Transcript for:
Enzyme Interactions and Protein Stability Overview

now dve yes yeah yeah okay one second so this one not okay see okay I think that's okay that's okay all right guys um think thank you so much all right guys let's have a seat all right good morning everyone [Music] um as a disclaimer I just finished a 2hour lecture so my voice is it's getting uh it's decayed so we'll see how long it will last otherwise we'll use some hand shaking sorry all right so um last week basically we we talked about the enzyme substrate interaction and let's see enzyme substate and we say there is the K on which is the K1 and the K off is K minus and we said the KD is basically K minus or the K off divided by K on by K1 and what you have to know is that the lower the KD the stronger the binding so one nanomolar is stronger than one micromolar okay okay and how do you get lower KD or lower you get you have to get very fast K1 or K on and very slow K off and the way this molecule bind let's say that I'm a receptor and this is the active side of the enzyme or the receptor right and is the drug molecule when you see a binding a binding of course there's interaction that happens but the binding is not a one way because if this stays and bind stays here that mean this is a calent interaction actually The Binding you have is like this right but what's a strong binding a strong binding has to be fast K on like quickly and slow K off you see that's how a lower KD like Fast K on slow K off but if you have slow K on and fast K off that's a poor binding okay or if you have fast on Fast off that's bad so you want fast on or fast K1 and very slow K off associate slowly that's why you get lower KD okay so now when I talk about give you an example of that my hand is a protein proteins are not static actually protein are alive they can move around and that's very important because that's important for the way they function and the way they interact with with different things and you can see for example they look at this myoglobin and they took different crystal structure different time points and you can see side chains and even like you know functional groups can move within the protein and that's part of the protein and that's the challenge if you when you're designing a drug based on a crystal structure one you are just looking the protein in this conformation and you are seeing the stuff that bind but the protein could be like this right that's why the future hopefully with molecular Dynamics or AI or the simulation could give you different conformation and see which drug will work better okay now a common thing about the proteins how would you guess the size of protein says the average molecular weight of an amino acid is110 you need to memorize this number and why is that because you know if you if you look at them you you guys will memorize the amino acid all of them and every amino acid will have carbon that has a molecular weate of 12 nitrogen 14 hydrogen right if you add it together roughly you get around 100 so what we will ask you for example in the example say this protein is 200 amino acids what's the average molecular weight you're going to multiply it by 110 and you're going to get Daltons because when we are talking about carbon when you say what's the mass of a carbon you see 12 12 is 12 12 what 12 Daltons okay and when you add 22 the, Daltons we refer to it in proteins as kilodalton or k k okay kilodalton kilodalton that's how you see it in the literature okay now we we discussed before is that you know this is how the protein this is the backbone and all the side chains coming and then you start bringing this to a threedimensional structure the side chains are important for function and structure both of them and you said the nonpolar residues such as these ones here are mostly on the interior of the protein why is that because they want to get away from water and that's why the hydrophobic effect remember guys when we talk about droplets of oil coming together the hydrophobic effect is the same principle here the charg amino acid arginine histadine lysine these are usually on the surface of the protein because they want to be closed to water they can form electrostatic and hydrogen bonding with the water make sense uncharged polar such as these amino acids are usually on the surface but sometimes they are present also inside the protein and these are the ones that are important for catalytic activity for entic activity as Dr CH going to discuss thoroughly with you now why because you cannot have ch2 ch2 we cannot have an alkal for catalysis right methane ethane these ones they don't have nucleophile orophile you want serine nule cadine right cine because they have electrons they can break a bond attack a bond right but what happen when these are inside these basically because they are how because they are polar how you are keeping polar material inside the hydrophobic pocket right they form hydrogen bond with each other and this hydrogen bond decreases their polarity to some extent make sense so inste of having Serene Serene that are polar and and B they just come together they lower the polarity and the pka in some cases as Chang going to describe all right so now we discuss what's what's the component of every polypeptide how the polypeptide will form a structure and basically we discussed where the amino acid side chain is going to be within the polypeptide now there's a New Concept that we're going to discuss now it's called protein stability or denaturation so when you look at a native protein this is the one that look the threedimensional structure you see it's a bunch of alpha Helix right this is called the active functional folded protein all the same folded active functional that mean the protein is intact when the prot is unfold you open it up you get unfolded or denatured protein this one you can call it natured if you want but we don't call nature right but theat unfolded cool that's what we call the unfolding so now protein stability what's the effect of every functional or every Bond or bonding mechanism that we discuss on the protein stability if you talk about hydrophobic effect say has the greatest influence on protein stability why because because of the entropy of the water molecule remember the important slide about the droplets of oil and I told you what dries these droplets of oil to come together is the entropy of the water molecule you need to go back to that slide it's the same principle here so hydrophobic interaction or hydrophobic effect basically is very important because of the entropy of the water molecule now this is important for both the structure the way the protein give you the structure but also the stability okay because when you are like an inch drop of oil is very difficult to bring drops of oil away from each other right because they like to stick with each other that's why both stability and structure okay what about hydrogen bonds we discuss hydrogen bonds thoroughly when we talk about the alpha Helix and we talk the beta sheet that's why hydrogen bond are Central feature of protein structure that's a protein structure they are very important for protein structure but what about protein stability they have minor contribution to protein stability why because bless you because when you are talking about protein structure how do you form an Alix you need to have the hydrogen bond within the backbone right when you have beta sheets has to be in between but when we are talking about unfolding let's say I have two right this is a Serene and another um let's say lysine right a Serene and lysine can form a hydrogen bond side chains and this will be a good exercise for you for next week we're going to give you a bunch of amino acid and tell you what kind of interaction happens between the side chain you're going to practice that so let's say I have a side chain of the Serene my left arm and a Hine my right arm they form a hydrogen bond right now obviously the strength of a hydrogen bone is about 20 angstrom right 20 Sorry by the way this is the ginger and when I used to sing singing concert in the past I mean this will help you to continue all right they should limit my the concept to two hours and I had mine in the morning but we'll continue with you guys so bear with me sorry all right so we started with Serene and Aline right so now you have 20 kilj per mole we discussed said that hydrogen B is strong so obviously if I break this Bond that's bad right the enthal is bad I'm losing that correct is that correct to some extent why because when I'm losing this hydrogen bond between the two this can still form hydrogen bond with water and the Serene can form hydrogen bond with water make sense so I'm breaking a strong bond but I'm forming two bonds with water so basically the net change in enthalpy is not much make sense so that's why basically they can form a hydrogen bond with water so that's why they have minor contribution to the protein stability make sense because remember Delta G is what determines if something Den nature negatively that mean Delta G should be negative spontaneous cool now what what about ionic interaction we said that when you have a solid plus and a solid minus like glutamic acid and lysine these are not hydrogen bond because they are solid ions they have electrostatic interaction we also call them ion pairs or salt Bridges as Dr Chan will cover cool these ones of course the bond is very strong it's like 80 K right well let's see the effect says minor contribution to the stability why is that let me explain it to you let's say I have a lysine a glutamic acid and a lysine right they have very strong enthalpy 80 Kil right now if I break this Bond obviously this will form a hydrogen bond and this will form a hydrogen bond right but the hydrogen bond is not as strong as a solid electrostatic because the electrostatic interaction is 80 KJ right but the individual hydrogen B 20 each correct so on the Delta H I'm losing Delta T is is is not is not supporting the folding right why the F what happen is that when I'm having a lysan and and an AR glutamic acid and Arginine the entropy here is very less but when they open they start moving around you see so basically the entropy is favorable because when they move around better entropy right in the unfolded stage but the enthalpy is unfavorable make sense so going from this stage when they are together the enthalpy is good is bad because they are stuck right but when I open enthal is bad but entrop is better so basically you are balancing Delta G and Delta H are balancing each other so you are not going to get a major difference in between the two states between folded and unfolded that's why they play A Minor contribution to stability as question so the question is that the enthropy is is is enough to compensate for enthalpy yes because remember we are not replacing enthalpy by nothing we are replacing with two or multiple hydrogen bond because even the co minus can form it's not like technically only one Bond if you look here sorry you see this one form two hydrogen bond right and this going to form one hydrogen bond so more or less yeah but good question any questions about this so you you see guys now we are moving fast if you review