 So now we proceed with the next talker, Professor Nevena Ilyeva from the Institute of Information and Communication Technologies at the Bulgarian Academy of Sciences. She works in the field of solving physical problems in biomolecule interaction modeling. She's interested in protein folding, geometric analysis of MD data of macromolecule binding, interactions in immune-active complexes, antimicrobial peptides and other molecular modeling problems. She's also active in the hyper-proteinase computing field, interested in developing HPC tools and techniques for molecular modeling. Professor Nevena Ilyeva is going to present us a talk titled in silico study of the anti-informatory action of hiperein within the COVID-19 context. So you can, that's our Professor Ilyeva. Thank you. Am I sharing the screen? Is it full screen? I think no. No. Yes, now it should be. It should be okay. Yeah, thank you. Thank you, Elena. First of all, I wanted to congratulate the organizers of this school for the really wonderful event, very well organized and with very versatile and fruitful program, interesting program. And of course, thank you for the possibility to present some recent results of our research, which is inspired, so to say motivated by the pandemic, which we all sit in at the moment, but it's actually relevant also in a more general context. So I'm going to present the results as you see from the title of the talk of some in silico study of the anti-inflammatory action of hiperein within the COVID-19 context. This is a collaborative work, research done in collaboration with my colleague Elena. Lilkova, our colleagues, Leander Litov and Petro Petkov from the Sophie University, Sanktrymentovsky, Miroslav Rangelov from the Institute of Organic Chemistry of the Bulgarian Academy of Sciences and Nadia Taldorov from the Institute of, okay, bio, okay, sorry, the name of the institute, you just had it in the previous talk. So, our interest in this topic was, of course, associated with the pandemic, which started well already a year ago, and it was actually the problem of how to manage to cope with the very bad caused by this very nice virus. You see, we have already heard some details about the SARS-CoV-2 virus, but the important point is that with its 29 viral proteins, it messes the work of over 300, well, 332 host cell proteins and is able to very, very rapidly reproduce once suited in the host cell in 10 hours, only about 10, about 1000 virions are burst into the host organism. Of course, in order, sorry, this went the other way, why I don't know, yeah, okay. So in order to manage to cope with the disease caused by this virus, the COVID-19 disease, it is necessary to understand the way how it hijacks the host immune system and how it manages to mislead the host immune reaction, so that it has some free time to spread in the organism before the reaction. Actually, the phases of the COVID-19 disease and the respective immune response shown here on this table indicate the point and the source of the trouble. In principle, the prolonged incubation period of the virus of the disease, SARS-CoV-2 disease, suggests that the virus has managed to develop countermeasures against the immune system. In the non-severe stages of the disease, the specific innate immune response and the adaptive immune response are reactive and for most of the, in most of the cases, the disease stops well, so an improvement starts at this point. But in some 10 to maybe 20% of the cases, the development goes in the direction of acute phase with acute respiratory distress syndrome and cytokine release syndrome characterizing the situation. It is actually this state of abundance of pro-inflammatory cytokines related with the interferon-stimulated genes, which causes actually the very heavy course of the disease and is actually the reason for lethal exit. So it is important to have a control over the development of the cytokine storm in its very early stages. It is attempted through immune modulators and cytokine antagonists. The key cytokines, which are involved in this overexpressed situation, so this abundance of cytokines in the affected organism, are interferon-gamma and IL-6. These are interleukin-6 IL-6. These two cytokines are the subject of our study and just a couple of words to introduce them. These are more or less of the same size cytokines signaling molecules. Here we see interferon-gamma in complex with its receptors. These spheres indicating the areas, the binding areas, so the place is on the receptors where the cytokine binds. It is a non-covalent homodimer with about 20 tons of weight consisting of two chains in antiparallel orientation of 143 amino acids each. It plays an important, really a key role in the immune signaling and complete absence, complete lack of this cytokine is a very dangerous situation. However, overexpression is not much better because it is associated with certain autoimmune diseases, among them the multiple sclerosis. There are two options to block the activity of this cytokine. One is by blocking the receptor via inactive mutated forms and the other is to block the cytokine binding sites. The other cytokine, which is of importance in the development of the cytokine storm, is interleukin-6 or IL-6, which is, as I said, about the same weight, almost 20 feltons, and it consists of several alpha helixes, which are helices which are