This page provides just a crude overview on antibiotics in general. If you want a bit more detail about structural studies: check the pages about antibiotics targeting the 30Sor50Ssubunit.
Antibiotics derive their name from the greek 'anti' (~against) and 'biotikos' (~life).
Penicillin
The discovery of the first antibiotic was - according to a wide spread tale - based on a mostly on a fortunate coincidence of chance and experiment. In 1928, Sir Alexander Fleming left the lab and went hiking for a long weekend. When he came back, he discovered that some of the cultures he had left, where contaminated with a blue-green mould. An easily reproducible experiment, as I just discovered looking into the fridge (I wasn't really looking for a mould, rather from that cold, fresh yeast product to dilute this dry topic at least virtually).
However, Fleming noticed that the bacteria and the mould were in a sense competing. The bacteria couldn't survive in the presence of the mould. A close look at the cultures revealed that the mould, named penicillinum notatum, lysed the cells. The baterial cell walls were destroyed by some fungal product, which became known as Penicillin. A similar discovery had been made earlier by Ernest Duchesne, who already in 1897 investigated the effect of Penicillinum glaucum on E. coli cultures. He showed, that the antimicrobial activity of the mould was also detectable in typhus infected animals. However, Duchesne enterred the army and died little later on tuberculosis, which prevented an earlier discovery of Penicillin. But even after Flemings discovery, it took another 10 years, before Penicillin became availlable as a therapeutic drug, which could be produced on an industrial scale. The rise of the second world war, allowed Howard Florey and Ernst Chain to proof the effficiacy of Penicillin, to save literally the lifes of 1000s of wounded allied soldiers and to receive - jointly with Sir Alexander Fleming - the Nobelprize in 1945.
Golden ages
The development of penicillin was followed by rapid discovery of other antibiotics, like the sulphonamides, the first broad spectrum antibiotic tetracycline, streptomycins, chloramphenicol and the macrolides, namely erythromycin. Therefore, a wide variety of efficient drugs where available already in the 60s and 70s to fight essentially any bactieral infection. Consequently, William H. Stewart, US Surgeon General, 1969 was urged to declare to the Amercian Congress: „The time has come to close the book on infectious disease.“
Warning by few scientists, that improper usage of antibiotics might lead to wide spread antibiotic resistance, were largely ignored. With fatal consequences ...
Non-therapeutic usage
One is tempted to believe, that antibiotics are mostly used to prefend (human) infectious diseases, but that's not the case. Antibiotics are used for such a variety of different applications, that it literally impossible to mention all of them. However, life stock propably consumes more antibiotics than any other application. Already in the 40s it has been observed that antibiotics increase the weight gained, though the reasons remain largely unknown. However, prophylactic doses of course help to prevent infections, which occur frequently in intensive farming. For the same reason, rivers and lakes are flooded with antibiotics, to increase the yield of aqua cultures like lachs, gamba or shrimp. Estimates of the amount of antibiotics used in agriculture vary a lot, however, most studies assume that 50-80% of all antibiotics produced world wide end up as animal feed or in other non-therapeutic applications. Which could easily be avoided as danish studies recently indicated, since the mass gain is only 4-5% compared to conventional agriculture
Drawback: antibiotic resistances
An important side effect of the non-therapeutic usage of antibiotics is the contamination of the environment with traces of antibiotics, which remain active especially in the soil for quite a while. Consequently, microorganisms are exposed to low levels of antibiotics for prolonged periods, levels which are unsufficient to kill the organisms, but sufficient to pose a selective pressure. If the low concentrations of antibiotics have any long term effect is completely open, but it's certainly something to be concerned about.
However, the strongest selective pressure is certainly present in hospitals. Coomunity aquired infections are therefore particularly dangerous for immuno compromised patients. Unfortunately, hospitals are at the same time heavily affected by outbursts of multiply resistent bacteria. Particularly dangerous are Staphylococcus aureaus bacteria. Typically 70-90% of the strains from this bug are resistant against several of the commonly used antibiotics, most virulent beng those which are resistant against methicillin as well as vancomycin. Vancomycin is frequently considered a last resort - if nothing else helps use vancomycin - if vancomycin doesn't help ... oops ...
