 Good morning the students. So this week we are discussing how we can use NMR spectroscopy in drug discovery. So based on the knowledge of protein-protein interaction, protein-drug interaction we ventured into the use of NMR spectroscopy in drug discovery. In last week we discussed protein-protein interaction. This week the first lecture was about how we can fish out some of the binders in the protein-protein interactions. So we discussed two kind of experiment when we are detecting on ligand, NMR experiments like STD, saturation transfer difference or water loxie or line width analysis is of paramount importance where when we can detect on ligand and find it out binders. So it is kind of a fishing out experiment. So there are so many small molecules which binds to a receptor or which is target and using these experiments we can find it out which molecule exactly binds to and with what strength it binds to the receptor or the target. On the other hand protein detected experiments can tell you the site on protein where ligand bind. For doing that we need to isotopically level these proteins with N15 or 13CN15 both. So if we do that and titrate ligand and whatever chemical perturbation happens or intensity decay happens using any of these probes we can find it out exact location where the drugs are binding. So that is about drug discovery. We are discovering out of many compounds whether it is coming from natural source or plant source or animal source or synthetic by our medicinal chemistry friend they are synthesized they have synthesized many molecules and which one of them is binder. What is the like what is the potency we can find it out using NMR spectroscopy. Now this week we are going to look at how we can start developing based on that knowledge whatever we have. So the first thing we can think of an SAR like structure activity relationship it is a like a it is classical titration based experiment that we can think of. So here is my protein right and let us see that it has some sort in binding site here is kind of a binding site which has a particular shape here is a star shape binding site and we need to find it out which molecules bind with what KD strength and using that knowledge can we start developing actually better drug. So SAR is a NMR method that on the use of that protein chemical shift can be used to see what where it binds. So there is a change in the chemical shift and essentially that will be used to screen the low affinity binders right and we can use such information the low affinity binders where they are binding to directly link now we can start attaching fragments and make a better affinity drug so that is the whole idea. So what we are going to do here taking a protein molecule we are isotipically labeling this protein and say there is a binding site where we are starting with a binder. So we see some of the shift that is happening here so suppose this peak shifted here this peak shifted here and these peaks shifted here other peaks said did not shifted right. So now we know that these are the binding epitopes on protein and we can find it out binder so even low affinity binders like this molecule binds with a KD of 17 millimolar okay. Now suppose there is another binding site which is here we can start again with a another set of compound and find it out which is binding here so like a different binding sites on same protein now next step we can attach it to find a better binder something like this. So here is the our titration experiment we took the N15 level protein we titrated with a molecule find it out all the binding position like here you can see the chemical shift shown here is directly telling this is not shifting right. So this is not shifting here you are seeing that there is a shift here these peaks are shifting here this is peak is shifting this is almost not shifting. So utilizing these we identified the binding sites so here is my binding site the molecules this molecule comes and binds like this okay so exact binding site we can find it out for one binding site. Suppose this protein has another binding site which say here now similar experiment I need to do with another set of compound find it out that what are the residue involved here which are binding. So that is the then like here so if we are starting with a with the another set of molecule you see a star shape molecule it is coming and fitting here the pentagon will not fit. So suppose this is the case now we have a better binder like here is say Kd of micro molar another ligand again LOA affinity ligand which binds at different site that is identified based on the chemical shift now this both ligand which was discovered in the previous case here which is say this shape 1, 2, 3, 4, 5, 6, 7, heptagon shaped ligand here is a star shaped ligand this binds with a micro molar affinity this binds with a millimolar affinity. Now we have found two ligand binding with two different affinity can we use these to optimize a better binders like can we attach both of these and have an analog which has like an even more binding site. So this is this is the idea that we are going we will be using in drug discovery case. So using SAR a structure activity relationship by NMR one can design a compound that binds to the catalytic domain of any protein like a straw maleicin. So here if you look at this guy was the first binder and this was the second binder we attached by some chemical bond right we use synthetic chemistry. Chemistry approach a medicinal chemistry approach using NMR based SAR we find the first binding molecule