Modern approaches to the development of anticancer drugs

Cancer Drugs
Polit.<url>: lecture by Vladimir Katanaev

We publish a transcript of a lecture by Vladimir Katanaev, Candidate of Biological Sciences, Head of the University of Constance group, Professor of Pharmacology at the University of Lausanne and a leading researcher, head of the Development Genetics group at the Institute of Protein of the Russian Academy of Sciences in Pushchino, on modern approaches to the development of drugs against cancer, delivered at the Polytechnic Museum as part of the project "Public Lectures <url>".

Neither I nor anyone else in the world has a cure that would help against cancer in general. Apparently, as far as the available package of our knowledge can tell us, such a medicine is impossible in principle, because each case of cancer is a separate disease. And even if we are talking about cancer of a certain tissue, for example, breast cancer, then in each case, unfortunately, this disease is unique and individual. We can develop a remedy that will work against certain groups of cancer cases, but it is impossible to find a remedy that would affect all types of cancer. After these introductory remarks, I will proceed to the lecture.

As you know, cancer is a terrible disease. In terms of the number of deaths from diseases on a planetary scale, cancer ranks third after infectious and cardiovascular diseases. In 2007, almost 8 million people died of cancer on our planet. It is clear that governments of different countries and private companies are investing huge amounts of money in the search for methods to combat this disease. For example, in the USA alone, since 1971, when the war on cancer was declared, more than $ 200 billion has been spent on research in this direction, however, unfortunately, this disease is still very difficult to treat. Here you can see the distribution by mortality: which forms of cancer are the most deadly – lung cancer is number one, accounting for 30% of all deaths from cancer, sex-specific forms of cancer are number two: breast cancer in women and prostate cancer in men. Next, you see that colon cancer plays a significant fatal role, and the other forms of cancer are distributed in descending order.

As you know, cancer and the process of cancer formation is a complex multi–stage evolutionary process, when a cell, reborn from normal to cancerous, accumulates multiple changes and various mutations. Many of these mutations trigger cascades (or pathways) of intracellular signal transmission, and I would like to start the discussion in more detail by telling you what intracellular signal transmission pathways are, and why incorrect activation of these pathways can lead to cancer formation.

In general, one more introductory word that I forgot to mention: in my report I will talk about three things that are related to each other. The first is cancer and cancer formation, the second is intracellular signal transmission, and the third is how modern humanity develops drugs, using the example of finding drugs against cancer. Since there are three topics, I will inevitably not be able to reveal any of them in their entirety, but the topics are related to each other, and I will try to give a coherent unity of the three topics, hoping that it will be more or less clear. If you have any questions for understanding, I ask you to interrupt me; I ask you to leave the substantive questions for a part after the end of the report.

So, in order for a normal cell to be reborn into a cancerous one, it is necessary that a number of changes in its genetic apparatus accumulate in it, and some of these changes trigger the activation of certain intracellular signal transmission pathways. What is intracellular signal transmission? Human cells and other organisms, and by and large unicellular organisms are required to communicate with the extracellular environment. They are obliged to receive certain information from the outside in order to be able to respond to it correctly. The cell is surrounded by a membrane, which serves as a certain barrier to the receipt of information from the outside. In order for this barrier to be passable for information, intracellular signal transmission pathways have been developed, which are schematically depicted here. There is a receptor on the cell surface – this is a protein that has extracellular and intracellular sites. Extracellular sites are responsible for binding to an information signal, which, as a rule, is a certain substance.

These substances can be either low-molecular, inorganic, organic, nucleotides, lipids, or rather high-molecular, for example, they can be proteins, nucleic acids, lipoproteins, and so on. After binding of the ligand to the receptor, a certain structural change occurs in the receptor, which makes the intracellular part of the receptor recognizable for certain proteins, which then transmit the signal further. As a result, the signal transmission system can be represented as a certain cascade of reactions that leads to the activation of a certain response program. The response program can be the start of transcription of certain genes, which in turn can lead to the stimulation of cell proliferation, that is, to their active reproduction; to cell differentiation, that is, the transformation of cells of one type into cells of another type, for example, the emergence of neuronal cells from epithelial cells or the emergence of differentiated cells (for example, muscle) from stem cells progenitor cells. At the same time, programs, that is, the ways a cell responds to extracellular information, can be more complex than simply activating the transcription of certain genes. For example, after receiving a certain signal, the cell can begin to migrate in the direction that is set by the source of the origin of the signal. This is done by cells of the immune system that fight bacterial infection cells, and, sensing the source of bacterial infection, they migrate in the direction of increasing the concentration of this signal in order to fight infection. There may be other forms of cell response to the received signal.

During the process of signal transmission inside the cell from its reception by the receptor to the launch of the response program, which occurs, for example, in the nucleus, a certain cascade (or chain) of reactions occurs in the cytoplasm, which can be schematically represented, as shown on the slide, when each of the transmitter proteins can be in two forms – activated and not activated – and when the receptor is activated by the corresponding ligand that came from outside, the receptor catalyzes the transition of the transmitter protein A from the inactive form to the active one, which, in turn, does the same with protein B and so on. This scheme is greatly simplified, because in reality we are dealing with a very complex network of activations and inhibitions of proteins from each other, when, for example, in this network there may be feedbacks – both positive and negative, in this network there may be cross-regulation of various signal transmission paths with each other. Nevertheless, in a simplified form, intracellular signal transmission can be represented exactly this way, and certain key signal transmitters can be identified in it (we will talk about them a little later). In eukaryotic cells, there are a small number of types of intracellular signaling pathways – about 17 of them. Further, this table can be divided into sections that will include those types of intracellular signal transmission that are active at an early stage of development, at a late stage of development, or in an adult organism.

In our lecture today, we are interested in those types of intracellular signal transmission that are mainly active at an early stage of development, because these are the types of signal transmission activation that trigger the development program, that is, when cells actively divide, actively change their cellular identity – this is something that does not happen in an adult organism or almost it does not happen, because in an adult body we do not want to increase our weight, we do not want our liver to suddenly double in size. All this works at the embryonic stages. So, if an adult organism, as a result of mutations coming either from communication with an aggressive external environment, when we receive carcinogens or UV radiation, or when mutations occur that occur during DNA copying, if these mutations lead to incorrect reactivation of these silent signal transmission pathways that were active earlier during embryogenesis, then this is just with a high probability will lead to the transformation of the cell into a cancerous one. Now I will show the "architecture", in a simplified form, of the five signal transmission paths that were presented on the previous slide, in order to give you a feel of what signal transmission is.

Here is the Wnt/Frizzled signal cascade, which we are actively studying in my laboratories. This cascade is triggered by a ligand called Wnt, which is a fairly large lipoglycoprotein. It binds to the Frizzled receptor, which in English is called "shaggy" – this name came from the fact that in drosophila, when a mutation in this gene was described, this mutation led to the fact that the orientation of the hairs and bristles on the body of the fly was disturbed, as if tousled. So, when the cascade is not active, the so-called axin-dependent destructive complex works in the cytoplasm of the cell. It consists of a number of proteins that are listed here. The task of this complex is to phosphorylate beta-catenin, an important protein that performs many different functions in the cell. When cytoplasmic beta-catenin is phosphorylated, this leads to its degradation through the proteasome. Thus, in the absence of a Wnt ligand, the concentration of beta-catenin in the cell is extremely low. When the Wnt ligand comes and binds to its receptors, this axin-dependent complex is restructured, beta-catenin is not phosphorylated, does not degrade, its concentration increases, it can go to the nucleus and start transcription of target genes.

The following example is a TGFbeta-dependent signal cascade. TGFbeta is one of the ligands-representatives of the whole family of protein ligands. They bind to a receptor on the cell surface, which is a dimer. Ligand binding provides a physical approximation of the two dimer components to each other, as a result of which the dimer is activated, activates some proteins in the cytoplasm, called SMAD, and the active form of these proteins also goes to the nucleus and starts transcription. All these five types of signaling cascade, which are active in embryogenesis, have transcription activation in the nucleus at the output, since they are responsible for launching development programs, and the programs are recorded in our genome.