It seems clear, that it won't hurt to have of access to new classes of antimicrobial compounds, which are not (yet) compromised by resistance. a precise knowledge of the molecular mechanism of the antibiotics (and the resistances) would certainly facilitate the developement of new drugs ... and that's precisely where structural studies of ribosome-antibiotic complexes start to be important ....
Action
Antibiotics and natural resistence
Antibiotics were originally defined as compounds which are produced my microorganisms to defeat other organisms. So antibiotics are essentially (natural) substances which inhibit growth or ultimately kill other organisms. Almost all antibiotics are still natural or semisynthetically derived compounds. Nature was more ingenoius than mankind, it seems ....
Naturally, organisms which produce such copounds must be immun against the activity of these substances. They are resistant, which means that a resistance mechanism exists for each and any antibiotic. Its just the question, if and how fast a competing organism acquires the resistance. In essence, there are three different modes of resistances:
Inactivation. Bacillus brevis for example produces an inactive metabolite of edeine, a universal antibiotic which killls pretty much all kind of cells. Streptogramins A are for example de-activated by an acetyl-transferase enzyme, which just adds an acetyl to an essential part of the drug, which prohibits binding.
Target modification. If the target of an antibiotic is different in the producing host, such that the drug can't bind to its target, the producing organism becomes resistant. Frequently, bacteria aquire resistance through mutation or methylation of the target site, a common mechanism for macrolide resistances.
Fast export of the drug out of the cell. Most organisms posses transporter proteins, which are designed to export substances out of the cell. The activity of tetracyclines or macrolides for example is frequently compromised by such efflux proteins, which export the antibiotic out of the cell, before it can cause any damage.
Basic action of antibiotics
The antimicrobial mechanism can also be divided into three non-exclusive groups, at least roughly:
Antibiotics blocking a functional centre of an enzyme. Thats the way, most ribosomal antibiotics work.
Antibiotics block through molecular minkry an important site.
Antibiotics induce and fix a specific conformation. Fusidic for example rigifies the elongation factor EF-G, so such at it can't leave the ribosome after translocation. Also thiostrepton is presumed to inhibit a conformational transition inside the ribosome.
Targets
An antibiotic must of course target a crucial element of the life cycle of the attacked organism, otherwise wouldn't do much harm. If this element is conserved among a large number of organisms, even better, the antibiotic will be a broad spectrum drug. Typical targets are ...
the cell wall
RNA- or DNA-polymerases
Peptid-Deformylases
Protein-Biosynthesis
und several more. For example ...
Penicillin, Methicillin or Vancomycin inhibit cell wall synthesis. These compounds block transpeptidases, enzyme which combine or glue the polysaccharides, which are the cell wall biúilding blocks. Without these the cell wall will be destroyed, the cell lyses, which ultimately kills the bug. Penicillins act therefore batericidal.
RNA-Polymerases are responsible for the composition of RNA-strands, for example during the transkription of the DNA into mRNA. Antibiotics like rifampicin bind to RNA-Polymerases and block their activity. This leads not to a desctruction of the cell, but stops their growth. These antibiotics act therefore mostly bacteriostatic.
Protein factory
Proteine-Biosynthesis is one of the major targets of antimicrobial substances. Actually, most of the recently developed antibiotics attack ribosomal protein synthesis. Protein-Biosynthesis is the process, which is responsible for the translation of the genetic code: the ribosome reads the copy of the genetic code contained on the mRNA and builds amino acid chains accordingly (see function for more details). Essentially all active cell components are produced by the ribosome. Inhibition of this process is therefore destructive for any organism.
The ribosome is a frequently named a universal or ubiquitous cell organelle, meaning that its function is vital for all living organisms. It's also universal in the sense, that the functionally important regions of the ribosome, the decoding and the peptidyl-transferase centres are highly conserved, eg structurally very similar. The universality makes the ribosome an equally interesting target for scientific studies and for antibiotics.
Some antibiotics utlize the high degree of conservation directly. Edeine or Sparsomycin for example bind to universally conserved regions of the ribosome, and consequently inhibit protein biosynthesis in all organisms. These compounds are therefore not extremly useful antiinfectives drugs, might still act as anti-tumor drugs, but also allow to study important aspects of ribosomal translation.
