 Hello students, we are going to focus today on a small molecule based analysis. In the previous lecture, we have learnt that the identification of small molecules which binds specifically to the proteins or hot spot regions of the proteins can become crucial in discovery and development of new pharmaceutical drugs and for elucidating the complex pathways in biological system. The primary goal of a small molecule screening in pharmaceutical industry is to identify suitable candidates on the basis of their binding to the selected target molecules. The first stage of the process often involves screening large candidate libraries to identify the promising candidates against a protein target. Usually the positive controls are used to test the suitable binding activities if a control analyte is available. The small molecules are often dissolved in organic solvents. Therefore, to maintain the analyte solubility, the concentration of organic solvent in samples and in the running buffer becomes very important. The response obtained from a small molecule analyte is inherently low because of their molecular weight. Therefore, the sensitive surface for a small molecule screening are prepared with high level of ligand ranging from 8000 to 10000 Ru for an average size protein. Similar to the previous lecture where we have performed the immobilization of proteins, in this lecture we will simultaneously immobilize two proteins, carbonic and hydrates and human serum albumin on two different flow channel of a CM5 sensitive surface. This chip will be used to demonstrate a small molecule screening where 8 drug molecules will be tested. We will now proceed with the immobilization and screening analysis of these small molecules on these two selected target proteins. So, let us have this lab experimental session now. In this session, we will understand the interaction of proteins with small molecules. So, to start with small molecule interaction, we have to start again with the immobilization. We go into binding or screening and we go into kinetic mode to determine the affinity of small molecules. Here we have opened a new immobilization wizard for the immobilization of human serum albumin and carbonic and hydrates on two different flow channels separated with two reference channels. So, the chip selected is CM5, flow channels per cycle is 1, on the flow channel 1 we leave it blank and flow channel 3 also we leave it blank, flow channel 2, human serum albumin is immobilized with a specified contact time and flow rate of 4 20 seconds and flow rate of 10 microlitre per minute. On flow channel 4, by amine coupling, we will immobilize carbonic and hydrates again with a specified contact time and flow rate mode for 720 seconds and 10 flow rate. We now go into the next tab, we will highlight the prime before run and the normalized detector and temperature will be at 25 degree centigrade and we go to the next tab and we have the rack positions with the required amount of reagents shown here. For the volumes listed on the table, we will now prepare buffers and reagents required for immobilization of these two proteins on the sensor chip. The proteins are prepared at working concentration of 25 microgram per ml for human serum albumin and 100 microgram per ml of carbonic and hydrates using an immobilization buffer of pH 5. We will be using 1.05 x PBS buffer as the running buffer which will be connected to the system followed by priming. EDC and NHS as prepared and suggested in the amine coupling kit for surface activation will be used in 1 is to 1 ratio. 1 molar ethanol amine HCl pH 8.5 is prepared for blocking the free ester groups on the surface. Lastly, we would require be a normalization solution to normalize the resonance signal in all flow channels to compensate for small differences between individual sensor chips. In preparation of these samples and reagents, we transfer the sample tubes to the appropriate rack and start the run as shown for beta 2 microglobulin immobilization in earlier lectures. We will hence move forward to discuss the immobilization results obtained from this run. From our experimentation, the following are the results for the immobilization of human serum albumin and carbonic and hydrates on two different flow channels. We see here from the immobilization results. Here you can see on the screen immobilization of human serum albumin by activation of the surface with EDC-NHS, passage of the ligand to the sufficient amount required, blocking with ethanol amine and immobilization. On the cycle 2 similarly you have activation with EDC-NHS mixture, passage of ligand and blocking of the surface with ethanol amine to reach an immobilization level of 11466. With this we are done for the immobilization of HSA which is human serum albumin and CA carbonic and hydrates on the surface of the chip and the chip is ready to go for the binding experiment. Small molecules to be screened for binding will include digitoxin, warfarin, naproxen, phenytoin, sulfonyl amide, carboxybenzene sulfonamide, furosamide and azosulphamide. These molecules are specific to HSA whereas rest are specific to CA. Farin will be used as a positive control for human serum albumin and furosamide for carbonic and hydrate. We will also use 5% DMSO in running buffer as negative control. Since small molecules are often soluble in organic solvents such as DMSO and so are drug molecules it can result in large bulk responses since reference flow cell has room for more DMSO than active flow cell with ligand. That is why we need to prepare a solvent correction curve by preparing solvent correction solutions with varying DMSO concentrations which will range from 4.5% to 5.8% DMSO. We inject these solutions over the active and reference surface and then correction curve which will result in corrected binding responses. We will now move forward to set up the binding as a wizard to screen small molecules for binding to human serum albumin and carbonic and hydrate. Here we will set up our experiment to check the interaction of some small molecules binding to carbonic and hydrate and human serum albumin. So for that we need to prepare a method and the method actually has the start of 5. One round of sample injection solvent correction which are 8 different correction solutions are passed on the surface and the DMSO effect on the responses is subtracted. Control samples like positive and negative controls were also included in this mode. In the low molecular weight screen mode we have the contact time of 60 seconds, dissociation time of 300 seconds, flow rate of 30 microlitre per minute and the molecules were flown on 1, 2, 3 and 4 channels with an extra wash