A very well–known receptor is the tyrosine kinase signaling cascade, which in some sense is similar to the previous one, because the receptor is also a dimer. The two components of this dimer approach each other when binding to a ligand, this activates the receptor, its tyrosine kinase activity, a number of proteins are activated, and at the output we also have the launch of transcription of target genes.

And finally, the Notch-dependent signal cascade. The receptor is called Notch, and binding it to a ligand ensures the proteolytic cleavage of this receptor, and thus its intracellular part is released and can go to the nucleus and launch development programs. It is interesting to note here that proteolytic degradation in the membrane occurs under the action of a certain complex of proteins that are also active in the cleavage of another medically significant protein, namely the precursor of Abeta-peptide. It is the incorrect activity of these proteins – presenelin and nicastrin – that leads to the fact that the Abeta precursor is broken down incorrectly, the Abeta peptide accumulates, which, in turn, leads to the development of Alzheimer's disease.

The activity of these signaling cascades underlies cancer formation in various tissues. Accordingly, it can be assumed that if we were able to develop substances that would penetrate into cells and block these incorrectly activated cascades, such substances could become the basis for the treatment of appropriate forms of cancer. The most famous example in this regard is the drug Gleevec, or Glivec in another way, developed and marketed by Novartis.

This drug, also known as Imatinib, affects the receptor-tyrosine kinase signaling cascade, a certain type of it. When I was thinking what example to give you to illustrate the process of drug development, I first wanted to talk about the development of Imatinib, because it is really a famous substance. For example, there are a number of different drugs produced by Novartis. You can see that Imatinib ranks second in the package of medicines sold by Novartis, and in 2009 the total number of sales of this substance worldwide amounted to almost 4 billion US dollars. It is interesting to mention what the total sales volume of this pharmaceutical giant Novartis is: in 2009 it amounted to 28.5 billion US dollars. Since in our country they are now actively saying that it is necessary to develop a big farm, it is interesting to compare the scale – what is a real big farm. For example, on Gazprom's website in 2008, I found that sales amounted to 82 billion US dollars. Novartis' drug sales are comparable in terms of sales. Accordingly, in order to create a big pharma in Russia, you need to create a company that will be comparable in scale to Gazprom – a very difficult task, although probably solvable.

Now I want to illustrate how the development of a drug takes place in the modern world. This process takes many years, it is expensive, and it is not always led from beginning to end by the same players, but nevertheless a variety of pharmaceutical companies follow the same path, which is indicated here. Here we see a number of steps that a certain substance must go through before it becomes a medicine. I also want to mention that every next step on this path is more and more expensive.

So, the first stage of drug development is finding, choosing the appropriate target on which this drug (substance) will act. Such a target may be a specific protein or a specific gene, about which it is reliably known that it is excessively active in a particular disease or form of cancer, and, accordingly, the identification of this factor allows us to proceed to the next stage, namely, to the development of substances that affect this excessively active protein. Very often, such a target is not just a specific protein, but a whole signaling cascade, which we will talk about later.

After the target is selected, the second stage is to find the so–called hits - these are the substances that in your test system, in your test tube, show the ability to inhibit this target protein. This stage of finding hits involves the development of a proper test system, which, as a rule, should allow for high-performance screening of substances. This means that you have 50 thousand-100 thousand – 1 million different chemicals – this is a library of compounds – and you assume that among these substances there are one or two that are able to perform the function you need, namely to inhibit your target protein. In order to find the right straw in this haystack, you must develop a test system that will allow you to screen all these substances in a high-performance way. Suppose you found these hits – one or two substances.

The next step is to optimize such lead-compounds. You have found a substance that in vitro, in laboratory conditions, is really capable of suppressing the activity of a certain protein that is overly active in a certain form of cancer. In order to prove that this substance can really be used as a medicine, you are obliged to test its effect on more complex models. You must make sure that this substance, getting, for example, into the patient's blood, will not kill him non–specifically - or will not decompose at a terrible rate and thus will not be able to perform the role of medicine. At the optimization stage, a chemical modification of the starting substance is carried out with the addition or removal of certain chemical groups, in order to change the starting substance in such a way that it does not lose its original positive properties for you, but also so that you get rid of its negative properties, for example, insufficient stability.

Suppose you have passed this stage and are now ready to proceed to the study of this substance in animal models. Naturally, before giving this substance to a patient, a person, you must make sure that it works on animals (mice, rabbits, dogs). We can't experiment on humans. We have to make sure that this substance at the level of the body will not suddenly be non-specifically toxic. We have to make sure that this substance works in animal models as we expect, that it is able to suppress the development of cancer, for example, in a mouse model, or that it is stable (does not decompose), and so on. Only after this stage is passed, you can proceed to clinical trials.

Clinical trials are divided into three and sometimes four phases. In the first phase, the substance that you have passed through all these levels is given to a small number of patients (or healthy volunteers) in order to clarify the mechanisms of its action and approximate its effectiveness, safety, required dose and side effects. 10-50 people. Very often these are volunteers – people who may be healthy, just willing to have the effect of this substance tested on them, and you want to check at this stage whether this substance is toxic in humans, whether it has any terrible side effects. Or even more often at this phase of clinical trials, the substance is given to patients who, unfortunately, are already terminally ill, that is, there are no normal means and methods at the hospital to save this person, and then the person agrees to use an experimental drug on him. I repeat, at this stage (maybe it will sound a little cynical), the goal is not to save a person, but to make sure that the substance does not kill him – this is the task of the first phase of clinical trials.

In the second phase, when you are convinced that the substance has the expected properties, a larger number of patients are tested, about 100-300, and here some details of how the substance should be given, in what doses, what side effects it has, how to minimize them, and so on are already being worked out. When this stage is passed, we move on to the third phase of clinical trials, when a very large group of patients is being tested – thousands, and here all kinds of side effects are already being investigated in detail, as well as a possible combination of your new drug with existing ones, because very often it turns out that the combination of two drugs has a much more effective effect than any of the these drugs separately. And only after all these stages have been successfully completed, the relevant authorities of different countries – in the USA it is the Food and Drug Administration (FDA), in Russia it is Roszdravnadzor – receive a package of documents from the manufacturer, which details all stages of the study of the substance: from its development to preclinical animal testing, to clinical tests, and all the details regarding its production. There can be no secrets here anymore. If earlier the company could hide exactly how it synthesizes a particular drug, then all these details are revealed here.

The relevant authorities evaluate the entire package of documents and decide whether to register this substance or not. This also sometimes takes a long time, and only after that the substance gets to the market, it can be bought at a pharmacy, or doctors can give it to patients in hospitals.

However, even at this stage, the manufacturing company continues to monitor the effects of this substance. Because very often it turns out that there are side effects that are noticeable for a duration of several years. When you conducted clinical trials for a year, everything was fine. But it may turn out that a certain substance will have a harmful property that will manifest itself 5-10 years after the start of its intake. And it often happens that popular medicines, which have spent a lot of money on the development and which have the right effect in the treatment of a particular disease, leave the market after a certain number of years, because additional details are revealed that indicate that this substance either has insufficient effectiveness or undesirable side effects.

Now I would like to take you through all these stages using the example of a specific substance that was developed by specific people against a specific form of cancer. I mentioned Glivec, a very famous substance that works effectively against the tyrosine kinase cascade. I would also like to tell you about the development of substances that affect the Wnt-dependent signaling cascade, because this is the topic of my laboratories' activities. But I will tell you about substances developed by different companies that affect those forms of cancer formation that depend on the activation of the so-called Hedgehog-signaling cascade. The fact is that if Glivec is already a very developed substance that is sold in huge quantities, and, for example, there is not a single effective substance that would affect Wnt–dependent signaling cascades, then the Hedgehog-dependent signaling cascade is an intermediate situation when the substance is not yet on the market, but has already been shown its effectiveness at different stages up to clinical trials.

So, the Hedgehog-dependent signaling cascade is so called because it is activated by a protein called Hedgehog, which means hedgehog in English. This name came from drosophila, because a mutation in this gene leads to an early defect in embryogenesis, and the embryo looks like a ball with bristles-needles instead of a normally elongated shape and looks like a hedgehog. In drosophila, worm, and human, this signaling cascade is extremely important at different stages of the development of the organism.