Action on the ribosome
The ribosome is always assembled from two different, independent subunits (see structure), which each performs different functions during translation. To form a functional ribosome, the subunits must be associated to form a 70S initiation complex, which requires active help of several cell components (like mRNA, initiator-tRNA, initiation factors). This step of protein synthesis is affected by compounds like Edeine or Pactamycin: edeine fixes a specific conformation of the small ribosomal subunit, such that the interactions with the mRNA and IF3 are hampered. The initiation complex can not be formed. and protein synthesis stops.
After succesful formation of the 70S initiation complex, protein synthesis is a cyclic process, which served to elongate the nascent chain, which requires cooperativity of elongation factors (EF). The elongation factors are resonsible for the positioning of tRNA molecules, and the translocation of tRNA through the ribosome, a process which is fueled by GTP hydrolysis. Antibiotics like Thiostrepton or Microccocin bind to a region of 23S rRNA, which is essential for GTP hydrolysis, and hence inhibit EF-G driven elongation, but also affect binding of initiation factor IF2. In contrast, Kirromycin, Aurodox or Fusidic Acid inhibit a conformational transition required for elongations factors EF-Tu resp. EF-G to leave the ribosome. The ribosomal stalls in a specific conformation with the elongation factor still bound, an excellent tool to obtain and study well defined functional complexes.
Antibiotics like Sparsomycin or Linezolid act in a similar way: they increase the affinity for tRNA molecules on the ribosome, such that the mRNA/tRNA complex can not be translated.
The end of the elongation cycle is signalled by a stop-codon on the mRNA. The ribosome is finally cleared of the nascent chain, mRNA and tRNA and the subunit subsequently separated, with the help of a number of co-factors like release, recycling, elongation and initiation factors. Though pretty much all of the steps are a suitable target for antimicrobial action, so far no antibiotics inhibiting termination or peptide release are known ....
Ribosomal tunnel and macrolides
During ribosomal protein synthesis the nascent proteins are essentially created as linear chains. Since the polypetide chains are not yet properly folded, they are would recognized as defective and are therefore in danger to get digested by preoteases. To protect the nascent protein, the chain is guided through a 100Å (1Å=0.1nm) long and roughly 15Å wide tunnel inside the large ribosomal subunit.
This ribosomal exit tunnel extends from the peptidyl transferase centre to the bottom of the 50S subunit, where different enzymes (chaperones) cooperate with the ribosome to fold the nascent chain into a properly assembled, functional protein.
Biochemichal experiments indicated, that macrolide antibiotics interfere at some point with the progression of the nascent chain, but usually without affecting peptide bond directly. The structure of the complex of the 50S subunit from Deinococcus radiodurans with different macrolides confirmed the biochemical data, and revealed the simple but very effective mechanism, by which macrolides inhibit protein synthesis.
Erythromycin blocks the tunnel, which guides the chain of amino acids through the ribosome. The ribosome can still produce short polypeptide chains in the presence of erythromycin, but synthesis of full length proteins is inhibited.
Erythromycin in red, 5S-rRNA in blue, 23S-rRNA metallic blue and proteins in gold.
Macrolides are composed of a few common building blocks. The always consist of a lactone ring with 14-16 members (though you can find 8 or 20 membered rings as well, they usually don't have any antibiotic activity), a desosamine sugar and different additional moieties (see below). The different dressings determine their bio-availability, acid stability and so on, but also influence the interaction pattern with the ribosome and the sensitivity to different resistance mechanism. Antibiotics like erythromycin also contain a cladinose sugar, but this moiety turned out to be dispensible. Without the desosamine sugar however, the antimicrobial activity approaches zero ....
Ketolides like ABT-773 or telithromycin, rather recently developed semi-synthetic derivatives of erythromycin, compensate for the lacking cladinose sugar by additional moieties, which lead to interactions with regions of 23S rRNA which are otherwise not involved in binding. The additional interactions are mostly involving the rRNA backbone rather than the bases, and are therefore much less sensistive towards mutations.
Azalides like Azithromycin have much better pharmacokinetic properties than most of the other macrolides, which leads to an improved antimicrobial activity. This effect is based on the introduction of an azo-group into the lactone ring, which reduces the hydrophobic characteristic of the ring and presumably improves the activity against gram-positive pathogens.
