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Serum albumin albumin (HSA) has two major drug binding sites located in domain-I (warfarin) and domain-III (diazepam) and one minor site along with fatty acids at the C-terminal chain of albumin (Fig 2). [4,5] Kragh-Hansen & Mandula In total, there are nine binding sites for metals and small molecules on albumin making it by far the most valuable carrier protein in serum . Principal function of HSA is to transport fatty acids (C10:0, C12:0, C14:0, C16:0 and C18:0 IE7E.pdb); and polyunsaturated fatty acids C18:1, C20:4 (1GNI.pdb). It is also capable of binding a variety of metabolites and hydrophobic drugs e.g. propofol and halothane (1E7A.pdb); warfarin (1ha2.pdb); thyroxine and bilirubin (1HK1.pdb); ABT-737 (1YSX.pdb); 3,5,3`,5`-tetraiodo-L-thyronine 'T4'; and with 3`,5`-dinitro-N-acetyl-LL-thyronine 'DNNAT' (2ROX.pdb); 3,3`,5,5`-tetraiodothyroacetic acid 'T4Ac' (1Z7J.pdb); bilirubin, thyroxine and hemin (1O9X.pdb), which results in temporary possession of drug molecules in serum.
Figure-2: Drug Binding site of Human Serum albumin.
Screening for the binding affinity of these molecules by computational and NMR methods prior to pre-clinical trials may provide important insight regarding pharmacokinetics. Computational screening of the ligands at the binding site of macromolecules have shown immense important in drug discovery process. In this study docking approach were implemented in order to find out binding strength of different ligands.
1.2 Protein ligand interactions by NMR
Pharmacokinetics and dynamics by NMR gives important insight in understanding of biological functioning. Such types of interactions by NMR spectroscopy could be measured in two ways; one way is to measure signals from the protein and an other way is that the ligand could be monitored. NMR based screening techniques depend on chemical shift perturbation, transferred nuclear-overhouser effect, diffusion and relaxation properties of the molecules. Recently few improvements have been done to screen the bound ligands directly through NOE pumping  Chen, and pulse field gradient (PFG-NMR) however diffusion based phenomena.
NMR is used as a screening, hit validation and lead optimization  Huth. NMR technique is now a rapid, reliable and robust method technique in drug discovery where biochemical assays are not available. Improvements in instrument and pulse sequence in recent years have added significantly to the drug discovery process. Digital recording, cryogenic probes, auto samplers and higher magnetic fields shorten time for data acquisition and improve spectral quality.
Drug discovery via NMR is applicable in hit finding (primary screening) hit validation (validation of binding site) hit optimization (structural analysis of complex structure and second site screening for further fragments). Hit optimization is also known as structure based drug designing which ultimately results into drug candidates  Shuker. The NMR based methods used in drug discovery are studied under two major subheadings, first: ligand observed signals and second: target observed techniques.
These are also classified according to parameter sensitive to ligand binding and other is site specific information about the residues in the structure that are involved in the binding. Site specific information has its own significance in drug development and NMR uniquely delivers such information in comparison to biological assay or other spectroscopic methods. For site specific information (SAR by NMR) target needs to be isotopically labeled. In fragment based approach binding site information is must which can be achieved only by use of labeled residues.
1.3 Target based experiments: Experiment based on chemical shift perturbation
Binding of ligand to the target leads to changes in chemical shift in NMR spectrum. For example formation of hydrogen bond decreases the electron density at the acceptor atom and hence generally leads to downfield shift. However anisotropy effect e.g. rings current effect also counts.
Changes in target's chemical shift are indicative of interaction with ligand. Multidimensional spectra are usually used as signal overlap and obscures analysis in 1 D spectrum. In these spectra, the shift of peaks due to interaction is observed. Obviously the use of 2D or 3D spectra results in increased experimental time. kd values also could be deduced if analysis is possible . If the displacement is related to the concentration of the ligand via titration, such type of experiments require a reference spectrum without ligand.
