 Okay. So we now welcome our second speaker, Professor G. Mugesh from the Indian Institute of Science Bangalore. Professor G. Mugesh is in the Department of Inorganic and Physical Chemistry and leads the group in Chemical Biology, Bioinorganic and Medical Chemistry. Professor Mugesh obtains his BSc from the University of Madras in 1990 and MSc from the University in 1993, followed by his PhD from IIT Bombay in 1998. He received an Alexander von Humboldt postdoctoral fellowship for research at the Technical University Brown-Schweig and after that a SCAD postdoctoral fellowship at the Scripps Research Institute. So Professor Mugesh has authored more than 160 publications in international peer-reviewed journals and has guided 35 PhD students. He is a fellow of the National Academy of Sciences, the Indian Academy of Sciences and the Indian National Science Academy, as well as the Royal Society of Chemistry. Professor Mugesh has received several awards of recognition, which include the Distinguished Alumnus Award of IIT Bombay, the Swarnagenty Fellowship, the Saasstras PNRO Award in Chemistry and Material Science, the SS Bhartanagar Award, the Infosys Prize in Physical Sciences and so on. So Professor Mugesh will be talking on artificial enzymes today and we look forward to a great talk. As usually you can write your questions on the chat and we will pass them on to the speaker at the end of the talk. Thank you. Over to you Professor Mugesh. Thank you very much for the introduction and great to meet you all. I would like to briefly talk about artificial enzymes and a little bit of our work in this area. I will take about 35-40 minutes and then leave some time for discussions in the end. So this is a topic, artificial enzymes. We all know about enzymes. We know they are biological molecules, typically proteins and that speed of chemical reactions that support life. So we call them biological catalysts. Small amount of enzymes are required because since they are catalysts, a small amount is required to mediate these biological reactions. So in this talk, I will touch upon few aspects of artificial enzymes and how they are designed and what type of functions they can carry out. So these enzymes play essential roles, as you know, in respiration, digestion of food, muscle and nerve functions, many other functions. So we know that they bind to molecules and alter them in very specific ways. And the human body contains hundreds of such enzymes. Some of them require metal ions. Some of them do not require metal ions. So they essentially use amino acids in the active site to perform such reactions. So one of the examples you are all very familiar with is amylase, which converts starch into sugar. And this enzyme present in saliva begins the chemical process of digestion. And food goes to starch and then glucose chains, maltose, glucose. And as you know, glucose is the most important source of energy in all organisms. And I showed one cartoon picture here, which is actually X-ray picture of your protein. As you can see here, the proteins are made up of amino acids. And we know there are 20 amino acids. And we know now 21st amino acid, selenocysteine and the 22nd amino acid pyrolysin. So these are genetically incorporated into the proteins. So this structure shows that the three dimensional structure of the protein, which you all might have studied about alpha helix, beta sheet structures, hydrogen bonding, how the proteins holds. When we look at the different types of enzymes, different organs have different enzymes. I have given an example here, and enzymes of DNA polymerase. And DNA polymerase mediate the DNA synthesis. Another enzyme which is present in brain is acetylcholine esterase, which is a key enzyme. As you can see here, acetylcholine is a nerve transmitter. And acetylcholine has to be converted to choline after a nerve transmission. Otherwise, it leads to nerve failure. And this is an enzyme, which is a hydrolase enzyme, which cleaves acetylcholine esterase to give acetate and choline, right? And then another key enzyme. So like this, we have hundreds of enzymes in human body. They mediate various biological functions. They are structurally different. They have different amino acids, combinations, and they mediate important biological functions. So in this talk, what I'm going to talk to you today, how one can understand the biological functions and structure of these enzymes and can design small molecules or peptides or proteins that functionally mimic these enzymes. And that is actually the heart area at the interface of chemistry and biology today. How do you mimic a biological function of an enzyme using small molecules? And these are called the artificial enzymes. When we carefully look at the enzymatic reactions, we know, sorry. No, no, please continue. I request the audience to please mute their microphones during the talk. Please continue. Okay. Yeah. So, you know, if you look at this, you know, process where enzyme and substrates bind with each other. So the substrate enters the active site of an enzyme, and it forms an enzyme substrate complex, and then the product gets released. So a chemical reaction takes place at the active site of this enzyme here. So the substrate binds, and then the chemical reaction takes place, then the products are eliminated. In organic reactions, we say that, you know, reactants react with the catalysts and the products are eliminated. So in this case, the catalyst is a bio catalyst. It's an enzyme that mediates the chemical reactions. And the reactions where the enzyme, where the reactions take place, the protein portion where the reaction takes place is called an active site. So since, you know, for many years, simple chemical catalysts have been designed to achieve some desirable features