However, the azo-group hardly contributes to the binding of azithromycin, just the fact that it's a 15-membered rather than a 14-membered ring affects the interactions slightly (in addition to secondary binding site, but that's a different site).
Macrolides and resistences: rRNA mutations
The interactions of the macrolides with the ribosome involve just a few nucleotides. Each of these interactions is therefore important for the effiacy of the antibiotics. Many resistances against macrolides are therefore based on modifications of the rRNA bases involved in interactions. Actually, we found an almost 1-1 correspondence between the nucleotides involved in interactions with macrolides and resistance mutations. One prominent example is the mutation or methylation of a single base (A2058 in E.coli), which leads not only to resistance against macrolides, but also against linsosamides and streptogramins B (therefore named MLSB resistance), which are all sharing interactions with this nucleotide.
The methylation of A2058 interrupts some of the interactions, and the addition of the methyl group doesn't leave enough space for erythromycin to bind, eg inhibits binding by steric hindrance. Mutations like A2058G, A2059G or C2610U act pretty much the same way.
Interestingly, A2058 is conserved in almost all eubacterial organisms, but differs in eukaryotes, archea (like Haloarcula marismortui) and mitochondria. They usually posess a Guanine instead of the Adenine. Which has a nice side effect: humans are resistant against macrolides (even if they would manage to enter eukaryotic cells).
Macrolides and resistances: Protein mutations
Despite the fact, that essentially all ribosomal antibiotics bind to rRNA rather than proteins, a number of mutations in ribosomal proteins have been found, which give rise antibiotic resistance. This has been observed for example for macrolides, chloramphenicol, pleuromutiline and several more.
The direct way to determine the precise mechanism of these resistance would be to solve the structure of accordingly mutated ribosomes, but this has not been achieved yet. The structures of the wild type ribosomes can still give a clúe, though not a proof how the mutations act. For example mutations in L4 or L22 give rise to macrolide resistance, though macrolides do not diretly interact with these proteins. However, both proteins bind directly to rRNA strands, which are involved in binding. So, alterations of the proteins probably lead to slight changes in the rRNA structure, which will affect the macrolide binding. Since these proteins are also involved in early assembly of the 50S subunit, it might well be, that the mutation introduce long range alterations, but thats just a speculation.
Electron microscopy reconstructions of ribosomes with mutated L22 indicate, that these kind of effects could be important. The results indicate that the loop of L22 obtains a rather different orientation in the mutant ribosome, such that the entrance to the ribosomal tunnel gets significantly larger, which could also mean that macrolides are still able to bind, but that the tunnel is now wide enough to allow the passage of the nascent chain even in the presence of the macrolides, again, thats speculative.
Resistance based on mutations of ribosomal proteins is generally not extremly effective. Otherwise, these kind of resistances would occur much more frequently, since ribosomal proteins are usually encoded by a single copy in the genome, whereas rRNA has - at least in many cases - multiple copies. To acquire resistance through rRNA mutations is hence much more difficult to achieve (all the copies need to be mutated simultaneously), but is still much more frequent resistance mechanism ....
RNA-World ??
Evolution has obviously favoured antibiotics, which are not so easy to defeat, so that the producing organism really gains an advantage over competing organisms. The production of the antibiotics is usually a rather costly business, even for bacteria or fungi. Most of the (natural and semi-synthetic) ribosomal antibiotics therefore bind exclusively to rRNA and not to ribosomal proteins.
There is of course another reason, why ribosomal antibiotics target mainly rRNA: most of the functional centres of the ribosomes - especially the decoding centre and the peptidyl transferase centre (PTC), the two most important sites on the ribosome - are composed exclusiverly of RNA. This observation lead to the notion, that the ribosome is a ribozyme, and supports the old hypothesis that ancient life was made of RNA.
The decoding site and the PTC are also a favorable target for antibiotics, because the sites are so crucial for protein synthesis and because the variations from organism to organism are very small (ribosomes function the same way in all organisms). Chloramphenicol, Clindamycin oder Puromycin are just three examples of antibiotics targeting the PTC. All three compounds bind essentially to the same region within the PTC, through a kind of molecular mimikry: they behave as tRNA bound amino acids and are therefore incorporated in a similar way. Which means that they leave no space for the true amino acids, so that peptide bond formation becomes impossible.
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