are grouped based on the target sites.

Fusion inhibitors: Agents which inhibit penetration of host cell interfere with receptor-mediated entry of virus in a cell. They obstruct binding, fusion and or penetration of HIV in human cell like Enfuvirtide mimics structure of heptad repeat 2 (HR2) region of 41 kDa glycoprotein (gp41) that binds to heptad repeat 1 (HR1) region and help fusion of viral envelope with the cell membrane. Binding of Enfuvirtide to the HR1 region prevents the HR2 region from access to HR1 resulting in inhibition of the fusion process. Amantadin is a tricyclic amine which inhibits penetration and uncoating, targeting matrix protein and haemagglutinin. It blocks cellular membrane ion channels and is effective against influenza A viruses.

Inhibitors of Nucleic acid synthesis: Nucleoside and non-nucleoside reverse-transcriptase inhibitors are 2 classes of antiretroviral drugs which suppress HIV replication by influencing action of reverse transcriptase. Nucleoside-Analog Reverse Transcriptase Inhibitors (NRTI) inhibit viral RNA-dependent DNA polymerase (reverse transcription) and are included in viral DNA nucleoside chain to prevent ongoing viral DNA synthesis causing chain-termination like Zidovudine. Zidovudine, the analogue of thymidine is phosphorylated by cellular enzymes to triphosphate form.

Protein processing inhibitors: Protease Inhibitors are substrate analogues for HIV aspartyl protease enzyme, engaged in processing of viral proteins. They bind to HIV protease active site and block it from further activity thus preventing the enzyme from cutting viral protein molecules to their correct sizes. This slows down viral maturation process leading to lack of functional virion formation. This protease doesn't occur in humans as it is virus specific protease.

Thymidine kinase substrates: Acyclovir (acycloguanosine) a chain terminator inhibits viral DNA polymerase. It enters cell through plasma membrane in form of the nucleoside and is then specifically phosphorylated inside cell by herpes virus thymidine kinase to the active form. It consequently obstructs DNA synthesis by hindering polymerisation.

DNA integration: Integrase inhibitors target HIV enzyme responsible for integration of viral genetic material in human DNA (integrase). Raltegravira 1-N-alkyl-5-hydroxypyrimidinone inhibits the insertion of HIV-1 viral genomic DNA into the host chromosome. It extends first phase viral decay and alters viral decay kinetics of HIV by significantly reducing the second phase and challenging current hypotheses of viral replication.

RNA synthesis inhibitors: Ribavirin interferes with viral DNA synthesis and can inhibit both DNA and RNA viruses. It introduces numerous mutations in viral RNA making it not capable to infect new cells. Neplanocin A is efficient inhibitor of S-adenosylhomocysteine hydrolase, and may also inhibit capping of mRNA.

Protein modification inhibitors: Castanospermine apotent inhibitor of lysosomal alpha- and beta-glucosidases may disrupt folding of few viral proteins by preventing elimination of terminal glucose residue on N-linked glycans. Neuramidase inhibitors like Zanamivir and Oseltamivir inhibit neuraminidase which is necessary for viral replication and are efficient against influenza A and B virus.

Protein synthesis inhibitors: e.g. Fomivirsen a phosphorothioateoligonucleotide slows down replication of human cytomegalovirus (HCMV) by binding to the equivalent sequence of the mRNA transcribed from the main immediate early transcriptional unit of CMV.

Immunomodulators: Interferons induce in the host cells ribosomes enzymes that inhibit viral mRNA like hepatitis B virus. Pavilizumab is a mononclonal antibody against infusion proteins of respiratory syncytial virus.

Antiparasitic Agents:

Antiparasitic agents are the class of drugs that are utilized for treatment of parasitic diseases like Helminthiasis, amebiasis, malaria. Parasitic infections belong to the class of infectious diseases which have protozoa or helminths as the infectious agents.

Antihelmintic Drugs: Antihelmintics are divided in classes based on the resemblance of chemical structure and method of action e.g. inhibitors of potentiation of inhibitory transmitters, tubulin polymerisation, uncouplers of oxidative phosphorylation, inhibitors of glucose uptake, muscle hyperpolarization, cholinergic agonists and inhibitors of enzymes in glycolytic pathway.

Inhibitors of tubulin polymerization: Mebendazole binds to beta-tubulin, interferes with beta-tubulin dependent glucose uptake stopping microtubule formation and effects glycogen depletion. It blocks the uptake of glucose leading to reduction of helminth's own glycogen with the resultant decrease in adenosine triphosphate formation. It causes degenerative modifications in the outer body covering among members of the phylum Platyhelminthes and intestinal cells of these worms by binding to the colchicine-sensitive site of tubulin, thus inhibiting its polymerisation or assembly in microtubules.

Potentiation of inhibitory transmitters: Macrocyclic lactones are the group of hydrophobic compounds with broad range antinematodal and antiarthropodal properties derived from soil microorganisms belonging to the genus Streptomyces. Inhibitors like ivermectineprinomectin, selamectin. Ivermectin start opening of gated chloride channels. They are potent against several immature nematodes and arthropods by binding to glutamate-gated chloride channel receptors in nematode and arthropod nerve cells. This results in opening of the gated channel leading to the influx of chloride ions with subsequent paralysis of the pharyngeal pumping method, body wall and uterine muscles of helminths.