of the enzymes. And this is how, you know, a pioneering work by Breslow published a science paper in 1982, who talked about, you know, the chemical catalysts. So can we make the small molecules which function as an enzyme? And recently, catalysts from synthetic genetic polymers, and this is an area that synthetic biology focuses on developing new artificial enzymes. And it is challenging to design artificial enzymes that can perform specific reactions inside the living cells. So the challenges are, you know, associated with the structure, and how do you introduce functional groups to perform chemical reactions at the active site, and then, you know, to design an efficient enzyme mimetic or enzyme or artificial enzymes that functions as an enzyme, particularly it will be challenging, you know, to design artificial enzymes that perform natural reactions. So the aim of the study at the interface of chemistry and biology involving artificial enzyme is to design artificial enzymes to regulate metabolic processes, cardiovascular processes, neurological processes under disease conditions. So this is where the artificial enzyme will be useful, because under disease conditions, these enzymes are non-functional, or the activity is not sufficient, or these enzymes are inactivated under disease conditions, can we use the artificial enzyme to substitute these enzymes inside the cells? And that is a great challenge, and the chemistry and biologists are working on the design of efficient artificial enzymes that can substitute an enzyme inside the cells. So I'm going to talk to you about very fundamental chemical process that takes place, because I'm going to talk about a set of enzymes, you know, that involves reduction of hydrogen peroxide. So hydrogen peroxide, if you look at this fundamental reaction, right, one of the most fundamental chemical reactions that takes place in human body, right? We breathe oxygen, oxygen is important for our survival, but the oxygen doesn't stay as such in human body, right? It can undergo various chemical transformations. And if you add one electron to molecular oxygen, it produces a one electron reduced species, which is known as superoxide. And this is produced by an enzyme called NADPH oxidase. So you add one more electron to superoxide, you get hydrogen peroxide, right? You are reducing further the molecular oxygen is going to hydrogen peroxide to electron reduction. You add one more electrons, you get hydroxyl radicals, because the hydrogen peroxide will be cleaved if you add one more electron. And you add the fourth electron, it goes to water. So if you look at the process, molecular oxygen is going to water by four electron reduction, but incomplete reduction of oxygen is also important. Superoxide is important by a molecule, hydrogen peroxide is important by a molecule. So this we call it as incomplete reduction, one electron or two electron reduction, but that's also important. But that level needs to be maintained. So you look at this picture, hydrogen peroxide bottle and you know, familiar with the hydrogen peroxide usage in the chemical laboratory. If you open this bottle with bare hand, what you see on the finger is a white color like this. What does it mean? It means that hydrogen peroxide oxidizes your skin protein and skin proteins become, you know, it creates a gentle white color on the skin. So if this is the case with the hydrogen peroxide, how hydrogen peroxide is produced in the body and how it is controlled in the body, right? So that is where the enzymes play key roles. So these are the enzymes which controls the reactive oxygen species. So when we say it's one electron reduction or two electron reduction, these produces the species which are known as reactive oxygen species because they are highly reactive in human body. They can damage biomolecules, they can damage DNAs, they can damage proteins, they can damage lipids, okay, it lead to lipid peroxidation, right? And these are called the reactive oxygen species. And there are certain enzymes that controls the reactive oxygen species like superoxide dismutase that controls the level of superoxide. That's why it's called superoxide dismutase because by dismutation reactions, superoxide is converted to hydrogen peroxide. There are other antioxidant enzymes like glutathane peroxidase, catalyst. So catalysts and glutathane peroxidase controls the level of hydrogen peroxide. So these are called the partially reduced oxygen species or reactive oxygen species. If there is an imbalance between the reactive oxygen species generating system. So in this case, the reactive oxygen species generating system is NADPH oxidases, superoxide dismutase leading to hydrogen peroxide. And if there is an imbalance between the reactive oxygen species generating under scavenging system, it lead to a redox imbalance in the system and that lead to oxidative stress. So oxidative stress is an imbalance between the generation of reactive oxygen species and the scavenging systems. And this is where the artificial enzymes can play a crucial role and bringing the redox imbalance to redox balance of controlling the oxidative stress in the cells and keeping this reactive oxygen species under control. So there are other peptide type molecules, you know, thyrodoxin, glutathyrodoxin, peroxidoxins. So these are small proteins or the peptides that can also control the reactive oxygen species in the human body. So I'm going to talk about a few examples. Hydrogen peroxide, hydrogen peroxide oxidizes the cysteine residue, you can see that, you know, what happens in human body. So hydrogen peroxide is of course, you know, is a important molecule and it's involved in signal transduction. If the concentration of hydrogen peroxide is high, it leads to oxidative damage. But if the