with 50% DMSO to prevent carryover effects. Stabilization time was 30 seconds. We verify this method and set up a run in which it will have a subtraction of active surface 2 was subtracted from the 1 reference surface, active surface 4 was subtracted to 3 and we have the following set of experiment setup here. We have 5 startups, one set of solvent correction of 8 runs injected here, 3 sets of control samples which include negative and positive control, samples like digitoxin, warfarin, naproxen, phenyl toin and a negative control were injected again and successively sample correction at every 30 cycles, 10 cycles or 20 cycles depending upon how you set up the experiment and finally another round of control samples injected. So, once we are done with that we highlight prime before run and this is how we prepare the template for the experiments with solvent corrections provided, buffer for the startup provided, various samples pipetted out in DMSO and warfarin, furosemite as positive and negative controls and 50% DMSO per wash in between each cycle. We will say next and the whole winding experiment including solvent correction takes 4 hours and 23 minutes and requires at least 200 ml of phosphate buffer saline with 5% DMSO included in the running buffer. Preparation of the running buffer, preparation of the samples, checking for the solubility, checking for the aggregation is very important when we setup our small molecule interactions. Immobilization level should be high, machine should be cleaned at least or desorbed one day prior instrument should be in a equilibrated with the running buffer for at least 6 hours before start of the experiment. As per the volumes listed in the table we will prepare the following samples and reagents. Running buffer will be 5% DMSO in 1.05 x pbs, positive controls and samples at 30 micromolar concentration in running buffer and running buffer will be used as negative control. A wash solution is prepared with 50% DMSO in water and lastly 8 freshly prepared DMSO solutions ranging from 4.5% DMSO to 5.8% DMSO are prepared for solvent correction. We will now work on the reagents required for the screening assays for binding of proteins, human serum albumin and carbonic anhydrase against several small drug molecules. In this protein small molecule study we will be using 5% DMSO in 1.05 x pbs as the running buffer which will also be used for the initial start up cycles. 8 different solvent correct solutions will be prepared ranging from 4.5% DMSO in pbs to 5.8% DMSO in pbs and these solutions will be prepared by mixing these solutions in different ratios as shown here. We will number these cubes as 1 to 8, 200, 400, 600, 800, 1000, 1200 and 1400 microlitre of 4.5% DMSO in cubes 100, 1200, 1000, 800, 600, 400 and 200 microlitre in cubes 1. We will mix these solutions vigorously as we are ready with other samples. We will be preparing all small molecule samples including Digitoxin, Warfarin, Penitone, Docky, Benzene, Sulfonamide, Pneurogenamide from 1.05 x pbs solutions of these drug molecules using DMSO solutions according to the table shown here. In ratio of 30 micro molar of small molecules will be used for this screening aspect. For washing we have also prepared 15% DMSO in water using 600 microlitre of DMSO of water in a 16 mm wire. Running buffer will also be used as negative control in this experiment. The agents have now been allocated in the required volume in these specialized tubes. We will now proceed including the samples at 30 micro molar concentrations, the negative control, the startup, 50% DMSO as wash solution and the solvent correction solution into the appropriate rack and then into the system to perform the protein small molecule screening assay. We have now including the wash solution, the sample startup solution and the negative control to start with our screening assay. Once the wizard is set up and the experiment is done for at least 4 to 6 hours we will open the data file to see for the data. So this is how the data looks like with all the control samples, sample, solvent correction and startups are overlaid on each other. Here we can see we have the reference surface, active surface as our human serum albumin. Again we have a reference subtracted surface 2 minus 1 and we have the reference surface 3, again an active surface for the carbonic anhydrase and the reference subtracted 4 minus 3 surfaces. When we go here on the overlay we see startup cycles, solvent correction, negative and positive control samples and various samples that were injected on the surface of the 2 active ligands. Here first we have to go on add solvent correction, check the solvent correction data and if the data is good enough we can say ok and the data is subtracted for the solvent effect of DMSO and we can see now in the all senseograms it shows 2 minus 1 corrected and simultaneously 4 minus 1 corrected with the solvent correction data that we have generated. Here another point is that we could evaluate the data in a bar chart mode, we have various startups, solvent correction, control samples and various components injected here and their interaction with the surfaces. Also similarly at 2 minus 1 corrected position we can see similar kind of a data with this we can conclude some molecules have no binding whereas some molecules have some amount of binding from the data and this indicates that the 2 different surfaces behave differently while interacting with the small molecules. This will end the session on solvent correction and binding of small molecules to 2 ligands immobilized on the surface. Different drug molecules bound to human serum albumin with different binding responses based on their respective affinity. In this case Digitoxin shows the highest affinity towards HSA followed by Valparin, Purozimide, Naproxen, Azosulphamide, Finitoin and others. The binding response for these molecules observed for human serum albumin correlated well with their expected affinity. Similarly different drug molecules also bound to carbonic anhydrase with different binding responses based on their respective affinity. In this case Azosulphamide shows the highest affinity towards carbonic anhydrase followed by Purozimide, CBSA, Sulfanilamide and others. The binding response for these molecules observed for carbonic anhydrase also correlated well with their expected affinity. In today's lecture you must have observed that different small molecules have different binding response and affinities for the 2 bound protein that is serum albumin and carbonic anhydrase. We will continue this experiment on protein small molecule interactions for kinetic analysis in the next lecture. Thank you.