Here you can see schematically how a mammal (in this case, a mouse) develops. Here are the time frames in which this cascade is important for the development of different parts of the body. This cascade triggers the very early stages of embryogenesis, when the anterial and posterial poles are designated in the embryo. At later stages of development, this cascade is activated during the development of limbs (arms and legs), during the development of the nervous system, at an even later stage – during the development of certain parts of the nervous system, and so on. So, for the development of the body, this signaling pathway is extremely important.

The "architecture" of this signaling path is schematically presented here. The key components are listed here – there are only four of them. Hedgehog activator is the ligand that "floats" in the intercellular space and binds to certain receptors on the cell surface, and the receptor that binds it is called the Patched protein. It is interesting that in these cascades, which work at the early stages of development, very often we have a chain of not activating reactions, but inhibitory reactions. Normally, Patched inhibits a protein called Smoothened, and Hedgehog in turn inhibits the activity of the Patched protein. At the output we have the Gli protein, which exists both in the cytoplasm and in the nucleus. It can exist in two forms – activator (activating) and repressor. The transition from the activator to the repressor form is determined by enzymes that cut off a piece of this Gli protein. This proteolytic cutting leads to the fact that this Gli protein becomes a repressor, and it suppresses the synthesis of certain target proteins of the Hedgehog-dependent signaling pathway. When Hedgehog arrives, the activity of Patched is inhibited, Smoothened is activated, and the Gli protein remains only in the activator full-size form, and in this form it passes into the nucleus and triggers the expression of certain target genes.

How can an adult organism have an incorrect activation of this signaling pathway, which can lead to cancer formation? There may be three such ways of incorrectly launching this pathway: in the first case, somatic mutations may occur in genes encoding either the Patched protein or the Smoothened protein. Both mutations lead to the fact that regardless of the presence or absence of the secreted Hedgehog protein, the cascade is activated. The second way to activate this signaling pathway in cancer cells is when, as a result of certain mutations, cells begin to super–produce Hedgehog, that is, it is synthesized by cells in excess quantities, enters the extracellular space and activates cells by the autocrine pathway. And, finally, the third, more complex option is when two different types of cells are involved in activating each other. One of the cells synthesizes certain growth factors as a result of mutations in excess, which activate neighboring cells, and those, in turn, after receiving this signal, begin to produce Hedgehog excessively, and then the signal is superactivated. All three pathways are highly likely to lead to the development of cancer cells.

This slide shows the areas of our body that are sensitive to the somatic activation of the Hedgehog-dependent signaling pathway and whose cells turn into cancer cells with a high probability. First of all, these are skin cells. Almost all cases of such a form of skin cancer as basal cell carcinoma depend on the mutational activation of this signaling cascade. Another very important example of a cancerous tumor triggered by the activation of this signaling cascade is medulloblastoma – this is a fairly rare form of cancer that affects children. This is a form of brain cancer, when the formation of cancer cells is stimulated in the cerebellum – this, of course, is fatal if not diagnosed in time and treatment is not started.

Here is our path of development of a substance from a hit to a drug, and now I will guide you along this path using the example of the development of drugs that suppress the Hedgehog-dependent signaling cascade. The first stage is to identify the target. In our case, the target was identified at the start. The entire Hedgehog-dependent signaling cascade is a target whose activity must be suppressed in order to suppress forms of cancer that are triggered by the incorrect operation of this signaling cascade, that is, stage number one has been passed on the move.

Stage number two. We want to find substances – our hits, which will have the ability to suppress the activity of the Hedgehog-dependent signaling cascade. In order to find these hits from the connection library, it is necessary to develop a high-performance screening system. Schematically, it is listed here. As a rule, it functions in multi-hole dies, consisting, for example, of 384 holes. In each well of this die there are several microliters (10-50) of a certain reaction mixture, which contains cells that are transfected by a special reporter. The reporter is more often a luciferase gene or a green fluorescent protein that stands under the promoter, which is triggered only when the Hedgehog-dependent signaling cascade is superactivated. That is, these cells do not glow until a Hedgehog-dependent signaling cascade has been stimulated in them. When it is stimulated, the cells begin to glow – this is how you measure the degree of activation of the cascade. All these works are carried out not manually, but by a special robotic system that pipettes the necessary substances very efficiently, very accurately and quickly, and then performs the necessary processing of this die (this may be incubation at a certain temperature, it may be rocking for better mixing, it may be filtration of components, washing and, finally, – retake in a special reader, that is, a device that will read your luminosity). All this is carried out by a robot that has hands, which takes a die and moves it back and forth. It is best to have three hands, so that everything is fast, and it would be possible to work with a large number of dies and joints. This robot, in addition to pipetting the necessary components into each of the wells, it also adds your low-molecular substances from the library there. As a rule, this is done in triplicates – the same substance is added to three wells so that the results obtained can be trusted. And the purpose of all this is to find the substance that will block the activation of the Hedgehog–dependent signaling cascade that you started by adding, for example, Hedgehog itself in excess quantities. Screening is performed in this way, and this stage was carried out by various academic laboratories and laboratories of several companies. Several examples are shown here. For example, the very famous Beachy laboratory in the USA, which has done a lot to understand the architecture of the Hedgehog-dependent signal cascade. They screened on a fairly small library of compounds – only 10 thousand, because the library costs money, and an academic laboratory cannot afford to screen a million compounds. But even such screening made it possible to find a number of hits, that is, a number of substances that, at a relatively low concentration, effectively suppress the activation of the Hedgehog-dependent signaling cascade in these cells. Also, a number of companies conducted their screenings with an already larger number of compounds and found quite a large number of hits.

Now we are moving on to the next stage, which was conducted entirely not by an academic laboratory, but by Genentech (California). Genentech has found a hit (its structure is listed here) that is very effective in inhibiting this signaling cascade. IC50 is indicated here – this is the concentration at which the cascade is suppressed by half. You can see that the concentration of 12 pM suppresses the cascade by half, that is, this substance is highly active. However, the following studies have shown that this substance has a number of qualities that make it impossible to use as a medicine, namely, this substance is poorly soluble in water, respectively, if you give it to a patient, then its maximum achievable amount in the blood will be low, in addition, this substance is metabolically unstable. These experiments were carried out on dogs that were given a radioactive labeled substance, and the half-life of this substance in the body plasma was measured. And it turned out that this period is too short, that is, the substance is metabolically unstable. And then the company carried out the so-called SAR (structure-activity based optimization), that is, the optimization of the initial hit by chemical means, which is based on the analysis of its chemical structure, in order to improve its properties. This SAR was conducted in two stages. At the first stage, the goal of optimization was to improve the metabolic stability of this substance (in the dog's body) while maintaining its high efficiency, and here it is shown what happened. This side group was replaced by a number of others, which are presented here in the table, that is, secondary variants of this substance were synthesized, in which one part was preserved and the other was changed. All these substances were tested on the initial test system and on dogs in order to detect metabolic stability. Substance number 15 was chosen, which had high efficiency and at the same time was highly stable. The next step was also a modification to improve the water solubility of the substance, and here this group was modified with such chemical groups as are shown here, and substance number 31 was selected, which had improved water-soluble properties. So, the "lead optimization" stage passed through an intermediate substance 15 to substance 31, which has a high efficiency of inhibiting the Hedgehog-dependent signaling cascade, is well soluble in water and stable in the body. And this substance was then carried through all the subsequent stages.

The next stage, as I told you, is preclinical testing on animal models. So, the company was developing a substance that should kill cancer cells. Before giving this substance to a person, it was necessary to show that on other organisms, on a mouse model, for example, this substance is capable of killing cancer cells. Very often, mouse xenografts are used for this kind of experiments. That is, a special line of mice (they are called nude mice), which has a suppressed immune response, human cancer cells are transplanted into certain areas of the body (often a certain number of human cancer cells are inserted into the tail with a needle). Since this mouse has a suppressed immune response, it cannot fight this transplant by immune means; human cancer cells feel comfortable and naturally multiply, as cancer cells should, and at a certain stage metastasize. And then a mouse that develops human cancer is fed a substance that is then planned to be used on human patients. In the case of the Genentech substance, several types of experiments were conducted when human cancer cells of various types were transplanted into a mouse, and the activity of a number of substances was investigated, for example, how much substance 31, as well as its precursors, are able to suppress the activity of the Hedgehog-dependent signaling cascade in human cancer cells planted in a mouse. And it turned out that substance 31, at a good concentration, suppresses this activity by almost 100%. Even more important studies are shown here when the volume of the tumor that developed in mice after transplantation of human cancer cells was measured. It turned out that if the mouse is not treated with any medicine, then in a week the volume of the tumor grows 5 times, if the mouse is treated with substance 31 in increasing concentrations, then the tumor stops growing and even regresses. Thus, these experiments were successful and allowed us to proceed to the first phase of clinical trials.

