TECHNOLOGIES
DRUG DISCOVERY
TODAY
Drug Discovery Today: Technologies Vol. 1, No. 3 2004
Editors-in-Chief
Kelvin Lam – Pfizer, Inc., USA
Henk Timmerman – Vrije Universiteit, The Netherlands
Lead optimization
New approaches for NMR screening indrug discoveryCesar Fernandez*, Wolfgang Jahnke*Novartis Institutes for Biomedical Research, 4002 Basel, Switzerland
NMR spectroscopy has become a powerful and versa-
tile tool in pharmaceutical research, particularly for
studies of protein–ligand interactions. During the past
few years, new NMR screening techniques have been
developed. Some of them aim to increase sensitivity,
which translates directly into higher throughput and/or
decrease of protein consumption. Other approaches
introduce completely new screening concepts and yield
qualitatively different information. A brief description
of some new NMR screening techniques applied to drug
discovery is given in this review, and their principal
advantages and drawbacks are discussed. These meth-
ods have made an appreciable contribution to drug
design, leading to the discovery of a large number of
high affinity ligands for biologically relevant protein
targets.
*Corresponding authors: (C. Fernandez) [email protected](W. Jahnke) [email protected]: http://www.novartis.com
1740-6749/$ � 2004 Elsevier Ltd. All rights reserved. DOI: 10.1016/j.ddtec.2004.10.003
Section Editor:Oliver Zerbe – Institute of Organic Chemistry, University ofZurich, Switzerland
Fernandez and Jahnke describe the latest developments in NMRmethodology aimed at improving screening. They comprehensively
describe the advantages and limitations of NMR-based screening and
compare it to other (mainly biochemical) methods. They particularlydescribe experiments and hardware developments for improved
sensitivity, such as labeling the protein with spin labels, introducing 19Fmoieties or 13C-methyl groups, affinity tags and so on. In their
discussion of hardware/processing, they introduce the concept ofcryogenic probes, surface microcoils and reduced dimensionality
experiments.Fernandez and Jahnke are members of the BioNMR team at
NOVARTIS. Both authors have outstanding experience in proteinNMR and worked on a large number of projects involving these
techniques for lead optimization.
scopy with its intrinsic high sensitivity to detect weak inter-
actions becomes the method of choice to pick up such low-
IntroductionNMR-based ligand screening (NMR screening, see Glossary) is
now a well-established methodology for drug discovery. As a
valuable complement to other available techniques, such as
high-throughput screening (HTS), combinatorial chemistry
and structure-based drug design, NMR screening is being
broadly applied in pharmaceutical research. A commonly
used strategy for fragment-based drug discovery consists in
building up high-affinity ligands in a modular way, starting
from small scaffolds or ‘‘fragments’’ and growing, linking or
merging them into high-affinity ligands. Because the indivi-
dual fragments generally bind with low affinity, they are
difficult to identify with conventional assays. NMR spectro-
affinity fragments by screening of a compound library. This
process has led to the discovery of numerous high affinity
ligands for several biologically relevant protein targets
(numerous practical examples are presented in the Related
Articles).
NMR screening methods can be divided into two cate-
gories: methods that observe target resonances and methods
that observe ligand resonances. In screening methods that
observe the macromolecular target, the parameters that are
typically monitored are the chemical shifts (see Glossary).
The advantages offered by these target observation methods
are substantial: the NMR assay is applicable to any class of
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Drug Discovery Today: Technologies | Lead optimization Vol. 1, No. 3 2004
Glossary
Chemical shift: probably the most important NMR parameter. It
defines the location of a NMR line along the radiofrequency axis in
the spectrum. It is measured relative to a reference compound and is
expressed in ppm (parts per million). Chemical shifts are very sensitive to
the chemical environment of a nucleus, and their magnitudes depend on a
large number of factors, e.g. chemical nature of neighboring atoms,
including atoms involved in tertiary contacts in folded proteins and in
intermolecular interactions in complexes; solvent properties and tem-
perature.
NMR probe: hardware designed to transmit and receive the NMR
radiofrequency signals, to and from the NMR sample. The principal
components are the radiofrequency coils.
NMR screening: identification of small-molecule ligands for macro-
molecular targets by observation of a change in an NMR parameter that
occurs upon their interaction.
