1. 285
NMR, already some 50 years old, has long been an invaluable
analytical method in industry for verification of chemical
synthesis and compound characterisation. The range of
molecular information accessible through NMR, however,
offers a far larger horizon of applications. Of these, ligand
screening by NMR has emerged as a very promising new
method in drug discovery. Its unmatched screening sensitivity,
combined with the abundance of available information on the
structure and nature of molecular binding, justifies the growing
interest in this dynamically expanding NMR application.
Addresses
*NOVASPIN Biotech GmbH, Mühlfeldweg 46, 85748 Garching,
Germany; e-mail: info@novaspin.de
†Institut für Organische Chemie und Biochemie, Technische Universität
München, Lichtenbergstraße 4, 85747 Garching, Germany;
e-mail : kessler@ch.tum.de
Current Opinion in Chemical Biology 2001, 5:285–291
1367-5931/01/$ — see front matter
© 2001 Elsevier Science Ltd. All rights reserved.
Abbreviations
HPLC high-performance liquid chromatography
MAS magic-angle spinning
NOE nuclear Overhauser enhancement
NOESY NOE spectroscopy
RDC residual dipolar couplings
S3 spin-state selection
STD saturation transfer difference
TOCSY total correlation spectroscopy
trNOE transferred intramolecular NOE
TROSY transverse relaxation-optimized spectroscopy
Introduction
NMR has gained widespread acceptance over recent years
as a most versatile tool for industrial drug research. The
relevance of NMR in this regard rests on two pillars — its
use as a powerful, universal and fast-screening technique
to detect intermolecular interactions with unparalleled
sensitivity, and its potential to yield unique information
with atomic resolution to guide structure-based drug
design. This review reflects this dual importance,
focussing not only on NMR screening techniques, but also
including structural applications and a brief survey of
NMR hardware and general methodological developments
that are contributing to advances in the field. The reader is
also referred to other reviews [1–5].
Basic developments in NMR
The application of NMR to industrial drug research is
greatly facilitated by recent hardware developments
(reviewed in [6]). Most important for screening purposes
are developments that reduce sample amounts and dead-
times for sample handling — notorious shortcomings of
‘wasteful’ NMR. Cryoprobe technology can substantially
increase the signal-to-noise ratio, which also translates into
lower accessible binding affinities. Flow probes alleviate the
need for NMR tubes and their time-consuming handling
[7••], enabling direct coupling with separation techniques
such as (HP)LC. Microcoil probes reduce the required
sample volume and also offer superior radio-frequency field
homogeneity [8], benefiting difference-based NMR
screening methods.
Screening techniques are also beginning to benefit from
recent fundamental advances in NMR that have already
proven of value in structure determination. These include
spin-state selection (S3), which is most often employed to
optimise relaxation properties (i.e. in transverse relaxation-
optimized spectroscopy; TROSY [9]), extending molecular
sizes amenable to NMR well beyond 100 kDa, primarily by
enhancing spectral resolution. S3 experiments are also
employed [10] to precisely measure residual dipolar couplings
(RDC) [11], a new set of unique structural constraints that
have proven useful in orienting (hydrogen-poor) ligands
bound to macromolecular targets. RDC result from partial
molecular alignment that can be brought about by adding
magnetically orienting media such as bicelles, filamentous
phages, and others [12]. Connectivities across hydrogen
bonds [13] also yield new structural constraints that can com-
plement the scarce nuclear Overhauser enhancement (NOE)
data in hydrogen-poor systems. Last but not least, various
new strategies for isotopic labelling of proteins, crucial to
NMR, have been developed (for a recent review, see [14]).
General aspects of NMR screening
NMR offers several key advantages for drug research:
1. NMR can detect the weakest ligand–target interactions
of any method so far and thus discover initial hits with
even millimolar binding constants.
2. NMR is a universal screening technique that, unlike
bioassays, requires no knowledge of a protein’s function and
therefore no target-specific set-up. Thus, protein targets that
have been identified purely on a genomics basis and so far lack
a functional bioassay can immediately be screened by NMR.
