new approaches for nmr screening in drug discovery

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-
Introduction
NMR-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

www.drugdiscoverytoday.com 277

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].



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).


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


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


http://www.spectroscopynow.com/


http://www.varianinc.com/

http://www.protasis.com/

http://www.protasis.com/


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


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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