Assignment:   The Use of Protein NMR in Active Site

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.

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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.

protein analytical tool has been used 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





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)


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 utilized 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










Figure 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 identified
through the whole complete process and the role it plays
highlighted in white and blue; Figure 111, highlights the various steps
involved in using protein NMR in active drug in drug discovery and its application.
(Yan Li et al, 2017;).


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)


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 magnetization 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.

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

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 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


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 using 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

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:)

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

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.

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.


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