Nuclear Overhauser effect (NOE): it is the fractional change in
intensity of one NMR line when another resonance is irradiated. NOEs
arise owing to dipolar interactions between different nuclei and are
correlated with the inverse sixth power of the internuclear distance. The
nuclei giving rise to these resonances must be located closely in space
(typically<5 A), either in the same molecule or in different molecules in a
complex. From the magnitude of the NOEs, interatomic distances can be
obtained, which constitute the basis for the determination of the three-
dimensional NMR structure of biomolecules in solution.
SAR-by-NMR: NMR-based method, in which small organic molecules
that bind to proximal subsites of a protein are identified, optimized, and
linked together to produce high-affinity ligands. The approach is called
‘‘SAR-by-NMR’’ because structure-activity relationships (SAR) are
obtained from NMR [2].
Saturation transfer difference (STD): NMR experiment designed to
detect ligand binding to macromolecules. Following saturation of the
protein with a radiofrequency field, the magnetization is transferred to
any bound ligand by intermolecular spin diffusion. Difference spectro-
scopy with a reference spectrum recorded without protein irradiation is
then used to identify the signals from bound ligands, whereas the signals
from the non-binding compounds cancel out.
T1r relaxation experiment: NMR experiment designed to determine
the transversal relaxation properties of compounds. Because the trans-
versal relaxation rates of the resonances depend on the molecular weight
of the compound giving rise to them, this experiment can be applied as an
‘‘NMR binding assay’’.
WaterLOGSY: NMR experiment that utilizes the large bulk water
magnetization to transfer magnetization via the protein–ligand complex
to the free ligand molecules. In this experiment, the resonances of the
binding compounds appear with opposite sign than those of the non-
binding compounds and can thus be used as an ‘‘NMR binding assay’’.
compound, with no upper limit in affinity (typically, com-
pounds with dissociation constants from mM to nM and
lower can be monitored), identification of the ligand binding
site is possible, and measurement of the dissociation constant
(Kd) can be pursued for millimolar and micromolar ligands
[1]. The main disadvantages of these techniques are the
requirement of large amounts of well soluble and isotopically
labeled protein, the need to deconvolute compound mix-
tures, and the upper limit for protein size. The ‘‘SAR-by-NMR’’
technique (see Glossary) belongs to the target-observe
methods [2].
For experiments that rely on observation of ligand reso-
nances, the choice of NMR parameters is more diverse.
These include longitudinal, transverse (e.g. T1r relaxation
278 www.drugdiscoverytoday.com
experiments, see Glossary) and double-quantum relaxation,
diffusion coefficients, and intramolecular and intermolecular
magnetization transfer (including transferred NOE, NOE
pumping, saturation transfer and WaterLOGSY experiments;
see Glossary) (for detailed reviews, see [3–5]). These experi-
ments do not require isotopically labeled proteins, the one-
dimensional (1D) experiments used by these methods typi-
cally require a shorter acquisition time than the 2D experi-
ments used in the screening methods with target observation,
the hits can be identified directly without the need to decon-
volute them from compound mixtures, and there is no upper-
limit to the size of target that can be screened (in fact, many of
them work best for larger proteins). However, a general
drawback of the ligand-observation methods is their inability
to detect high-affinity ligands. In strongly bound ligands,
slow dissociation rates prevent transfer of the properties
of the relative small fraction of bound ligand molecules
to the bulk unbound ligand molecules, which are in
effect the ones observed by the NMR experiments. Competi-
tion-based experiments (see below) can eliminate this
drawback.
In the past few years, several new screening methods have
been developed. Some of them have been devised to combine
the advantages from both, ligand-detected and target-
detected techniques, and to overcome intrinsic limitations
of available methods. A general trend is observed to design
NMR experiments and instrumentation that yield increased
sensitivity, leading to higher throughput and/or decrease of
protein consumption. At the same time, novel NMR techni-
ques that allow measurements on larger molecular targets
have been recently developed [6–9]. This has extended the
applicability of NMR to studies of soluble proteins with
molecular weights well above 50 kDa [10], of integral mem-
brane proteins in micelles [11,12] and of macromolecular
assemblies [13]. A brief description of recent advances in NMR
screening techniques is given in the next sections.