hydrogen peroxide concentration is optimum, it leads to apoptosis, global changes in gene expression proliferation. So hydrogen peroxide, you know, has two roles. One is that it leads to oxidative stress. Another one is it's a lifespan determinants. So if you look at this picture, what is happening here at the molecular level, superoxide is produced by NADPH oxidases from molecular oxygen. I mentioned it is a one electron reduction of molecular oxygen or it can come from mitochondria, one electron reduction of oxygen generating a superoxide. So the superoxide level is perfectly controlled in the human body by superoxide dismutase, right? And that leads to hydrogen peroxide and hydrogen peroxide formation initiate the redox signaling in the cells. For example, peroxide redoxin, glutopulothane peroxidase catalyst that control the hydrogen peroxide level, keeping enough amount of hydrogen peroxide to initiate the signaling pathways. The signaling pathways is initiated by oxidation of the protein thiol to protein sulfenic acid. So this portion of the scheme is called the redox biology, right? So you look at the other side. Hydrogen peroxide can react with free metal ions like iron 2 plus. We have plenty of iron in human body. When it reacts with iron 2 plus, it generate hydroxyl radical and this lead to DNA damage, lipid oxidation. And this is called oxidative stress. So we can say that hydrogen peroxide is necessary for cell signaling, but one need to differentiate the redox biology from oxidative stress. And this is a fine balance of hydrogen peroxide level that either leads to redox biology or to oxidative stress. And you may ask an interesting question, right? When, for example, when we put iron along the water and oxygen, we know that resting takes place, right? And oxidation of iron, right? It lead to resting. And human body has about 4 to 5 grams of iron. Only 1 milligram is lost per day. And why no resting takes place in human body? Because human body has iron, human body has water, human body has oxygen. Why there is no resting? This is the reason why iron in biology is tightly regulated. If iron can lead to resting, if iron gets resting in human body, it lead to diseases. In a simple way, if you say, resting of iron in human body can lead to diseases, like neurodegenerative diseases, cardiovascular diseases, right? And this is what I'm going to talk about today, you know, artificial enzymes. So the first example I want to talk about to you today is an enzyme called glutathione peroxidase. And many of you would not have heard about glutathione peroxidase, although you know there are enzymes which can take care of hydrogen peroxide in the human body. Glutathione peroxidase is unusual. So this has an enzyme, this has an amino acid, which is selenocysteine. And this is the structure of the protein. And you look at the structure here, the selenocysteine is considered as the 21st amino acid, right? And commonly the textbook describes 20 amino acids as genetically coded amino acids. But selenocysteine is considered as the 21st amino acid. And this enzyme used this unusual amino acid for chemical reactions. And you look at the amino acid selenocysteine, which is hydrogen bonded to two other amino acids in the active site, tryptophan and glutamine. And we call this as a catalytic triad, right? Because the catalytic activity of an amino acid at the protein at the enzyme active site can be enhanced by non-covalent interactions. So these non-covalent interactions are hydrogen bonding. And this enhances the nucleophilic reactivity of this selenium IT, right? And you look at this picture. So what you see here, a chemical reaction that takes place is then a peroxide is converted to an alcohol by using a glutathione, which is a which is a tripeptide present in the human blood in large concentration, in millimolar concentration. So this enzyme used this glutathione as the cofactor to perform this reaction. So it's a catalyst, right? Glutathione peroxide is a catalyst. So catalytic amount is sufficient nano molar concentration. So if you have a hydrogen peroxide, hydrogen peroxide is converted to water, if you have an organic peroxide, it is organic peroxide is converted to an alcohol. So in this process, glutathione is oxidized to glutathione disulfide. So it's a redox reaction, right? So the peroxide get reduced to alcohol and glutathione get oxidized to glutathione disulfide. So in this way, the redox balance in human cell is maintained. And if you look at the catalytic cycle here, you can see selenium is of course important as part of the selenocysteine. Hydrogen peroxide is converted to water and glutathione peroxide is used glutathione and it takes that to oxidize glutathione and which is reduced back to reduced glutathione by another enzyme called glutathione redactase using NADPH, which is a redox cofactor, right? It's very intriguing reaction using a selenium, right? We know selenium is a toxic, right? But how human body use selenium, a toxic element, you know, for physiological functions. So the toxicity depends on the concentration, right? And selenium deficiency is very common, right? People have thyroid problems where selenium is important. Hyperthyroidism, hypothyroidism related to selenium deficiency, right? So in this case, the GPs deficiency is directly induces an increase in the oxidative stress and its over expression rescues the cells, right? So that means if you can make a small molecule synthetic compound that can functionally mimic glutathione peroxidase, we can rescue the cells from oxidative stress. So this is the simple concept people use to understand, you know, the glutathione peroxidase, the importance of glutathione peroxidase and mimicking the function by synthetic artificial enzymes. So what is special about