Chemical shift perturbation is most commonly tracked in 13C and 14N HSQC(Heteronuclear single quantum coherence) spectra, although HNOC () spectra also have been used. Labeling is another tedious job; 15N labeling is comparatively cost effective. While 13C methyl group labeling of valine leucine and isoleucine give better resolved chemical shift on binding because apparent signal intensity of methyl groups is three times of the amide protons. 15N-HSQC gives insight of the hydrophobic region whereas methyl labeling discloses binding nature of hydrophobic region.
Protein observed experiment needed labeling of the target molecule; especially the binding site must be uniformly labeled. For protein 15N labeling allows changes that occur upon binding to be detected very quickly and enables identification of the binding interface once [15N-1H] correlation map has been assigned. In most of the cases hydrophobic site buried therefore specific 13C methyl labeling may also be used, which is claimed to be more sensitive as, amide moieties represent hydrophilic areas while methyl groups are usually located in hydrophobic regions, Comparison of [15N-1H] HSQC and [13C-1H] HSQC screens may thus reveal additional information of interest.
1.3.1 SAR by NMR
[8,9] Shuker & Szczepankiewicz Chemical shift mapping is another way to characterize binding epitope if sequential alignment is available. If two fragments are to be combined as proposed SAR by NMR technique, these fragments are fine tuned separately to give higher affinity ligands, these two fragments bind to the same target but at different binding sites. The subsequent interaction of the two results in high affinity ligand. SAR by NMR is also known as fragment based drug discovery.
In vitro studies sometimes weaker interactions thereby another interesting approach 15NHSQC was applied on bacterial slurry. 2D spectra sometimes are not sufficient to visualize the binding process because of remaining signal overlaps. If binding site is known, site specific labeling could give better resolved spectra.
1.4 Ligand observed experiments
It is based on 1D NMR spectra and therefore are comparably fast allowing higher throughput screening of mixture of ligands without the need of deconvolution, as long as signals of ligand do not overlap. Ligand observed experiment is based on the analysis of changes in signal amplitudes as observed in 1H spectra for ligand signals in the presence of target. Provided that the complex is in fast exchange on the NMR time scale, the observed parameters for the ligand are the population weighted average of the free and bound form. Non-interacting substances are not influenced. In principle, all experiments in ligand observed experiments give a response in the form of a reduction (disappearance) or increase (appearance) of signal amplitude. The intensity of effects depends on the molecular weight of the target. Reference sample recorded in the absence of the target, and the test sample, which contains the target molecule, the signal amplitude detected can be described by an exponential law
I(Ï„ )=I0 . e-keff.Ï„
Here keff describes the dynamic process (e.g. the transverse relaxation rate R2, or the diffusion coefficient D) and Ï„ describes a time constant typical for experiment setup. keff can be as follows,
Increasing the population (kb) of the bound state, which will change the signal detected more drastically if the ratio of kb to kf is strongly dependent on the molecular weight of the target.
1.5 Experiments based upon changes in relaxation properties of ligands
Transverse and longitudinal relaxation of proton spins is mainly promoted by dipolar interactions between proximate units. The relaxation efficiency depends upon distance separation of the interacting spins and on the reorientation of the antinuclear vector. The latter is determined by the motional properties of the molecule, which is rigid, and in isotropically tumbling molecules is described by an overall rotational correlation time. Binding of the ligand to the target leads to dramatic changes in overall tumbling for the bound fraction of ligand, whereas the changes for the target can be neglected. Usually weak binding will lead to significant changes in line widths of ligand signals. The overall correlation time can be estimated from MWtarget using the strokes-Einstein equation. Alternatively, it may be estimated from the equation obtained by fit of experimental data, )
N denotes the number of amino acids and T the temperature  Daragan.