very competely bound in a very compact conformation to each other. The level of this cytokine under normal conditions is very, very low, close to zero, but it can increase 1000 times under, in the cases of infection, inflammation, or cancer, and this is the reason why it is used as a biome. And for example, when in the blood test, the C-reactive protein is measured, it is a direct relation to the levels of interleukin-6. It is important because it plays a modulating role for the coordination of the B and T cells, the regulation of the B and T cells involved in the immune response, and the inhibition of the IL-6 signaling is considered to have therapeutic potential in cancer, including diseases, infections, and well, in particular, COVID-19. The signaling pathway of interferon gamma is, as I said, the signaling complex, the biologically active complex is the cytokine associated with its receptors. With interleukin-6, it's a bit more complicated because here is a picture of this molecule, this cytokine. First, it binds to the alpha receptor, IL-6 receptor alpha, then two such structures bind to a third receptor, omnipresent on the surface of many cells, GP-11030. Two such triple structures hexamerize form the actually biologically active complex that initiates the signaling. There are different options to stop or to interfere in this process. First is to block IL-6 itself and, for example, an antibody named cytokine map does this, or it is attempted to block the receptor, alpha receptor, analyzing antibody is allowed as biological. The problem with this approach is that there are side effects, there is increased risk of infection and lack of specificity of this action. Another option would be to prevent the formation of the triple complex, and that way stop the formation of the biologically active signaling molecule, and our approach is oriented also towards, in this direction. Instead of using antibodies, a feasible alternative in this case, so for interacting with cytokines and in one or another way preventing them from exerting their biological function, are glucose aminoglycans. So, for example, heparin. Heparin is a polymer, a polysaccharide from 3 to 30 kilodaltons, it's a very uniformed in the length of the chain structure. It's actually a variably sulfated repeated repeating disaccharide units, you see them here, an example, and the sulfate ion actually carries negative electric charge minus two, and this repeated construction actually explains the most important property of heparin, and it is the high density negative charge of this molecule. It is very highly high charge density and makes the molecule extremely high biological active, biologically active, it binds to over 700 proteins, which is kind of a record among these molecules. In the medical practice, however, it is used not the heparin, the unfractionated form, but the low molecular weight heparin, the fractionated form, which is by definition a bit more uniform. At least 60% of the chains are short, so have a molecular weight below eight kilodaltons. The preference is given to this molecule because it has more predictable pharmacokinetics. And from now on, when I say heparin, I will mean exactly the low molecular weight version, the fractionated version. It is known, there is experimental evidence that heparin binds to both the wrong gamma and interleukin-6, and due to this, it should have some influence on their activity. The objective of our study is to understand the molecular mechanism of the anti-inflammatory action of heparin due to the binding to this. And our hypothesis is that actually through binding to these cytokines, heparin impairs the formation of the two biologically active complexes of interferon with its receptor, and IL-6 with the receptor IL-6 alpha, and the GP-130, which is sometimes even called IL-6 beta receptor. And by this impairment of the formation of the two biologically active complexes, heparin actually impairs, prevents the activation of the respective signaling pathways. So the approach is a series of in silico experiments consisting, of course, in reconstruction, in production of the necessary structures. For example, the full length interferon gamma homodimer, investigation of the structures, complexion, and identification, the contactorious and binding sites, and simulation of the binding, heparin binding itself, followed by structure analysis and visualization. The molecular dynamic simulations performed are with the package GROMACs. Here is a protocol, well, the typical protocol used in these simulations. And for the purposes of this study, we have performed the series of simulations of interferon, A-perform, and in complex with representative hexazacharites, polyzacharites, well, representative heparin molecules, which are, as you see, very long altogether to microseconds of simulations. And shorter simulations but still long enough for the purposes of this study of interleukin and complex with heparin and with receptor and heparin. So for the structure of interferon gamma, we have used our earlier result for reproduction of the full structure, because in the crystallographic databases, the flexible C-terminal domain is missing. So for IL-6 and the complex, we have used structures from PDB with the IDs given on this site. And a slide, and for the molecular for representative heparin molecules, so low molecular weight