the the the lectures before and you review the equation Delta G equal Delta H and understand what's negative means bonding and this is just kind of following up if you don't understand this one that mean you didn't do a good job in reviewing the hydrophobic effect and the electrostatic interaction and Delta G and Gibs feere energy so go back and cover that very well and if you have a Sil question you can come and ask me all right what about disulfide disulfide bonds they are calent and they are actually import mostly for extracellular protein why because it said dulfi are rare in the intracellular protein because the cytoplasm has so much glutathione remember we said there's 5 Millar glutathione inside the cell and that will break any disulfide bond but definitely disulfide is a strong bond so if I have a molecule that has disulfide it's going to be stronger right not going to fall apart this is a real calent Bond right so technically why if you if you think about why the body kind of just evolved this mechanism where I say you know what I'll just keep DFI for the outside think about it which is a more damaging or harmful environment the blood for example or inside the cell let's say I'm drinking lemon and ginger it's changing the pH of what your stomach and if you drink so much of it by the end of the lecture you're going to have acidity in your blood right so the blood depending on what you eat what you get get stung with that's the very harmful like chaotic environment right so you want the stability to be in outside the cell right inside the cell we have so many enzymes and proteases that's protecting that inside so it's not much needed that's why glutathion would rather I'm not worried about the stability of the protein I have chapiron and other things that can take care of it but I want to make sure that I have a protective mechanism for antioxidation or antioxidants right I don't want toxic chemicals to come in the S and destroy it right but of course if we don't have oxidation or peroxides or all these things yeah why not that the body would have evolved to have dulite also inside the cell right but the the plus from just stabilizing inside the cell compared to losing a major antioxidant is not worth it so we keep it this way make sense all right now another way to stabilize these uh interaction something called metal ions and metal ion are part of something called zinc finger now a zinc finger looks like a stretch of protein you see a polypeptide that has a beta beta sheet like two strands and an alpha Helix right and it looks like a finger like the shape of it right so zinc finger is between 25 to 60 residual which is amino acid uh arranged around one or two zinc ions you see the zinc ion now this zinc ion is a positive charge so obviously it form it need to form how would it bring like you see if you look at this stretch of peptide it's like this but if you want to bring it in a in a finger shape it's strained in a way right because you are bending it and what's the stabilizing effect for bending it is the electrostatic interaction between the plus charges of the zinc and partial negative charges on two Hines and two 16s you know because they have Delta minus on them so Delta minus Delta minus on these functional groups will give you this kind of stabilized so basically it's an enthalpy stabilization of this make sense all right why the body use this one let's say for example my body I'm a protein and I want to interact with the DNA you could put a zinc finger which is my hand on on the protein and this will go and interact with the DNA you know so we use as a way of communication and we talk about protein protein interaction remember when you talk about transin and artino binding protein this is protein DNA interaction which is very important for transcription and other functional grou functional activity inside the cell and that's Dr Chan going to be discussing this with you guys okay any questions about this all right now what about protein denaturation how would you denature a protein you can denature it by heat and when I'm talking about protein it could be a biological protein green fluoresence protein a steak tofo whatever protein you can destroy it by heat most proteins unfold or melt at temperature higher than 100 and why because the heat yes the question is that is thetion a reversible or irreversible depending on how much the natur you add and there's some examples where you can return we'll discuss that but of course if you burn your Stak that's a one-way process you cannot regenerate it right but certain protein you can okay all right I'm hungry acids or bases well why acids or bases because if you change and you see it on Monday next exercise when you change the acidity or the basicity of the solution that's why we taught you pH and PKA you're going to change the ionization so if I take glutamic acid which is like Co minus right and line they will form salt salt bridge right but if I put them in the stomach or an acidic environment the glutamic acid going to be Co correct and the uh line going to be still plus correct when you have Co minus like let's go back this is interesting because prep you guys for the the exercise one at P 7.4 there's static interaction there's a salt bridge there is Ion pair but at PH2 what kind of bond you going to have think about it while I'm sipping on my Cher would you still get ionic interaction at PH2 always you have to study the ionization what's going to happen with the ionization what's the ionization of Lysine going to be PKA 9.5 right at PH2 is going to be more even ionized so going to stay the same what's the P of the side chain of glutamate four right at PH2 is going to be what two is lower than four it's going to be protonated what you going to get here you're going to get H so what happened do you get electrostatic interaction no what do you get hydrogen bone make sense which is weaker now imagine you have acidity all over the place or basicity that's going to change the ionization and obviously that's going to mess up the the protein stability make sense I'm talking about heat why heat breaks the protein because you have vibrational energy that will break the protein and then if you have heated so much you're going to start breaking the backbone of the protein and if you break it heat it so much you're going to burn it all the carbon going to be charcoal basically okay all right the last one is detergent and this is a very common biological determent called SDS and you can find it in some phace products sodium dudical sulfate if you look at this one we call it detergent because it's an Ile and we discuss with you guys what's a it has the sulfate which is very polar and it has the backbone which is very liopic this is a detergent that's why this will affect the hydrophobic interaction because you see this could form Myles and detergent it's like a soap basically okay all right now protein can be denatured also by reducing agent if you have disulfide in the molecules you can add these common uh D the reducing agents called Di or per to ethanol they will break the disulfide bond and lastly you have these two chaotropic agents they called guanidinium ion which is part of the amino acid arginine and you have Ura which is basically a byproduct of metabolism that you guys will discuss with Dr Chan both of these molecules they they affect multiple things but they affect both hydrogen bond and hydrophobic interaction and that's why when you start breaking this bonding the protein will fall apart okay now what about this real example in real life when you cut a banana or an apple you start getting this Browning this Browning is basically what happen is that there's an enzyme let's go here dopamine is the hormone of happiness you know we have a picture of dopamine on the you see if you look there the the second picture see that's the hormone of happiness and we're going to study a lot about it most of the drugs for psychosis or for depression you want to increase the dopamine even cocaine and all this good stuff that's some people and just they increase the dopamine level inside so we are seeking dopamine okay and dopamine is interesting it's a molecule that looks like there very polar but dopamine get oxidized because called the catola which we'll discuss later because anything that has a phenol or two hydroxy get oxidized quickly something called quinone and these molecules once you have them they start polymerizing okay polymerizing because this are very strong electrophiles so basically there is an enzyme that's called polyphenol oxidase there is some dopamine in the banana okay and basically when you cut the banana the fruit will activate this enzyme and that enzyme will start working on dopamine converted into this electrophilic species M sorry converted to this electrophile and what happened with why this is electrophile because you see they have two carbonal group next to each other right and then you get a nucleophile from other proteins and you get Mel something called melanin which is the the pigment that you know so you see you form an electrophile from dopamine and then a bunch of side chains from lysine and everything start binding and forming a polymer this polymer is almost like clotting for the plant this is something to seal the plant and protect it from further damage can you imagine so the banana is thinking oh cool I'm going to protect myself now don't tell me oh my God I feel bad for the banana because we end up with with nothing to eat right I mean can't say that okay can be vegetarian whatever but banana we have to live with with that okay but down the line do they feel do I have no idea but we know that they have an enzyme and that enzyme converted into a protective mechanism now how would you prevent this one by heating if you heat the banana What's happen you kill the nature the polyol ACD if you add acid you remember sometimes you squeeze the lemon in the they don't why because you are decreasing the ph and you are denaturing or decreasing the activity of this particular one right and last one reducing the amount of oxygen now this is not decreasing like the mechanism of of of reducing the oxygen or putting in the sealant is not damaging the protein is what doing what you are preventing the oxidation still you get it so you need to understand the difference between the three adding lemon juice or cooking your banana you are destroying the enzyme time removing the oxygen you are preventing the oxidation of damine in the pl yes question uh your your colleague is asking would freezing it works yes that's a very good idea and actually very interesting we should add this one but that's very freezing because you are decreasing the entic activity you are not damaging the protein right that's very interesting I mean I I yeah because I think if you remove it from the fridge right it will start very cool interesting you have question also good excited about it that's very cool good good thinking any questions about this all right so now one of your colleagues asked with the protein renat let's see what happened let's say that this is an experiment they took this native active protein and you can see it has how many dulfi bonds they y that has what 1 two 3 