The effective relaxation rate is very sensitive to the chemical shift difference between free and bound state. Since proton usually have different environment upon bindings, the changes in relaxation rates are not uniform for all signals. The effects due to enhanced transverse relaxation can be experimentally determined from a CPMG pulse experiment. Determination of T1p by ROSEY experiment is more attractive. ROSEY suffices to record relaxation -weighted experiments to reveal binding.
1.6 Diffusion editing experiments
Affinity NMR utilizes the changes in translational diffusion to probe an interaction of a ligand with a target  Johnson  Lin  MA  Lin. The diffusion- editing experiments are based on the fact that upon binding of ligand to the target the hydrodynamic radius rH of the ligand increases dramatically and hence the diffusion rate decreases. This is principal property of molecule, and hence all signals from the ligand will be influenced in the same way.
In principale, the hydrodynamic radius may be calculated when the structure is known. However, such calculation contains large errors due to uncertainties in the extent of the hydration shell. One can use the following empirical formula to estimate hydration radii compatible with NMR result
Which can be found under www.protein-solutions.com/calc.htm
With the help of stokes -Einstein relation, the translational diffusion coefficient may be calculated according to
Relative decrease in the diffusion coefficient of the ligand upon binding to the target indicates interaction. If there is large difference in mass the change in diffusion coefficient is large.
Diffusion constants are most conveniently measured with the bpPFGLED ( ) sequence. Therein, magnetization is stored as polarization along the z-axis during the diffusion delay in order to reduce loss due to T2 decay furthermore, an additional z-filter at the end of sequence allow for decay of eddy currents caused by the pulse field gradients. In order to yield optimal results, the lock circuit should be highly damped during the measurements.
The signal decay due to diffusion in series of 1-D spectra is given, in line with eq. by
The experimental parameters (gradient strength (g), gradient length (Î´), and diffusion delay (T) are selected to optimally suppress the signals of nonbinding compounds and to allow for the detection of resonances of the biological target and bound ligands.
1.7 NOE based Techniques
Recently, a number of NMR screening techniques based on the Nuclear Overhauser effect (NOE) have been developed.
1.7.1 The saturation transfer difference (STD)
The saturation transfer difference (STD) technique  Lin  Neuhaus  Meinecke uses the difference of two spectra. The STD spectrum itself is recorded with saturation of target resonances (mostly the "methyl hump" of the protein is saturated). A second experiment in which irradiation is performed far-off-resonance, is acquired as a reference. The "STD module" contains the following elements viz irradiation-excitation-relaxation filter (optional). The relaxation filter can be used to efficiently suppress residual target magnetization prior to acquisition. It is well known that the steady state NOE leads to highly efficient spin diffusion for large molecules and aggregates  Lin, therby usually precluding its use for structure determination of high molecular weight molecules.
Hence, upon saturation of target signals, efficient spin diffusion leads to almost complete saturation of all target resonances via intermolecular spin diffusion. In the case of binding, intermolecular spin diffusion leads to saturation of ligand resonances as well. In the reference experiment, all signal intensities remain unchanged, since no target signal is irradiated (off resonance irradiation). Substraction of STD from the reference experiment yields only signals of binding compounds, because their signal intensities have changed.
Preferably, subtraction is done in an interleaved manner by phase cycling the receiver phase concomitantly with switching between on- and off-resonance irradiation such that only the difference spectrum is recorded. STD can be used to screen single ligands for binding, but it may be also applied for the screening of compound mixtures. In this case, the "STD module" can be combined with other 2D experiments (e.g. STD -TOCSY) in order to unambiguously identify binding compounds without the need for further deconvolution of the mixture.
Although STD is most commonly applied in solution state NMR, it can be used advantageously in HR-MAS NMR  Jens. In HR-MASS NMR target is immobilized on controlled pore glass (CPG). Spin diffusion is much faster for immobilized targets, and therefore the STD effect is much more pronounced when compared to the solution state. The use of HR -MAS ensures enough resolution to identify binding compounds via their 1H NMR spectra. Since the free ligands are observed, STD works best in fast exchange regime, i.e. with kd values in the milimolar or upper micromolar range. One drawback of the STD method is the need for target resonance isolated from all irridated signals. Thus, in principle, all ligands spectra have to check for signals residing in the irradiated spectral region.