3. NMR concomitantly enables a determination of
binding constants [15].
4. NMR spectrally separates individual components,
allowing the direct screening and deconvolution of
mixtures from natural sources or combinatorial chemistry.
5. In addition to the mere binary binding information,
NMR returns crucial structural information for both target
and ligand with atomic resolution for the subsequent
optimisation of weak initial hits into strongly binding
drug candidates.
Applications of NMR in drug discovery
Tammo Diercks*, Murray Coles* and Horst Kessler†

2. NMR screening exploits the fact that many NMR observ-
ables change even upon temporary binding because of
global and local effects (see Figures 1 and 2). The effects
of binding may be monitored either on the target or on the
ligand and are summarised in Table 1. The observed
molecule primarily dictates inherent limitations such as
sample availability (possibly with appropriate isotope
labelling), solubility, stability and molecular size.
Ligand-observed screening
Detection with these techniques generally takes place on the
dissociate, free form of the small ligand (implying a minimal
Kd of ca. 10–7 M), allowing the direct identification of bind-
ing species from mixtures. The unobserved target molecule
is not subject to size limitations and may even be immo-
bilised. In general, isotopic labelling is not required. Many of
these techniques rely on size-dependent effects, which can
be difficult to resolve for weak binding (implying a maximal
Kd of ca. 10–3 M) and small relative molecular size differ-
ences between ligand and target. These effects can be
selected for in 1D filtered spectra, in which tuning the filter
selects for different ligand affinities. Alternatively, the size of
the effect could be evolved in a second indirect dimension,
yielding pseudo-2D edited spectra. The information for
structure-based ligand optimisation obtainable through these
techniques is more limited than for target-observed tech-
niques. It may, however, reveal a ligand’s binding epitope, its
bound conformation, and even contacts to other ligands
binding at adjacent sites.
Use of molecular diffusion
Detection of transiently binding ligands by their decreased
diffusion coefficients is an established screening technique
(for exhaustive reviews see [16,17•]) Pseudo-2D diffusion-
edited experiments (diffusion-ordered spectroscopy,
DOSY), where molecules are separated according to their
individual diffusion coefficients, are particularly useful for
deconvoluting mixtures. Alternatively, 1D diffusion filters
can be combined with standard 2D correlation experi-
ments, such as in the diffusion-modulated gradient COSY
(correlation spectroscopy) [18] and diffusion-encoded total
correlation spectroscopy (TOCSY; DECODES) [19], to
elucidate spin systems and facilitate mixture analysis.
Diffusion filtering or editing might also be enhanced by
combination with other screening techniques, such as
NOE pumping [20,21] (see below). In a promising appli-
cation for combinatorial chemistry, diffusion filtering has
recently been used in solid-state MAS (magic-angle
spinning) NMR to identify and distinguish resin-bound
molecules from impurities [22].
Use of relaxation
Screening based on binding-induced slowing of global
dynamics, leading to a general decrease in NMR relaxation
times, is conceptually very similar to diffusion-based
screening. The diffusion filter is replaced by an inversion-
recovery delay, a spin-lock period or a CPMG echo to filter
for the different types of relaxation times, T1, T1ρ and T2,
respectively. One-dimensional relaxation-based screening
usually requires subtraction of a reference spectrum of the
target-free ligand mixture, and measures must be taken to
avoid artefacts due to binding-induced lineshape distor-
tions, chemical-shift changes and residual target signals
[23]. T2-based ligand screening may also be performed by
comparing spectral linewidths [24•] that increase upon
binding. Complications can arise from superposition of
line-broadening due to chemical exchange or slow
286 Combinatorial chemistry
Figure 1
Global NMR consequences of ligand–target
interactions. Observation is restricted to the
ligand where the effects are most
pronounced. Their strength depends on
molecular size differences and binding
kinetics.
kA
kD
+
Complex properties
similar to
target properties
• Slow Relaxation
• Fast diffusion
• Positive NOE
• Fast relaxation
• Slow diffusion
• Negative NOE
Small ligands ComplexLarge target
Current Opinion in Chemical Biology
Figure 2
Local NMR effects of complex formation may be observed on the
ligand and/or the target. Structural information is available as effects
are mostly localised to the binding site.