As I said, the Hedgehog-dependent signaling cascade is notorious for its ability to trigger such forms of cancer as basal cell carcinoma and medulloblastoma. Two types of first-stage clinical trials were conducted. It shows the tests that were carried out on patients suffering from basal cell carcinoma in the advanced stage, who were practically not helped by other treatments. A group of 33 patients was used, and they were given this substance in different doses, and half of all patients showed a significant improvement in their condition, and most of the second half showed stabilization, that is, there was no further deterioration in their condition. Here you can see photos of cancerous skin tumors of two patients before and after treatment with this substance. You see how terrible the form of cancerous tumors is, you see that the substance obviously improves the situation, although a complete cure has not been achieved in these experiments. Thus, these phase one clinical trials have been very successful. The company also conducted phase one clinical trials on another form of the disease, medulloblastoma. Here the situation is in some sense unique, because one patient was examined (this is completely not standard), who was near death and no treatment helped. Medulloblastoma, as I have already said, affects children and adolescents. This patient was diagnosed very late, at the age of 22. He was diagnosed with medulloblastoma– a tumor of the cerebellum. The tomography shows that this patient had a powerful metastasis throughout the body. He was treated in all possible ways, surgical intervention was impossible, because the form of the disease was too neglected. Both chemotherapy and radiotherapy were performed, other well-known medications were used, but everything was useless, and the patient was in extremely poor condition, exhausted, with a lot of weight loss, constantly suffering from pain, and in the hospital it was decided to try to test the effect of this medicine on him. It turned out that within two months, while this medicine was given to him, he stopped experiencing severe pain in his bones, quickly gained weight, left the hospital, returned to normal life, began going to work, running in the morning, but it lasted only a few months. Here you can see his tomography after a successful phase of treatment. However, after a few months, the disease returned – metastases appeared everywhere again, and after a short time the person died. Further research showed a very important thing, namely, the researchers found out the reason why the disease returned. Cancer cells are special, and each case of cancer formation is an evolutionary experiment, when a cell accumulates a number of mutations that allow it to defeat the body's defense systems. And in this particular patient, cancer cells developed a mutation in the gene encoding the Smoothened protein, which was a direct target of this substance (that is, it has been proven that this substance physically binds to the Smoothened protein and inhibits its activity). So, the cancer cells of this patient developed a mutation that led to the Smoothened protein becoming insensitive to this substance, it stopped binding to it, one amino acid substitution occurred, which made these cells insensitive to the drug, and they multiplied again and killed the person. In this case, a cruel irony is presented, because all previous methods of treating this patient stimulated, in fact, the maximum mutational activity of cancer cells, because the person was treated with chemotherapy and radiotherapy, because this was the only thing available. You know that these methods of exposure significantly increase the mutational ability.

Now this substance is in the second phase of clinical trials against several forms of cancer at once. I was at the report of the head of these studies; he believes that they will be able to bring this substance to the market in about 2 years.

I have completed my report and now I want to go through the main points. I hope that I was able to convey to you the idea that the process of turning a normal cell into a cancerous one requires the accumulation of a number of changes, many of which trigger certain intracellular signal transmission pathways that should not be active in an adult organism. They were active during embryonic development, and in an adult organism, if they start up again, it just leads to cancer with a high probability. And finally, I showed you the drug development process. On average, this process takes about 10 years and costs about a billion US dollars, and it is clear that big pharma is able to carry out this process from beginning to end, however, other players may be involved in this process at different stages, for example, academic laboratories can play at an early stage. I said that each next stage is more and more expensive, and academic laboratories and small startup companies cannot conduct clinical trials because of their extreme high cost. However, they can conduct the substance up to preclinical tests on animal models, and if everything goes well, then these substances are then sold to large pharmaceutical companies that already have enough funds to carry the substance through all subsequent stages. Very often it turns out that pharmaceutical companies buy substances developed by smaller firms. I have read several examples of Russian companies buying up substances brought to this stage. It is also possible to participate jointly at various stages of this process. We are working in our laboratory at an early stage, we are interested in finding substances that in the future can become drugs against forms of cancer that depend on the activation of the Wnt/Frizzled signaling cascade - this is primarily breast cancer and colon cancer. Many other firms or laboratories have advanced further, but no one has yet reached the end in this direction through the Wnt/Frizzled cascade. However, the Hedgehog-dependent signaling cascade has shown greater success in this sense, and it is really hoped that soon a drug that saves people from these forms of cancer (for example, basal cell carcinoma) will appear on the market.

Thank you for your attention, I will be glad to answer your questions.

Discussion of the lecture

Boris Dolgin: About the development of large-scale pharmaceutical production in Russia. You, after all, roughly understanding the situation with us, would rather, as an expert, advise – to do it, not to do it, if to do it, then how? Is the question clear?

Vladimir Katanaev: Yes, the question is clear, I think it is necessary to do, to strive for big pharma to appear in our country, but how to do it is another question, here it is necessary to combine individual activity of private companies and the right state policy. For example, socialism is still in Cuba, and the drug development system there aims not to make a lot of money, but to develop drugs, and this system has proved effective in developing a number of drugs that Cuba is now actively selling around the world, except for the United States, which has imposed an embargo on Cuban drugs. The right combination of public policy and the possible participation and activity of private players and private investors is necessary. This is exactly what is happening now, because, I know, there are several companies in Russia that have set a goal to become a big pharma. Maybe this is a somewhat overstated goal, but you need to strive for it, and at least become average players, not Novartis level, but ten times more modest – it will also be very cool.

Boris Dolgin: The state's assistance at the first stages is understandable. If there are any recommendations from your side, taking into account the American experience in the degree of rigor, the complexity of the last stage?

Vladimir Katanaev: Do you mean clinical trials?

Boris Dolgin: No, registration.

Vladimir Katanaev: I think (although I don't understand the details of the registration process very well) that registration takes place according to more or less similar rules in all countries, they are optimized and work everywhere. Although, it may be that the process of registering medicines in Russia encounters additional difficulties that are characteristic of the Russian bureaucracy, but I do not know about this.

Boris Dolgin: They talk about additional difficulties not in Russia, that in general this is too long, too problematic process in developed countries, and due to this, at some stages they tried to transfer this process to Russia or other countries where this process is easier.

Vladimir Katanaev: The fact is that even if the substance is registered in Russia, this does not mean that it can be sold in the USA. Rather, it definitely means that it cannot be sold in the US until it is registered there.

Boris Dolgin: So you need registration in each of the countries?

Vladimir Katanaev: Large pharmaceutical companies are actively conducting clinical trials in Russia, including Novartis has a very large clinical trials department in Russia. Other firms are also actively playing in this field. This is due to the fact that clinical trials in Russia or other developing countries are cheaper than in Western Europe or the USA. Therefore, many big pharma play on this site.

Boris Dolgin: Accordingly, this is some kind of opportunity for our scientists to earn extra money and conduct their own research.

Vladimir Katanaev: So far, this is an effective opportunity for hospitals, hospitals and doctors, because they, by participating in these clinical trial programs, receive funding from Western firms. So, it cannot be said right off the bat that the activity of Western firms in the field of clinical trials in Russia gives us some special chance to create a big pharma. It can be, but not necessarily. I think it is more correct to develop other stages in our country.

Boris Dolgin: Thank you. Now we have an additional opportunity to ask questions to the lecturer in advance online. I have four questions. Although we indicated Vladimir's regalia, but someone thought that he was a doctor. Why can smoking cause cancer? How many years can you smoke and not think about such consequences for the body? It's not for you, I think.