New methodologies for NMR screening
Reporter screening
As discussed above, the main issue with most ligand-detected
experiments is their limited affinity range, which is often
restricted to 3–4 orders of magnitude in Kd, within the range
�1 mM to 100 nM. One approach that has recently been used
to overcome this constraint is competition-based experi-
ments, such as NMR reporter screening [14–16]. By this
method, not the binding to the protein target of the test
compounds is directly observed, but the ability of a test
compound to displace a known ligand that is added to the
mixture of protein and test compounds as a ‘‘reporter ligand’’
or ‘‘spy molecule’’, which binds to the protein with medium
affinity (Fig. 1). Bound reporter ligands can be readily dis-
tinguished from unbound ligands by their resonance signals
as seen in conventional 1D 1H or 19F spectra, proton T1r
Vol. 1, No. 3 2004 Drug Discovery Today: Technologies | Lead optimization
Figure 1. Principle of NMR reporter screening. In this experiment, the
resonance signals of a reporter ligand (triangles) are detected in the
presence of the target protein and test compounds in 1D proton spectra.
(a) Without protein target, the signals of the reporter ligand are sharp, as
commonly observed for small, unbound molecules. (b) In the presence of
the target protein but in the absence of test compounds, the reporter
ligand is bound to the target protein with moderate affinity, and its
relaxation rate is increased, as evidenced by the severe line broadening of
the reporter ligand resonances in the NMR spectrum. (c) After adding
test compounds that bind to the same site as the reporter ligand with
higher affinity, the reporter ligand is displaced from the binding site, so
that it becomes unbound. Unbound reporter ligand can be readily
distinguished from bound reporter ligand by its sharp resonances in
the NMR spectrum (peaks colored blue, compare with spectrum in (a)).
If test compounds are added that do not bind to the protein target, bind
more weakly than the reporter ligand, or bind to a different, non-
competitive binding site, the test compounds would not displace the
reporter ligand from its binding site, and therefore it would remain
bound to the target. This would be manifested by broad lines in the NMR
spectrum of the reporter ligand, as seen here in (b).
relaxation, WaterLOGSY, or saturation transfer difference
(STD) spectra.
Reporter screening has several additional advantages when
compared with conventional NMR screening methods based
on detection of ligand signals (Table 1). First, it allows obser-
vation of high-affinity ligands that are commonly detected as
‘‘false negative’’ by other ligand-observation NMR methods.
Moreover, it only detects ligands that bind to the protein
active site, while not detecting unspecific binding. As an
alternative, if fluorine-containing reporter compounds are
used, 19F NMR spectroscopy can be applied, which can
increase the throughput and significantly reduce signal over-
lap in the NMR spectra [17,18].
Spin labels for NMR screening
Spin labels can be used to identify and characterize inter-
molecular interactions. Recently, several applications of spin
labels for NMR screening have been reported [19]. They are
based on observation of relaxation enhancement of ligand
resonances caused by its proximity (typically up to 15–20 A
apart) to a spin-labeled group, for instance, a paramagnetic
organic nitroxide radical such as TEMPO. The spin labels can
be covalently attached either to the protein, or to a first
ligand.
The SLAPSTIC method (spin labels attached to protein side
chain as a tool to identify interacting compounds) (Fig. 2) is
an extension of the traditional T1r relaxation experiments,
where one forces amplification of the bound state relaxation
properties via covalently attached spin labels to protein side
chains, such as lysine, tyrosine, cysteine, histidine and
methionine [19,20]. As a condition, at least one residue of
this type should be as close as possible to the binding site, but
must not interfere with ligand binding. SLAPSTIC is a very
sensitive NMR screening technique: it allows a reduction of
protein demand by 1–2 orders of magnitude compared to T1r
relaxation experiments on non-modified protein targets.
Second-site NMR screening can be efficiently performed
with the use of spin labels [19,21]. This method utilizes a spin-
labeled compound as a first-site ligand. Screening this com-
plex with a library of compounds allows identification of
compounds that bind simultaneously with the first, spin-
labeled ligand, in a neighboring binding site (second site).
Second-site ligands can then be identified from quenching of
their NMR signals by the spin-labeled first ligand. It is impor-
tant to remark that this relaxation enhancement will be
manifested, if and only if both ligands bind simultaneously
to the target protein and at neighboring binding sites. This is
of special interest during the drug discovery and optimization
process, because linking two ‘‘fragments’’ under these con-
ditions can generate compounds with significantly higher
affinity than the affinities of the two individual compounds.
The principal advantages of this approach are its robustness
to identify second-site ligands and its high sensitivity.