Surface protection
Current Opinion in Chemical Biology
Intermolecular
magnetisation
transfer
Chemical shift perturbation
Perturbation of local dynamics{

3. conformational motions (on the microsecond and
millisecond timescales), which are often relevant for
induced-fit during intermolecular interactions and damped
upon binding. Thus, a size-dependent decrease in T2
times might be compensated for by a flexibility-dependent
reduction in line-broadening.
In contrast, the recently proposed concept of transferred
T1 times [25] relies on the fact that relaxation rates
measured on a fast-exchanging ligand in the free state also
contain contributions from its local dynamics in the bound
state, as immobilisation of flexible regions of the ligand
generally increases relaxation rates. Transferred relaxation
rates may therefore be seen as a dynamic analogue of
transferred NOE (see below). Individual differences in
relaxation rates must be compared with their average in
order to distinguish local from global dynamic changes
[25]. Another complication arises from local relaxation
contributions because of molecular anisotropy.
Intramolecular magnetisation transfer: transfer-NOE
Transferred intramolecular NOEs (trNOEs) are well
established as unique sources of structural information on
the bound ligand (for a review see [26]) where spin-diffu-
sion should be suppressed to obtain optimal structural
precision [27,28]). The observation of trNOE has proved
to be equally useful in the fast screening of ligand
mixtures [24•], forming the basis of ‘bioaffinity NMR’
[29]. The technique relies on the size-dependence of the
intramolecular NOE, which shows slow NOE build-up
with weak positive maxima for free ligands and rapid
build-up with strong negative maxima for the bound state.
If dissociation of the ligand occurs quickly enough (i.e.
Kd > 10–7 M), a sufficient percentage of observable free
ligand will retain intense negative trNOE as a ‘memory’ of
the bound state and thus quickly indicate binding. If the
residence time is too short (i.e. Kd > 10–3 M), however,
trNOE will build up too weakly for detection [30].
Routinely, trNOE are recorded as 2D NOESY (NOE
spectroscopy) spectra with short mixing times favouring
build-up of trNOE over direct NOE. Relaxation filtering
suppresses residual target signals. Notable are applica-
tions of trNOE-based screening [30,31•,32] and recent
experimental improvements [33].
Screening by trNOE imposes upper limits on the ligands
(< 1 kDa), which have to fall into the positive NOE
regime. For borderline cases, this regime may be extend-
ed, for example, by increasing the temperature [31•].
Another problem is the possible cancellation of positive
direct and negative transferred NOE, which could be
resolved by more lengthy 3D experiments such as the 3D
TOCSY–trNOESY [34], or by resorting to other tech-
niques such as saturation transfer difference (see below).
Intermolecular magnetisation transfer
The intermolecular magnetisation transfer is always posi-
tive and cancellation cannot occur. For its measurement,
non-equilibrium magnetisation must first be created asym-
metrically (i.e. on one component only, usually the drug
target). It is then transferred to the binding partner during
a mixing period. The intermolecular magnetisation trans-
fer results in altered signal intensities for both components
and can finally be monitored on either the starting or the
receiving molecule (usually the ligand). Remaining target
resonances can be suppressed (e.g. by isotope filtering or
relaxation filtering). Subtraction of a reference spectrum
(recorded without initially disturbing the magnetisation) is
usually required to reveal these intensity changes and
entails the risk of subtraction artefacts.
Asymmetric non-equilibrium magnetisation on the target
molecule may generally be created by selective irradiation,
requiring the existence of a unique NMR frequency band.
Applications of NMR in drug discovery Diercks, Coles and Kessler 287
Table 1
NMR observable effects of ligand binding.
