Assignment: The Use of Protein NMR in Active SiteMapping.Nuclear Magneticresonance spectroscopy (NMR) is an analytical technique that is based on usingthe known chemical constituent of a compound to distinguish it from otherunknown compounds. The ability of this technique to distinguish the differencein molecular structure of substances and the information it provides about thedynamics and interactions of molecule in the smallest possible unit of a mattermakes it an indispensable tool in the process drug discovery, development anddelivery. This chemical analytical method is very sensitive to its environment,so can give very minute information about how the smallest fragment of amolecule binds to a target molecule, protein or its complexes. Informationabout the exact binding site or interaction between the fragment and thereceptor of interest is also highlighted. Hence, this technique is a very vitaltechnique in the Pharmaceutical, forensic, quality control industry. Thisanalytic technique also has its application in the field of research where itis used to determine the purity, quality, quantity and structure of the unknownwhile confirming that of the known substance.
The combination of thisanalytical chemistry technique to Protein in the biological science is what isknown as Protein NMR. IntroductionProtein NuclearMagnetic Resonance has been used extensively to study enzyme mechanisms, analyzingstructures of proteins, nucleic acid and its complexes This technique is alsoemployed in studying protein ligand /protein interactions and the dynamics ofprotein. In the field of drug development, the study of protein and itscomplexes are of utmost importance as they play vital role in physiological andpathological conditions and process hence the importance of thoroughlyunderstanding their catalytic process and how they bind to their substrate. ProteinNMR in active site mapping thus, is the application of NMR in the region of anenzyme where substrate molecules bind and undergo chemical reaction as well aswhere its residues forms temporary chemical bonds with the substrate. This regionin an enzyme is known as the active site.
The mapping of active sites is quitecrucial in the field of pharmaceutical science or drug discovery. The detailedknowledge of the site of a target receptor for drug discovery and theunderstanding of the protein dynamics in the targeted site will maximize theefficacy of the proposed drug by giving a clear and precise understanding ofthe protein -ligand binding information and protein-ligand/protein interaction(Yan Li et al,2017). These interactions aid the design of new drugs forinstance enzyme inhibitors, by providing in depth details of the size on theactive sites, how many subsides are present, their properties, how they cometogether and bind chemically.
The understanding of this unique interaction isalso a tool for comparison in active site mapping, where it is employed to compareprotein active sites and their structures in more details so as to design drugsthat can exactly match into the enzyme substrate complex using the key and lockanalogue for enzymes. This proteinanalytical tool has been use in lots of studies to investigate enzyme behaviors,their mechanisms as it takes less time an effort to acquire structuralinformation of compounds and DNA when compared to other methods like X-ray crystallography,florescence and IR spectroscopy, hence the ever growing importance of active sitemapping using Protein NMR.(Yong et al.2012)19FNMRstudies has be done to clearly distinguish structural and functional featuresof its recent application in active site mapping out of galactose binding-protein, transmembrane aspartate receptor, the Che – Y protein dihydrofolatereductase , elongation factor-TU, and D-lactose protein as seen in dehydrogenase, that demonstrate the utilityof 19 F NMR in the analysis of protein conformation state even in particles that are so large orunstable for full NMR structuredetermination.
(Mark A.D, et al 2010).Thesekind of studies depends on the chemical shift pattern of FNMR as this method isvery sensitive to change in its environment due to the presence of fluorine 19,as well as the existing weak Vander Waal force of bond as well as the presenceof the local electrostatic field. Figure 1.
Overview of applications of NMR in drug discovery NMR spectroscopy canprovide critical information at early stages of hit validation andidentification. NMR measurements for binding studies can represent a key stepto eliminate false positives from high-throughput (HTS) campaigns, to validateputative hits from in silico screensor to identify novel scaffolds in fragment-based programmed. NMR and X-raycrystallography can also provide unique information to subsequently guidehit-to-lead optimization. ADME-tox, absorption, distribution, metabolism,excretion and toxicity (Pellecchia M el at: 2002) This review willmainly concentrate on saturation transfer difference (STD – NMR) method whichis a solution state nuclear magnetic resonance spectroscopy technique used intarget- based drug discovery, hit identification, validation and leadoptimization which is a tool that is extensively utilised in drug developmentprocesses as seen in our review of this method in the biological studies of new urease inhibitors. Fig2 flowchart showingdrug discovery process Fig3 showing theprocess in Protein NMR Process Figure11.This is a flow chart showing the different level of application of NMr inthe process of drug discovery from when the target is identifined through thewhole complete process and the role it plays highlighted in white and blue;Figure 111, highlights the varous steps involved in in using protein NMr in active drug in drugdiscovery and its application.
