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Introduction The key strategy of drug development in the contemporary era involves the screening of compound libraries. The process of HTS becomes useful only when the results of screening analysis are visualized. This visualization is accomplished by performing high quality HTS assays which employ certain radioactive, fluorescence or other biophysical methods to see the changes. Assay methods have to be developed and optimized according to the need of experiment. Knowledge of basic principles of varying array of assay techniques is crucial to prolific screening of targets in time and cost effective manner. Biophysical Assay Methods Primary assays are usually done in doublet or even singlet to select the hits. Once some positive result is obtained after the screening of hundreds of compounds in primary assay, the obtained hit is selected and precise secondary screenings are done [1]. Biophysical assays are a bit complex but they give very beneficial and reliable results. Biophysical methods of detection in high throughput screening are of three types: Nuclear Magnetic Resonance (NMR) Surface Plasmon Resonance (SPR) X-ray Crystallography Nuclear Magnetic Resonance (NMR) Various pharmaceutical and research groups have incorporated NMR into HTS due to its natural synergy with medicinal and combinatorial chemistry. The general methodology of NMR assay involves following steps: The compounds are screened for their binding affinities with 15N labeled proteins. Binding affinity is measured by observing changes in amide chemical shift in protein binding site. Once the lead compounds are identified they are optimized to make up the deficiencies in appropriate binding. Targets are then screened for the second ligand. Second ligand is also optimized to make it bind appropriately with the target. 3D-structure of protein-ligand complexes is generated in order to determine size of linker. The optimized inhibitors are then linked by a linker to generate final inhibitor for the target protein. As an example of this method, a library of 32,000 compounds was screened against RGS4 (a regulator of G-protein signaling). The 50 most potent primary hits were analyzed by NMR for confirmation. One compound was confirmed as an inhibitor of RGS4 at a unique binding site [2] NMR is frequently used for optimization of kinase inhibitors. Surface Plasmon Resonance (SPR) HTS assay can also use surface plasmon resonance as a detection tool. This technology involves binding of inhibitors to surface attached protein targets. Polarized light falls on the target and valence electrons in a solid are oscillated by this incident light. Resonance is achieved when the frequency of incdent photons become equal to the frquency of oscillating electrons. Signal is observed as a sharp shadow in the reflected light from the surface. The angle of reflection depends on the mass of the slois at the surface. The change in resonant angle (from I to II in figure) can be monitored non-invasively in real time as a plot of resonance signal versus time. This technique gives very useful information regarding rate of association and dissociation of inhibitor to the substrate [3]. ”" X-ray Crystallography X-ray crystallography provides an insight into the 3-D structure of the target or inhibitor-target complex with the elucidation of position of atoms as well the chemical bonds between them. Beam of X-rays fall on the crystal structure of the compounds and are subsequently reflected depending on the nature of compound under observation. Imatinib, a tyrosine kinase inhibitor targeting Bcr-Abl target protein exploited X-ray crystallography. An inhibitor of p38 Map kinase provides an example. The leads can be optimized depending on the data provided by the X-ray analysis. ”" Conclusion In reality, only one identification techniques in high throughput screenings is not enough for lead optimization. Instead, a combination of techniques is required to provide reliable results. The selection of techniques depends on the nature of compound being screened and the principle of technique being used. For example, NMR is good for the inhibitors which bind weakly to its substrate while X-ray is better for inhibitors which make string association with the target. Consequently, the knowledge of all the possible identification techniques is worthwhile in the exploration of beneficial drugs! References Martis E.A., Radhakrishnan R. High-Throughput Screening: The Hits and Leads of Drug Discovery- An OverviewJournal of Applied Pharmaceutical Science 201;1(1): 02-10. Stockman BJ, Dalvi C. NMR screening techniques in drug discovery and drug design. Progress in Nuclear Magnetic Resonance Spectroscopy 2002; 41:187–231. Cooper MA. Optical biosensors in drug discovery. Nature Reviews Drug Discovery 2002July; 1:515-528. Related Posts HTS Kinase Assays Virtual Screening and HTS Related to Biophysical Assay Methods in HTS HTS Kinase Assays The most imperative advantage of high throughput screening is it’s easy to comprehend and quick to perform assays yielding prodigious outcomes. One of the most ... The Process of HTS The urge to unveil the hidden truths of life, to reveal the concealed and to unleash the interred, has been native to human since birth. ... HTS and Drug Discovery A man of Stone Age if brought into the contemporary world will not deem in the innovations made so far even after visualizing them. But ... We has established long-term and stable relationships with more than 10,000 customers from pharmaceutical and biotech companies, universities and research institutions. 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