four so how many cines you going to have eight of course right because two cyes will form disulfide and you can cter so when they add Ura the Ura will break the hydrophobic interaction right and the hydrogen bond but they have to add the beta to ethanol to break the disulfide bond if they don't add the beup to ethanol they're not going to get the disulfide you need to know these things okay now what happen is that they unfold it but once they remove the Ure and the BET up to ethanol and added oxygen to get oxidation right they found that the protein went back to the exact thing this is quite amazing because if you look at it you have 816 they could randomly actually right why they are coming back to the same exact location why not they interact with other proteins right and form intramolecular disulfide why they don't miss up that's basically the renaturation happens for certain protein not for everything and protein regeneration okay when the primary structures intact the main thing if you look we kept the primary backbone intact if you break the backbone that's with excessive heat or excessive thing nothing happens obviously you have to keep it but also what H what what the idea from this experiment is that there is something in the primary structure that will tell the protein how to fold into the right configuration and that's the billion dollar question that I told you AI is trying to solve now right if you can predict how this will form this will be amazing because we don't have to get Crystal structures and all the tedious work just get the primary sequence and see what they've been working on it some of it is working some of it's not but we didn't T where oh my God by looking at the dinosaur genes I can translate into a protein and figure out what's the shape of the protein we didn't reach reach that one yet but once we reach it it will be a major breakthrough in the scientific field all right now how does this work how does the process how does the protein structure dictate it as I told you we don't fully know but we know there are processes of basically protein technology doesn't like me okay all right if you look at this slide basically yeah you have the unfolded so when you have let's say the body when the ribosome right the ribosome is making this polypeptide chain you can see at the beginning we start forming secondary structure and what's the first one you have Alpha Helix beta sheet Alpha Helix beta sheet two alpha Helix okay that's secondary then after that the secondary start coming together slowly till you reach almost subdomains in a way like you have like you know one polypeptide become like two balls that's what we call the molten globule okay and once you reach to the molting Global that's when you have the hydrophobic effect like I make like two small balls that are hydrophobic and then once you reach this one this is basically the folded monomer basically the two come together and that's called the hydrop obic collapse hydrophobic collapse is almost like a hydrophobic effect and why this process is very driven and very spontaneous because once I have two hydrophobic together what happened to the water I get the water out the entropy increase similar to the droplet of oil make sense so you have to imagine that these two are almost like two doubles of oil they have to come with each other and what dries the hydrophobic collapse is the hydrophobic effect which is basically the higher entropy of the water of the system any questions about this you see guys because you understand what's the the droplet of oil this becomes almost translation of the whole thing right becomes easier now that's one thing so the hydrophobic effect or collapse is a major determinant of protein structure and driving it but there's something called also molecular chapiron these are like Protectors of proteins inside the body because if you look the body is not making one protein at a time there's thousands of proteins being made within this cell and how do you prevent two hydrophobic molecules from collapsing Within each other right so the body has a lot of proteins inside the cell called chapiron they protect these hydrophobic segments of the proteins till they form this particular one okay that's from the that's not you guys that's the other lecture and once I form the hydrophobic I mean I excluded all this once I form this one I'm good the protein doesn't aggregate why because I kept all the hydrophobic segment inside that protein make sense but where's the fear the fear is at this stage right if two proteins come close to each other the hydrophobic from A and B could come together right and that's why the chaperon is protecting them that's why we call them chaperons all right what are molecular chirons are essential proteins that bind to unfolded or partially folded polypeptide right where they assist in the folding of the Native structure but they prevent the improper Association of the hydrophobic segments as I explained to you this is an example of a chapiron that's called heat chock protein 90 and you can see this this chapon has two arms flexing arms so basically if it comes close to hydrophobic interaction it comes closer to it and protect it and wait for it to form and then release it so it's like almost chaperoning and protecting that's why we call it right these are very important now obviously if I'm moving my hand Dr Al is moving his hand what moves the hand of course muscles but what driving the energy ATP I'm using ATB to move right that's why we use energy we eat sugar we eat protein to give me ATP these proteins also they use ATP to get them moving okay now these are very very important the H choke protein 90 because they required to recover heat denat proteins or to prevent misfolding under environmental stress okay when you are in an extreme heat the body overe expresses all of these heat shock proteins right because the body wants to protect the protein inside your cell right so the body will up regulate this one to protect it again so obviously if you look at people from different places on the earth you will find the levels of expression of HSP different from one place to another okay now for this particular one hsp90 is very very important in cancer and this is actually a target for many cancer medications and clinical trials as well okay why this is before we go so talk about uh before we end this this lecture when we talk about cancer cancer is basically multiple diseases not one but in many cases you have a certain protein inside the cell that's going Rog basically overexpressed protein higher amount of it start growing and then driving the cell to grow uncontrollable in an uncontrollable fashion okay so obviously you want to Target this particular protein but this protein also is present in normal cells as well right and that's what the classical example of chemotherapeutic that that will Target the normal cell and the active cell how would you get selectivity is basically the cancer cell is dependent on that protein much more than the normal cell because your normal cell doesn't grow as fast obviously when you have protein the same HSP if you have HSP in normal cell and HSP in the cancer but since the cancer is growing at a much faster rate if you block HSP you're going to affect the cancer cells much more than the normal cell so it's not going only to the cancer it goes everywhere and for that reason you get side effects for example in organs or tissue that grows very rapidly so for example the hair because oil is growing you hit the HSP in the hair follicles and all these things then you get toxicity in the esophagus because you see your mouth and grows if you cut your tongue grows quickly that's why the people start having issues with esophagus anything that you know or the stomach or all these things make sense how would you get selectivity only if you target that cancer medication to the cancer cell only and there is that called targeted cancer therapy which we will discuss also in this class later on make sense any questions about this so a normal anti-cancer Agent P tax all they target everywhere the selectivity comes from the dependence of the cancer cell more on the protein than the normal so any uh questions about this lecture guys okay so the next one is okay okay so now we discuss protein structure we discussed how the protein forms why protein is stable how the protein is unstable now we're going to start talking about why this is relevant to Pharmacy students so we're going to talk about the protein denaturation as a disease but also protein as a therapeutic can you use a protein to treat the patient and if the protein goes wrong in the wrong direction can form serious diseases as we're going to see shortly so there something called amiloidosis and protein Therapeutics let's start with amiloidosis i me this is a big word it says extracellular deposition of normally soluble proteins in certain tissues in the form of insoluble fibrous Aggregates called amids so basically in a simple way certain proteins they start forming Aggregates non- selective unfolding right and these unfolded proteins this start clumping on certain organs and causing diseases all these are amid diseases Alzheimer you have the amid beta protein and tton you have this protein Li mad cow disease you see this one pron protein all these are ameloid diseases different protein giving you ameloid that cause different disease make sense let's see an example of Alzheimer one of the most devastating neurod degenerative disorder Alzheimer neurodegenerative what does that mean that mean it gets worse over time it's a progressive decline of memory because these neurons are dying as we're going to see shortly affect about 10% of population over age of 65 and this is serious because we are an aging population we are living longer the health SC is advancing so we're going to see much more serious issues of Alzheimer than our previous generations right there's of course environmental and other factors that we need to figure out the food or diet or exercise and that's something still a big debate sadly there's no effective treatment currently available there's a couple of antibodies that came to the market the last couple of years but they are not reaching like a something meaningful it's just basically slowing things to some extent how did they start they start in actually early 1900s so basically the first kind of recorded diagnosed patient her name is Austin derer she had Alzheimer people didn't figure out that she has Alzheimer some people in the past they used to think that these people are like like touched by a by by witch or some crazy things or they go in a different word and it was crazy thoughts right but this physician after this patient died he took her brain and analyzed it and his name was Alzheimer okay so Alzheimer is the physici who diagnosed the first known case of Alzheimer okay Dr Alzheimer so when they took at the brain of of a normal patient the one that you get a normal person the one you see to the left and they look at the brain of an Alzheimer PA patient they found that the brain of the Alzheimer patient is smaller in size okay now when somebody tell you this brain is smaller size what does it mean that mean