Vladimir Katanaev: Yes. Smoking is not allowed at all

Boris Dolgin: What is the situation with medicines against the queen of cancerous tumors – melanoma? Next, more medical things, and can you say something about melanoma?

Vladimir Katanaev: By and large, no. Different forms of skin cancer depend to varying degrees on the activation of the Hedgehog-dependent signaling cascade, so what I told you today applies to certain forms of melanoma, but I can't say anything more specifically. I can say about the basal cell carcinoma, which I mentioned: if it is diagnosed at an early stage, it is cured very simply – surgically, and the neglected form requires medical treatment, which is being developed by different companies
Boris Dolgin: The third question is completely medical, and the fourth is mildly medical: I wanted to hear about modern strategies for choosing methods of treatment by an oncologist. Can you say something about this? How do you see from your non-medical place what the doctor is facing now, does he have a special choice?

Vladimir Katanaev: There is certainly a certain choice, but the situation is that the most effective method of treating any form of cancer is surgical, if this cancer is diagnosed at an early stage and if we are not talking about different forms of blood cancer, where there is nothing surgically cut out. Accordingly, if a patient goes to the hospital with a form of cancer that is available for surgical treatment, then the oncologist has no choice – he will go to the surgeon and they will cut out this tumor. Then the standard package of exposure is chemotherapy. The substances that are being put on the market now are in some sense still pioneering therapeutic agents, and I do not know how well oncologists from different countries, maybe in the Russian outback, will be aware of certain drugs, as well as how they can be available in this Russian outback.

Boris Dolgin: Well, outside of the Russian hinterland, do you happen to know what new drugs are in Russia?

Vladimir Katanaev: I am convinced that Imatinib (Glivec) is actively sold in Russia. The fact is that it affects its own forms of cancer, and first of all it is leukemia, and I am convinced that in Russia it is also used in the treatment of these forms of cancer.

Dmitry: Tell me, please, are you aware of the successes of Chinese scientists in this direction, since recently there have been publications that they have made significant progress in a number of drugs in this direction as well? Thanks.

Vladimir Katanaev: What I know about the activities of Chinese researchers is that the scientific justification for the use of Chinese traditional methods of medicine is very popular in China. And this makes some sense, because very often researchers are trying to figure out what chemicals are behind certain natural mixtures that have traditionally been used in Chinese medicine. And sometimes it really turns out that it is possible to identify the active substance and show that it works effectively against certain forms of cancer on model systems. I'm not talking about the patient now, but about working in a test tube. Bringing substances isolated from traditional mixtures to a formal cancer drug, as far as I know, has not happened, but it may happen.

Lev Moskovkin: The issue of registration is one of the key issues in the new law on the circulation of medicines and its heated discussion in the Duma. I am not an expert, but after listening to this discussion, I made the opinion that the Soviet system was simpler and more efficient, and now there is a trade war, just like in all other capitalist countries without exception, and here, in this law, a very strange thing appeared, because Golikova and Khristenko they pushed their way through, they turned out to be corrupt patriots, but not much, it's not that simple there. My question is this. Due to the fact that you presented a brilliant technology, I had great pleasure listening to all this, but the schemes are general, and the question is related to the fact (provocative question) that there are other ways, for example, the study of the causes of immunodeficiency, and not only acquired. Pharmaceutical companies, in my journalistic experience, are not interested in this. They are not interested in people not getting sick. I really liked the way you gave the Cuban example, because it is the key one. Thanks.

Vladimir Katanaev: Thank you for your remark, but I think there is nothing to answer here, because you said things that are understandable. Representatives of large pharmaceutical companies, when they speak at various conferences, often openly say: "You see, friends, we have to make money." That is why a large number of diseases that are not interesting for pharmaceutical companies still do not have medicines, because the modern system (with the exception of the Cuban example) of drug development implies that these drugs should then be sold for such an amount that would pay off the investment in development (and the investment is gigantic). Therefore, pharmaceutical companies, on the one hand, can be understood, and on the other hand, there is a problem of so–called neglected diseases - "abandoned" diseases. And here, both in Western Europe and in the USA, active cooperation of private pharmaceutical companies with the state is beginning to take place, because the state is beginning to play a more active role in the development of drugs against such diseases, which are initially uninteresting for big pharma, because the market is very small. I hope that such cooperation will be worked out, and in the future ways will be found to develop medicines even for low-profit diseases in some sense.

Asya Kazantseva: You said that thousands of different substances are needed in order to create the perfect medicine, but is there any way to model substances on a computer in advance that can be a starting target for sorting? 10-20-100, but not hundreds of thousands of plots. Thanks.

Vladimir Katanaev: The question is very good, and you are absolutely right. This approach has a right to exist, moreover, it is actively used. It has its limitations, because for such computer modeling it is required to know the specific three-dimensional structure of the target protein against which you want to find your medicine. That is why large pharmaceutical companies have their own very large crystallization departments, and very often there is a synergistic approach when high-performance screening is combined with rational design at various stages. For example, the first stage often occurs when libraries are screened and the source hits are found, and the second stage – their optimization – often involves computer modeling, which allows you to predict which groups can be planted on the source substance in order to improve the original good properties and get rid of the original bad properties. Computer modeling is actively used here.

Boris Dolgin: In other words, continuing the idea, if we know more about targets, we will be able to use computer modeling from the very first stage.

Vladimir Katanaev: Yes and no. Another limitation of computer modeling as an approach is that you limit yourself to one protein in advance. If we were talking about the Hedgehog-dependent signaling cascade, using computer modeling, you would say this: "Well, I think that the Gli protein, for example, the fourth of this cascade, is the most important, and the development of substances that will bind to it is the key to finding effective drugs." You crystallized it, solved its three-dimensional structure, put molecular docking specialists behind supercomputers. They found the right substance for you, you synthesized this substance, confirmed its activity. Then you give it to a cell culture or an organism, and it turns out that it is not effective enough, less than you expected. And if you initially use not one protein as a target, but the entire signaling pathway, then your chances that you will find an effectively working substance that you can bring to the medicine increase.

Boris Dolgin: You can do the same thing based on one protein

Vladimir Katanaev: That's right, but very often when you conduct screening using a whole signaling pathway as a target, you find very unexpected things. Namely, you find a substance that effectively suppresses this signaling cascade, and then you begin to investigate what it does act on. And it turns out that it does not act on the first, not on the second, not on the third, not on the fourth of those proteins that you expected, but on a protein that was completely new and unknown in its participation in this signaling cascade. However, the substance actively affects it, and it turns out that this protein is actively playing in this cascade, and you then bring this substance to the medicine. This happens very often. I am not against computer modeling, but it is necessary to combine, and this is done by pharmaceutical companies.

Andrey Letarov, Institute of Microbiology of the Academy of Sciences: Could you clarify a little background regarding the role of early signal transduction pathways in the etiology of various forms of cancer? To characterize this question in one phrase, let's imagine that we have obtained a certain mammal by genetic engineering methods, in which, at the end of embryonic development, all five pathways are irreversibly inactivated. Will it be healthy, happy and completely insured against all forms of cancer?

Vladimir Katanaev: The question is very good, extremely important. I believe that you yourself know that the answer is negative, such an organism will not be happy and, apparently, will not be viable, because these five signaling pathways are active to varying degrees in different parts of the adult organism. For example, the Wnt-dependent signaling cascade is reactivated in pregnant women in the mammary gland. An increase in the number of milk-producing cells, which are then necessary for feeding the baby, occurs when the Wnt-dependent signaling cascade is started again. Here the moment is very subtle, because, on the one hand, this cascade is necessary for the normal functioning of human life, and on the other hand, if it is reactivated or activated not when a woman is pregnant, then she has a high risk of developing cancer, and 50% of all cases of breast cancer are just associated with activation of the Wnt-dependent signal cascade. There are other examples, the same Wnt-dependent signaling cascade must be active in neurons at a low, but not zero level for proper remodeling of axons, dendrites, and synoptic contacts in general. If the Wnt/Frizzled-dependent cascade is completely cut off, this is abnormal, and one of the hypotheses of the mechanism of the destructive effect of Abeta-peptide on the development of Alzheimer's disease is that Abeta-peptide inhibits the Wnt-dependent signaling cascade, which is normally active in a low form in neurons. There are other examples: our immune system is obliged to renew cells, and so on. So the problem here is complicated. These cascades cannot be turned off completely. It should be possible to reactivate them. There is another good example. During tissue regeneration, it is necessary to restart the same signaling cascades that were active during the development of the organism. In various organisms, including mammals, during the regeneration of heart muscle tissues after a heart attack, for example, the activation of the Wnt/Frizzled-dependent signaling cascade occurs, and it is believed that in order to stimulate regeneration, it is necessary to stimulate this cascade, but very carefully so as not to cause cancer formation in other places. Thus, a clear balance is always needed between sufficient activation and insufficient activation or re-activation.

