Methods/ at cysteine residue which results in the presence


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:).

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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.

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 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

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 – 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;).  LeBlanc J.L et al, 2012: ) (Liu J.J et
al, 2012:).

So 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.

a target

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;)


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.