NMR screening based on methyl group chemical shifts
As an alternative to NMR screening by observation of protein
target resonances in 2D [15N,1H]-HSQC spectra, Fesik and
coworkers suggested to monitor 13C/1H chemical shift
changes of methyl group resonances in 2D [13C,1H]-HSQC
spectra [22]. Application of this method demands labeling of
the methyl groups with 13C. Because uniform 13C-labeling of
proteins is relatively expensive, a protocol for cost-effective
selective labeling of valine, leucine and isoleucine(d1) resi-
dues in proteins has been proposed by the authors [22].
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Drug Discovery Today: Technologies | Lead optimization Vol. 1, No. 3 2004
Table 1. Overview of five NMR screening technologies
Technology Reporter screening Spin labels NMR screening with
methyl group resonances
3-FABS Affinity tags
Pros - Affinity range extended to
high-affinity ligands
- Relatively high
sensitivity
- Relatively high
sensitivity
- Relatively high
sensitivity
- Relatively low protein
concentrations needed
- Possibility to discriminate
specific from unspecific
binding
- Very robust in identifying
compounds that bind
simultaneously at
neighboring binding
sites
- Works very well with
larger proteins
(MW > 50 kDa)
- Possibility to determine
IC50 values from
measurements at
variable inhibitor
concentrations
- Possibility to rank ligand
affinities using titration
series - Possibility to obtain
distance information and
orientation of ligands with
respect to each other and
with the protein
- Valuable complement
to experiments based
on amide group
chemical shift changes,
especially when the
ligands bind to
hydrophobic surface
areas of the protein
Cons - Hit identification requires
deconvolution of the
mixture components
- Chemistry is required to
introduce spin labels in
compounds or in
protein side chains
- 13C-labeling of the
protein methyl
groups is required
- Chemistry is required
to introduce CF3
groups in
compounds
- The sequence of one
of the protein partners
has to be modified
(addition of a ligand
binding domain)
- Need to identify suitable
reporter ligand(s) - Only applicable to
enzymes
References [14–16] [19–21] [22] [24,25] [26]
In NMR binding studies that monitor methyl group che-
mical shift changes, the sensitivity is increased about three-
fold compared with the corresponding experiments based on15N/1H chemical shift observation. In addition, selective
methyl group labeling on a perdeuterated background is
advantageous for screening high molecular weight protein
Figure 2. Principle of the SLAPSTIC experiment. Small, unbound
organic compounds in solution have typically sharp resonances (spec-
trum at the top). When they bind to a paramagnetic spin-labeled protein
target, their signal intensity is drastically reduced or completely
quenched by paramagnetic relaxation enhancement (spectrum at the
bottom).
280 www.drugdiscoverytoday.com
targets (MW > 50 kDa). Finally, NMR screening or binding
assays that monitor methyl group chemical shifts offer a
valuable complement to experiments based on amide group
chemical shift observation, especially in cases were the
ligands are located close to valine, leucine or isoleucine
methyl groups.
3-FABS
All NMR screening techniques discussed so far are binding
assays that detect ligand binding rather than enzyme inhibi-
tion. Functional screening of enzyme inhibition can be per-
formed by NMR spectroscopy if substrate and product
concentrations are monitored as a function of time. This is
a well-established NMR technique (reviewed in [23]).
Recently, an improvement was proposed in a method called
3-FABS (three fluorine atoms for biochemical screening)
[24,25]. This method allows rapid and reliable functional
screening of compound libraries, performed at protein and
substrate concentrations comparable to the ones utilized by
standard HTS techniques. The experiment is only applicable
to enzymes and allows measurement of accurate IC50
values.
3-FABS monitors 19F signal intensities rather than 1H sig-
nals. Signal overlap with test compounds is thus drastically
reduced. 3-FABS requires labeling of the substrate with a CF3
moiety (Fig. 3). During the assay, the enzymatic reaction is
performed with the CF3-labeled substrate and quenched after
an established delay that depends on the enzyme and the
reaction conditions. Fluorine NMR spectroscopy is then used
Vol. 1, No. 3 2004 Drug Discovery Today: Technologies | Lead optimization
Figure 3. Schematic diagram showing the principle of the 3-FABS
method. First, a fluorine-containing moiety, like CF3, is introduced in
the substrate as a ‘‘sensor’’ or reporter group. The chemical modification
of the substrate by the enzyme (here represented as addition of a
chemical fragment denoted ‘‘A’’) induces changes in the electronic cloud
of the CF3 moiety. This results in distinct chemical shifts for the product
and the substrate 19F signals, and therefore chemical shift changes in the19F NMR spectra (bottom).
to monitor the substrate and the enzymatically modified
reaction product. Because of the high intrinsic sensitivity
of 19F chemical shifts to the chemical environment, modifi-
cations of the substrate during the enzymatic reaction are
readily detectable, even when the CF3 moiety is distant from
the reaction site. The high sensitivity of 19F NMR spectro-
scopy, the 100% natural abundance of the isotope 19F and the
presence of three fluorine atoms result in 19F signals of high
intensity.