you have less cells and the cells that mostly make the the brain are what neurons right so obviously when you have less neurons they found that these less neurons are actually the memory neurons right that's why when you start death of the neurons in the brain you start losing memory slowly slowly and gets worse over time make sense so what basically what when they took sectioning from this smaller brain they found these dark species that they call at that point ameloid it's insoluble material that you don't find it in the normal tissue they call it amid because they thought it's a starch ameloid is actually the original name for Amo is a starchy material now starch is the starch a protein no starch is a carbohydrate sugar right but the name continues but when we mean what what we mean now by ameloid is a protein based disease but the word amid by itself is a starchy material because starch is not soluble in water right you know but this like polymeric sugar all right so now that if you zoom in on the molecular biology on the biochemistry of this you find that actually how does this plaque you see this plaque it's a bunch of protein clustering so the whole process is that we have billions of of of of neurons inside our body okay and on the surface of many of these neurons you have a proteins called am amid precursor protein you see this protein got a ameloid precursor protein we all have it inside our neurons inside our brain you see this is a transmembrane domain and has this so there are two enzymes in the brain one is called beta secretas and what is called gamma secretes they cut this AP and they form 40 amino acids and that 40 amino acid is called beta ameloid okay you need to memorize this beta ameloid is a 40 amino acid we all produce beta ameloid all the time constantly we keep producing beta amid for a reason that we don't know yet we have theories it could be antibacterial or something else but we we don't know the exact reason why we are forming it okay now when we are young when we are healthy we have a way to use beta Amo and break it and get rid of it but certain people they have a mutation in the beta secretes or gamma secretes that actually produce much more of the beta amid than the body can deal with and what happen when this sticky 4 amino acid you have a lot of it it start forming the beta Amino plaque so the black is the bad one not the beta ameloid okay the aggregated hydrophobic interaction structures cool certain people with aging even if they don't have the mutation why when we age we start becoming more vulnerable for Alzheimer because our protasis the cells the chaperons inside our body or the proteins they are not as efficient as before that's why we start building up this plaque over time usually when you have the genetic mutation you get the Alzheimer at a much earlier stage and it's more devastating rapidly when you get with aging a slower progression but become now obviously how would you how would you treat this disease that's something I just taught it to the p2s you know in the med cam in the third semester for them how would you treat Alzheimer based on this what would you do like you know you're going to start thinking as a mkim we give you some kind of int Target what beautiful Target the mutation so basically you could Target beta or gamma secret as Inhibitors and these are in clinical trials yes good job what else yes beautiful some way to treat to break the B Amid and that's the only the drug only drug that was recently approved it's an antibody that goes and break this and that's the multibillion dollar drug Eli and everybody's working great idea what else one last one let me give you a hand this is bad this is not bad yes beautiful you saying keep them from coagulating and that's why we call aggregation Inhibitors coagulating we call it aggregation so there's aggregation inhibitor that's the stuff I was working on okay uh when I was at stord that's part of my my research that we were finding small molecules that will go in the brain and basically preventing this from happening okay now the interesting thing about this when you look at this plaque why this plaque is so insoluble why start clumping on the neurons because they found that it forms something called beta sheet beta structures and what's a beta structure is a bunch of beta strands you see the beta strands but they start stacking in a fibral kind of form around the axis you see that if you look from the top it's like beta beta beta strand going into a fibral and become long and what happen when these beta structures are close to each other they form strong hydrogen bond and they exclude the water molecule right and when you have them in that kind of confirmation they become insoluble they are not beta sheet they are different they're called beta structures compact fibral of this C okay now the interesting thing about this one if you look at the Alzheimer it could be a random structure or it could be Alpha Helix could be beta sheet depending on the disease but all of them eventually will give you something similar beta structure so even an alpha Helix cic protein will form a beta structure and form amid so all the amid or most of the amid that you're going to find they're going to have fibrils which what they call this one which are made from beta strands or what we call beta structures remember beta structure is Bad Bet sheet is normal any questions about this all right so now another disease another Amo disease that actually we discuss today is called transin amiloidosis or a protein called based on the protein called transin which we discussed earlier okay now TTR or trans tytin is a homotetramer how many subunits you going to get four how many types one type right this protein is made by the liver and secr in the blood it's also made in the brain and we showed you earlier that this is the protein that transport vitamin A inside our body correct now TTR is the most abundant protein inside our brain which is quite interesting now we were interested in TTR when I was at Stanford because there was this theory that TTR in the brain the native form prevent the aggregation or culation of beta Amo to the aggregate okay the native TTR is basically an gation inhibitor which is amazing right so the ni was working on finding small molecule that will increase the production of TTR in the brain but that's a little bit more challenging we were working on a me method basically to bring TTR to the bind to this SP artificially with a chemical okay that was exciting idea and people were excited but the problem with this is that our molecules were getting a little bit bigger and what's the problem of bigger molecules getting across the blood bur barrier for the blood bur barrier you want molecules that are small and liopic right for us we were able to prevent this and get like extremely powerful activity in the peripher in the test tube right but obviously if it doesn't go in the brain it's not going to help and that was but at that point I was saying you know what I I me somebody would say the project failed and you work very hard like for couple of years like 24 hours a day right and you were weakend and stuff but then I thought okay you know what I started working with this protein and I designed an aay will allow me to find molecules that b to this protein and I told my adviser at that point we were working in Alzheimer it was a neuroscience lab I told him you know what I'm going to repurpose my research into something else because if you look at trans tytin by itself it's a homoa right we have it folded but if you have genetic mutations that destabilize the tetar you're going to get a lot of the monomers and this monomer actually form amid black or beta structure similar to Alzheimer you see so the tetramer has to go to monomers and the monomer will unfold and form aggregate that will go to the heart or the neurons in the periphery because this is made by the liver right so the body is making this destabilized protein floating in the body breaking apart and forming plaque on the heart so this is almost like aoid of the heart and this was quite interesting story because something that will prevent Alzheimer of the brain right when it's native and functional it forms black of the heart when this arm function was quite interesting to us now at that point in 2000 that was in 2008 2009 not many people were interested in this disease because they thought there's only 5,000 patients they thought the mutation only that's affecting it okay but what they realized later down the line that there's almost 500,000 because even aging population they have ameloid plaque on their heart and it's a major disease that we didn't know exist even we were not sequencing or goog for these people okay so the idea is that if you have this for example mutation the Val to when I ask you guys you need to memorize the three letter single letter that's how physician now talk about it in their reports right so valin to methine single mutation the person will die in the age of 30 right for a few years they get paralyzed and poly neuropathy completely shut down if you have the v122i these people they will have two three years and then they will die from heart failure why because the plaque keep building on the heart in the past how would you help them you have to go and remove the liver because the liver is producing the protein so you get the liver transplant but if you have a bad heart what do you do you have to change the heart so you have to change both but if you change the heart only the bad liver going to mess up the heart you see so the patient was a mess to get like a couple of million bucks of transplant and waiting for both liver and heart was not feasible so people start looking and you go to a village for example and we finish the story before we take a break you go to a village in Portugal where the poly neuropathy was actually very bad I mean there's a lot of people in Portugal that they have high penetrance and the penetrance basically comes to a family the family you have like few siblings most of them they will die at 30 and one person will survive then people start studying this population and you go to small villages there you know the inter family marriage is very popular so the genetic pool is not diverse right so they start studying them and they found okay why this family for example this guy live completely normal and these families most of them are dead they found that actually there are also something called called protective mutation this is a mutation that actually stabilize the protein and this particular mutation actually when they study is called ponine to methine at position 119 one change you see made this protein even more stable than the normal protein so if you are normal let's say you live 70 years old if you have one of these mutation you die in the 50s or 30s if you have the t119 and you get lucky you live 10 years longer than the normal population it's even a protective effect than the normal population yes again the prevalence why it's higher in those populations because you said about heat the heat thing molecules earlier is more it's separate levels from each ethnicity so that's also causing the problem right but now that we now