Global effects of binding Ligand observed Screening Applications References
Translational dynamics Diffusion rates Affinity NMR [17•,19]
Rotational dynamics Relaxation rates
Line broadening [23,24•]
Sign of transferred intramol. NOE Bioaffinity NMR (trNOE) [29,30,31•,32,34]
Molecular orientation Residual dipolar couplings *
Local effects of binding Ligand- or target-observed
Chemical shift perturbation Chemical shifts SAR-by-NMR (HSQC screening) [44•,48,49,51,53•]
Local dynamics perturbation Relaxation rates *
Surface protection Water exchange rates *
Paramagnetic surface probing *
Intermolecular magnetisation transfer Via scalar couplings *
(e.g. intermol. H-bonds)
Via nuclear dipolar couplings Bioaffinity NMR (STD) [32,35,37•]
(intermol. NOE) (Reverse) NOE pumping [20,21]
WaterLOGSY [38]
Via electron dipolar couplings
(paramagnetic relaxation) SLAPSTIC [39•,41•]
*No application to NMR screening reported yet. Intermol., intermolecular.

4. Initially localised, it is spread across the entire molecule by
spin diffusion such that magnetisation may be picked up
by a ligand binding anywhere on the surface. Alternatively,
non-equilibrium magnetisation may be created symmetri-
cally, followed by asymmetric suppression making use of
the large size difference between target and ligands. For
instance, a diffusion filter would suppress magnetisation
on the ligands, whereas a relaxation filter would suppress
magnetisation on the target.
Saturation transfer difference (STD) [35] as the special
case of intermolecular steady-state NOE is observed after
frequency-selective irradiation of the target and subtrac-
tion of a reference spectrum with off-resonance irradiation.
Although usually run as 1D spectra, STD can be coupled to
standard 2D correlation experiments such as TOCSY, COSY
and HMQC (heteronuclear multiple quantum correlation) to
alleviate spectral overlap [32,35]. The distance-dependence
of the observed signal intensities identifies those nuclei
closest to the target–ligand interface. This ‘epitope mapping’
[28,36] may be seen as a ligand-based complement to the
‘SAR-by-NMR’ approach. A remarkable application extends
STD screening to solid-state NMR [37•], in which the target
was immobilized on controlled-pearl glass, facilitating its
recovery. From a comparison of STD integrals, it was
possible to assess relative binding strengths of the dissolved
ligands. A reference spectrum with pure target-free resin
must rule out direct ligand–resin interactions.
NOE pumping [20] employs the scheme of symmetric
magnetisation inversion, followed by asymmetric suppres-
sion. Classically, the ligand magnetisation is suppressed via
diffusion filtering, and its recovery directly indicates inter-
molecular magnetisation transfer from the target, hence
binding. A suppression of remaining target magnetisation
prior to acquisition greatly facilitates identification, but the
diffusion filter employed may entail serious intensity losses
through concomitant relaxation of the target magnetisation.
This problem is alleviated in the reverse case (reverse
NOE pumping) [21], in which ligand magnetisation is
preserved while target magnetisation is suppressed via
relaxation or isotope filtering. The ligand magnetisation is
then reduced because of transfer onto the target, and signal
reductions observed on a ligand thus indicate binding.
These reductions, however, are only revealed after subtrac-
tion of a reference spectrum, which also takes account of
unspecific magnetisation losses through T1 relaxation.
WaterLOGSY [38] exploits the surface layer of target-
bound water molecules as a pool of selectively invertible
magnetisation that can be transferred onto binding ligands.
After simultaneous selective water suppression and
relaxation filtering (for target suppression), only signals of
binding ligands are detected.
Paramagnetic spin labels
Magnetic spin interactions can be enhanced drastically by
introducing paramagnetic spin labels. The induced NMR
effects — resonance shifts and line broadening — can
thus be observed over larger distances (up to some 20 Å),
shorter contact times, and at lower protein concentrations.