Affinity tags
Protein–protein interactions can be detected by a novel NMR
reporter system based on affinity tags [26]. In this approach,
one of the binding partners is fused to a ligand-binding
domain, where a medium-affinity, low-molecular weight
reporter ligand is bound. Protein–protein interactions are
then monitored via changes in the NMR relaxation of the
reporter ligand: because the parameters of the reporter ligand
spectra depend on the molecular weight of the protein–
ligand complex (among other factors, e.g. affinity constant,
and protein and ligand concentration), changes of these
spectral parameters can be used to probe changes in the
molecular composition of the ternary protein–protein–ligand
complex. One of the principal advantages of this technique is
the relatively low consumption of unlabeled proteins.
NMR instrumentation and experimentation
All NMR techniques currently applied for drug discovery,
including the ones discussed above, take advantage of recent
advances on NMR experimentation and instrumentation.
Particularly, technologies that improve throughput or allow
reduction of sample consumption are very attractive. Some of
them are discussed briefly in the following paragraphs.
The introduction of cryogenic probes (see Glossary) (http://
www.bruker-biospin.com; http://www.varianinc.com) has
been a significant step ahead in NMR technology. By cooling
the radio-frequency coils and preamplifiers to �30 K, the elec-
tronic noise can be significantly reduced, which results in a
signal-to-noise ratio increase by a factor of 2–4 (depending
mainly on sample conditions). This translates in reduction of
measurement times by factors of 4–16, or reduction of protein
consumption by factors of 2–4, to achieve equivalent signal-to-
noise ratios. In the context of NMR screening, these numbers
represent considerable saving of resources, because NMR
screening involves preparation of a large amount of NMR
samples. By contrast, cryoprobe technology opens the possibi-
lity to perform measurements at even lower concentrations,
which allows detection of ligands with lower affinities and
compounds with low solubility.
A further approach to increase the probe sensitivity has
come from microcoil technology. Although microcoils have
been commercially available for several years for work with
small molecules, they have been so far scarcely used for
biomolecules. A recent publication showed that a newly
introduced CapNMR microcoil probe (MRM Corp.; http://
www.protasis.com), working with sample volumes as low as
5 ml, can provide useful NMR spectra of biomolecules [27].
Despite of the higher mass sensitivity of this probe (approxi-
mately 7.5 times higher than a conventional 5 mm probe),
they need much higher protein concentrations than 5 mm
probes, which is a drawback for practical work with typical
protein samples. Future developments, perhaps the further
improvement of cryogenic microcoil probes, will probably
extend the application scope of these probes for routine NMR
work.
Recent advances in the NMR field, particularly in techni-
ques that speed up NMR data acquisition, have a considerable
potential for NMR screening. During the past few years,
extensive activity has been going on with the aim to speed
up multidimensional experiments, provided that the inher-
ent sensitivity is high enough. Among the most promising
‘‘fast multidimensional NMR techniques’’ [28], as they are
coined, are GFT-spectroscopy [29], single-scan multidimen-
sional spectroscopy [30], Hadamard spectroscopy [31] and
projection reconstruction techniques [32]. To date, the
majority of these techniques have been tested using small,
well-behaving proteins or peptides, where they could
increase data collection speeds from 1 to several orders of
magnitude. However, it remains to be seen whether these
new methods will indeed dramatically speed up the charac-
terization of larger protein molecules and have a significant
impact in the process of drug discovery. In the future, with
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Drug Discovery Today: Technologies | Lead optimization Vol. 1, No. 3 2004
Outstanding issues
� NMR screening of integral membrane proteins, particularly GPCRs,
and molecular machines, is still very challenging.
� In spite of recent developments in NMR sample handling and
automatization (see, e.g. http://www.tecan.com), further automatic
NMR screening solutions are highly desired.