that is binding at a millimolar range then we found another molecule that is binding at micro molar range. Now we attach these two and we find a molecule which is by joining these now binding with a very high potency of nanomolar range right. So now the molecule that we are discussing a straw maleicin one is a metallo MMP right matrix metalloprotease which has implication in cancer. So one can find it out the protein with a two different binding site the two ligands separately by SAR by chemical shift perturbation and now we can join them by these bonds to find a more potent molecule that has a potency of essentially nanomolar right. So here a exposition of a simple titration experiment where you use for screening weak ligands and structurally directing these ligands by chemical linkage to have a more potent compound and that is how one can approach in drug development stage right. So first thing what we did we discover the binders in the first approach by titration. Then using our chemical synthesis or medicinal chemistry we attach these two weaker binder to come up with a strong binder of nanomolar affinity and that is how we are developing a more potent drug ok. So that is the simple concept. Let us discuss some of the advanced concept of the NMR based screening. So one of them is called shape screening how we can screen it. So it is a basic strategy in the shape screening essentially to access the binding of a fairly small but diverse library of low molecular weight scape fold to target to have a drug target using NMR techniques that are based on observing the ligands. So what we are doing again let me dissect all those shape screening essentially we are taking a small set of library of various compounds and now we are detecting on the ligand side like using STD experiment that we have seen and finding it out what are the binders and then probably we will start developing from there. So ligand based experiment so I hope you remember STD. What we are doing here is recording an NMR spectrum of this small molecule we are having some peaks here then we added our like the receptor and we irradiated the receptor signal. The effect of that irradiation RF irradiation is transferred to the binders right it will not transfer to the non binders because they are not in contact of the protein. And then some of the peaks say this peaks so low in intensity. So if you take the saturation transfer difference only this peak will be shown these two peaks will be almost zero you know that this is the binding site on the ligand. So we are going to use this STD approach STD like approach and observing on ligand and then we are screening the molecules that is called shape screening. So here using shape screening we find a chemical moiety that can bind to our ligand side say here our protein target is protein kinase P38. Now we find a moiety which binds with a low affinity of Kd of 2 millimolar it is a quite weak right. So now then we find it out what is the essential moiety can we start like a playing around using some chemistry and find it out like do some modification like here you attach something like here you attach another group or attach another group and in this case you can change some of the group like here nitrogen can be changed to say sulfur compound then you attach few of these. So if we do such kind of chemistry by attaching few moiety and then again doing STD kind of experiment to find it out what is the next binding, next binding a strength. So by doing this all chemistry medicinal chemistry approach again combining that with a shape screening using ligand detected experiment one can find it out now it is the molecules that were developed can bind with a fairly strong strength which is going to be up 200 to 300 micromolar. So you see like a almost 1000 fold or at least 100 fold binding capacity we have increased. Now let us go ahead. So now what we did with this K fold that are derived mainly from this shape like a shape what I mean here is a binding site. You know the kind of a shape of a binding site now you are finding a key that can fit here and that is we screened that shape and then shape or framework that are commonly found in the known drug. So initially we had a weak binding heat say Kd is in micromolar but that is important to find the framework and then in follow up a strategy the shape heats can be used for starting plate of platform for virtual screening. We can start modifying using the virtual screening like a computer based drug design and medicinal chemistry what we will fit it there that binding becomes stronger. So even looking at the structure the binding pocket shape of the binding pocket and the initial framework you can start designing the molecule in computer which will tightly fit into that. Then you go and synthesize this molecule and then again you come back to do NMR find it out what is the real Kd. So this iterative approach of finding a shape where this molecule is binding finding initial plate form or the shape of the molecule developing that into a better binder first using computational approach then using synthetic approach coming back again to STD NMR finding it out what percentage or what fraction it has improved that is how you proceed in this kind of drug development. So by doing this last example I showed then design of an mitrogen like design of an inhibitor for mitrogen activated protein kinase P38. So on the basis of this initial finding a library of analog can be developed like just now we see lots of lots of compound can be synthesized here we can change