heparin molecule, we have used based on literature studies, representative molecule, which is a hexazacharite with a net charge of minus nine. And the structure of this representative molecule here, its composition is represented by this sequence. With this set, we start with our results on, sorry, yes, on binding simulations of interferon. We have used this initial conformation, we have placed four molecules in a close proximity to the cytokine for representative hexazacharites, so heparin molecules. And in all three simulations, the details vary from simulation from one to the other, but the general pattern is the same, all four hexazacharites bind to the cytokine and are on the surface in the areas which exhibit a positive charge density, which is expectable because of the high negative charge density of the zacharites. What is important for this whole process? First of all, it is important how the binding affects the structure of interferon molecule. Here are the secondary structure plots, this top left corner is the interferon and the other three are the binding simulations. Well, it is difficult to follow details on these four plots, but what is evident is that if changes occur from this to the other compared to these other three situations, they are here in the lower part of the tables, which correspond to the region from 120 to a second above, and this is exactly the flexible C-terminal domain. So the secondary structure in the other part of the molecule, the globular part of the molecule remains highly unchanged while certain changes appear in the C-terminal domain. The dynamics of the binding process can be followed by investigating the time course, the development in time of the pair contacts between interferon and the hexazacharites. Here is a result, if average plot, for the three binding simulations, shown separately on the right panel. In the left panel is the average over these three simulations. What is seen is that very rapidly from the very beginning, pair contact formation starts, and it is associated, it corresponds to the binding of the first two zacharites. When two zacharites are bound, the third is attached with a slow, small delay, and then we have kind of saturation. What is the point here? The point is that with two zacharites attached to the molecule, the charge of the whole complex becomes neutral. And with the third hexazacharite attached, it is already negative. It has negative net electric charge. And in order to have the fourth sugar bound to this initial complex, it is a bit more time needed because then actually the molecule searches, explores the space for areas with a locally positive density. For example, we will see this on the contact plots. We will see this where these areas are. The contact map of this context shows this is the frequency, the occupation frequency of the context for the last 20 seconds of the simulations from blue to which is zero to lila or whatever, which is one increasing occupancy. We see that actually these contacts are formed in the region, 240, 20 to 144, so the c-terminal domains. Also, between 86 and 29 amino acid, there is tetrapeptide, which is a positively charged part of the molecule as well. So the ability of the complex was investigated by studying the hydrogen bonds formed between the heparin and the four sugars. And we see that bound state is stabilized through a relatively high number of hydrogen bonds, 7, and this means that these constructions are very stable. On this slide is shown the little surface of interferon alone. And here is the complex with the heparin molecules. Here we have a Bible moments depicted of the interferon and of the complex of interferon with the receptor. We see there ideally parallel orientation, and of course for this, this absolutely nice charge distribution is crucial. This substantially distorted charge distribution so electrostatic potential surface upon heparin binding makes actually practically impossible, such co linearity and the formation of the process already on the electrostatic reasons. So do we expect to reduction of the biological activity of the interferon through this binding is shown at best on this plot where we investigated the secondary the solvent accessible surface area of heparin interferon shown here with black for the molecule which is in the preform so alone. And here are the results from the three binding simulations so we have here substantial decrease of the solvent accessible surface area which means that the biological activity will be reduced for the ill six molecule, the separation is different. Here is a picture of the complex ill six ill six receptor alpha and the GP 130. In this structure, the binding sites have been identified so they involved amino acids, mostly responsible and binding. The point is that ill six is neutral is a neutral molecule. There is no a priori aspect that electrostatic would play a substantial role in the binding to happen in, however, in the binding simulance we see here is ill six molecule and here is the heparin molecule representative heparin molecule this is the same picture from another in another. What we see is actually that heparin very well perfectly well accommodates itself in a big pocket, so to say in a pocket which is in blue is the area of the ill six molecule, which is positively charged so it actually accommodates itself in the positively charged