selenocysteine? Why nature, you know, took all the way, you know, all the trouble to make selenocysteine and incorporate a selenocysteine into protein, which is, which is a very difficult process, but still nature does it. Why? Because the pKa of a selenol group in protein is much lower than the pKa of a thiol group in selenol and then that is lower than the hydroxyl group in serine, right? All three are amino acids. I'm talking about three amino acids here. Change, the change is only one atom, right? The oxygen is changed to sulfur and which is changed to selenium. So the one atom change, how it affects the pKa, right? It goes from 13 to 8.3 to 5.2. What does it mean? This means that, you know, the selenol group in, in selenocysteine is a powerful nucleophile, right? Selenolate is a powerful nucleophile. So it can perform chemical reactions much more efficiently than sesine and serine, okay? So with this in mind, people have tried to synthesize small molecules because, you know, making molecules is not very difficult, right? You can make molecules in the laboratory, but making them with a specific purpose is difficult. And this is one of the small molecules made with the purpose, right? And which is called epsilon. A small molecule, you look at the structure, it's, you know, two benzene drink here and you have a five-membered heterocycle. But what you see here is a, is a direct selenium nitrogen bond. And this compound has undergone several clinical trials as antioxidant, anti-inflammatory, anti-atrochlorotic, antithropedic, etc., okay? So selenium deficiency, this is a selenium compound. Why, why people focused on selenium? Because the enzyme itself contains selenium in the active site. So therefore, one needs to make selenium-based compounds to mimic the glutathione peroxidase, right? If you want to artificially mimic artificial enzymes that mimic the glutathione peroxidase, that should have a selenium, right? Selenium deficiency is detrimental for normal growth and development in mammals. And a narrow range of chemo-protective, chemo-therapeutic and acute toxic dose levels have been studied very well. And the most toxic selenium compound is sodium selenide here, right? And people who have selenium deficiency, they are treated by giving brazil nuts. Brazil nuts have 8 to 83 microgram of selenium per gram, one of the highest selenium-containing natural products, right? Brazil nuts. So, but if you look at the metabolism of the selenium in human body, it's a bit complicated and do not worry if you don't understand the chemical processes. But I want to show you, you know, the selenium metabolism in the cells and why the selenium compounds can functionally mimic glutathione peroxidase and some of the metabolic pathway can be used to eliminate them, you know, to modify them to eliminate. And these are some of the selenium species which are produced, you know, inside the cells. So, the chemistry, the molecular design is very important. If you look at, you know, the structure, very, very unique structure. So, there is a cleavable selenium nitrogen bond here. So, when you add a nucleophile and the selenium is an electrophilic center, so if you add a nucleophile, the selenium nitrogen bond can get open and that initiate the chemical processes. And when we substitute the selenium with the sulfur, the compound is inactive. Just one atom change, right? You have substitute the selenium with the sulfur. There is no activity. This indicates that the selenium is essential for the activity. So, what is the catalytic mechanism because as a chemist, you know, we all study, we want to know what is the molecular level mechanism, right? When something shows some activity, right? A compound shows certain activity, we want to know what is the molecular level mechanism, right? Because that is important. That is that way of, you know, the chemist addressing the reactivity, right? So, in this case, you can see this epsilon which ring opens here selenium nitrogen bond. It forms a covalent intermediate, then it goes to another intermediate and this process continues. And you can see multiple, all of these have been isolated. I am not going into the details of how we do the characterization. But what I would like to tell you is that the mechanism of epsilon, which has undergone several clinical trial, you know, has been very well established now. And what people have found is that why the cyclic compound, right? Why is cyclic is important, right? And these people correlate with a reversible inactivation pathway of the PTP1B. PTP1B is an enzyme. It is a tyrosine phosphatase. Protein tyrosine phosphatase 1B, you know, this cleaves the phosphate group from cysteine residues. So, if the cysteine tyrosine, sorry, the tyrosine residues, tyrosine get phosphorylated and the deposphorylation is important, right? Kinesis, phosphorylate tyrosine residues, they have tyrosine kinases. But the phosphatases are the enzymes which cleaves the phosphate group from tyrosine and catalyzing these reactions. So, this is a unique enzyme. And what they have shown is that the cysteine group at the active site of these enzymes undergo a reversible oxidation. You can see cysteine sulfenic acid. I mentioned this initiative there, redox signaling, right? Sulfenic acid. And then, you know, the reversible cyclization involves the formation of a sulfenelamide. And if you look at this five-membered ring and the five-membered ring here in epsilon, it's quite similar, right? And here too, a sulfur nitrogen bond is cleaved here. And here, selenium nitrogen bond is cleaved here. But when you oxidize this, it goes to the cyclized form. So, a reversible inactivation pathway, you know, gives a clue why this compound is less toxic, because although selenium is considered as a toxic element, why