Nuclear what is known as Protein NMR. Introduction

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)

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 the existing weak Vander Waal force of bond as well as the presence
of the local electrostatic field.





Figure 1. Overview of applications of NMR in drug discovery





NMR spectroscopy can
provide critical information at early stages of hit validation and
identification. NMR measurements for binding studies can represent a key step
to eliminate false positives from high-throughput (HTS) campaigns, to validate
putative hits from in silico screens
or to identify novel scaffolds in fragment-based programmed. NMR and X-ray
crystallography can also provide unique information to subsequently guide
hit-to-lead optimization. ADME-tox, absorption, distribution, metabolism,
excretion and toxicity (Pellecchia M el at: 2002)


This review will
mainly concentrate on saturation transfer difference (STD – NMR) method which
is a solution state nuclear magnetic resonance spectroscopy technique used in
target- based drug discovery, hit identification, validation and lead
optimization which is a tool that is extensively utilised in drug development
processes as seen in our review of this method in the  biological studies of new urease inhibitors.















Fig2 flowchart showing
drug discovery process






















Fig3  showing the
process in Protein NMR Process










11.This is a flow chart showing the different level of application of NMr in
the process of drug discovery from when the target is identifined through the
whole 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 drug
discovery and its application.(Yan Li et al, 2017;).


Materials and
Sample preparation STD-NMR Experiment

Jack bean (Canavalia
ensiformis) urease (EC, 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 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 – 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;)


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.