 Welcome to today's lecture. Now we have given you glimpse of how to determine the peptide structure and you have some idea like why it is important. Next is like what to do with this structure is known. So if you look at the whole drug industry is after getting a target for protein because you know proteins are workhorse for our body. Anything and everything that you imagine is done by protein. We are walking done by protein. If we are our heart is beating it is all protein action. If our eye movement eye movement is happening because of protein. So your oxygen is carried to different position it is by protein. Protein is very important target and therefore whole pharma industry is looking for target. One target that can particularly affect a protein which is corrupt in a disease case. So therefore the protein drug or protein ligand interaction becomes very important because proteins are a proteins are communicator, proteins are workers, proteins are everything. So protein ligand interaction is very very important for the pharma industry. But pharma industry actually is generally does not work like our lab. So in lab we can take one thing and go in detail and study very like slowly to what function is happening. But pharma industry many times requires a quick way to find it out whether this molecule will work or not. Should I invest so much time, money and energy in identifying those molecules. Therefore a high throughput high throughput process is always desirable for pharma industry to find it what is what will work and what will not work what we should discard it. So NMR over the years has emerged as one of the important parameter in drug discovery and today we are going to monitor this aspect of NMR spectroscopy how you can use the NMR in drug discovery especially suited for pharma industry. So as I said it is all about protein drug or protein ligand interaction. Protein is bigger molecule ligand it is smaller molecule how we can understand if this ligand interacts with a protein and suppose we are giving a soup of ligands which ligand will interact with a protein. So that we can take this optimize this make a better efficacy drug. So now we got all the different parameters that can be used to identify or to monitor the property of ligands. So here NMR can monitor different physical property that can exist between a protein and a ligand and that can be used for finding a better optimized ligand in drug discovery protocol. So let us start as we said ligand is a small molecule. So small molecule means it is a slowly relaxing molecule and if it is slowly relaxing then its diffusion is very fast. So in solution it is tumbling very fast. We had previously discussed small molecules that means it will have positive Hanoi and then will be like there are not too many protons here. So spin diffusion is also going to be very very slow. So we have a negligible or negligible spin diffusion whereas protein generally this is many KD. So this is say few Dalton 1000 Dalton or so. So it will be 100 kilo Dalton. So proteins are bigger molecule. So bigger molecules that means they have a fast relaxation and since they are big so they will be diffusion very slow in solution. They will have a negative Hanoi bigger molecule if you remember our previous classes we have discussed that big molecule will have negative Hanoi and then small molecules will have positive Hanoi. And there will be lots of spin diffusion because the proton network is very dense and that is how spin diffusion will be quite a bit. So now suppose this ligand interacts with protein and make a complex. So now ligand is small protein is big. So the resultant complex is going to be big. So if this resultant complex is big we have a fast relaxation it is similar like a protein. It will have a slow diffusion because molecule now overall molecule has become big and it will have also negative Hanoi and it will have fast spin diffusion. So it will acquire all the property of a protein molecule. Now what we are going to do is a concept that we have discussed earlier. So if you remember we have said that if two spins are somehow interacting through a space you put out this spin the effect of this perturbation will be seen on that. Like polarization transfer we had discussed earlier. So similar concept is going to be used in something experiment called saturation transfer difference NMR spectroscopy or it is in short form it is called STD, saturation transfer difference. So what we are going to do? Suppose our ligand which has some protons and protein which has some protons so chemical shift of this proton H is delta A and this chemical shift is delta B. Now if suppose this is binding in a micromolar range and it is in fast exchange so ligand is binding going off in a microsecond time scale or fast exchange. So that is the complex that we are going to do. So now suppose we do some experiment as some trick suppose they are two are interacting and we saturate selectively the signal of this. Saturate the signal of proteins the effect of that saturation will be seen on the chemical shift of ligand. We are saturating protein signal we are seeing effect of that saturation on ligand signal if these two are