In the transferred NOE (tr NOE) experiment, a 1-D or 2-D NOESY experiments is recorded. The intermolecular NOE build-up to be used as parameter arises from the bound state but is observed via the free ligand, requiring rapid exchange between bound and free states.
1.7.3 NOE pumping
The NOE pumping technique uses intermolecular cross relaxation  Aidi. The pulse sequence consists of a diffusion filter element for nonlabeled samples with a subsequent NOE mixing period. In the diffusion filter, ligand magnetization is effectively dephased while protein magnetization is largely preserved. During the NOE mixing time, both intra- and intermolecular cross relaxation occur. Ligand signals are easily distinguished from protein resonances which are largely preserved. During the NOE mixing time, both intra and intermolecular cross relaxation occurs. Ligand signals are easily distinguished from protein resonances because of their much narrower line widths and their different NOE build up curves.
1.7.4 Reverse NOE Pumping
In reverse NOE pumping experiment  Aidi, a relaxation (T1p /T2) filter is followed by NOE mixing time. During the mixing time, polarization is transferred from the binding ligand to the protein. The signals of the binding ligands are modulated by longitudinal relaxation as well as by intermolecular cross relaxation.
A second reference experiment is recorded using the second sequence. Therein, the order of two elements is interchanged and the resulting ligand signals are mainly modulated by T1 relaxation during the mixing period. Substracting the FIDs from the two experiments (the RNP and the reference spectrum) results in a spectrum containing only signals of binding ligands.
1.7.5 Water LOGSY
Dalvit has obseved when ligand binds to the protein the intermolecular water-ligand NOE will be negative [21,22]. This is because of dramatic change in the correlation time of the ternary water-ligand-protein complex compared to that of the water ligand complex. Bound water is quite often found at the interface between the protein and the ligand. The residence times of water in protein cavities lies in the range between few ns to few 100Âµs  Gottfried, long compared to the effective correlation time of 0.3ns, at which the intermolecular NOE changes its sign. Intermolecular cross-relaxation rate depends on the water residence time. The water LOGSY technique is thus similar to transfer NOE technique, but with the difference that the water-ligand NOE indicates binding.
1.8 Limitation in ligand based screening: Ligand based method is failure in case of high affinity ligands. Because strong interactions possess a long residence time in the binding cavity of the macromolecule, their exchange rate is very low. This slow exchange results in the loss of the information about the bound state as the nonequlibrium magnetization that is created on the ligand decays before it can be detected by simple ligand based techniques and leads to false negative.
1.9 Hit validation via ligand based screening: High affinity ligands could not be detected by simple ligand based technique. A known medium affinity binder (reporter ligand) is added to the solution of the target. If other ligands (screening ligands are present in the solution as well, the reporter ligand will be displaced according to the affinity of screening ligands. With competition based experiments only ligands that bind to the same site as reporter ligand are detectable, also making this method relevant to the hit validation process  Dalvit  Dalvit  Jahnke. All competition experiments can be used to estimate dissociation constant of screened ligands if the kd of reporter ligand is known. An NMR titration has to be performed with the ligand of interest while the concentration of the target and reporter ligand kept in fixed. The change of the intensity of the reporter ligands signals and subsequent fitting to corresponding equations gives the required information.
