Assignment: exact binding site or interaction between the fragment

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 thoroughly understandingtheir catalytic process and how they bind to their substrate. Protein NMR inactive site mapping thus, is the application of NMR in the region of an enzymewhere substrate molecules bind and undergo chemical reaction as well as whereits residues forms temporary chemical bonds with the substrate.

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This region inan 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 of theprotein -ligand binding information and protein-ligand/protein interaction (YanLi et al,2017).

These interactions aid the design of new drugs for instanceenzyme inhibitors, by providing in depth details of the size on the activesites, how many subsides are present, their properties, how they come togetherand bind chemically. The understanding of this unique interaction is also atool 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-raycrystallography, florescence and IR spectroscopy, hence the ever growingimportance of active site mapping using Protein NMR.

(Yong et al.2012)19FNMRstudies has be done to clearly distinguish structural and functional featuresof protein as seen in  its recentapplication in active site mapping out of galactose binding- protein,transmembrane aspartate receptor, the Che – Y protein dihydrofolate reductase ,elongation factor-TU, and D-lactose 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 STD-NMR ExperimentJack bean (Canavaliaensiformis) urease (EC 3.5.

1.5), urea, Dulbecco’s Modified Eagle Medium (DME),cycloheximide,di-sodium hydrogen phosphate, mono-sodium di-hydrogen phosphate Unichem(India). Mous, and phenol were obtained from Sigma-Aldrich (USA). Deuteratedmethanol (CD3OD),and deuterium oxide (D2O) were purchased from the Armar Chemical (Switzerland) 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 °Cligands. The reaction mixture was in excess of 100folds of ureaseconcentration. They were dissolved in 13.3% of CD3OD, and 86.7% deuterated phosphatebuffer (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. STD-NMR spectra wererecorded with 32 scans (NS), and eight dummy scans. For each experiment, 90°pulse was calibrated separately. Gaussian selective pulses of 48ms length withan excitation bandwidth of 140 Hz, separated by 1 mms delays were used. Tosaturate the protein selectively, on-resonance irradiation was provided from 0to ?1 ppm (protein resonances), while off-resonance irradiation was provided at30 ppm. Difference spectrum was obtained by subtracting the on-resonanceirradiation spectrum from off- resonance spectrum. This was followed by dockingstudies that involve the study of the molecules present and how they interactwith each other so as to establish their identity, molecular structure and howthey bind to the proteins present.

These facts highlight the kind of inhibitionand the kind of interaction that is existing between the ligand and the proteinat the atomic level(Scopes 2002;)(Meng et al,2011;).  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 ofurease inhibitory activity by STD- NMR technique was done using Saturationtransfer differential NMR which is a ligand resonance based spectroscopicmethod that is undoubtedly one of the most widely used NMR Spectroscopic techniquedue to it’s ability to establish a binding relationship between the inhibitorsand protein as seen in this experiment. This technique uses the advantage of theability of the protons of the inhibitors which are in close contact to thetarget protein so receive high value of Rf saturation hence promotingdifferential signal in STN-NMR spectroscopy hence displaying this signalreceived from the environment with great intensity between receptor protein andligand molecule. This is an edge that the ligand resonance spectroscopictechnique have over the target based NMR technique as this method explores theproximity of the inhibitors to the protein and the intense signals generated tomake deduction we were able to established from this experiment that the wholemolecules were interacting with the enzymes (Jalaluddin A.et al 2017:). ligandNMR  as seen in this study, tend toobserve signals from ligands, no isotopic labeling is required for targetprotein, thus experimental method takes less time than target based NMR methodand can be used to determine dissociation constant either by the use oftitration experiment or be observation of changes in the width of a ligandinduced by protein binding (Yan Li et al.

2017;).The Docking  studies was able to affirm enzyme inhibitoryactivities F- NMR experimentalon the other hand is a target based method employed for the investigating ofprotein-ligand binding interactions in drug delivery mainly use in fragmentscreening, as the 19F nucleus has a natural abundance of 100%(83% of thesensitivity of 1H) and a massive chemical shift of dispersion (Didenko, J t etal, 2013;). Since “F- atom is not naturally present in biological systems,which means there will not be any background signal observed or detected (Horst.

R. et al, 2013:) (Kitevski – LeBlanc J.L et al, 2012: ) (Liu J.J et al, 2012:).So a target proteinwas first labeled in the bacteria system by adding 19F– labeled amino acid inthe culture medium, then purified after which it is modified by using 2-bromo-N-(-4-(trifluoromethyl)phenyl) acetamide (BTFMA) resulting in a very rapid 19F spin and because it isligand resonance spectroscopy a 19f atom was introduced in the ligand to enableits  observation through chemicalanalysis due to the 19F atom’s  chemicalshift being very sensitive to its environment and the changes that occurs init  as a result of the weak Dan Der Waals  bond and the presence of electrostatic field(Didenko T. et al, 2013:) Hit- identificationsteps is then adopted to identify, screen and validate the inhibitor as it is avery sensitive technique that is able to break down compounds with similarstructures to aid their detection by comparing the chemical shift change.

Thehit identification step was carried out using F-NMR method as this technique isalso use for this purpose in fragment base drug delivery in three differentways, which are; the comparison of the 19f-labelled compound with libraries ofavailable ligand –observed experiment with the aim of screening the19F- labeledcompound against libraries of available screened compounds to establish theligand size and chemical diversity with the view of using it for furtherdevelopment. More so, as biomedical assays are mainly use for primary screen inprotein NMR active site mapping, this method is then employed to confirm hitsscreens from HTS campaigns (Gee C.T, et l 2016:) as the 19f-labeled target isdistinguish from every other compound present in the normal HTS library as theyall do not possess 19F-labelling and in this system of identification theresidue from the labeled atom is usually close to the active site to enable structuraland biochemical characteristic to be studied, the  presence  of a fluorinated compound makes ease of studyof substrate by the use of F-NMR methodThis assay isdesign in such a way that the changes of the substrate on breaking down must becarefully observed to monitor the disintegration of the target protein, so asto be able to record and determine its ability to test a screened compoundaccurately as this is used for the hit identification and confirmation of thefluorinated substrate. The advantage ofthis method over the other is that even though ligand-observed experimentscannot be use for the identification of binding site this method can be used attimes due to the presence of the 19F labeled atom that aids in identificationof residue that are vital for binding in the presence of 19f assigned atom.

This methods of identification and confirmation also tends to produce positivefalse results in ligand – observed experiments due to the problem ofnon-specific interaction and aggregate effects (Zega .A, 2017;)Conclusion:Conclusively, protein NMRspectroscopy in active site mapping is an indispensable tool with wide range ofapplication in early stage of drug discovery, through all the phases ofmanufacturing till it is displayed on shelf owing to its methods, such asSTD-NMR spectroscopy, and its ability to adapt molecular docking techniques toits advantage. This characteristics of this technique aids its precision indrug screening and the ease of its application as well as the fact that it doesnot require a lot of data and its less time consuming when compared to otherNMR methods employed in this field. Furthermore, the knowledge that this methodprovides about the presence and the kind of enzyme present in a target site asseen in the study of the new urease inhibitor, the intensity of the bondbetween the active site and the inhibitor is very important for the formationand design of new drug, hence aids in producing drugs that binds to its receptor