Nuclear Magnetic resonance spectroscopy (NMR) is an analytical technique that is based on using the known chemical constituent of a compound to distinguish it from other unknown compounds. The ability of this technique to distinguish the difference in molecular structure of substances and the information it provides about the dynamics and interactions of molecule in the smallest possible unit of a matter makes it an indispensable tool in the process drug discovery, development and delivery. This chemical analytical method is very sensitive to its environment, so can give very minute information about how the smallest fragment of a molecule binds to a target molecule, protein or its complexes. Information about the exact binding site or interaction between the fragment and the receptor of interest is also highlighted. Hence, this technique is a very vital technique in the Pharmaceutical, forensic, quality control industry. This analytic technique also has its application in the field of research where it is used to determine the purity, quality, quantity and structure of the unknown while confirming that of the known substance. The combination of this analytical chemistry technique to Protein in the biological science is what is known as Protein NMR.
Protein Nuclear Magnetic Resonance has been used extensively to study enzyme mechanisms, analyzing structures of proteins, nucleic acid and its complexes This technique is also employed in studying protein ligand /protein interactions and the dynamics of protein. In the field of drug development, the study of protein and its complexes are of utmost importance as they play vital role in physiological and pathological conditions and process hence the importance of thoroughly understanding their catalytic process and how they bind to their substrate. Protein NMR in active site mapping thus, is the application of NMR in the region of an enzyme where substrate molecules bind and undergo chemical reaction as well as where its residues forms temporary chemical bonds with the substrate. This region in an enzyme is known as the active site. The mapping of active sites is quite crucial in the field of pharmaceutical science or drug discovery. The detailed knowledge of the site of a target receptor for drug discovery and the understanding of the protein dynamics in the targeted site will maximize the efficacy of the proposed drug by giving a clear and precise understanding of the protein -ligand binding information and protein-ligand/protein interaction (Yan Li et al,2017). These interactions aid the design of new drugs for instance enzyme inhibitors, by providing in depth details of the size on the active sites, how many subsides are present, their properties, how they come together and bind chemically. The understanding of this unique interaction is also a tool for comparison in active site mapping, where it is employed to compare protein active sites and their structures in more details so as to design drugs that can exactly match into the enzyme substrate complex using the key and lock analogue for enzymes.
This protein analytical tool has been use in lots of studies to investigate enzyme behaviors, their mechanisms as it takes less time an effort to acquire structural information of compounds and DNA when compared to other methods like X-ray crystallography, florescence and IR spectroscopy, hence the ever growing importance of active site mapping using Protein NMR.(Yong et al.2012)
19FNMR studies has be done to clearly distinguish structural and functional features of protein as seen in its recent application 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 utility of 19 F NMR in the analysis of protein conformation state even in particles that are so large or unstable for full NMR structure determination.(Mark A.D, et al 2010).These kind of studies depends on the chemical shift pattern of FNMR as this method is very sensitive to change in its environment due to the presence of fluorine 19, as well as
Jack bean (Canavalia ensiformis) urease (EC 220.127.116.11), 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). Deuterated methanol (CD3OD), and deuterium oxide (D2O) were purchased from the Armar Chemical (Switzerland)
The measurement of urease inhibitory activity by STD- NMR technique was done using the afore mentioned technique, that is very popular in drug discovery and possess high sensitivity hence often used for ligand –observed NMR screening methods. In this experiment, Gaussian RF pulse was applied to the most up field protons of the target protein which when saturated is then transferred throughout the molecule by spin diffusion. At the final stage of this process the bound ligands received magnetisation through cross relaxation and enhanced signal intensity is displayed (Atia-tul_Wahab et al.2013:).
The sample for this experimental process is prepared with Jack bean (Canavalia ensiformis, EC3.5,1.5) using deuterated NMR buffer to prepare(20uM) of urease solution, which is then stored at 4 °C ligands. The reaction mixture was in excess of 100folds of urease concentration. They were dissolved in 13.3% of CD3OD, and 86.7% deuterated phosphate buffer (4 mM, pH 6.8).
This was followed by STD-NMR screening experiment performed on Bruker 400MHZ NMR spectroscopy at 298K Stddiffgp19 pulse 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 was 8.0 and 4.0.
STD-NMR spectra were recorded with 32 scans (NS), and eight dummy scans. For each experiment, 90° pulse was calibrated separately. Gaussian selective pulses of 48ms length with an excitation bandwidth of 140 Hz, separated by 1 mms delays were used. To saturate the protein selectively, on-resonance irradiation was provided from 0 to ?1 ppm (protein resonances), while off-resonance irradiation was provided at 30 ppm. Difference spectrum was obtained by subtracting the on-resonance irradiation spectrum from off- resonance spectrum. This was followed by docking studies that involve the study of the molecules present and how they interact with each other so as to establish their identity, molecular structure and how they bind to the proteins present. These facts highlight the kind of inhibition and the kind of interaction that is existing between the ligand and the protein at the atomic level(Scopes 2002;)(Meng et al,2011;).