this compound is less toxic in human because of the reversible cyclization pathways. So, a variety of compounds have been designed to mimic the function of, these are considered as artificial enzymes for glutathione peroxidase. A variety of compounds have been designed last two to three decades to understand the structure activity relationship and also their ability to catalytically reduce hydrogen peroxide and their ability to control hydrogen peroxide level in human body, right? You can look at the chemical design, right? You have a cyclic compound here. You have a compound with a tertiary amide structure. You have a compound with secondary tertiary amine structure. You have another set of compounds without this methoxy group, a secondary amine group, a secondary amide group. So, these, all these compounds have selenium in the active site because the glutathione peroxidase contains selenium in the active site of the enzyme. So, this is how, you know, you modify the compounds, introduce new functional groups, introduce new structures to these molecules to modify and modulate the activity of these compounds in human cells, right? And so, you can perform chemical reactions in the laboratory, you know, multi-step reactions to make this molecule. So, this is part of, you know, drug development, right? In pharmaceutical companies, what they do? They do multi-step synthesis to make an artificial, a synthetic compounds for biological activities and drug development. And this is where we synthesize molecules in the lab. And in this case, this 4A to D were synthesized in the laboratory by keeping the structure of epsilon in mind because epsilon has been shown to bind to protein reversibly, right? To avoid that binding, we removed the carbonyl group here, but you can see these compounds. So, the molecular design involves a fine balance of the chemical reactivity, biological activity and the toxicity. So, the rational design to improve the GPX activity of epsilon has led to the development of new type of molecules as shown here for functionally mimicking glutathione peroxidase. So, this is the catalytic mechanism. The catalytic cycle involves the formation of a selenoxide, a selenol and a seleninic acid species, and then it is reproduced in the catalytic cycle. So, you can use NMR techniques to characterize the intermediate, you can isolate them, you can purify them by column chromatography, that is what we do in synthetic chemistry lab, right? You make molecules, purify them, characterize them, make sure that what you are working with is the right compound because even small change in the substitute and can alter the function of these compounds in human cells, right? This is how we do. So, what, you know, the experiments that have been done to understand, because any molecule that you make in the lab, if you want to show that this could be considered as a drug, so the first step is to test the compounds in human cells, right? Whether first thing is whether these compounds can enter the cells because the human cells have multiple mechanisms for cellular update, some compounds can be taken up by the cells, some compounds cannot be taken up by the cells. There are strict regulated pathways to take up this, take up the small molecules or the peptides proteins. So, we wanted to check first whether these compounds can be taken up by the cells, okay? And as you can see here, these are the human cells, cancer cells of course, you know, that's easy to handle in the lab to study the biological activities. As you can see here, these cells are untreated cells. These cells are the one treated with the hydrogen peroxide. If you go back to the beginning of my talk, hydrogen peroxide is signaling molecule, right? But higher amount of hydrogen peroxide can lead to DNA damage. It can lead to protein damages. It can lead to lipid peroxidation. So, if you want to functionally mimic the enzyme glutathrin peroxidase with artificial enzymes, you need to show that these compounds act on hydrogen peroxide inside the cells. As you can see here, this is the compound under hydrogen peroxide. The cells, you can see that it's quite similar to the untreated cells. This is the one with the, you know, the Fourier with the hydrogen peroxide. Again, it's similar to the untreated cells. So, what does it mean is that these compounds do react with the hydrogen peroxide and to control the hydrogen peroxide level inside the cells, just like the enzyme glutathrin peroxidase. You remember the glutathrin peroxidase cycle I showed earlier, hydrogen peroxidase is converted to water by using glutathrin as the cofactor. And these compounds just substitute this enzyme glutathrin peroxidase. And this is amazing, you know, how small molecules functionally mimic a huge protein, right? And function similarly in human body, right? And this can be, you know, it goes to water and this complements the effect of catalysts in human body, okay? These are all fluorescent experiments. So, how you be monitored these experiments in the laboratory is that use a fluorescent microscope to monitor this fluorescent, this is a fluorophore, fluorescent probe that reacts with the hydrogen peroxide and enhances its fluorescence. So, when the fluorescent is enhanced, that means that hydrogen peroxide level is high in the cells. So, that can be brought back to the normal level by using a synthetic or artificial enzymes. So, these compounds can also prevent DNA damage. You know, DNA damage, as you know, you know, Nobel Prize has been given to, you know, to people who studied DNA damage, DNA repair mechanisms few years ago. And DNA can undergo damage. It can be either a single strand break or a double strand break. And you know, double strand break is more difficult to repair, because there is no complementary