Soluble paramagnetic reagents have classically been
employed to search for metal ion binding sites and probe
solvent-accessible surfaces. Intermolecular binding, which
blocks part of the solvent-accessible surface, may be
detected and localised in a similar manner.
In a recent application to second-site screening, a
confirmed binding ligand was spin labelled and used to
search for ligands concomitantly binding to the target
protein at an adjacent site [39•]. The method is analogous
to the observation of transferred inter-ligand NOE
contacts [40], yet offers larger specificity and sensitivity,
establishing connectivity between more distant binding
sites. The need to synthesize spin-labelled ligands without
affecting their natural binding properties is, however, a
major drawback. A blind test must furthermore rule out
that ligands interact in the absence of the target.
In the SLAPSTIC method, the target protein is spin-
labelled to assist in identifying binding ligands [41•]. The
line-broadening of ligand signals upon binding is thus
strongly emphasized and allows for a reduction in protein
(and ligand) amounts by one or two orders of magnitude. It
is critical to spin-label the target near, but not within the
binding site, as this would invariably lead to altered
binding properties. Moreover, no structural information
can be derived from this method.
Use of residual dipolar couplings
Alignment of the target molecule also causes alignment of
the ligand upon temporary binding. RDC may thus be
transferred from the bound to the isotropic free state if
dissociation is fast enough (Kd in the millimolar range)
[42,43]. In contrast to NOE data, the structural information
available from RDC does not suffer from spin-diffusion
and is therefore precise even for large, immobilised targets.
RDC yield information on both the bound ligand’s
structure and its orientation relative to the target without
the need for assigning target resonances as demonstrated
in two recent applications [42,43]. This technique has not,
however, been applied to ligand screening yet.
Target-observed screening
Observation on the target, rather than on dissociated
ligands, is neither restricted by the ligands’ size nor by an
upper affinity limit. It also immediately reveals different
binding sites, enables direct distinction between specific
and unspecific binding and provides a wealth of structural
information for ligand optimisation. This information,
however, can only be extracted if the spectral assignments
for the target are known. Inherent limitations often
preclude observation on the large target molecule, although
relaxation-optimised techniques (TROSY) may be of
benefit. The amounts of protein required, which must be
isotope-labelled, can be reduced significantly by employing
288 Combinatorial chemistry

5. cryoprobe technology and screening larger mixtures of
compounds [44•]. Mixtures of ligands can not, however, be
directly deconvoluted.
Chemical-shift perturbations
Most target-observed screening applications rely on
chemical-shift changes as indicators for intermolecular
binding. After assignment, these changes can be mapped
upon the protein structure, revealing its binding sites
[45,46] and guiding structure-based ligand optimisation.
This forms the basis of the ‘SAR-by-NMR’ approach
[47,48], in which optimised ligands are constructed by
linking weakly binding fragments. The approach contin-
ues to be successfully used in drug development [49–52].
Standard screening using 2D 15N,1H correlation spectra
has recently been complemented by 13C,1H HSQC-based
screening on proteins bearing 13C-labeled methyl groups
[53•]. The selective labelling, using inexpensive starting
materials, avoids the detrimental effects of 13C–13C
homonuclear coupling while greatly reducing the cost of
sample preparation. This application benefits from the
higher proton multiplicity and slower transverse relaxation
of the methyl groups. Consequently, intensities were
reported to be on average three-times higher. On the other
hand, screening based on methyl groups suffers from
assignment difficulties and lower spectral dispersion. Note
that the distribution and accessibility of (polar) amide and
(non-polar) methyl groups are not equivalent, possibly
leading to different screening sensitivities.
Equating chemical-shift perturbations with spatial proximity
to the ligand may be misleading, especially in the presence
of large shielding anisotropies in a ligand or because of con-
formational changes in the target. Therefore, an alternative
has been proposed in which the binding site and the bound
ligand’s orientation can be determined more reliably [54]
without the need for a complete re-determination of the
complex structures. It is based on the comparison of chemi-
cal-shift changes in the proximity of the binding site
induced by a series of closely related ligands. This can be
seen as trading synthetic for spectroscopic efforts.