Andrey Letarov, Institute of Microbiology of the Academy of Sciences: Excuse me, but the second part of the question? The first part of the question was, will this mammal be happy, but will it be rid of all forms of cancer, that is, do all forms of cancer depend on it?

Vladimir Katanaev: All forms of cancer depend on certain types of signaling pathways, but I focused on these five, but then there are thirteen more and, in principle, there are forms of cancer that depend on the activation of cascades that go further in the list in the table, so – no, such an animal will not only not be happily, but it will also not be immune from the development of cancer. It will be insured against the development of certain forms of cancer, but there will be those that activate other forms of cancer.

Richard Bunko: I wanted to ask you what is the result of the work? You were talking about exactly three signaling systems. In my opinion, at the very beginning of the lecture, you linked your models of signaling systems specifically with transcription. Am I speaking correctly or not?

Vladimir Katanaev: There is a transcription at the output.

Richard Bunko: And only then do you associate the transcription disorder with the growth of the tumor.

Vladimir Katanaev: That's right

Richard Bunko: For a growth tumor, this is not a result. In your lecture, you pointed out the excellent results of drug sales, but did not link them with cancer mortality. Dear organizers of the lecture, you should not worry about buying expensive medicines. It is not yet known whether there will be a result from them. You are constantly comparing signal paths with the norm. The model was not initially chosen very well. Pay attention to your advice.

Boris Dolgin: What is the essence of your thesis? What do you suggest?

Richard Bunko: A model. Signaling pathways do not correspond to the connection with the development of tumor growth. There's something completely different going on there. This is very important, if you don't want to do it, you don't have to.

Vladimir Katanaev: I would like to briefly answer, as far as I understood the question or thesis. Cancer is a very complex multi–stage process, and other mechanisms that are not mentioned in my report today are inevitably involved in the development of cancer. It is impossible to tell about everything in one hour, and I deliberately did not set such a goal. I was deliberately talking about one mechanism or group of mechanisms that inevitably trigger cancer formation. There was a question: can there be cancer formation without activation of signaling pathways? This cannot be, however, the activation of signaling pathways does not exhaust the entire process of cancer rebirth. There are other mechanisms, for example, angiogenesis plays a huge role, that is, when a cancerous tumor around itself stimulates the formation of blood vessels that will feed this cancerous tumor after it reaches a certain size. A huge number of people are working towards the development of angiogenesis inhibitors – it is also for the fight against cancerous tumors. There is a field for the activities of various scientists here.

Question from the audience: I have three small questions. First, about the Cuban model. I'm curious, I haven't heard anything about her, if you can – a couple of examples. Is it an oncological drug or something else?

Vladimir Katanaev: No, the most famous drug is a monoclonal antibody, not an oncological one. Unfortunately, I don't remember what disease it was developed against. (Nota bene: The most famous achievements of Cuban biotechnology are the meningitis B vaccine and a number of anti–cancer vaccines, for example, against lung, prostate, colon cancer.)

Question from the audience: The second question. Looking back, there are chemotherapeutic drugs that are often used, they are extremely toxic. I wonder if they were being tested now, would they have passed according to the current drug safety criteria, or have the criteria changed now and are already being compared with what they are, and then they were accepted because there was no alternative?

Vladimir Katanaev: I can only express general considerations on this matter. Quite rightly, and Boris also raised this issue, that the rigidity of the requirements for the registration of a new drug increases over the years, and there is an opinion that a number of famous drugs that have been effectively used on the market for years, if they were introduced now, would not have been registered. For drugs used in chemotherapy, it can be assumed that it would be more difficult to withdraw them, it would not be possible to withdraw them at all – I can only speculate. I believe that they would have been able to be removed anyway, they still work, and the fact that a person suffers from them, it is clear that you need to choose between immediate death and prolonging life for several years.

Question from the audience: Thank you very much. And the last question: at your chemical processing, at what stages information is disclosed, even if not about the production, but about the structure itself, that, for example, in order to publish an article, there is already, as a rule, it is necessary to disclose the formula, the structure of the drug, and so on. That is, at this stage we will have to somehow take care of the protection of intellectual property. How is it all coordinated and how is it happening?

Vladimir Katanaev: This issue has been developed and worked out. Before an article with open data is published, a patent application is filed, and only after that it is published. The same is true for any technological developments, whether in biology or in other fields of science. This is a proven process.

Natalia, Institute of Chemical Diversity: I have a few questions, because the topic is close to my professional activity. The first question concerns the choice of a target. You talked about the advantages of choosing a whole signaling cascade over its individual components-proteins. I wanted to ask about safety and correct to what extent such a risky action on fundamental cellular activities, proliferation, affects possible side effects, and, perhaps because of this, what are the difficulties associated with the development of substances specifically against the Wnt signaling pathway.

Vladimir Katanaev: I am convinced that there is a situation where it is much more effective to choose a specific protein as a target. For example, this was done when the drug Glivec (Imatinib) was developed. It acts on a specific modified form of kinase (called BCR-ABL), and there was no need to use a whole signaling cascade as a target, because in the case of leukemia, a specific mutant form of kinase is formed, which is very convenient for use as a target for the development of substances that inhibit it. You have touched upon another point, which is even more important, namely, how safe are substances, drugs that affect certain forms of signaling cascades. This is a matter of colossal importance. I don't know if you paid attention to my penultimate slide, where I showed the state of affairs with the second phase of clinical trials conducted by Genentech. There, for example, three forms of cancer were indicated, against which this substance is currently being tested in the second phase. But there was no mention of medulloblastoma, which almost always occurs due to excessive activation of the Hedgehog-dependent signaling cascade, namely, as a result of a mutation in the Patched gene. And although it was shown in the first phase of clinical trials that the substance is active in the treatment of this disease, but the problem is that medulloblastoma develops in children and adolescents – this is an organism still developing, and it is shown, for example, in mice, that if they block the Hedgehog-dependent signaling cascade at their childhood stage and youth, then this leads to irreparable violations and defects in the further development of the body and its functioning. Therefore, it turns out that this drug against medulloblastoma has no prospects, because if it is given to children, even if the child has cancer suppressed, the side effects of this drug will be so bad that the patient will not be able to continue to exist normally. I don't know how much I answered your question.

Natalia, Institute of Chemical Diversity: Thank you, quite. I have one more question. On which drugs, on low–molecular or high-molecular, the attention of pharmaceutical companies is now increasingly focused, what is the future for - protein molecules or low-molecular substances.

Vladimir Katanaev: I think, both for those and for others. Pharmaceutical companies are actively developing both. If we talk to representatives of the department of low molecular weight substances, they believe that the future belongs to them, on the other hand, in the same company Novartis or other pharmaceutical giants there are large departments where they develop so–called biologics – large molecules, protein first of all, sometimes these are antibodies (monocline or specially genetically modified) that physically block binding a specific ligand with a specific receptor. The future belongs to both, they do not compete with each other.