In principle all ligand observed techniques can be extended to a competition type of experiment. However experiments using simple 1D spectra are specially worth considering, e.g. WaterLOGSY, STD, and 19 F screening, which rank among the most powerfull ligand based screening techniques
1.10 Fluorine based screening: A comparably new technique is the screening of ligands that contain 19F-labels, for example, in the form of fluorinated aromatic or trifluromethyl groups  Peng  Dalvit  Dalvit The 19 F nucleus has some unique features that make it an attractive probe. Once the 19F library is characterized (knowledge about all 19F chemical shifts) the standard mixture can be used for screenings. It has high gyromagnetic ratio (Î³F~ 0.94 Î³H) and occurs at 100% of natural abundance making it very sensitive NMR nucleus. It has broad chemical shift diversion, allowing the use of large mixtures without signal overlap. Upon binding, the chemical shift and the linewidth of ligand 19F signal is strongly affected (as result of the large chemical shift anisotropy (CSA) of the 19F nucleus). Hence the acquisition of 1 D spectrum with and without the protein molecule is sufficient to screen the binding.
Ofcourse the need for 19F-labels seems to be a profound drawback. However, about 10% of all drugs on the market already contain fluorine modified for metabolic stability. Additionally the small fluorine atom could be replaced if there is no loss of binding affinity.
1.11 Limitations of NMR based drug discovery: There are two major limitations in NMR based screening, one is that it requires large protein sample. Another limitation is poor solubility of biomolecules. Hydrophobic ligands are often required to enable binding to a hydrophobic pocket of the target. To achieve a reasonable throughput in ligand -observed experiment, the concentration per substance in mixture has to be around 100ÂµM or 0.5mM in cryo and conventional probe technology respectively. Experiments are typically performed at tenfold reduced protein concentration. The required high concentration of ligands limits the number of compounds that can be tested simultaneously, since the total amount of ligands is normally limited for reasons of solubility and stability of protein
Screening of the binding affinity of the molecules under study by computational and NMR methods prior to pre-clinical trials may provide important insight regarding pharmacokinetics. Computational screening of the ligands at the binding site of macromolecule has shown immense important in drug discovery process. In the present study docking approach were implemented in order to find out binding strength of ligands.
Protein ligand interactions i.e pharmacokinetics and dyanamics by NMR gives important insight in understanding of biological functioning. NMR based screening techniques depend on chemical shift perturbation, transferred nuclear-overhouser effect, diffusion and relaxation properties of the molecules. Recently few improvements have been done to screen the bound ligands directly like NOE pumping  Chen, and PFG-NMR both are based on diffusion phenomena  Wu.
Diffusion property of molecules is very useful in identification of changes in chemical environments of ligands and macromolecule. Diffusion based NMR methodologies can be used to evaluate protein ligand interaction in solutions because change in diffusion directly reflects the molecular size and hydrodynamic properties of molecules. The diffusion rate is measured by pulse field gradient nuclear magnetic resonance spectroscopy (PFG-NMR), which is based on the attenuation of individual proton resonances under the influence of field gradients. The amplitude (I) of the signal is directly related to the rate of change in diffusion diffusion coefficient (D), according to fallowing equation,
Intensity of NMR signals in the presence of gradient pulse is I and I0 in the absence of gradient pulse, big delta âˆ† is the time period for translational diffusion, Î³ is the nuclear magnetogyric ratio, g is amplitude and Î´ is duration of gradient pulse.
Diffusion-assisted NOE experiment is another method introduced by Chen and Shaprio in 1998 as a fast active drug screening technique. In the pulse sequence (Figure-2), a diffusion filter (LED, longitudinal eddy current delay) is first applied to prepare a state in which free ligands are filtered out while signals from the macromolecule remain. Bipolar gradient pulse pair (BPP) minimize spectral artifacts and chemical exchange modulation. To observed signals from bound ligand, a NOE experiment (two 900 pulses separated by mixing time tm ) follows immediately after diffusion filter, which leads to magnetization transfer between spins that are close to each other in space i.e. from macromolecule to bound ligands.
In the present study we implemented two diffusion based techniques, PFG-NMR and NOE pumping along with computational screening at the two specific drug binding site of albumin as well as blind screening were applied for curcumin and curcumin bioconjugates. For protein ligand interaction serum protein and tryptophan was used as a model system as a binder and glucose as anon binder.  Jiangli