base pair to repair the double strand break, at least the single strand break. And there are biological mechanisms that that can repair the DNA, which are damaged by reactive oxygen species. So, you can see here, this is the DNA damaged, you can see it through due to hydrogen peroxide, untreated cells. The cells treated with the hydrogen peroxide, DNA damage is noticed in this case. But treating the cells with our compounds, you can see that the DNA damage is prevented, which means that the synthetic molecules, which we call it as artificial enzymes, can reduce hydrogen peroxide to non-toxic water in the presence of glutathione. Therefore, it functionally mimic the enzyme glutathione peroxidase. Okay, so you may ask a question, what happened to the glutathione peroxidase already present in the cells? As you know, the hydrogen, the enzymes are present in low concentrations, like nanomolar concentrations. So, when you add hydrogen peroxide to the cells, the enzyme which is already present in the cells is not sufficient to take care of the hydrogen peroxide. Therefore, you see DNA damage, but supplementing that with the compounds, you can see that DNA damage is prevented. This is very, very interesting work, how you can make very, very small molecule. Few atoms, not molecules with few atoms can mimic an enzyme, which is huge with hundreds of atoms. So, we also study the chemical reactivity, see how the bond breaks, how the bond forms is important in chemistry. How you study the bond breaking and bond forming reactions. So, you look at the finer details, you look at the molecular levels, you can see that a nucleophilic thiol can attack at either selenium or sulfur, when you have a selenosulfide intermediate in the catalytic cycle. This is an intriguing question that people asked several years, how glutathione peroxidase overcome this problem, because the nucleophile can attack at the selenium and sulfur, but the attack at the selenium is much more preferred, because selenium is more electron deficient than sulfur or more electrophilic than sulfur. So, but in the natural enzymes, the attack happens at the sulfur, not at the selenium. How the enzyme overcome these difficulties. So, how the enzyme use the peptide backbone to overcome these difficulties. So, computational studies can give insight into the reaction mechanisms. So, if you look at a disulfide bond in a cysteine and you have a cysteine, you can reduce the disulfide bond by adding two electrons, two electron and two proton, you can cleave the disulfide bond in protein to produce the cysteine residue. And therefore, the cysteine residues are target for many small molecules. So, if you can, if you want to inhibit a cysteine containing enzymes, people use an electrophilic reagent, a nucleophilic attack of cysteine at the electrophilic center, you can make a covalent bond. And these are some of the, some cysteines are with disulfide bonds and some cysteines are free cysteines, they are target for nitrosylation, disulfide bond formation, yes, polymer metallurization or that, you know, there are also active site cysteines which are present in these cases. So, if you have a reactive cysteine and you make an artificial enzyme which can react with the cysteine, you are going to inhibit that enzyme. So, that is the problem with Epsilon. One question that you may ask, you are saying that Epsilon has undergone several clinical trials, why Epsilon has not entered the market yet? So, one of the drawback of Epsilon is its reactivity towards the free cysteine residues in protein. And what is interesting is, this inhibits the enzyme glutathione reductase which recycles glutathione, you know, which reduce glutathione disulfide to glutathione, which is very, very important process in redox, you know, in redox signaling. But this compound inhibits the enzyme. So, therefore, the redox recycling of glutathione disulfide to glutathione is not happening. And we have shown for the first time that, you know, the reaction of Epsilon with glutathione reductase is one key reason why Epsilon is not used as a drug. So, you can overcome this difficulty by changing this carbonyl group. This is what I mentioned about, you know, the structural design in small molecules, how you can find tune, just changing one or two atoms, you can change the reactivity completely, right? So, in this case, what we did, we removed the carbonyl group from here and introduced a methoxy group here as I showed earlier, right? So, that compound, you can see this, you know, the one I showed you earlier, this compound, right? You don't have the carbonyl group here, but you put a methoxy group. So, you still make the cycle without a carbonyl group. And this is really challenging, right? You need to understand the chemistry behind the cyclization process to do that. So, you see often reports like this, you know, you see a science daily report which says, human trials are just rescued in drug, could be safer treatment for bipolar disorder. So, it says, initial human trial promising for failed drug epsilon, why they call it as a failed drug, because as a glutathrin peroxide is mimetic as an artificial enzyme, it failed, because it reacts with enzymes which have 16 residues. Okay, it says, epsilon, abandoned as a stroke treatment, has a successful first human trial as scientists aim to repurpose it as a treatment for bipolar disorder. So, epsilon has gone from identification as a potential bipolar treatment to human trials in about two years, incredibly fast for any drug discovery process. And epsilon is part of the National Institute of Health Clinical Collection. This is a chemical library of bioactive drugs considered clinically safe, but without proven drug, proven use. So, if someone, you know, any of you would like to take epsilon