Experimental For F-NMR Technique
Purification of the target protein is usually the first step, followed by the modification of the protein of target by 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 “F spin ( Horst et al, 2013;) (Kitevski et al,2012:) ( Liu J, J et el, 2012;) making it possible for chemical analysis to be carried out , which is normally the last step before the process of Hit identification. (Norton et al, 2016;)
Hit identification is carried out at this stage to for the purpose of screening F- labeled compound using ligand – observed experience known as FBDD,that usually has an existing library or in the absence of this library one can easily be made-up by adopting similar rules to those use in usual fragment library to sustain ligand size and chemical variations. F- NMR as a target based protein spectroscopy can be used to affirm the hit screening from HTS campaigns in which 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, are labeled with Fluorine atom. This technique is then preceded with the identification and validation of the targeted resonance in the presence of the fluorinated substrate.
In this review we have looked at the use of protein NMR in active site mapping by using biochemical assay, then followed by the use of STD-NMR which is a ligand resonance based technique, for the primary identification of urease inhibitors. Then followed by molecular docking studies to validate the biochemical experiment as well as to estimate the relative binding affinity between the ligand and receptor. F-NMR which is a target based resonance, coupled with hit identification methods were also use to observe targeted ligand, screening were carried out, confirmation of the primary screen with the use of the F atom and its identification and validation in the presence of the fluorinated substrate was achieved in this experiment
The measurement of urease inhibitory activity by STD- NMR technique was done using Saturation transfer differential NMR which is a ligand resonance based spectroscopic method that is undoubtedly one of the most widely used NMR Spectroscopic technique due to it’s ability to establish a binding relationship between the inhibitors and protein as seen in this experiment. This technique uses the advantage of the ability of the protons of the inhibitors which are in close contact to the target protein so receive high value of Rf saturation hence promoting differential signal in STN-NMR spectroscopy hence displaying this signal received from the environment with great intensity between receptor protein and ligand molecule. This is an edge that the ligand resonance spectroscopic technique have over the target based NMR technique as this method explores the proximity of the inhibitors to the protein and the intense signals generated to make deduction we were able to established from this experiment that the whole molecules were interacting with the enzymes (Jalaluddin A.et al 2017:). ligand NMR as seen in this study, tend to observe signals from ligands, no isotopic labeling is required for target protein, thus experimental method takes less time than target based NMR method and can be used to determine dissociation constant either by the use of titration experiment or be observation of changes in the width of a ligand induced by protein binding (Yan Li et al.2017;).The Docking studies was able to affirm enzyme inhibitory activities
F- NMR experimental on the other hand is a target based method employed for the investigating of protein-ligand binding interactions in drug delivery mainly use in fragment screening, as the 19F nucleus has a natural abundance of 100%(83% of the sensitivity of 1H) and a massive chemical shift of dispersion (Didenko, J t et al, 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 protein was first labeled in the bacteria system by adding 19F– labeled amino acid in the 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 is ligand resonance spectroscopy a 19f atom was introduced in the ligand to enable its observation through chemical analysis due to the 19F atom’s chemical shift being very sensitive to its environment and the changes that occurs in it as a result of the weak Dan Der Waals bond and the presence of electrostatic field (Didenko T. et al, 2013:)
Hit- identification steps is then adopted to identify, screen and validate the inhibitor as it is a very sensitive technique that is able to break down compounds with similar structures to aid their detection by comparing the chemical shift change. The hit identification step was carried out using F-NMR method as this technique is also use for this purpose in fragment base drug delivery in three different ways, which are; the comparison of the 19f-labelled compound with libraries of available ligand –observed experiment with the aim of screening the19F- labeled compound against libraries of available screened compounds to establish the ligand size and chemical diversity with the view of using it for further development. More so, as biomedical assays are mainly use for primary screen in protein NMR active site mapping, this method is then employed to confirm hits screens from HTS campaigns (Gee C.T, et l 2016:) as the 19f-labeled target is distinguish from every other compound present in the normal HTS library as they all do not possess 19F-labelling and in this system of identification the residue from the labeled atom is usually close to the active site to enable structural and biochemical characteristic to be studied, the presence of a fluorinated compound makes ease of study of substrate by the use of F-NMR method
This assay is design in such a way that the changes of the substrate on breaking down must be carefully observed to monitor the disintegration of the target protein, so as to be able to record and determine its ability to test a screened compound accurately as this is used for the hit identification and confirmation of the fluorinated substrate.
The advantage of this method over the other is that even though ligand-observed experiments cannot be use for the identification of binding site this method can be used at times due to the presence of the 19F labeled atom that aids in identification of residue that are vital for binding in the presence of 19f assigned atom. This methods of identification and confirmation also tends to produce positive false results in ligand – observed experiments due to the problem of non-specific interaction and aggregate effects (Zega .A, 2017;)
Conclusively, protein NMR spectroscopy in active site mapping is an indispensable tool with wide range of application in early stage of drug discovery, through all the phases of manufacturing till it is displayed on shelf owing to its methods, such as STD-NMR spectroscopy, and its ability to adapt molecular docking techniques to its advantage. This characteristics of this technique aids its precision in drug screening and the ease of its application as well as the fact that it does not require a lot of data and its less time consuming when compared to other NMR methods employed in this field. Furthermore, the knowledge that this method provides about the presence and the kind of enzyme present in a target site as seen in the study of the new urease inhibitor, the intensity of the bond between the active site and the inhibitor is very important for the formation and design of new drug, hence aids in producing drugs that binds to its receptor and exert a physiological effect as well as highlighting Professionals on pathological issues.