even R2 group we can add some methyl at some position, ethyl at some position or some other group and see what how the binding is approaching improving. So that is why we have a range of binding it can vary depending upon how tightly it binds. So on the basis of these initial finding of binders using STD approach we can develop and library of analog with further derivation and using this initial core structure we can obtain the high affinity ligand. So what substitution we can do? Substitute something the core framework we are keeping same we are attaching something like this is also you saw in the last slide this is also kept intact but there here we are attaching another ring here we are attaching another ring with fluorine. Now fluorine is a very interesting molecule in the next classes I am going to tell you this can be used for tracing the drug right when you develop a drug you have to find it out where it is going. So fluorine is a very interesting nuclei in terms of enamel and it has many other role in the medicinal chemistry. So this fluorinated compound one can develop few of these fluorinated compound and you see all these derivatives that we have developed or the synthesized now my strain has increased the potency has increased. So now it is 10 to 200 nanomolar we started with a millimolar but developing on that framework based on the shape of the binder we have come up to a 200 to 100 to 200 of nanomolar. And here the simple thing was used simple thing of one dimensional NMR was used for a screening identifying making it high affinity analogue starting from a weak binding weak binding heat or weak binding heat where these like then we started developing sorry weak binding was this 2 millimolar these two rings 5 member rings perazine rings and here 6 member ring with a nitrogen. So we are starting developing and by developing all these we have found analogs that can bind with a very high affinity of 10 to 200 nanomolar. So that is a shape based screening. Now another approach that one can think of an NMR solve. So solve is called structurally oriented library valence C engineering. So here we are now looking at the protein molecule and we are engineering a binder right. So looking at the protein molecule and even we are not totally labeling it we will do some smart strategy to engineer a drug molecule binding molecule. So how we are engineering? So first we need to have some structural information. So structurally oriented library valence C engineering right. So here say this is my binding site. We need to have some proofs here. So NMR solve is a drug design strategy that provides information for the construction of focus library you are not going to do a blind screening like many approach is done that you take it one like compound or 10,000 compound just randomly screening based on the high throughput and you find some thousands then you come to 100 then you come to 10 then two molecules are working at least in vitro and then you do not know what happens. So here NMR can cut down that screening. So now we are designing based on these information you are not blindly screening it you are using some information that are available based on the protein binding site or the ligand binding site that we saw in the last slide. Here this strategy provides information for the construction of focus library. So where the information is coming from the binding site. So let us look at how binding site is providing. So basically this NMR solve exploits the fact that there is a large family of protein that have a adjacent binding site and one of them is conserved. So many of these same family of protein they can have a similar binding site or they have a additional binding site and that information needs to be like here say conserved family proteins. Here you see one binding site looks common but the another binding site here is a star shape, here is some other shape, here is a triangular shape. They have at least one binding site which looks quite conserved right. So now what we are going to do exploit which is conserved binding site let us see what is there. Now for doing that we need to put some of our probes. What are our probes or some of the what are our probes so it can be amino acids, it can be moiety in amino acids, it can be protons which are there. So we need to label them and we are looking only at these and this gives the idea about the binding site. So once we have the idea about the binding site we can come up with a ligand that will probably fit here. Now we already started with a initial head. Next we are going to develop that. So depending upon what is the second binding site we can screen it and then start attaching. So let us see how it happens. So what we are going to do here are my probes, these are probes. They provided information and information is coming from the chemical shift change. So we can have either say N15 level protein or 13th level protein or both level protein and we just recorded the correlation spectrum like whatever we are discussing and then we find what are the residue that are involved here right so on a structure. So we did titration we find it out the residue involved here and those residue are going to be conserved in other family protein as well. Now next we have to find it out what is at the like what is at the joining site between these two. So we need to have a probe here as well. Now once we have a probe we can design even the molecule that will fit here and next will be designing for another binding site. So let us see. So we find the orientation of the first