that uh people are living longer than there's people with the mutation are also now exist existing more yes so basically if you have that these D stabilizing mutation like the v30 and the V1 122 they make the Tet weaker so they fall apart more so you get more of the monomer and you have too much more of the monomer similar to amid Beta start forming plaque but this population what they are very rare that's stabilizing they found why some people live longer even their family died earlier by random they found that this is actually stabilizing and very few people less than 0.1 whatever perc in that population they actually live longer than the normal population what happened here is that when they St it and there was crystal they found that actually this mutation does something one hydrogen bond extra can you imagine between the red and the yellow and between the green and the stuff this one mutation there are two serin Moes you see serin what's the side chain of serin oh right they are far away almost like four angr can they form a hydrogen bond no because we said the hydrogen bond is three irr right this mutation actually bring the serines by a couple of anrom and then they can form a hydrogen bond this hydrogen bond make the protein more stable than the wild type people live longer see the power of a hydrogen bond is just couple of angstrom make you live longer or get screwed basically in this kind kind of life that's where genetics is extremely powerful right so now what happen is that scientist start saying okay can I artificially stabilize the protein by adding a small molecule because technically small molecule how they B to protein by F bonding right and that's where the therapeutic approach for doing this is can we find something that's similar to the molecule and that's something we will discuss after the break now it's 1050 we'll be back at 11: yeah sure sure um for lecture two and when the D nature becomes umed again is the function 100% yes when okay yeah but not for everyone for not um and then in terms of like e e e e e e e e e e e e e e e e e can you guys hear me okay can you guys hear me okay on the back oh okay good all right guys we're going to start now okay ah so we said that we want to find medicine or drugs that will bind to the protein and stabilize it because if the protein can stabilize itself with a single hydrogen bond we can do something to mimic that so uh this was L by a scientist at scripts Research Institute in La Hoya in San Diego and the molecule that bind to the protein is called teamus Theus was acquired by fiser and in 2019 was the first drug approved for the cardiopathy part of this disease you know this affect the heart and this was actually the first drug that's called aggregation inhibitor okay because inhibit this drug teamas bind to the protein and prevent the step from going to the amid that's why it's called aggregation or amiloidosis inhibitor now this is one of the most expensive cardiovascular medicine in history it costs about 220,000 a year and the patient has to be taking this every day for the rest of their life okay because the body keep producing TTR every day every two days the half life of T is two days so the body keep producing and you have to keep stabilizing it with this molecule okay now when we were working one of the molecules I discovered when I was at Stanford this molecule called A10 A is for alach and G is for gri which is my colleague at Stanford and this is the picture of the trans tytin protein you see and that's a picture of how two molecules if you have two active site we call it two molecules of AG 10 this is a zoom in picture here and you see this is the skeleton format of AG 10 this is how a of 10 binds in the pocket of the protein okay so if you look here you have two molecules this is how teamus Works binds and this is how a 10 binds when we looked at The Binding stability we take this and we see which one will stabilize the protein more we find that teamas stabilize only 60% of the protein in blood because you know this protein is a blood protein is transported so 60% for tus but when you look at age 10 it was 100% stabilization that was quite interesting and we didn't know the answer until we get the crystal structure which is the stuff that we have here so if you look at the crystal structure you see the carboxilic acid of h10 at physical pH is going to be what Co minus correct and then when you look at the structure you find two lysine residues so what do you have when you two have two lysine from identical unit and a carboxilic acid you have two ionic interaction or two Salt Bridges make sense but what the interesting thing is that if you look at AG 10 has the perzo the two nitrogen and you see the two nitrogen are forming hydrogen bonds with what Serene two Serene Moes these are the two serines that form hydrogen bond in the mutation the t119 M remember guys I told you theine were far away so basically what age of 10 form two strong hydrogen bonds two point8 angstrom so artificially instead of the mutation forming the two sering together a bridging them in this kind of structure make sense so we call Ag 10 unique and 100% because it mimx the mutation make sense which is was quite intriguing to us now if you look at teamas it has a carboxilate but the problem the car which is shorter theous or AG 10 you see theous shorter right so the carboxilic acid was not long enough to form electrostatic interaction with the gine that's why it formed through a water molecule you see the Red Dot so the water is act so it's not a strong electrostatic interaction yes what is that oras no even if they did that chain extension you change the drug so it doesn't bind anymore one change of a carbon or any molecule is not a drug anymore it changes everything right could be better or worse so this Bond was very weak but that's not the major thing where is the strength that we are getting from the hydrogen bond here right which is the one that makes the mutation now if I ask you can do form hydrogen bonds with the Serene at the bottom does it have noof or NOS that we discuss no so the F cannot form hydrogen bond at the bottom because it doesn't have nitrogen or oxygen or fluoride or sulfur right the famus has two chlorine and two chlorine are hydrophobic so the hydrophobic interaction is not as strong right so it's entropy more than enthalpy okay so we are inaly driven binding very is strong and the interesting thing about this one is basically you have to think if you want to stabilize something you want the enthalpy right because basically if I take age of 10 it's grabbing the two tetramers away from the top and grabbing them from the bottom right that's why it doesn't fall apart but if you look the Famas it's just weaker interaction at the top that's why we're getting 100% And they get 60% okay any questions about this so now obvious ly we started this and we realized that this molecule has a drug- like properties it's orally available it's relatively nontoxic this journey took more than 10 years right in 20089 we started 2013 we published the molecule it took us about three years to convince people that we have a better drug at least in vitro because we didn't have clinical data and nobody wanted to fund us I mean for a while everybody's saying what the heck you are going against fiser and there's 5,000 patient only now fiser they said you know what they told the government I want to make a billion dollar because I bought the company for 600 million right and you have 5,000 patient 200,000 person you get a billion dollar but what they realize down the line that there's hundreds of thousands so everybody start working on this and in 2016 we get somebody who fund us actually to continue because the market become bigger now you expect the market 10 to 15 billion which is like enormous and the sale from f is five billion which is like unheard of for a rare disease so we I started this company called idas Therapeutics and idas basically you can just read about it is it took our molecule we went public and basically started the clinical trial this is me and my daughters and my wife we went to NASDAQ that was 2018 so and you can look at the video is interesting you know because like you see you go to NASDAQ and they do all this like of shows and acting this was a milestone because this basically allow us to get the funding to do the clinical trial clinical trial cost hundreds of millions of dollars at the end of the day we started in 2016 finally last year we get phase three completed and successful and actually we are expecting approval in November of this year so before you guys end you're going to see some hopefully something that's going to come out with u with a major it's very very rare to have someone in Academia developing a drug you see so I think this is something we are super excited about it and if it works it's going to make a big difference I believe because the data that we have it just cut the mortality and the cardiovascular hospitalization by 50% which is huge for the these patients you know that these patient two three years they are dead and we have some people on the drug that they've been living for The Last 5 Years and by doing like this to be honest with you look at it is just the reward that you hear we did a study in Japan like for smaller in the US it was 600 patient but in Japan like 90 people and after five years all of them are alive and you look they said seriously I mean this is like the impact of it of course the financial is a different story right but the impact when you change human life in that is something crazy but for for me I didn't even fathom it yet because not approved yet but we hope that it will be so we want your prayers the other good thing is that when there's another drug coming to the market is going to drop down the price competition is always good for the patients right now the government is paying the 200 nobody has 200,000 right the government is chipping in and fiser is basically you have to have copay only I think few thousand bucks okay all right so now this is the story of of of TTR but we learn also that there's other proteins that have similar mechanism and that's an example of a protein called Alpha cicin and thus A protein that form ameloid but where in Parkinson's disease you see but if you look if you look at TTR structure is it beta sheet or Alpha helis Beta sheet right the beta strands are not but looks beta sheet correct so this form Amid and if you look at Alpha culin is a homotetramer also but it's what Alpha helis so if I break pul how many I'm going to get four subunits one type cool and this basically what this is interesting video from big guy at U Harvard who was very famous in the amid field how do you get that I know there's a way that you can get the voice okay guys you look at the sub caption assume that you are watching it with your father and you don't want to to listen or mother anyway so long story short let me tell you what's the thing you guys can watch it again me see here on there okay so okay yeah okay so basically they thought that this protein the alpha cic is a tetr but they found that actually to be able to form ameloid it has to go into monomers and then the monomers will form amid make