� Automatic analysis and assignment of NMR spectra is still very
demanding, and the currently available software does not seem to
work reliably with every protein target.
� In typical drug discovery projects involving NMR spectroscopy, signal
sensitivity is still an issue. Development of NMR techniques and NMR
instrumentation that increase sensitivity will always be desired.
Related articles
Coles, M. et al. (2003) NMR-based screening technologies. Drug Discov.
Today 8, 803–810
Peng, J.W. et al. (2004) NMR experiments for lead generation in drug
discovery. Prog. NMR Spectrosc. 44, 225–256
Stockman, B.J. and Dalvit, C. (2002) NMR screening techniques in drug
discovery and drug design. Prog. NMR Spectrosc. 41, 187–231
Fejzo, J. et al. (2003) Application of NMR screening in drug discovery.
Curr. Top. Med. Chem. 3, 81–97
Pellecchia, M. et al. (2002) NMR in drug discovery. Nat. Rev. Drug. Discov.
1, 211–219
Jahnke, W. and Widmer, H. (2004) Protein NMR in biomedical research.
Cell. Mol. Life Sci. 61, 580–599
Zerbe, O., ed. (2003) BioNMR in Drug Research, Wiley
Related links
� Spectroscopy Now: http://www.spectroscopynow.com
upcoming advances in NMR instrumentation, some of
these techniques might become very useful tools for NMR
screening.
Comparison of technologies
The technologies compared include reporter screening, spin
labels for NMR screening, NMR screening monitoring methyl
group resonances, 3-FABS and affinity tags for study of pro-
tein–protein interactions. All these methods have advantages
and disadvantages and should be regarded as complemen-
tary. Table 1 lists the most relevant characteristics of these
techniques.
Conclusions and outlook
NMR screening is nowadays widely applied for validation of
hits identified in other assays and for the discovery of high-
affinity ligands for biologically relevant macromolecules.
NMR spectroscopy offers several advantages, like applicabil-
ity to any soluble protein, without the need for prior devel-
opment of a target specific set-up, robustness against
detection of false positives, and versatility, which is shown
by the vast number of experiments or ‘‘assays’’ that are
possible and the large number of spectral parameters that
can be monitored. Moreover, NMR screening techniques can
be applied at any point in a drug discovery program, be it for
hit finding very early in the program, long before an HTS
enzymatic assay is developed, or for hit validation, or during
the process of lead optimization.
In the drug discovery process, the interplay of NMR with
other screening methods is likely to be of increasing impor-
tance, because these techniques complement each other very
well. Particularly powerful are methods that combine NMR
screening with HTS, mass spectrometry, X-ray crystallogra-
phy and in silico screening, to exploit the benefits of each
technique. Particularly, X-ray-based screening has the unri-
valled advantage of providing direct structural information at
atomic resolution of the protein–ligand complex, which can
be immediately used for further optimization of the com-
pound. Usually, collection of this information by NMR spec-
troscopy, if possible at all for the system studied, is very time-
consuming. By contrast, NMR screening is relatively fast in
delivering a hit list for the target, whereas X-ray-based screen-
ing requires availability of good-quality crystals that are
suitable for soaking or co-crystallization. Furthermore, X-
ray screening can miss numerous ligands if the particular
protein–ligand complex does not crystallize.
A challenging and exciting frontier for NMR screening lies
with those macromolecules that resist structure determina-
tion by conventional methods. These targets include integral
membrane proteins, particularly G-protein coupled receptors
(GPCRs), macromolecular complexes, and molecular
machines, such as the ribosome. Another potential applica-
tion of NMR in drug discovery will probably be ‘‘in vivo’’ NMR
282 www.drugdiscoverytoday.com
screening, where NMR is used as a tool to monitor protein–
ligand interactions within living cells [33,34]. An attractive
feature of this method is that proteins can be studied in the
presence of other proteins and endogenous small molecules,
which represents more appropriate physiological conditions.
Further developments in NMR instrumentation and meth-
odologies will lead to higher throughput and application of
NMR screening technologies to even larger protein targets. As
NMR hardware and techniques continue to improve sensi-
tivity and resolution, NMR screening will become a powerful
tool for designing novel therapeutics that target the most
challenging biological systems. Without any doubt, further
technical progress and innovative methods in the NMR field
will have a profound impact on drug discovery in the near
future.
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