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Antimicrobial agents encompass substances that are bactericidal, antiviral, antiprotozoal, antiparastic and antifungal1. Antimicrobials were originally discovered in nature, for instance from plants or fungi, and many have been chemically modified by man to change the 'character' of the chemicals to suit our purposes, or because resistance to them develops. In any case they are substances are used by man to selectively target the differences between man and lower organisms, in an attempt to inhibit a micro-organism's growth, and ward off infection.
Humans differ from viruses, bacteria and fungi at a cellular level. There are aspects unique to all of the above, and by exploiting these it is possible to develop substances that can target a specific feature of one of the organisms, and by inhibiting or changing the feature it is possible to kill only the organism required, even when exposing all of the organisms to it.
Prokaryotes and eukaryotes separated from each other in evolutionary terms around 1.50 billion years ago2. Consequently they have evolved down different paths and therefore have different structures and mechanisms present within their cells. Some general differences are described below in Table 1.
Table 1: A table illustrating the general differences between Prokaryotic and Eukaryotic cells.
No nucleus and no membrane bound organelles
True nucleus and membrane bound organelles present
Smaller and less complex 70S ribosome
Larger and more complex 80S ribosome
Structure: Plasma membrane present in addition to a peptidoglycan cell wall, and in some cases a capsid.
Plasma membrane only
Circular naked DNA strand, free in cell. (Genome size is much smaller)
Linear DNA complexed with histones into chromatin, and packaged densely into chromosomes
The process of translation and transcription also differs, using different molecules in different manners; there are also a wide range of metabolic processes present in prokaryotes, many of them foreign to eukaryotic cells and hence potential targets for antibiotics.
Fungi are eukaryotic and so differ from humans less than bacteria and viruses, however there are still differences that can be used in antifungals to specifically target their death.
The archetypal antimicrobial drug is the antibiotic, whereby the term antibiotic is generically used in reference to antibacterial drugs. They target aspects of bacteria foreign to eukaryotic human cells, such as the structure of the bacteria, bacterial protein synthesis, RNA polymerase and DNA polymerase.
There are many types of antibiotics, but the first discovered in 1928 by Alexander Fleming, was a drug called Penicillin, that was named after the fungus Penicillin notatum, from which it found to be produced in2.
Penicillin is the source (and member) of the 'Î²-lactam' class of antibiotics1. All members (aside from penicillin) have been modified chemically at Penicillin's R-group, which modifies its properties slightly and creates a slightly different drug.
Essentially however, the Î²-lactams action against bacteria centres on the differences in cell structure, in this case the presence of a cell wall3.
Almost all bacteria have cell walls in addition to a cytoplasmic membrane (except for instance Mycoplasmas and Chlamydias), unlike human cells whereby the contents of the cell are separated from the exterior by a simple lipid bilayer membrane2. The presence of a cell wall is a difference that is exploited by Î²-lactam antibiotics.
Both Gram-positive and Gram-negative bacteria have a rigid layer of peptidoglycan within their cell walls, a structure that is responsible for maintaining the integrity of bacterial cell walls3. The basic structure of peptidoglycan is chain of repeated glycan tetrapeptides, which is arranged in sheets. The glycan chains are cross-linked by peptides, which form strong glycosidic bonds to hold them together4.
Î²-lactam antibiotics bind to, and acylate, bacterial transpeptidase enzymes, which usually act to cross link the peptidoglycan sheets4. By irreversibly binding to these bacterial enzymes (which are also referred to as penicillin-binding proteins - PBPs) Î²-lactams will inhibit their activity, and prevent PBPs from completing their actions of cross linking residues in peptidoglycan3.Cell wall formation is hence inhibited, the peptidoglycan layer is weakened, damaging the integrity of the cell, resulting in bacterial cell swelling and lysis4.
The affinity of Î²-lactams for bacterial transpeptidases can be illustrated simply, whereby transpeptidases can be observed to covalently bind to affinity columns containing Î²-lactam antibiotics such as ampicillin4.