sense so same mechanism what TTR has so the idea what they were saying how would you find a therapy for this Alpha cicin you find need to find a molecule similar to A10 and I apologize for this but you guys can watch the video okay basically you need to find a molecule that will stabilize the tetar form this tetar is not toxic only the monomer has to form ameloid and the basically the ameloid is basically what become toxic Mak sense but the interesting thing is that the structure of the ameloid that comes from this protein and the structure of the amid that come from TTR is very very similar they are all beta structures make sense so TTR is beta strands native Native beta strands form beta structure amid this one an alpha helical structure but form beta Str at the end cool that's what we meant that beta structure could come from a native alpha or a native beta make sense any questions about this all right okay so now we gave you protein structure then we discuss protein stability right and then we talk about protein disease like when a protein basic such as TTR a native protein goes Rogue and basically form amid or when you you have basically a protein such as ancin right could form disease the last part now is that when we use protein as therapy as molecules that will cure disease now this started with the famous insulin one of the first few examples of polypeptide that was used as a medicine insulin is about 50 amino acids so some people call it polypeptide some people call it the protein technically above 50 is considered protein lower than 50 amino acids is called peptide okay so technically insulin is a peptide now in the past when people have diabetes they were extracting insulin from animals from pigs from cows and they will inject it into the human right because that will lower their insul but that's a little bit consuming time consuming and costly and everything right so the recombinant DNA technology which started at UCSF and Stanford in the 70s and acquired by genetic which become a major company in the Bay Area for you know protein Therapeutics now genetic recently was acquired by R so it's part R phac skills they found a way basically to do molecular cloning and genetics to produce this and basically in a much more massive scale and that kind of open the field for antibodies and other Therapeutics as we're going to discuss shortly now how does the recombinant D technology CA molecular cloning this is different than cloning a human or a goat or a sheep or whatever right this is a nice interesting link for people who are interested what's the difference between cloning a human and cloning a gene let's say I want the gene of insulin I take the sequence of the gene of insulin from the human and I take the DNA and I put it into a vector it's called the plasmid that's something that will allow me to transfer this gene into bacteria or something that grows rapidly so if I take the Gene and I put it bacteria the bacteria will grow the gene so rapidly because you know bacteria can grow in hours right faster and when the bacteria grows it start producing a lot of this protein I can extract it from the bacteria or the cells that we are talking about Mak sense that's the molecular cloning we are talking about and Dr Chang is the expert in this he he does a lot of this kind of work with meleon cells or equoli Etc now when you look at protein Therapeutics the classical molecules that most of the stuff you find it in a in an outp pharmacist are molecules like A1 and teamas you see their structure looks like a chemical organic molecule nonnative we don't have the structure of aen inside our body oramas these are chemicals we make right novel chemicals that nobody ever made before okay but proteins because they are native they are just every protein Therapeutics is the same composition 20 amino acid different combination different size right so the advantage is they have the high high diversity because again 20 amino acids different length you can have billions of variation right they have high specificity why because there have so many side chains and they can form hydrogen bond electrostatic right they have lower toxicity why because they are innate we are proteins we are made from them right a has a Florine as you know perol we don't have perol inside our body right but they are faster clinical development because you know again the toxicity profile is very predictable the metabolite also are very predictable you break it with an esterase right you don't for form toxic or hyperoxidized protein species right however the disadvantage they are poorly Oral B availability why they are protein they get chewed chewed up right by the stomach by the proteases they have short EnV of half life we are we have a lot of proteases inside our body to protect us from other things but mostly to control the function of our enzymes remember what I told you why the body picked proteins as a control for hormones because you can control the hormonal activity by breaking that enzyme quickly right yes so itly blood and prot it I don't know like based protection yes so your colleague is asking that mean you saying if you want this to work you have to put it directly in the blood and put something with it to make it more protected 100% And that's there's a section that we going to talk about which is exciting good thinking so you see I like this I mean you your colleagu is basically saying what's what we can address this problem and that's what we want from you guys I mean to have to start using the brain for what to expect and then when you see it it become makes sense yes sure is your colleague is saying wouldn't be putting it in the blood directly would be costly yes so when we're talking about insulin you cannot take oral insulin you have to take it in the past used to be but you can get subq so subcutaneous is more or less closer putting it in the parental system so most of these Therapeutics are parental Administration IV or subq you don't find antibodies giving oral because if you if you have a very expensive antibody and you put oral it's like eating protein shake right and it's going to be chopped up right you're going to waste it cool so all this is basically has to be parental Administration and there's a huge field of making insulin or analoges that are oral because this will be amazing for people you don't have to poke yourself so many but the challenge is it's very polar so it's going to get poor penetration across the gut gut membrane but also stability but they are working The Big Field good good questions short in half okay so now when you look at these proteins you have insulin which is basically these are three monomers of insulin you see they are alpha helicus everyone is about 50 angstrom and what's the molecular weight 6 kilodalton because you multiply it by 110 you're going to get six Kil look at this protein it's called pigrum basically in the body we have a hormone or a protein a protein sorry called granulite Colony stimulating Factor we all have it this is the one that produce in or M induce the production of wide blood cells so for somebody who's going through cancer therapy their white blood cells goes down so you give them this analogue that's more stable version of the natural hormone right natural protein and this basically will produce more more white blood cells for these patients to increase their immunity and you can see the size about 18 kilo you can see the shape the size is different tells you how the size and the shape could affect the biological function if you look at antibody the examples the immun globin G these are the largest protein inside our body 150 kiloan antibodies is one of the largest if not the largest proteins right that are basically soluable protein and you can see because bind two things as we're going to discuss it has the y-shaped conformation cool now albumin is the another protein albumin the size of it is about 60 kilodalton TTR and albumin they are very similar size about 60 kilodalton so for reference okay so now since antibodies are the most clinically tested now in the clinic anti-cancer agents you have so many clinical trials of antibodies for various cancers why because because it's very easy to develop them to have specificity as we're going to see so definitely we need to study their structure this is the immunoglobulin base you know they have different type of antibodies right but we are talking here about the immunoglobulin G basically structure so if you look at the antibody it has two portion called the variable and constant and we will explain it in details antibodies they have a y-shaped conformation they have two light chains these are the one in brown you see these are two light chains and they have two heavy chains the ones on Blue so if you break this one you're going to get four unit this is almost quaternary structure right you're going to get four units two types one type of light and one type of heavy how do they interact with each other with hydrogen beta sheet everything in addition to that they have D sulfide bonds and why there sulfides allowed because most of these are in the blood looking for antigens looking for chemicals that are damaging and toxic to us make sense so you see if you look at the light chain every light chain has two domains remember when you talk about domains it's the same polypeptide chain but like almost glob has two domains called the variable light and the constant light you see if you look at the heavy you have the variable heavy and you have three constant constant one constant two constant three make sense so basically if I look if I have have for example I'm the y shape antibody only the by hands are the variable the rest is constant so all mean all antibodies in the body they have the same constant the only difference is that they're variable which matters right because if I'm grabbing things I want the legs and stuff to be the same only I change the hands so I can grab different things right that's why what you call so you see you have variable light variable heavy but then constant one constant domain but you have three constant for the heavy chain make sense all right any questions about this all right now within if let's say my arm is part of the variable all what matters when I grab things is my hand hands right so the tip top of this every variable is called the hyper variable region or the antigen binding domain and that's what matters that's why the the variation happens at the top service of the variable light and variable heavy both of them cool so these ones are called frag anti fragment of the antibody B binding and this is fragment constant FC constant is constant because constant among all antibodies are we okay with the structure so far any questions Okay cool so now if you look here this said these constant or the legs for me right here they bind to something called neonatal FC receptor this is a receptor that you have it on many cells many cells they have a receptor that can grab the the legs basically or the constant right and the same receptor actually bind to Albin you see the Neal FC receptor also bind to Albin what's the advantage of this if I take the antibody by itself you will have a half half life of one day