interact and this precise concept is used in a technique called saturation transfer difference. So now saturation will be transfer if they are in exchange and they are in proximity if they are not in exchange or if they are not interacting they are not in close proximity then effect of saturation on protein will not be transferred on the ligand and they will be not interacting. So that is what we are doing. So the KD of such kind of process should be in micromolar and size difference between ligand and target should be at least 15, at least 15 fold. What I mean if your ligand is say 1000 Dalton your protein should be more than 15 to 20 kilo Dalton if it is more it is better otherwise the effect will not be seen. So one KD is ligand and 15 to 20 kilo Dalton each protein then we can do these experiments which is called STD and binding of this should be around micromolar range. So you have to have some idea of binding. Now protein is taken very small concentration you do not need very large concentration of protein you just need large concentration of ligand. So if you take protein concentration say in few micromolar and ligand concentration in millimolar we can do these experiments. Now what we are saying since this concentration is very low so we will not get any signal coming out of the protein we only get signal coming from the ligand. Now say this is a my signal from protein and this is my signal from ligand I just brought it for but this signal we are we are not seeing it just for representation I plotted it. Now we are irradiating the signal from protein which is delta B is irradiated saturated. So saturation means you are making the population here and here equal that is so that means this peak will disappear after saturation. Now since these two protons are in exchange and also in close proximity the effect of saturation particularly at this position you are going to see here therefore the intensity of this peak will decrease, intensity of this peak delta A will decrease. So now by saturation of peak of a protein effect is seen on the ligand peak and that actually will show up in reduced intensity of the ligand and this method was developed by a group in Germany and published in Angokam. So this is called STD. So let us discuss what actually we are doing here. So we are saying say this is protein signal if we take in high concentration. So this is protein signal. Now we have to saturate a signal which is not in ligand because we are selectively saturating now protein signal. So suppose we are saturating somewhere here quite offensively like minus 1, minus 2 ppm and effect of this saturation we are going to see on the ligand signal. So this is say our reference spectrum for the ligand 2 equal intensity peak we are getting. Now we saturated the protein signal and all the protons of the ligand which were in close proximity of the protein their intensity will decrease. So here if you look at this intensity has decreased this has not changed. Now if you take a difference of these two then we know that okay this will cancel it out and only signal from here will appear. So we know that this proton is interacting this proton is not interacting. Similar you can take then another example here is a reference spectrum. If there is no STD means no interaction then you do not get any effect and therefore if you take a difference you do not get any signal okay. So that is what you see if it is interacting in the difference spectrum you will see some peaks if they are not interacting that means there is no peaks. There is no peak that is what easily just by recording 1D spectrum of ligands in presence of protein you can find it out whether this ligand is binding to particular protein or not. So things to remember we are collecting two experiment one is reference spectrum where we are not saturating protein signal one where we are saturating protein signal and recording the spectrum. Now we are taking the difference of these two reference spectrum and the saturated spectrum and if peaks appears that means we are with this ligand bind to that protein and we are getting therefore a signal. So we need to saturate protein peak usually this is methyl is student and that we can find it out just by knowing where the ligand signal is not. The pulse sequence is something like this okay. So two experiment is done one saturated one non-saturated and this is done by for saturating then you just apply a 90 degree pulse then this is for removing the protein signal and essentially record it. So saturation followed by 90 degree and then recording in one experiment you don't saturate in another experiment you saturate take the difference and the difference peak will tell whether it is binding or not. So just for an example I am showing it say here is my ligand which is binding to a protein. So in first experiment we recorded proton NMR spectrum and we get the chemical shift for each of these protons right ABC you can see here. Now then we saturate a signal from protein and then subtracted that signal from here. So if you look at this, this, this, this they are anyhow coming from solvent they are almost vanished and here also this peak and this peak vanished. However DEBF these