The Î²-lactam antibiotics hence clearly target the cross-linking of peptidoglycan in the cell walls of bacteria, a feature not present in human( or eukaryotes cells), and because of this specificity Î²-lactam antibiotics are relatively non-toxic to humans and yet have bactericidal properties4.
As mentioned in the introduction above, human cells are eukaryotic cells and bacterial cells are prokaryotic. Due to this, human cells have ribosomes of a different type to bacterial cells. Human cells have an 80S [i] ribosome (made up from a large subunit of 60S and a small subunit of 40S) and bacterial cells have a 70S ribosome (with large and small subunits being 50S and 30S respectively)2.
Chemical Footprinting studies5 have indicated a single high affinity binding site for tetracyclines in the 30S ribosomal subunit. Chemical footprinting involves modifying ribosomal RNA (rRNA) chemically and using reverse transcriptase (which synthesises a complementary strand, but stops at the exact base modification) to monitor ligand binding sites to the ribosome and its subunits5.
The bacterial 30S small ribosomal subunit contains 16S rRNA and is made up from around 21 proteins5. It is to one of these proteins that tetracyclines bind to6. More accurately they prevent the association of aminoacyl-tRNA to the A-site of the bacterial 30S ribosomal subunit5. The A site, also referred to as the 'acceptor' site, is where the new amino acid-tRNA complex first attaches to form a peptide bonds in elongation of the petide chain6. Photocrosslink experiments7 demonstrated that tetracyclines block the A-site and inhibit tRNA binding, meaning that protein synthesis cannot occur, and the bacterial cell will die as it cannot perform many of the tasks required for cellular maintenance and metabolism7.
Interestingly, mitochondria should also in theory be affected by antibiotics targeting the bacterial 70S ribosome. Mitochondria are present in human cells due to an ancient endosymbiotic evolutionary event8. Gene sequence data supports the theory that, in this event a bacterium survived endocytosis into the cell, and the energy provided from the bacterium from respiration conferred an evolutionary advantage, allowing this type of cell to prosper and flourish8. What remains of this in modern eukaryotic cells is the mitochondria present today. Mitochondria present in human cells thus have a prokaryotic 70S ribosome, so in theory any antibiotic that target bacterial protein synthesis should also affect mitochondrial protein synthesis. However tetracycline is still medically useful as mitochondria are not affected at concentrations used in antibiotic drugs2.
Viruses are very different from other organisms simply due to the fact that they are not considered to be truly alive. Viruses are little more than a strand of RNA or DNA enclosed in a protein shell (capsid). They can only grow and replicate within living cells, and will not replicate when placed in a nutrient medium, unlike other microbial life9.
Viruses are very small and have a very small genome size compared to cellular organisms. In simple retroviral genomes for instance only gag, pol and env genes are present9. Thus, viruses have evolved to 'hijack' enzymes, energy, metabolites, and replication machinery from the cells they infect. Consequently there are multiple steps present in the viral lifecycle that are not only different than in human cells, but can be targeted by antiviral drugs.
Steps to consider as targets in the viral life cycle are9:
Viral attachment to the cell. If this can be prevented then it would be of good use as a viral prophylaxis
Viral penetration into the cell. This may occur via direct fusion with the plasma membrane or through receptor-mediated endocytosis
Viral 'uncoating' to allow replication of genetic material
Viral genomic replication
Synthesis of progeny virions
Viral escape from the cell
These are steps that are targeted not only be antiviral drugs, but also by components of the immune system to prevent and impede infection. A successful antiviral drug should target unique facets of the viral lifecycle, such as specific receptor interactions during viral attachment, viral assembly pathways unique enzyme function for instance: reverse transcriptase, RNA dependent RNA polymerase, viral proteases and viral DNA polymerases10.
Herpes and Acyclovir
Herpesviruses are classified as a Baltimore type I virus, meaning they have a dsDNA genome, which is converted to mRNA and then to protein before it's effects can be manifested9.