Intermolecular magnetisation transfer
The general scheme for intermolecular magnetisation
transfer experiments (see above) can also be employed for
target-detected screening. In one concept [55], target mag-
netisation is selectively suppressed by relaxation filtering
after symmetric excitation and then recovers by intermolec-
ular transfer from binding ligands. Prior to observation on the
target, the ligand signals are filtered out by selective multiple
frequency suppression. This, however, precludes the direct
screening of ligands with more complex 1H spectra.
Screening for macromolecular interactions
Screening for macromolecular interactions is a special case
in which the ‘ligand’ and ‘target’ have comparable molecu-
lar sizes, and size discrimination is impracticable. Other
than that, many of the techniques mentioned above can be
and have been applied to the problem. For instance, STD
was used to probe protein–nucleic-acid interactions by
saturating well-separated protons of the latter [56]. A varia-
tion of the scheme scans for protein–protein interactions
[57] with molecular distinction made by perdeuterating and
15N-labelling only one partner. The unlabelled protein
remains susceptible to saturation of the aliphatic protons,
while intermolecular STD is observed on the 1H,15N
moieties of the labelled protein. The low proton density in
this perdeuterated species precludes efficient spin-diffu-
sion and the STD is largely confined to the binding site.
Design of NMR screening libraries
In contrast to other screening techniques, NMR screening
requires relatively high sample concentrations and sampling
times. With its unprecedented sensitivity (detecting interac-
tions with Kd up to 10–2 M) and atomic resolution, however,
NMR is particularly suited for guiding the ‘bottom-up’
construction of complex ligands from small, weakly binding
molecular scaffolds. In line with this, the principles for com-
posing a dedicated ligand library are availability, good
solubility, low molecular mass (i.e. < 1–2 kDa), and limita-
tion to some 102–103 compounds of maximal diversity. For
mixture screening, batches should contain (non-reacting)
ligands with isolated NMR signals for easy monitoring.
Various attempts have been made to construct libraries using
consensus fragments identified from statistical analyses of
known drugs (e.g. see [24•,58,59]) and protein-binding
ligands [60], and from fragmenting an existing lead
compound [52]. Pre-selection of small ligands for NMR
screening can also be based on the scoring results obtained
from virtual docking [61].
Conclusions
NMR screening is now being recognised by industry as a
powerful technique for lead discovery. The underlying prin-
ciples have mostly been discovered and developed long ago
by academic scientists, such that the novelty of NMR
screening lies mostly with its applications. In this review, we
have offered a classification of the available NMR methods
and outlined possible future developments by a reduction to
basic NMR properties and principles. The distinction
between ligand- and target-observed screening techniques
is most important. In practice, a dual strategy that combines
the complementary strengths and detectable affinity ranges
of both techniques should prove advantageous. Most impor-
tantly, ligand-observed techniques may produce false
negatives in cases of very strong binding where exchange
between target-bound and detected free ligand is too slow.
These techniques nevertheless have clear advantages for
pre-screening large libraries as they implicitly deconvolute
ligand mixtures, require no isotope labelling and pose few
limitations on the target. Consecutive target-observed ‘fine-
’screening of the identified binding ligands can then rule out
unspecific binding, identify different binding sites and
modes, and guide structure-based ligand optimisation. By
Applications of NMR in drug discovery Diercks, Coles and Kessler 289

6. screening a carefully composed small library of basic
molecular scaffolds, the pre-screening step may be omitted
for amenable targets.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
••of outstanding interest
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2. Moore JM: NMR techniques for characterization of ligand binding:
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3. Roberts GCK: NMR spectroscopy in structure-based drug design.
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mixtures for aggregation and solubility is required to prevent clogging of the
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Applications of NMR in drug discovery Diercks, Coles and Kessler 291