peaks appears that means this ligand interact with a protein at DEF these 3. So this moiety interacts with the protein this moiety does not interact with protein. So precise in beautiful way just by recording 1D spectrum we can find it out binding epitope of the ligand to the receptor and that can achieve achieved with a very, very high sensitivity in a very quick manner just 1D, 1D means few seconds. In few seconds we identify which moiety of the ligand is interacting with a protein that is what is STD for another example let us take a mixture of these two and we want to find it out which one binds. So if you look at here the mixture has only like only one difference. So difference is essentially here this just everything if you look at it same right all the moiety is same so only this CH3 is shifted here. So 6, 7 changes there. Now these two ligands you are given a mixture and you want to find it out which one of them is actually binding to the protein whether methyl at 6 position or methyl at 7th position that is what we have to identify which one is binding with. So we recorded a spectrum the 1D proton spectrum and we can identify the red are coming from the 7 methyl triptophan and 6 is coming from 6 methyl triptophan. We record a spectrum we identify these peaks then we record an STD spectrum means by saturating protein peak recording 1D spectrum and we find that here are some peaks which are appearing. So here if you look at these peaks are appearing. Now we can find difference spectrum is a mixture of protein which is HSA and 6 methyl triptophan and 7 methyl triptophan and then corresponding now you can find it out the spectrum. So if you look at here this is binding. So if you look at this methyl seems to be strongly interacting like 6 methyl triptophan seems to be strongly interacting with HSA compared to 7 methyl triptophan and one can get other protons as well. So this 7th does not interact 6 interact. So now from the mixture of these 2 compound just they are isomer position is different 6 to 7 one can find it out that which one interacts with the protein which does not interact and that is just within a few second one can identify. So now that was we started with 1 we will now look at mixture of 2. Similar thing you can do have a mixture of 3 or even 4. Suppose we have a mixture of these many compounds and then one of them is binding depending upon what is the activity site and how ligand can fit it. So we keep increasing the saturation time and now what will happen that a binder the binder will show the STD peak here and nonbinder will not show. So nonbinder there will be no peak coming from these 2 shapes only for this will be shown here. So binder will show a peak nonbinder will not show a peak and therefore even from the mixture of the compound you can find the appropriate ligand. So you see now this can be used a throughput screening method for finding it out which ligand is binding to protein and which does not bind to protein. And now that can even like here was a ligand and if you look at the nonbinders like here it is a just flat peaks that means something like this is not binding. Reference spectrum for these guys are like this and reference spectrum is very well spread. So here you can see each of these peaks are present in STD spectrum. Again reminding you STD spectrum is a difference spectrum where we are saturating and where we are not saturating. So the non saturation minus saturation is giving peak means these molecule like blue molecule may bind and here the other red molecule or pink molecule is not binding therefore you do not see peaks in this region where the reference spectrum has a peak and this does not have. So if you look at this is just a very small molecule concentration of the molecule we are taking 80 micromolar of say binder or the ligand and 2 micromolar of protein that is what you need just for finding it out. So with a very low concentration of ligand protein now you can and from the mixture of the compound one can find it out a binder and a nonbinder and therefore it is a high throughput method for screening of a drug molecule. Now other thing that we have said can also be used for finding it out whether it is binding or not. So if you remember I said there are many parameters that can be used like here. So one thing was ligand being a smaller molecule relaxes so protein being a bigger molecule relaxes fast. So if that is the case can we use this relaxation property to identify whether this molecule is binding or not. That means relaxation property which one T2 will transfers relaxation rate and transfers relaxation rate if you remember it is encoded in the line width. If a molecule is relaxing fast that means lines are broad if relaxing slow lines are sharp. So here is a line for protein and here is a line for ligand. Now we are going to exploit this phenomena of line shape to find it out whether this is the ligand is binding to protein or not. So as we have seen before line width is directly related to apparent molecular width, high molecular weight, broad line, small molecular weight, sharp line. So a small molecule like 100 Dalton to 1000 Dalton the order of magnitude lighter than the typical