Natalia, Institute of Chemical Diversity: Thank you very much – and the last short question. From your point of view, at what stage is it most profitable for a company to invest money for drug development if it wants to turn into a big pharma: start from screening or buy ready-made drugs at the first or second stage, or expand sales of substances that will then allow financing the development of new ones? Thanks

Vladimir Katanaev: It depends on which company we are talking about. If we are talking about Chemrara, then it is clear that the base of Chemrara was and is the synthesis of huge libraries of chemical compounds, and it is quite natural for such firms to enter pharmaceutical activity with screening of chemical compounds at a relatively early stage. On the other hand, if we are talking about some investment funds that may not even have a screening kitchen and so on, but they have money that they are ready to invest, they will probably be interested in buying substances that have already passed a number of stages, and then they will find a subcontractor, so that he would conduct clinical trials for them. It depends on which player we are talking about, what his base is, what his goals are. Academic labs can only play at the earliest stage. They can create some innovative test systems, find unexpected, previously unknown targets and then conduct screening, on their own or in cooperation with other companies, and hope that this substance will be picked up by big pharma at the next stage.

M. Sargsyan: I wanted to find out what they spend such a large amount of money on at the second stage, because, of course, patients are not paid. When tested on animals, it is clear that for their purchase. Is it really so much money spent on salaries and the purchase of reagents?

Vladimir Katanaev: Here again, I can only speculate, since I myself have never participated in clinical trials and do not plan to. Patients probably don't get paid, really, but hospitalization costs a lot of money, and very often the patient needs to be conducted from the beginning of the test to its end with all possible detailed studies, analyses, full monitoring of various activities (cardiovascular system; condition, ratio and number of blood cells, and so on) - apparently, all this is very expensive. I don't know how it is in Russia, but in Europe and the USA, the very fact of hospitalization is very expensive if you get, God forbid, to the hospital. I have seen the financial bills that the hospital issues to the insurance company. In this account, the lion's share of the amount is occupied by the very fact of hospitalization. It costs a lot, and why, I do not know. There are, apparently, objective reasons, there is, perhaps, a monopoly collusion in some sense. However, it costs a lot.

Konstantin Lvovich: If you approach this problem in the most general way, then you have revealed only half of the topic, and you have not touched on the other half at all. I would like to ask a question about this. People with cancer are being studied, healthy people are also being studied, in particular, old people who have lived to a hundred years – the wealth of the nation, because they apparently have effective defense mechanisms. Thanks.

Vladimir Katanaev: Yes, I deliberately touched not even half, but even a smaller part of the problem. Regarding your question: yes, of course they are being studied, and here it is not only relevant to research related to the development of cancer and the fight against it, but also to the problem of aging in general. There are pan-European large-scale programs where the largest possible groups of individuals across Europe who demonstrate healthy longevity are tracked, their genetic composition is tracked by certain polymorphisms, genes that are believed to be important in various manifestations of human activity, their relatives and descendants are tracked. I recently listened to the report of the coordinator of a very large European project (they received the maximum possible funding). This project involves laboratories-representatives of almost all European countries, where the goal is, in particular, to track the fate of individuals who are still young, at least not old, but relatives of those individuals about whom it is known that they lived to a ripe old age and were healthier than the standard an elderly man. During this program, it is supposed to monitor a wide variety of metabolic, genetic, biochemical, cellular manifestations of these people. The program coordinators hope that this program will be extended until these people grow old, so that they can track them to the very end and maybe then find out why some people can live to old age and not get cancer.

Vladimir: The question of the diversity of cancer. Let's imagine that you have developed a successful drug, brought it to the market, but, as you yourself said, only 50% of breast cancer will respond to the drug. Do you have any idea how you can divide a group of patients into those who will respond and those who will not, before giving the medicine to them for use? Is it possible to screen patients and purposefully prescribe medication, that is, there may be a general provocative personalization of drug therapy.

Vladimir Katanaev: This is a very important and correct question, and in principle, when applied to other diseases, not only to cancer, the idea of personalizing medicines is gaining more and more popularity, at least expressed in the sense that each patient needs to develop and select a certain dose, that is, if the whole group of patients is given the same if the dose is the same, then the idea is that before you give the medicine, you need to conduct certain studies, analyzes, when this patient needs to be given a dose twice as much as it should be according to the norm, and this one, on the contrary, a dose of two times less is enough. This also applies to a combination of medications. This idea is gaining popularity. I cannot say how widely it is used in everyday medical practice (I think not widely), but it will probably be used more and more.

Olya, Faculty of Biology, Moscow State University: Already during the questions, the topic of the importance of the work of embryonic signals in the cells of an adult normal organism was raised, that is, it turns out that accurate targeted delivery is needed, and a lot of its various variants are being developed now. Which one do you think is the most effective.

Vladimir Katanaev: This is an extremely important issue, which I have completely and deliberately not touched upon in my report. This is a topic of great importance, for the research of which huge efforts are spent by both pharmaceutical companies and laboratories. In order to kill a certain form of cancer, you need to create a certain concentration of the healing substance around the cancer cells. This, apparently, will be better than creating the same concentration throughout the body, where this substance will block the signaling pathway that normally should work (for example, in other cells). The possibility of such targeted delivery will reduce side effects, their probability and strength. Which methods are the most optimal for targeted delivery – in my opinion, there are no optimal ones at the moment, there are developments, but it is obvious that for each form of the disease, the method of targeted delivery will be different, because if we are talking about cancer, then each form of cancer is a unique disease. For example, if we are talking about blood cancer, then, of course, it is necessary that the substance be distributed throughout the blood, but if we are talking about a localized form of another type of cancer, then we can talk about more targeted delivery there. For the skin, it can be ointments. But it is clear that ointments will not work for brain cancer. Here, each form of cancer needs its own optimal form of delivery.

Olga, Lomonosov Moscow State University: In your report, you focused on the creation of a new drug, what do you know about the mechanisms for improving existing cancer drugs, in particular, antitumor antibiotics in our country? This is the first question, and the second is rather philosophical, so I'll ask it right away. What do you think about the prospects for the development of pharmacology for the treatment of cancer? How is it that cancer cells develop mechanisms of multidrug resistance to almost all known drugs, that is, it gradually happens one way or another? How to solve this problem, and is it possible to solve it at all? Thanks.

Vladimir Katanaev: Your two questions are very closely related to each other. I will answer with a concrete example to illustrate how this can be approached, and how this problem is already being approached. I mentioned the drug Glivec, but we can also talk about a substance that affects the Hedgehog-dependent signaling cascade. When patients begin to be treated with a certain substance, it turns out that there is a certain proportion, the proportion of people who do not respond to this substance. Maybe it's because their initial mechanism of cancer formation is different, they got into the wrong 50% if we are talking about the mammary gland, but it often turns out that insensitivity is caused by the fact that cancer cells already have a specific molecular resistance to a specific substance, a specific point replacement in the protein that makes this substance inactive. Moreover, if a patient is treated for a long time, and it is not possible to quickly overcome cancer cells, but only to suppress them, then over time the probability that a mutation in the target protein will appear, making these cells insensitive to cancer drugs, increases. This problem has been addressed by pharmaceutical companies, and they are developing so-called inhibitors of certain cascades of the second generation. Molecular drug design is very actively used here, when the crystal three-dimensional structure of the initial protein of a certain kinase is known, the crystal three-dimensional structure of the mutant kinase is known, which is insensitive to the starting substance. Then, by the method of molecular docking, it is analyzed why the substance no longer acts, and how it can be changed in order for it to act on this altered form. Such activity is being carried out successfully. Pharmaceutical companies are bringing second-generation inhibitors to the level of clinical trials, but while this activity is catching up, that is, patients are treated first, then it turns out that some patients do not respond because their target protein is changed, then everything goes back to the laboratory, a substance is being developed that will be able to inhibit the altered substance, and so on next. Maybe it makes sense to try to simulate in advance by computer what changes may occur in the future in the original target protein that can make this protein insensitive, and then immediately develop a package (panel) of substances that will affect different possible forms of mutant kinases or other target proteins. Whether such a proactive approach is used in the pharmaceutical industry, I do not know, probably not, since they need a financial justification for certain studies. If there is a certain group of patients whose target kinase is modified, then they start looking for drugs against it. They don't do it in advance yet. Maybe they will.

Question from the audience: I have two more related questions. You say that the use of a drug that blocks the Hedgehog pathway leads to the death of cancer cells that do not carry mutations. Why is this happening? There are quite a lot of cells in the body where this pathway is inactive, and they are not going to die for this reason. And a related question. Is it possible to find among the products of genes that activate this pathway, or the products of proteolysis that occur during signal transmission, some molecules that could be used as markers that somewhere, in some place there is hyperactivation of this pathway and, therefore, it is necessary for the development of means, as a last resort – for the diagnosis of cancer?