charged pocket. The, there are no substantial structural changes in this case as well. The interests are actually polar determined by the charge distribution and the amino acids, which are involved in the interaction. The binding heparin binding are highlighted here and these are all amino acids which actively participate in the binding site for the molecule. In particular, in binding site one these are these two Arginine sent in the binding site to other amino acids depicted upon binding to heparin. The, the two Arginines are already involved in heparin inter ill six binding, so they are not so to say free freely available to form the complex with the receptor. Here is the change of the solvent accessible surface area of the amino acids, which are involved in the binding sites of ill six, and we see the blue color is the molecule with orange is in complex with heparin. Here for ill six and here for for ill six plus ill six receptor alpha. This is for which the change in the in the surface of accessible surface area was highest so exceeded the standard deviation and again we have amino acids which are involved in the binding site formation, mostly affected by heparin binding. This is who assume that upon heparin binding actually the possibility of ill six to bind to its receptor elf and then to the GP 130 is diminished. And an important point here is that magnesium ions so to the land bias positive a an important role in stabilizing this complex, in particular, by in this stabilized complex becomes mostly affected also this 31 time design residuum which is very important for the biological activity of the action I'm coming to my conclusions, take home what were the results of our investigations here is actually what was written in with words shown also in the picture for bet rising so to say. Interferon acts very actively binds actively now to inter in heparin binds to interferon gamma to inter look on six and it also to the complex ill six ill six receptor alpha, forming various is shown here in this intermediate stage, and by forming this table complexes, it prevents the formation of biologically active signaling complexes on with its receptor and the triple complex ill six ill six receptor alpha receptor beta. This leads to the conclusion that he a potent and inflammatory agent due to its ability to engage with two of the key cytokines in the development of the cytokines storm. The action of heparin does not depend actually on the virus type and even in general more general on the course of the inflammation process itself. In addition to the well known and the coagulant activity which is the reason to apply heparin in the treatment of COVID-19. It exhibits anti-inflammatory, anti-vial activity so it's a three fault activity. And an added benefit is that actually heparin is a well known Medicaid World Health Organization list of essential medicines so it is very well studied side effects well known so in this sense it's a safe suggestion for a treatment procedure. I want to thank to our colleagues from biological experimental biological colleagues and the funding agencies and of course for the computational resources which made possible this research. Thank you for your attention. Thank you. So I see that there are several questions in the chart in the chart. The first one is, is there any reason why you were using the version 547 and not the more recent one. I, sorry, I was reading the second question. Well this the study was started when this was the most recent version and we haven't repeated the long simulations with a newer one, and we would not expect to have substantial changes why should it be a change because of. We do not expect to have substantial changes. I'm reading in the chat actually about the hexasahorides how did you decide that the hexasahoride with such specific composition is a good representative, a good representation of LMVH is a complex mixture. Well, how did we decide it as I said literature study when you make simulations you have to change to select a representative molecule, it is not reasonable to make several molecules together. What is important is actually we have tried actually two different structures I must say, but for two different representative structures, but the results are actually the differences are. So, the important point is to have electric charge which should be able to make the molecule visible for the interferon in particular in the case with the interferon. And yes, it is also associated with the computational resources available of course one should not forget that. This is also an important point, and I think there was maybe one more question. Yes, have you planned to validate your results experimentally how feasible. Is it. Yes, we have experimental validation of the results about interferon gamma. It is that I haven't demonstrated this results because of the scope of the school, but we have experimental validation of the block of the influence of the influence of the copper in binding on the biological activity of interferon gamma for interlook and six we rely on data from the literature, but we have not yet, we have not, we haven't planned, we do not plan on experimental studies. Also, because of practical reasons that is, it is difficult to find somebody to perform this research, it is not very commonly used in the studies molecule, it turns out. Okay. Thank you.