and use that for new disease, you know, this is available because the safe, the safety and efficacy of epsilon has been very well demonstrated in human cells. So, therefore, many of the preliminary experiments can be avoided. Recently, that's what people have done, you know, targeting mycobacterium tuberculosis at transpeptidase with a cysteine reactive inhibitor, including epsilon. What they have done, you know, this enzyme has a cysteine residue, use epsilon to covalently link that it forms a selenosulfide bond here. In this way, it inhibits this transpeptidase, you know, mycobacterium tuberculosis. And very recently, you can see that the crystal structure of SARS-CoV-2 main protease provides a basis for design of improved alpha-keto amide inhibitors. So, one of the best characterized drug target among coronaviruses is the main protease, M-PRO, which is called M-PRO. Essential for processing the polyproteins that are translated from the viral RNA, that inhibiting the activity of this enzyme would block viral replication. No human proteases with a similar cleavage specificity are known, so therefore, such inhibitors are unlikely to be toxic. So, this is where, you know, people immediately got into epsilon because epsilon is known to bind to cysteine residues irreversibly, right? So, what they have done, this is again a nature, you know, accelerated article peer review because you have a pandemic, you know, you identify a compound, a chemical compound that is potential for treatment, you know, you see that, you know, these are peer reviewed so quickly and published so that people will be able to use them. See, this is structure of M-PRO from COVID-19 virus and discovery of inhibitors. So, screening of 10,000 compounds identifies epsilon as one of the six potential COVID-19 therapeutics. You can see here, it inhibits this enzyme M-PRO by forming a covalent cysteine epsilon covalent bond, right? So, you see how the chemistry, the how science evolved, right? People started studying earlier why epsilon failed as a drug and then you see that that use that as a strategy to design new type of inhibitors for this virus viral proteins, right? And molecular characterization of epsilon binding activity to SARS-CoV-2, there are further reports which states that the catalytic site is blocked by epsilon, right? And there is a recent report by Professor Kilimanthee is that you can see that, you know, it's a target for main protease and this epsilon has multiple functions. It reduces ROS, it decreases the cytokine neurofills, it brings down the ferritin, ILF, IL6, okay? These are the key biomarkers involved in lung injury or thrombosis, you know, vascular inflammation and so on. So, a small molecule, you know, it's just a few atoms, you know, the number of studies that have taken place with this compound is enormous, right? And trying to understand, you know, how one can, you know, design small molecules, okay, to functionally mimic an enzyme. So, before I conclude, what I would like to summarize is that reactive oxygen species is important in biological functions, but that level goes up, it can lead to variety of diseases, neurodegenerative cardiovascular and other related diseases. But the enzymes that act on reactive oxygen species, it's not so efficient under disease conditions, whether one can generate artificial enzymes to mitigate the reactive oxygen species affecting human body. And recent studies show that it is successful, recent studies in fact highlight the importance of artificial enzymes. One can design artificial enzymes to functionally mimic the natural enzymes to control the redox balance, to control the oxidative stress and to control the reactive oxygen species. And people terms this as an oxidative use stress and oxidative distress, because certain stress is important for the cells to function. And oxidative stress is important, right? So, therefore, it's a time to distinguish oxidative stress, oxidative use stress from oxidative distress. The oxidative distress is the one I mentioned about the bad side of hydrogen peroxide. And the oxidative use stress is the good side of hydrogen peroxide. So, we are looking at the same molecule, but two sides of the same molecules. How do you deal with such molecules using synthetic compounds or enzymes in biological systems? So, with that, I thank all the students who work in my lab who carried out work at the exciting area of artificial enzymes, the interface of chemistry and biology, funding agencies and collaborators. So, with this, I thank you all for your listening. And if you have any questions, I would be happy to address them. Thank you. Thank you, Professor Mogesh. Thank you for a very exciting talk. So, we'll just see if there are questions on the chat. Yeah, you can please write your questions on the chat and I'll read them out for Professor Mogesh to answer. So, yeah, so there's one question has already come from Subali, who is the leader of IJSO. So, there doesn't seem to be a specific codon that codes selenocysteine specifically. Yet how does the cell ensure that the protein glutamate peroxide gets selenocysteine at its active site in every protein? Yeah, it's a very interesting question and that's why I mentioned, you know, nature takes all the trouble to introduce selenocysteine. It is basically a serine which is introduced at the active site first. Then, you know, the serine is converted to selenocysteine by a series of enzymes. And one of them is selenocysteine. So, the phosphate group is introduced to the serine to activate it and then it is converted to selenocysteine. So, therefore, not in every protein, only in glutamate peroxidases, there are 10 isoforms of them. Some of them are selenoproteins, some of them are cysteine-containing proteins. There are three major