Mechanisms of action of antibiotics

Within the framework of a joint post-science project and Peter the Great St. Petersburg Polytechnic University, we publish a text by Candidate of Physical and Mathematical Sciences Andrey Konevega, dedicated to the research of the protein biosynthesis apparatus and the development of new classes of antibiotics.

Antibiotics are small molecules of natural, semi–synthetic or synthetic origin that inhibit the growth of bacteria. The mass production and use of antibiotics began during World War II, in 1943. 

Most of the known antibiotics are produced by fungi or bacteria, which themselves are resistant to the antibiotic and provide themselves with a competitive advantage over other bacteria. The discovery of antibiotics was a revolution in medicine and provided humanity with several decades of relatively safe existence.

Mechanisms of action of antibiotics

Certain types of antibiotics act as inhibitors of the protein biosynthesis apparatus and its central part, the ribosome. The ribosome is a kind of factory, a large molecular machine weighing more than 2.5 megadaltons and with a diameter of about 200 angstroms, on which proteins are collected in the cells of all living organisms. Ribosomes have been studied since the 1950s, but today this field of research is experiencing a new birth. The interest in studying the protein biosynthesis apparatus of prokaryotic (bacterial) cells is due to the fact that bacterial ribosomes are the target for many types of antibiotics that are used in therapy.

Antibiotics bind to the ribosome and inhibit, that is, slow down or prevent individual reactions that are catalyzed by the ribosome. They can compete with the binding site for a natural ligand or block a specific ribosome conformation. Individual structural elements of the ribosome have conformational mobility, which allows it to interact with native substrates and provide a complex process of protein biosynthesis. But individual antibiotics can inhibit certain reactions. Due to this, protein synthesis stops or begins to occur with errors. As a result, the wrong proteins are produced, and this leads to the death of the bacterial cell.

Scientists have managed to make great progress in protein biosynthesis research precisely because a large number of inhibitors are currently known. By inhibiting individual reactions, it is possible to obtain new information about the molecular mechanism of action of ribosomes. On the other hand, it is possible to find out the molecular mechanism of inhibition. For some antibiotics, for example, a molecular mechanism has been proposed, which has been studied in vitro. This mechanism had an inhibitory effect at concentrations several orders of magnitude higher than those actually used in therapy. As a result of experiments, it was possible to detect another reaction, which is inhibited by an antibiotic in low concentrations. If we have many stages, it is clear that the antibiotic acts on one of them. But since it is difficult to check all the reactions, it is difficult to find out exactly where the antibiotic has its destructive effect. Checking all reactions requires considerable time and resources for research. There are several levels at which the effects of antibiotics can be investigated. The easiest way is to grow bacterial cells, add an antibiotic to them and see how these cells died. But with this approach, it remains unknown how this happened.

Protein biosynthesis apparatus

There are a number of approaches to separate the processes that have been affected in the cell. As a rule, these are processes related to DNA replication, transcription or translation. If it turns out that these are processes related to translation, then we can use the arsenal of methods that have already been developed – in vitro methods, when we use purified components of the protein synthesis system, that is, ribosomes, transport RNA, protein elongation factors. In this case, only known components are added to the system, and we know exactly what happens in such a system. And then we can add an inhibitor and analyze the reaction it affects.

The range of methods is quite wide. The recent boom in research in the field of protein biosynthesis occurred in the 2000s, when the first complete spatial structures of ribosomes appeared, which were obtained by X-ray crystallography. For these studies, the Nobel Prize in Chemistry was awarded in 2009. At this point, the researchers were divided into two camps. One group believed that there were no outstanding issues left and it was possible to curtail their research and start doing something else. And another group of scientists believed that everything was just beginning, because the molecular mechanism was unclear. Prior to obtaining spatial structures, functional studies resembled the study of a black box. We took a ribosome, added substrates to it, matrix RNA (information carrier), transport RNA, and at the output we received a polypeptide. What happened in the middle of this process is unclear. With the advent of structural information, scientists for the first time got the opportunity to do experiments in a more targeted way. Now we know where which protein is located, where which nucleotide is. So, we can assume how this or that functional center works, make directed mutagenesis and check how it will affect certain reactions.

Immediately after the appearance of the first spatial structures, structures appeared in which the ribosome was in combination with antibiotics. And then the first understanding was born of where this antibiotic is connected, in which center, what it can affect. Then these biochemical, biophysical studies found a new life. The structural methods that have developed have had a tremendous impact on the study of ribosomes. Later, cryoelectronic microscopy methods appeared, which now also allow obtaining the spatial structure of such large, macromolecular complexes with very high resolution – about 2.5–3 angstroms. Cryoelectronic microscopy methods are gradually replacing crystallography, they have already surpassed it in a number of parameters. Now we can assemble a functional complex, see where the antibiotic is bound, and make assumptions about its molecular mechanism of action. It is important that now obtaining structural information takes days and weeks, and not years, as it used to be. This is, of course, a colossal scientific and technological progress.

Bacterial resistance to antibiotics

According to a World Health Organization report published in 2014, about 23,000 deaths in the United States (and about 25,000 in the European Union) each year are associated with infections caused by bacterial strains that are resistant to antibiotics. Infections cause significant economic damage (direct and indirect), amounting to billions of dollars.

The first antibiotics in the clinic were used in the 40s of the last century. Later, new classes of antibiotics appeared, and several years later bacterial strains resistant to the antibiotic were discovered.

This stability has a number of reasons. Firstly, bacteria can mutate. Mutations occur in ribosomal RNA or in some protein factor and change the properties of this ligand. At the same time, the inhibitor, that is, the antibiotic that binds in this or a neighboring place, simply ceases to have such a significant effect. After that, the mutant strain gains a competitive advantage and begins to multiply.

Bacteria, as evolutionarily ancient organisms, have learned to adapt to difficult living conditions. They have effective methods of developing resistance to antibacterial agents, to antibiotics, so as soon as an antibiotic appears in therapy, after some time we should expect that a resistant strain will appear and these antibiotics will cease to work.

About 9 years have passed since the beginning of the widespread use of tetracycline and before the appearance of the first reliably resistant strains to it. And for methicillin, only 2 years have passed from the start of use to the detection of resistant strains. In recent history, the emergence of resistant strains for some antibiotics has been documented the next year after the start of their use. It is almost impossible to put the final point in this struggle. The only thing that can be done is to pay serious attention to the development of new classes of antibiotics in order to constantly replenish the arsenal and develop new drugs.

Development of new antibiotics

Most of the antibiotics that are currently used in therapy are either natural substances or some derivatives of natural substances. And only a few antibiotics are completely synthetic substances invented by chemists.

There are several approaches to the development of new antibiotics. Basically, new inhibitors are trying to find by screening those substances that produce microorganisms – either bacteria or fungi. Since the probability that such substances are synthesized is high, you can simply sort out those substances that are secreted by bacteria or fungi. Most of the antibiotics known today are found in this way. An alternative method is rational design, that is, using data on the structure of the ribosome, the structure of the active center and trying to come up with a molecule that will interact with one or another functional center and inhibit reactions. In addition, you can combine both approaches.

About the author:
Andrey Konevega – Candidate of Physical and Mathematical Sciences, Senior Researcher at the NIC "Nanobiotechnology" of Peter the Great SPbPU

Portal "Eternal youth" http://vechnayamolodost.ru  26.04.2016