Acyclovir is an antiviral 'pro-drug' discovered in 1974, partly developed by Gerturde Elion, that acts on viral DNA polymerase11,12. It is considered a pro-drug as it must be chemically modified within cells before it becomes active.
Acyclovir is a partial guanosine nucleotide, chemically named acycloguanosine. It is selectively converted into acyclo-GMP (guanosine monophosphate) by viral thymidine kinase (such as those present in HSV or VZV) at a rate 3000 times more effective than human cellular thymidine kinases, making it a very virally specific drug11. Acyclo-GMP is subsequently further converted into acyclo-GTP which is a potent inhibitor of viral DNA polymerase and results in chain termination (due to no 3'OH on sugar group) and the specific death of virally infected cells11,12.
So by exploiting the differences between viral and cellular thymidine kinases, acyclovir is able to inhibit viral DNA polymerase, preventing viral replication to reducing viral infection.
Retroviruses and Reverse Transcriptase Inhibitors
Retroviruses are Baltimore type VI class viruses13. They have a diploid ssRNA genome that is converted to dsDNA by reverse transcriptase9 (incidentally discovered by David Baltimore and Howard Temin in 197513) before viral proteins can be produced.
Reverse transcriptase is an enzyme unique to retroviruses13. This distinctive feature of retroviruses is an ideal target for antiviral drugs to impede and to prevent or halt retroviral infection. Anti-retroviral drugs, such as those to treat HIV, target this in a number of ways13.
Nucleoside Reverse Transcriptase Inhibitors (NRTIs) and Non-Nucleoside Reverse Transcriptase Inhibitors(NNRTIs), are antiviral drugs that also inhibit the elongation of the viral nucleic acid chain13. NRTIs, like acyclovir, act as specific chain terminators2, however NNRTIs such as Efavirenz (EFV) are small molecules that bind a hydrophobic "pocket" of HIV-1 RT and denature it, preventing its correct functioning due to the conformational change14. However as HIV has an extremely high mutation rate, with ~ 1 new mutation introduced to every new HIV copy13. Substitutions, point mutations and the formation of 'quasi-species' contribute to the rapid development of resistance. So, although we can target HIV, it is a very difficult virus to treat successfully, and multiple drugs are often used to decrease the chances of virological rebound1,2.
Antifungals are an interesting type of microbial drug, because as fungi and human cells are both eukaryotic, finding unique differentiating cellular processes or structural differences to target is more of a difficult task1. Targeting a facet that is shared between the two would kill both cells, so with antifungal drugs is important to maintain drug selectivity and hence minimise toxicity to human cells1.
Drugs targets are not impossible to find however, a fact exhibited by for instance the Polyene compounds. Polyenes such as Amphotericin B, are natural products of the bacteria Streptomyces nodosus, and exhibit fungicidal action of a wide range of fungal species15. These substances predominantly target ergosterol, a fungal-specific sterol, used to increase fluidity in the fungal phospholipid bilayer15. The phospholipid cell membrane is a component common to both fungal and human cells, and although the sterol present in human cells is cholesterol, polyenes do bind to and target this also, however at a lower rate15. Unsuprisingly, when polyenes are prescribed (for instance against Candida albicans causing oral thrush) side-effects are caused, and caution must be used when prescribing how much, as anaphylaxis can be caused which is potentially fatal. Polyenes are amphipathic molecules, meaning that they are both lipo and hydrophilic, which is a feature thought to be important to its mechanism of action. They bind to ergosterol, causing impairment of the selective permeability of barrier function, resulting in the loss of cations from the cell, causing cell death15.
Most other antifungals also targert ergosterol, either directly or in its production and trafficking to the cell membrane, the echinocandins target glucans present in fungal cell wall, but to date there are not many other targets than that. Similarity between cells is not the primary problem in developing antimicrobials however, the primary predicament would probably be the development of resistance. Evolutionary pressures on the micro-organism will mean that those that mutate or gain genetic information and change may survive, and it is because of this that antimicrobial drugs will always need to be improved, and new drugs will always need to be developed.