one day is long because you know most pepti like few minutes right but usually when they look at the half life of an antibod is 21 days three weeks why is that because basically these antibodies and Albin the same thing they bind to the unal SV receptor and they get protected inside the cell they keep recycled inside the cell you see so basically my hands will grab the cell the cell will take me inside protect me for some white and release me rather than allowing other proteases and enzymes to break me and make sense so the Nal a receptors are receptors present on many cells inside the human body they bind to the FC part of the antibody of all antibodies and they bind also to Albin human serum albumin and they prevent their excretion and degradation that's why the half life goes from one day to several weeks about three weeks typically could be two weeks any questions about this okay now it's very important why is that because if you look I told you Albin is the most abundant protein you have a concentration of 600 microl right if you combine and about 60% of the human blood proteins are Albin if you combine Albin with IG is about 90% of the serum content that's huge so you see the body is smart say I'm making something that 60 kilon something that 150 kilon right multiple subunits very expensive material right and I'm just producing 90% of the plasma because I need to protect it I need to use it for functional activity to protect the body Albin is the protein that transfer many of the drugs that you guys going to be discussing because it's hydrophobic so hydrophobic molecule going to stick to Albin and get stays in the body so for that reason we de the body evolved to have the Neal FC receptor to prevent this expensive material from being exed and degraded quickly make sense any questions about this all right well now when you look at monoclonal antibodies these are very more than 500 approv or clinical development the number is much higher than now it's just they're exploding so much and most of them are based on the immunoglobulin G and you can see here you see can see see the the light which one the light the green you see and the variable is this is the variable light constant light and then the heavy you see one two three four right four domains same okay so now let's say that you know I have a cancer cell and a cancer cell has a protein that you know only in the cancer cell and I don't want to give it like I don't want to hit it with a tax Soul or HSP inhibitor I want to give an antibody that will block block and attack this cancer cell so if this cancer cell has a different protein than others I can technically develop an antibody to attack this right correct this this receptor so what happen is that what people do they will take this anti canc the protein on the cancer cell and they can immunize right a to produce the antibody because you don't produce the antibody you cannot produce it in human obviously because that's unethical right and dangerous so if I take this protein and inject into a mouth I'm going to get an amazing antibody it's called murine right this will be perfect this has all the pieces that but can I give this antibody to human it will bind to the cancer receptor right and block it and kill the cancer cell can I give a murine antibody to human now if you have a peanut allergy what does mean that your body will recognize even a penis tiny bit of the penis and make crazy thing what's an allergic reaction or immune reaction is that you are reacting to a foreign subject that's not new to you right and when you inject something we don't have urine antibodies inside so the body GNA have an allergic reaction you're gonna cure the cancer but you're going to kill the patient from a severe immune reaction make sense so what you have to do you have to make a human version of that right you cannot generate the human directly sadly if you immunize a mouse you cannot IM you can you can generate it if you give to human right and that happens when you get the old V vaccine vaccine for influenza remember when you get the flu vaccine they will give you like deactivated part of the virus and then the human body will produce the human right but that one is just deactivated or dead virus is okay right but you cannot experiment with new cancer proteins make sense so how would you make this they said you know what you just said that what matters is the variable right the constant is the same so why don't I take the variable from the urine and I attach it to the constant and that's why I get chimeric so when you see chimeric is about 70% humanized that was the earlier stages when we were still new in the technology but the technology improved and you said you know what why do I need to care about the variable because what matters is the hands right the hyper variable and why don't I just take the sequence from here I can genetically know it right by structure by Etc and just stick it to the rest of the human and that's why you get human so when you wear the word humaniz it's 90% human 10% yes excellent the question is that would you get some allergic reaction this yes you get allergic it's not 100% because you get allergic to this small part right so that reason we generated now that the technology move to get fully human and there are two technologies here the idea said you know what when you give we can we can just immunize the mouse and that's fine because we can isolate the antibod St stud it and then give it to human if it's good so the way if I take the gene for the IG from human and put it in a mouse so the mouse will be producing human IGG right and that's what's called transgenic Mouse platform so you take the gene of IGG from a human and you put it in the mouse so when you add the antigen to Mouse it will produce a human IG okay make sense and that's what's called transgenic Mouse and then you get of course a bunch of proteins and you have to isolate and purify but for me it's just this is kind of the level that I want you to know if you need more information Dr Chan or you can Google would be important okay another way is that said you know what the human beta cells has different combination of variable you can just basically study this because the body is ready to make any antibody you see when you get allergic to any new material quickly the body produce these right but we cannot depend on that what happen if we take millions of combination of the hyper valuable region and try to express it and see which one will bind to the protein of Interest right now that's a little bit challenging to do it in a test tube or in in a bacteria cell but we know that there's something called bacterio fages These are viruses this viruses they whatever you put inside them they will Express on their surface and it's called bacterio fages right and you can grow them so rapidly so basically you can use a technique that Dr Chan also uses because he's the molecular biologist I'm the chemist basically you can allow these viruses to make millions of viruses with different hyper variable right and basically what you are doing then after that you screen which of these millions of viruses will bind to your protein of interest and that will allow you to get the hyper variable region and what you do after that you take the hyper variable and you stick it to the rest of the anti Mak sense so this basically is almost a high throughput screening but it's not like a chemical you have to do like you know robots or whatever it's like done in a t in a certain molecular biology approach that's easier to do Mak sense doesn't make time yes yeah your colleague is asking which is preferred to be honest with you I don't know personally I think both are used but but that's something probably you could Google but I'm not the expert to ask yeah yeah but yeah you can just probably Google it yeah okay any questions about this okay now what are the limitations for antibody Therapeutics EXP expensive because again the process of generating with either takes time limited to cell surface remember these are very large molecules they are not going to cross the the cells for most of the cases so they're going to Target receptor on the surface of the cell not orally available they are proteins poor penetration across the blood brain barrier large some of them they do but because they're very big it's difficult to cross the blood brain barrier and large side does not get to tumors very well now when we are talking about a cancer cancer is not one cell cancer is like millions or billions of cells on the top of each other right that's called the tumor tumor is like multiple layers so when you have an antibody the antibody comes to the mass and start binding very effectively and killing the cells on the surface but the problem is that you cannot go deep into the tumor because the tumor is like has a lot of blood and less less of oxygen and it's very viscous material right so it's very difficult for a large antibody to go three deep and cure the solid tumor very well that's why many of the anti-cancer antibodies that work very well work for blood cancers but for solid tumors the success is limited yes it's also because cancer cells have variation structure it can't uh UL that cancer C have have more more variable structure no in terms of yeah they have variable and that's why we're targeting like let's say they have a different receptor and that's why targeting this but I think the penetration is mostly in that when the the cancer grows it doesn't grow like a normal cell it grows in an unorganized way and you have multiple layers and then there is limitation of the blood flow the oxygen so it's very very viscous so the antibody has to diffuse to get to the core of the cancer and kill it so you have a lot of dead cells in the surface it's more complicated physically yeah yeah good all right so now how can we make these antibodies even much more powerful is that because you can see I told you there's an enzyme there's a receptor the antibody will block it and block its activity right but can we make this even more so there's something say you know what okay let's say that this is uh a cancer cell and this is a normal cell the cancer cell has this hair two protein okay the normal cell doesn't the normal cell doesn't have it okay so if I take an antibody the antibody going to bind to this it's not going to bind to the normal cell which is good right but what happen if I attach a toxin something very toxic to the antibody antibody will take that toxin only to the cancer cell that's called targeted cancer therapy make sense you cannot take that toxin which is basically this guy ridden by itself if you take this guy by itself it's going to kill both normal cell it's going to passively diffuse and kill everything but when you attach to antibody the antibody will take it only to the cancer cell that has that receptor and this is another big feel that if you work Hospital you're going to find a lot of these in the impatient because these most of these are IV administered makes sense and it's complicated but for somebody who's dying they can go and right take this the risk is versus reward this called antibody drug conjugates okay and one example is basically genetic have this drug