Assignment: known chemical constituent of a compound to distinguish

Assignment:   The Use of Protein NMR in Active Site

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.

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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 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 protein as seen in  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

 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) STD-NMR Experiment

Jack bean (Canavalia
ensiformis) urease (EC, urea, Dulbecco’s Modified Eagle Medium
(DME),cycloheximide, di-sodium hydrogen




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.

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