Vladimir Katanaev: The first question is: why are these cells dying? Because they initially began to multiply due to the fact that their signal cascade was activated, and they changed their essence in such a way that, at least for their reproduction, they require constant activation of this cascade. If the cascade is suppressed, then reproduction is suppressed. It often turns out that cells do not die, in any case, this is not a direct and immediate response of cells to the suppression of the cascade. They stop reproducing, their dying is already a more difficult regulated process, depending on other indicators. On the other hand, for certain types of cancer cells, it has been specifically shown that preventing the work of the signaling cascade in these cells leads to their apoptosis. Why these cells die, and not those that do not activate this cascade, is because they are different, they depend on this cascade. Specifically, the molecular mechanism of this phenomenon will differ from line to line. I can't give you a specific general answer. The second question was about markers. This is a very important topic that is being actively researched. We talked about a breast tumor and that in half of the cases in the development of this tumor, activation of the Wnt-dependent signaling cascade is observed. The methods that show that there was activation in this case, but not in this one, which I know are histological methods. They are not applicable to the convenient diagnosis of the patient's analysis. Such methods should be developed, and I think they are working on it, there are certainly some successful examples when certain forms of cancer are able to secrete a certain substance, by the presence or absence of which we could conclude that this particular signaling cascade is activated in them. I can't think of specific examples like this right now, but I think they are, but in general, probably, the problem has not been solved, because it is possible to effectively determine whether this signaling cascade was active histologically, often post mortem, that is, when tissue analysis and immune staining for certain proteins are performed, which indicate the activation of this cascade. (Nota bene: although tissue biopsy is widespread.)

Boris Dolgin: Is there a task in this direction at all?

Vladimir Katanaev: Of course. I want to tell you about a simple example. In the case of forms of blood cancer (various leukemias), histological analysis is easy to carry out, that is, blood is taken from a person, and his cells are analyzed, and clear confirmation can be made that, for example, the BCR-ABL protein is activated in these white blood cells, this has been done for a long time, but this is a separate case when no deep intervention is required in order to conduct a histological analysis. In other cases, it is much more difficult.

Sergey Golovachev: As far as I understand, all the traditional methods of cancer treatment that currently exist (chemotherapy, radiotherapy, surgical methods) are a fight against the investigation. I got the impression that the model you mentioned is a step forward in this regard, because an attempt is being made to understand why these cells are being reprogrammed. And at the beginning you gave three cases why this happens, and as far as I understand, it is still not clear and has not been studied. On the other hand, you say that each form of cancer is caused by a combination of various factors. Is there a prospect of finding a single cause? What is the position of science, you as a biologist, about some deep cause of the formation of all forms of cancer? Because it's either just a chain of coincidences in each specific case, and trying to identify some kind of law is like predicting a quantum method, it's a chain of events, probabilities, and nothing more. Thanks.

Boris Dolgin: Is it even possible to talk about cancer as a single phenomenon?

Vladimir Katanaev: In this sense, no. Cancer cannot be spoken of as a single disease, and a substance that would cure all forms of cancer does not exist and cannot exist. What we know about life and how the cells of the body work allows us to conclude that there can be no such substance. This does not mean that it is impossible to develop substances that will be active against certain forms of cancer. And here the question arises as follows: does it make sense to develop a substance that will work at best in half of breast cancer cases, knowing that the second half of all examples of the disease will not respond to this drug? And, apparently, the answer is positive: which, of course, is worth it, because if we manage to save at least half of all patients, it's already very cool. For some other forms of cancer, it will turn out that the search for a deterministic cause will allow you to identify an even more modest proportion of certain forms of cancer, for example, maybe your medicine will be active against 10% of cancer of a certain tissue. Does it make sense then to develop such a substance? I think it does anyway, because there will be human lives behind all this, which, perhaps, it will be possible to save. It's not that you shoot an arrow at one target, you have a lot of targets, and the more targets you hit in the course of your activity, the more people will eventually be saved, but there will always be those you can't save.

Alexander: Could you comment on the percentage of successful output of activity at each of the stages (for example, the laboratory examined 100 drugs and the output of 1%), and also how much development costs at each of the stages, and what time frame (for example, the analysis of one hit per year)? Can I clarify?

Boris Dolgin: It feels like you are going to write a business plan.

Vladimir Katanaev: Here, of course, everything varies from case to case. The total average duration of the process is about 10 years. The total average cost is billions of dollars (more than 1, less than 10). The percentage of culling is at least 90%. You find 10 hits – and you will be very lucky if you take at least one of them through the lead optimization stage. The probability that this substance will successfully pass preclinical trials and then clinical trials is low. For example, you are responsible for the national system of drug development, you should be aware that you need to conduct all research as massively as possible, that is, not have 10 hits, but 100, or even better – 1000. And of these, suppose you conduct 10 animal models and one clinical trial – then you have won. If you started with one, then the probability that you will successfully reach the very end is low. This is a discouraging realization for people who are trying to create a hit from their laboratory research and then bring it to a drug. Very often, scientists do not realize that the substance that works perfectly in their hands and on their models has no chance of becoming, for example, a drug against brain cancer, simply because it cannot overcome the blood-brain barrier in any way (well, it has such properties). These things need to be kept in mind when large-scale tests are planned, and, of course, bigpharma is well aware of all this. About the cost. As I said, the cost increases at each subsequent stage, but here everything again depends on the specific situation: it is more expensive to conduct experiments with a thousand mice than to conduct experiments with cell culture in Petri dishes. It also happens that the first stage is expensive. For example, you want to scan a million substances (for example, to buy a million substances from a Chemrar) – it will be very expensive.

Egor, MSU: I also have a related question. You say that it is getting more and more expensive with each stage, but how does the profit vary in comparison with companies that are engaged in development at early stages? This company will benefit less. For how long? What is the proportion of formula, and what is the proportion of clinical trials? How much do they pay for the formula, and how much do they pay for clinical trials conducted by a large company?

Vladimir Katanaev: I can't name specific figures, because I don't know. We can say in general: the profit that startups and small companies can count on when they successfully carry the substance through the early stages up to animal models. The profit they can make by selling their substance to bigpharma, which then goes further, it will be immeasurably less than the possible profit of bigpharma if bigpharma then successfully conducts this substance through the clinic and brings it to market. The difference in profit is at least an order of magnitude, but you have to put up with it.

Boris Dolgin: If you do not resort to the help of a large pharmaceutical company and hope that you will do everything yourself, everything can come down to zero.

Vladimir Katanaev: Yes.

Mikhail, MSU: You cited the phrase BCR-ABL. As far as I remember, such rearrangements, translocations, often occur a second time, when some oncology is treated with chemotherapy or radiotherapy, then secondary leukemias arise, which, as a rule, are already incurable. My question is: do any medications currently work on poorly studied cascades that arise from chromosomal translocations?

Vladimir Katanaev: Naturally, such a canonical, dogmatic example is the drug Glivec, which works against BCR–ABL, as well as against other forms of ABL kinase that arise as a result of chromosomal rearrangements. There are chromosomal rearrangements caused by some viruses, which also leads to the fusion of the viral protein Gag, if I am not mistaken, with ABL, which also leads to the appearance of constitutively active forms of ABL kinase. I do not know if there are other situations in cancer formation that are not related to ABL history, when chromosomal aberrations lead to reliable, from the point of view of cancer, opportunities for further cancer rebirth, so I can't remember right off the bat. In principle, you know perfectly well that chromosomal rearrangements (aberrations) at the late stages of cancer formation occur frequently, have a mutagenic effect and, of course, contribute to the accumulation of additional somatic mutations. Other examples, as in the case of myeloid leukemia, when a specific chromosomal rearrangement leads to the formation of a specific mutant form of protein, which is observed in 100% of cases of this form of cancer, I do not know, probably this is a unique case.

Boris Dolgin: Thank you very much.

Portal "Eternal youth" http://vechnayamolodost.ru26.05.2011