this antibody that Target the hair to receptor what they did they put this very toxic agent and they put a Linker now it's very important that the Linker has to cleave inside the cell once this antibody goes and bind inside the cell the antibody doesn't passively diffuse the antibody will bind to the receptor and then the receptor will Endo side size okay yes beautiful if this breaks in the blood that's going to be massive toxicity and that's the problem of many adcs actually they cause like toxicity of the blood cells and white blood cells Etc so now you can have a protasis that will be break but what else like if I ask you there's something present inside the cell but not outside that you could use it as a Linker there's something inside the cell that you can use it for targeting remember this is something inside the cell but not outside we talk about of course there's a lot of things inside the cell yes not Char protein but yeah part it is a protein it is it a something that deals with the protein something I said something a lot of it is present inside the cell something that has five Millar of stuff inside the protein inside the cell glutathione okay good so you have glutathion inside the cell what would you design the Linker so that it will only only break inside the cell what would you put between the drug and the antibody sulfur not sulfur what yeah close what you put dulfi Bond so many of these adcs they have a dulfi bond because inside the blood you don't have a reducing environment the dfat is stable right but once the antibody with the dulat go inside the cell the glutathione will break it so disulfide are actually linkers in drug targeting and delivery make sense cool all right another example is that let's say that I have protein of Interest this is a protein and I want to make make it stay longer in the body what I can do I can use what make the antibod stay longer is the FC the legs of the antibody right the FC what happen if I put this protein of Interest attached to an FC right it will bind to the receptor and it will increase the half life of this Protein that's how we make to answer your question that's how we make things stay longer in the body right and one example is this called FC fusion protein you see the FC from the general constant I attach it to this protein and that's for tumor necrosis Factor one example is called itan recept used for romatoid arthritis let's look at the mechanism of basic mechanism of rato AR you have the tnf receptor this is a receptor that bound to the cell and you have the tnf cumor necrosis Factor Alpha is a li the protein that binds to the receptor and then Downstream effect will cause romatoid Al aritis so if you want to prevent romatoid arthritis what do you want to do you want to prevent the tnf Alpha from binding to its receptor cool there are different ways but one way if you take the receptor and make a decoy receptor like make an isolated version of the receptor you see that's called a deco receptor that Deco receptor will grab this tnf but since this Deco receptor is not attached to the cell membrane is not going to give you biological function makees sense so I make a fake receptor basically that will take this one and prevent it from binding to the real receptor that's why we call it decoy receptor did you guys get this one any question about this okay so that Deco receptor basically is what they have here basically right this is this one will bind to the tnf Alpha and the FC will make it stay longer in the body and then prevent it from binding to the actual tnf Alpha that's why you are shutting down this process and that's why this is a drug for romatoid arthritis okay now the last example says some proteins you know sometimes it's very difficult when you when you get the sequence you get the hyper variable region right and you get like you know the variable is in the fap you get the fap which is the one that bind to the antigen and you try to put it in an FC region but there is solubility or stability issue because not everything will fit I mean you can't put the the hands on every leg right in that case you need to find a creative way we said that for any protein inside our body you can have prot is you can have enzyme that will break it but also you have renal filtration and we said anything that less than 30 kilon 30 Z would get filtered through the kidney right so if you look at the Fab Fab is smaller in size so technically if you take just only the variable region here right from this antibody it's going to bind very well to the receptor and it's going to go deeper in the cancer cell right but that's the problem with that it's not going to last in the body so how I'm going to increase its size to avoid that threshold of the filtration I add something called polyethylene glycol it's a polymer now you put hundreds of this and this one you put in the water 12 so basically I have like the FP which is the size of this guy and I put a polymer the size of my hand so this becomes too big and avoid renal filtration so basically pulation is another approach to increase the size of proteins and make their half life longer by lowering their renal filtration any questions about this just okay a fra fragment antigen binding that's the fraction that bind to the antigen FC the constant that's the one that bind to the receptor and increase the half life how's my voice doing still good oh thank you thank you all right now we discuss different mechanism how to increase the serum half life of proteins we said that FC fusion protein we give you what FC right we discuss conjugation to pig or they call it pilation we also sometimes you change one amino acid if you take insulin it's a 50 amino acid insulin still can get tral filtrate but there's proteases that break the insulin okay and that will terminate the action so they change the structure of insulin to something called lantis would change the solubility but also PR vent protolysis okay and that's why lantis you can take it every 24 hours but regular insulin you have to take three times a day so you can change the amino acid where the proteas break it put stck bulk or charge to prevent the proteases lastly you can have glycosilation glycosilation is adding a sugar to the protein if you look at arthr perotin this is a hormone or A protein that will motivate the production of red blood cells inside our body but arotin is not very long acting so what do you do you add more sugars on the surface of the protein and that will cause this analog called dartin which will increase the half life why sometimes not about of course you increase the size but sometimes when you have an ant a protein there's antibodies that will attack it and the nature thing but when you have more sugars on it you protect it from even normal proteases and normal antibodies as well any questions about this yeah yes so basically tnf Alpha is a natural Lian that bind to the actual receptor and cause romatoid arthritis one way to do it is that you bring a decoy protein similar to the receptor that one is called this guy okay this one alpha helic structure that looks similar to the receptor itself but it's not attached to the to the cell right and if I give that one it will be floating here with an FC if I give this guy here you see you see this alha helical structure is identical to these things but it's going to be floating here and then the tnf going to bind to it and basically you block this step make sense that's why we call decoy it's like a fake like to to pull it away from make sense did that answer your question okay all right now the last thing basically is uh what happened with small peptide small peptide they're going to get renal filtration that's a major issue so what you can do you can there's some insulin that you can put fatty acid on them right so there's glps and Insulin what happen when you have a fatty acid fatty acid will bind to Albin you remember guys when we talk about Albin we said Albin has a lot of fatty side fatty pockets and what happen is that albumin the size of Albin is 60 kilodalton so if you take a 50 amino acid which is 5 kilodalton right and you put a fatty acid chain on it it will stick to Albin and then it prevent Ral filtration and that's why the half life becomes longer and many of the drugs inside your body they have longer half life because they also latch on Albin which will prevent their metabolism and excretion you could also cyclize sometimes you know you have you know all the amino acid polypeptide we talked about are linear right n Toc if you take the N Toc and cyze it you get also more stable peptides and the last one is something that you know we develop in our lab and that's what we've been working for the last few years is something that we are using reversible uh TTR binding the problem with Albin let's say the current technology is only Albin but Albin you cannot use it for everything because Albin sorry the the fatty acid conjugation sometimes when you put it to certain proteins the solubility decreases for for insulin it's a lot poly you have 50 amino acids so even if you put a C12 carbon it balance it but if you have like a 10 amino acid right like GnRH you cannot put like a 15 carbon because that definitely is not going to dissolve right so for us we thought okay can you have a hydrophilic can you attach a hydrophilic molecule to to the peptide that's a bad idea because that will make renal filtration faster but we found a very creative way so the idea that we came up with if I take this small peptide this is an anti-cancer agents called gonadotropin releasing hormone the half life is like few minutes okay so if I take the protein the blue one by itself it's going to be chopped by proteases and going to be Ral filtered right it's like one kilo Dart in the size of it cool what happen if I attach this peptide to a 10 remember AE 10 the one I told you for therapy what happen inside the blood I make a dumbb basically I take the GNR will be secret and blocked what happen if I attach it to AG10 which is my hand inside the blood this dumbb going to bind to transitin non-covalent right and what happened what's the size of transitin 60 kilodalton right so I'm moving and changing something that's 1 kilon into something that 60 kilon right but the good thing this is temporary this is non-covalent and we design it in which this you have one nanomolar binding to the anti the cancer receptor but only 300 nanom to TTR so when there's nothing it's binding to TTR but once it gets closer to the Target the tug of war is won basically by the receptor okay but obviously if this goes by the the kidney this is not going to go so this is something that we are very very excited about and this is something we we we have a lot of Grants and Publications on this is a very very cool technology the beauty about this thing is actually AG 10 is very polar so we are maintaining the polarity of this molecule you can give it as solution and I think this will make be transformative so we're waiting to for this to be applied to many other things as we going to talk about later okay so I think for today we'll stop at this stage because the last part of this lecture is is talking about you know covid antibodies so we'll see you guys on Thursday