binding site a common ligand that we mix. So we already by exploiting this binding site by putting specific levels there we know the idea of this binding site and these drugs at the binding site number 1 will fit to the all members of the family. So half job is done right. So one find a good binder here we can just translate that binder to another family member of that family of proteins right. So the orientation of the most common ligand mimicking the binding site of a protein determined and that provides the basis for designing the linker here just and for linker design we need to have another probe which is coming from the amino acid that is located here and then we are like that will be essentially direct my ligand to this side. So now we are developing a framework here based on the information which is provided by this what is the linker. Now this will direct that my next binder will be coming here and attaching to a particular site. So now NMR data for protein 1 can be used to guide synthesis of a combinatorial bi-ligand library. So using this we can synthesize. So here is my first molecule you also find a binder sorry a linker and then we synthesize a variety of molecules. We make a small library which has a bi-dentate and this bi-dentate like we have one time this shape another time this shape third time this shape. So two things helped us one the information of the first binding site, second the linker this exclusively came from the information that we obtained from the NMR. Now utilizing these we synthesize a combinatorial library and then we did the activity essay to finding it out what can best bind to this place. So such approaches has been used in fragment based drug design this is called fragment based drug design we having two binding sites. So here is a fragment the binding site one can be find it out with HSQC binding site two can be find with HSQC again experiment and then one can essentially we find these two molecules and we can map on the tertiary structure then we synthesize a linker and using again in 15 level you can find it out that binder. So this actually approach was used to identify a binder for BCL-XL and BCL2 compound and this is the compound that is still in clean culture ABT737, ABT199. So based on this fragment based drug design like attaching these ligands actually the drug was designed and now it is in the clean culture. So 3D structure based arsenic 3D structure was used for doing this. The another approach in the drug design can be seen is called Interligent NOE, ILOE. So what we are doing here say we have a two binding site right here I used in fragment based drug design what I used in the information based on the protein side we had a N15 level protein we titrated it find the two binders then attaching it using some information which is there and found a better molecule. In the second approach what we will be doing is using the NOE between two ligands. So here is my first ligand that is binding to the first site, the F2 is a second ligand binding to a second site. If they are close enough they can do they can show the nuclear overhouse are effect because they are in close proximity. So now we can like here is my F1 ligand here is my F2 ligand and if we record an NOE between them we find the NOE which is coming here. So in this case we have started with an unlabeled protein target unlabeled ligand we just look at the small molecule that is binders the drug molecule the NOE. Now this information provided that this particular portion of ligand 1 is in close proximity of a particular portion of ligand 2. So this interligand NOE will help us in essentially joining this. So structural analysis of the fragment based interligand NOE will help us that where we can do the chemistry to join this. And now this will be called a bidentate ligand you can again go for N15 labeled protein target and find it out how better it is binding. So we have like if you look at carefully what we are using the NMR spectroscopy with the chemistry, medicinal chemistry and some biochemistry to label the protein, some combinatorial chemistry to synthesize the ligand and after combining all these approach we can synthesize a better potent molecule the bidentate compound and one can validate again using NMR spectroscopy. So this is kind of a approach was used to synthesize the molecule and that gave us a strong binder molecule that are being used nowadays for the drug designs. To sum up now what we learnt basically in drug design essentially we have to start with a framework either on protein detected experiment or on ligand detected experiment. So we start with a framework and then they start developing it if needed if your protein has two binding site you identify those two molecules use some information coming from the protein site attach them and make a better binder or a better potent drug. So we are starting with a millimolar of binding affinity and one can reach up to nanomolar of binding affinity and that nowadays are used in designing and developing drug. So I hope in the last two lectures I gave you some idea that NMR has a profound impact on drug design and development. In the next class what I intend to do is to give you an overview how NMR can be used for monitoring the drug fate like once you consume or once anybody consume the drugs how it is passing through different organs and can we use that information the NMR based information to understand the drug metabolism. So that is what I intend to do in the next class. Looking forward to have you in the next class. Thank you very much.