protein of kilo Dalton, kilo Dalton protein will have a broad line. So a small molecule sharp line of few hertz and then proteins have broad line of say few like 100 of hertz, 10 of hertz or 100 of hertz. So if a small molecule binds to protein now as we have seen. So this whole molecule will now tumble slow and behave like a bigger molecule therefore line will become broader. So line sharp for small molecules protein line broad and complex will be line broad and that is what we will have. So if here is my small molecule this is my protein this will have sharp line like this and typically this should have broad line like this. So now if the complex formation happens because of complex formation now we are looking at the line of say ligand which was earlier sharp line now because now it is slowly tumbling it gives to broad line. So line width increases because of binding from narrow to broad. So it was earlier fast tumbling now it becomes slow tumbling. So tumbling also changes line also changes and that gives an idea now this ligand is binding to protein. So upon complexion one ligand lines will become broader and one can identify just looking at the line whether this binder or not binder. So like here nonbinder lines remains same if suppose this was binder lines becomes relatively broad and just looking at the line one can find it out whether it is binder or nonbinder just by looking at the line one can find it out binder or nonbinder. So now just we had looked at NMR analysis of ligand protein interaction here if we will concentrate it say free compound we have a free line. So Kd should be in typically micro molar range here micro molar range Kd should be and size difference between ligand and protein as I was saying it should be minimum 15 fold. So ligand line width will change upon binding and as you keep titrating so you have a say 500 micro molar of ligand and just 5 micro molar of protein. Now you keep titrating it so if you keep titrating what will happen you just look at this line follow this line. Now this line was quite sharp here two lines are sharp if you increase here ligand to protein now if we are coming one to one line become really broad. So this says that now this protein ligand interaction has dramatic increase in the line width happens not at the low protein concentration include that binding is not there. So if you increase here say ligand and if you go to ligand protein concentration line becomes dramatically broad and that says that this ligand is interacting with a protein great. So application of STD so what all you can do by saturation transfer difference you can essentially determine the Kd of the ligand dissociation constant. So you can find it out association constant dissociation constant kinetics K of and K on and then one can find it out any type of receptor in protein either it is soluble or even membrane integrated you can find the ligand for a receptor whether it is integral membrane protein or a soluble protein one can identify and even proteins which are embedded on the surface of whole cell you can analyze by STD and MR and find it out whether there is a ligand for that or not. And one good thing is that you need very less protein concentration just say about few micromolar even picomolar is good enough for obtaining the STD and MR signal. So that is what you can do by STD and MR application. So I hope this concept can be used in pharma industry to identify it. So I will go further and then we find it out now you kind of direct detect the ligand binding to a membrane protein in the living cell. So this topic because this is very very interesting you see the now soluble proteins are okay you can find it out but billions of dollar revenue is just by targeting G protein coupled receptor GPCRs though they mitigate the all signaling and 30% proteins are actually membrane protein many of them like GPCRs and all those. So they are difficult to investigate what is the receptor ligand interaction because these proteins are not easy to purify for making it soluble and looking at the binding affinity they are difficult to purify, difficult to reconstitute and get it for a structure determination. So but STD offers a very quick methods without perturbation of the protein in moment to find it out how what will be ligand protein receptor interaction. So difficult so all the difficulty of associated with membrane protein can be tackled by STD and MR. So what one can do here we can use the atomic level binding interaction of cell surface protein in the living cell but we are looking at the ligand all the time and this is done by something called saturation transfer double difference SDD. So this is SDD NMR. So now I am going to explain you what actually SDD NMR is saturation transfer double difference for studying the membrane embedded protein or protein immobilized on the cell surface and finding the binding epitope on the ligand how there we are going to do this. So this is very interesting hope to see you in the next class and I am going to explain in detail how you can use this SDD technique to finding it out these kind of interaction of whole cell. Thank you very much and looking forward to see you in the next class.