selenoproteins in human body, glutamate peroxidase, iodothyrin and diadenase, which convert T4 to T3, right? Thyroid hormones and, you know, hyperthyroidism is associated with the enzyme. So, in these cases, it is the serine, then later on serine is converted to selenocysteine by a series of process. Okay, thanks. The next question from Professor Jhulabaprak from IVO. So, she has three questions. The first is, what blocks selenium absorption? Yeah, selenium absorption is basically regulated by the metabolic process of selenium. Selenium absorption, the transporter, you know, the defect in transporters, which can block the selenium absorption in selenium-deficient regions. People, in fact, identify the mutations that can block the selenium absorption. In other case, you know, selenium deficiency in water, selenium deficiency in water can the less amount of selenium in water that we drink can lead to selenium deficiency. And the side effects of epsilon is the binding of epsilon to the various proteins. You know, one of them I mentioned as the key protein glutathione redactase, but the covalent bond formation between epsilon and the cysteine that lead to the side effects. And since these are clinical trials with specific diseases and it's not being used as a medicine, the common side effect is not known. So, one of them possibly that it affects the redox signaling as well. How does epsilon help in prevention of memory impairment disease? It's not very clear, although epsilon has been used as an immunomodulator. There is no clear evidence that it prevents memory impairment. And people have also tried in neurological disorder, for example, but I don't think any successful study is available to understand this. Okay. Thank you. I don't see any other questions yet, but let's wait maybe. Yeah, we can wait, yes. Yeah. So, if you have a question, you can write on the chat or you can raise your hand and unmute and ask the speaker directly. Are there any other questions? Yeah. So, again, a question from Prasabaprat, how do you administer epsilon? Yeah, it is a good question. And as of now, the clinical trial, all this IV injection, that's how administered because it's epsilon is not water soluble and it's dissolved in some solvent and dilute it with the buffer and that's how it's administered. Okay. Amritansh from IBO, again, could you please provide an insight to how enzymatic mechanisms attract? Yes, enzymatic mechanisms, people use specific biomarkers or fluorescent probes either spectroscopically or microscopically can be tracked. And some of the processes are ultrafast processes. Therefore, ultrafast spectroscopy is useful. And the type of, you know, the enzymes that we study, you know, they have metallo metals in the active site, metalloproteins, one can use spectroscopy to track them. And in some cases, inhibitor studies have been useful to understand the enzymatic mechanism. So that means, you know, you identify the amino acids which are involved and you design a specific inhibitor and see whether the activity is blocked or not. And in some cases, the isolation of intermediates, you know, trapping the intermediates and then getting structural information like X-ray or, you know, cryo electron microscope can provide additional information about the structure if you can trap, you know, some of the starting material or the intermediate. And that's how, because it's not possible to probe a entire chemical mechanism by a single technique. So multiple techniques to be used to probe each step of the enzymatic mechanisms. Okay. Thanks. There's a question from Arithra from IGNSU. Is making a direct copy of biological enzymes very difficult? So we are, that's why we are looking into artificial enzymes. Yes. And making the, you know, enzymes can be generated in, for example, you know, we can, we can do it in bacterial cell expression of these enzymes. For example, even glutathenproxidase. We can express glutathenproxidase in the cells and we can in vitro and purify them. But the problem is the uptake, the cells don't take glutathenproxidase because these are all proteins, they cannot enter the cells. Therefore, we are looking at a small molecules that can enter the cells, not getting degraded, because if enzymes are taken up directly, they go to endosomes and then lysosomes and the lysosome cleave them into fragments like small amino acid peptides. So they no longer be working in the inside of the cells. So therefore, we are looking at small molecules that can be taken up by the cells. Okay. So there are, of course, challenges, you know, very interesting question. This is, you know, there are challenges using enzyme as a drug, right? These are the difficulties. So that's why people are looking at the small molecules. Are there more questions? If you have a question, please feel free to write on the chat or raise your hand and ask. Okay. I don't see any more questions coming. So maybe, yeah, maybe another 10 seconds we can wait. Yeah. Okay. So thank you very much for delivering this excellent talk. And for finding time to do this for us. It would have been wonderful to have you here at the Home Mother Center and meet all the students. But unfortunately, this year, we are forced to again, making an online event because many of the students have their semesters going on. So thank you. Thank you again for doing this. Thank you. Yeah. Thank you. And and we'll, of course, look forward to you to join for the award function. So for everybody, what we'll do now is that we will take a brief break of about 20 minutes. And the award function will begin at 12 o'clock, but do not leave the room. You can stay in the room and just keep your microphone muted and maybe your camera off. And we will again resume this at 12 o'clock with the award function. Okay.