Inhibition of glucose uptake: Blocking the uptake of glucose, the main source of worms' energy would lead to starvation and final death of the parasite. This may be attained by modulating the cytosolic or mitochondrial enzymes. Praziqunatelan isoquinolone analog increases cell membrane permeability in vulnerable worms and flukes leading to loss of intracellular calcium, paralysis and contractions of the parasite's muscles and consequently expulsion of the parasite. Mebendazole is vermicidal in action and causes degeneration of parasite's cytoplasmic microtubules and thus cautiously and lastingly blocks glucose uptake in vulnerable adult intestine-dwelling helminths and their tissue-dwelling larvae leading to depletion of parasite's store of glycogen.

Cholinesterase Inhibitors: Organophosphates bind to enzyme generally in charge for breaking down acetylcholine after it has passed the message by the synapse. It makes the cholinesterase unavailable for breaking down the acetylcholine thus the neurotransmitter continues to cause neuron to convey electrical charges leading to overstimulation of nervous system causing death. Trichlorfon irreversibly binds to active site of acetylcholine esterase, thus inactivating it. This leads to loss of control by parasite over its nervous system causing paralysis.

Cholinergic agonists or parasympathomimetics: are nicotinic receptor agonists that combine with acetylcholine receptors and reproduce the effects of parasympathetic stimulation by causing spastic muscle paralysis because of persistent activation of excitatory nicotinic acetylcholine receptors on body wall muscle of nematodes e.g. imidazothiazoles and pyrimidines. Antihelmintic activity of imidazothiazoles is attributed mostly to their cholinomimetic activity, whereby they stimulate ganglion-like structures in somatic muscle cells of nematodes. The outcome of this is continued muscular contractions leading to neuromuscular depolarising blockade causing paralysis.

Muscle hyperpolarisation: Drugs such as Piperazine targets nervous synaptic transmission. Piperazine, a pharmacological analogue of the natural inhibitory transmitter obstructs neuromuscular transmission in the parasite with hyperpolarising the nerve membrane leading to flaccid reversible paralysis of body wall muscle. Extent of muscle hyperpolarisation by piperazine relies on concentration of extracellular chloride reducing once the fraction of the chloride ions is replaced by sulphate anions.

Antiprotozoal Agents:

Anti-protozoal drugs are utilized to treat infections or diseases caused by Protozoa. Commonest diseases caused by protozoa are Trichomoniasis, Malaria, Trypanosomiasis or sleeping sickness, Amoebiasis, Toxoplasmosis etc. These drugs demolish protozoa or prevent growth and skill to reproduce. Anti protozoal agent can target either the enzymes found in both host and parasite but are crucial only for the parasite, enzymes found completely in parasites, common biochemical functions found in both parasite and host but with different pharmacological properties or show selective toxicity for parasite as compared to host.

Enzymes found only in parasites:

Enzymes for dihydropteroate synthesis: 6-hydroxymethyl-7,8-dihydropteroate synthase (DHPS) is the necessary protein in lower eukaryotes, prokaryotes and plants utilized in synthesis of folate. Folates are necessary cofactors in production of methionine, glycine, thymidylate, purines etc. for organism.

Plasmodia: produce majority of the folates from starting, and therefore inhibition of dihydropteroate synthase by such drugs leads to reduction of deoxythymidine triphosphate and decreased DNA synthesis. In treatment of human malaria caused by P. falciparum, sulfadoxine has been utilized in combination with pyrimethamine. Pyrimethamine is the antifolate drug which reduces dihydrofolatereductase. Sulfonamide group of compounds like sulfadoxine and dapsone, reduce dihydropteroate synthase which catalyzes the condensation of paraaminobenzoic acid (PABA) with 6-hydroxymethyldihydropterin pyrophosphate to yield 7,8-dihydropteroate.

Pyruvate: ferredoxinoxidoreductase: are present in anaerobic protozoa like Entamoeba spp, Giardia and Trichomonas. This enzyme can decrease nitro group of metronidazole to form cytotoxic reduced products which bind to DNA and proteins. Nitazoxanide and Tenonitrozole are antiprotozoal agent which interfere with action of Pyruvate:ferredoxinoxidoreductase.

Nucleoside phosphotransferase: All protozoan parasites studied thus far are deficient in de novo synthesis of purine nucleotides. Purine nucleoside phosphotransferase found in Leishmania can phosphorylate purine nucleoside analogs like allopurinol riboside, formycin B, 9-deazainosine, and thiopurinolriboside converting them to corresponding nucleotides. These nucleotides are either further converted to triphosphates or finally included in nucleic acids or become inhibitors of other necessary enzymes in purine metabolism. T. vaginalis, T. foetus, and Giardia lamblia appear to be deficient in de novo synthesis of both purines and pyrimidines. Pyrimidine and purine salvage pathway becomes crucial for the parasites.

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