(Yan Li et al, 2017;). Materials andSample preparation phosphate, mono-sodiumdi-hydrogen phosphate Unichem (India). Mous, and phenol were obtained fromSigma-Aldrich (USA Deuterated methanol (CD3OD), and deuterium oxide (D2O) werepurchased from the Armar Chemical (Switzerland) STD-NMR ExperimentJack bean (Canavaliaensiformis) urease (EC 3.5.1.
5), urea, Dulbecco’s Modified Eagle Medium(DME),cycloheximide, di-sodium hydrogen ). Methods/ExperimentalThe measurement ofurease inhibitory activity by STD- NMR technique was done using the aforementioned technique, that is very popular in drug discovery and possess high sensitivityhence often used for ligand –observed NMR screening methods. In thisexperiment, Gaussian RF pulse was applied to the most up field protons of thetarget protein which when saturated is then transferred throughout the moleculeby spin diffusion.
At the final stage of this process the bound ligands receivedmagnetisation through cross relaxation and enhanced signal intensity isdisplayed (Atia-tul_Wahab et al.2013:).The sample for this experimentalprocess is prepared with Jack bean (Canavaliaensiformis, EC3.5,1.5) using deuterated NMR buffer to prepare(20uM) ofurease solution, which is then stored at 4 °C ligands.The reaction mixture was in excess of 100folds of urease concentration.
Theywere dissolved in 13.3% of CD3OD, and 86.7% deuterated phosphate buffer (4 mM,pH 6.8).
This was followed by STD-NMR screeningexperiment performed on Bruker 400MHZ NMR spectroscopy at 298K Stddiffgp19pulse program was used for STD-NMR experiments. Saturation time was 1.0–2.0s,while interpulse delay (D1) was the same as D20 or D20 + 1. Loop counter was8.
0 and 4.0. Experimental For F-NMR Technique Purification of the target proteinis usually the first step, followed by the modification of the protein of targetby using compounds containing fluorine like 2 bromo-N-(- 4 – trifluoromethyl) phenyl)acetamide (BTFMA)at cysteine residue which results in the presence of a protein with active “Fspin ( Horst et al, 2013;) (Kitevski et al,2012:) ( Liu J, J et el, 2012;) making it possible forchemical analysis to be carried out , which is normally the last step beforethe process of Hit identification. (Nortonet al, 2016;)Hit identification is carried out atthis stage to for the purpose ofscreening F- labeled compound using ligand – observed experience known as FBDD,that usually has anexisting library or in the absence ofthis library one can easily be made-up by adopting similar rules to those use in usual fragment library to sustainligand size and chemical variations. F- NMR as a target based proteinspectroscopy can be used to affirm the hit screening from HTS campaigns inwhich a chemical assay has being used as the primary screen (Gee C.T et al, 2016:).
The proteins of targets, which are normally close to the active site, arelabeled with Fluorine atom. This technique is then preceded with theidentification and validation of the targeted resonance in the presence of thefluorinated substrate. Results:In this review we have looked at theuse of protein NMR in active site mapping by using biochemical assay, thenfollowed by the use of STD-NMR which is a ligand resonance based technique, forthe primary identification of urease inhibitors. Then followed by moleculardocking studies to validate the biochemical experiment as well as to estimatethe relative binding affinity between the ligand and receptor.
F-NMR which is atarget based resonance, coupled with hit identification methods were also useto observe targeted ligand, screening were carried out, confirmation of theprimary screen with the use of the F atom and its identification and validationin the presence of the fluorinated substrate was achieved in this experimentDiscussion; The measurement of ureaseinhibitory activity by STD- NMR technique was done using Saturation transferdifferential NMR which is a ligand resonance based spectroscopic method that isundoubtedly one of the most widely used NMR Spectroscopic technique due to it’sability to establish a binding relationship between the inhibitors and proteinas seen in this experiment. This technique uses the