NMR-based screening: a powerful tool in fragment-based drug discovery

RSC Publishing presents an excerpt from:

Exploiting Chemical Diversity for Drug DiscoveryEdited by Paul A Bartlett (University of California, USA)Michael Entzeroth (S*BIO Pte Ltd)

www.rsc.org/biomolecularsciences

RSC Biomolecular Sciences

2102

0676

From the theoretical and operational considerations in generating a collection of compounds to screen, to the design and implementation of high-capacity and high-quality assays that provide the most useful biological information, this book provides a comprehensive overview of modern approaches to lead identification.

Hardcover | 2006 | xxiv + 402 pages | £119.95 ISBN-10: 0 85404 842 1 | ISBN-13: 9 780 85404 842 7

This is the latest book in the RSC Biomolecular Sciences series. For further information, visit www.rsc.orgbiomolecularsciences

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http://dx.doi.org/10.1039/b605309m

http://pubs.rsc.org/en/journals/journal/MB

http://pubs.rsc.org/en/journals/journal/MB?issueid=MB002006

NMR-based screening: a powerful tool in fragment-based drug discovery{

Jochen Klages,a Murray Colesb and Horst Kesslera

First published as an Advance Article on the web 5th May 2006

DOI: 10.1039/b605309m

1 Introduction

NMR has become a powerful tool in the pharmaceutical

industry for a variety of applications. Instrumental improve-

ments in recent years have contributed significantly to this

development. Digital recording, cryogenic probes, autosam-

plers, and higher magnetic fields shorten the time for data

acquisition and improve the spectral quality. In addition, new

experiments and pulse sequences make a vast amount of

information available for the drug discovery process. As a

consequence, new screening technologies involving NMR have

become indispensable for modern drug discovery. These

developments have been summarized in several recent

reviews.1–12

The drug development process is usually divided into the

following familiar phases. Initially, a target is identified that

may be involved in initiating or blocking a certain physiolo-

gical effect. Most drug targets are proteins, but RNA and

DNA also can serve as targets for drug discovery. When a

suitable target is identified, it is screened for compounds that

may cause the desired biological effect. Assays involving

binding to the target, either directly or in competition with

labelled ligands, are often used in place of functional (cellular)

tests. The molecules that are identified as hits from binding

screens are validated for the desired biological effect, e.g.

inhibition of enzymatic activity. The remaining leads are then

optimized by structural refinement to increase the affinity and

selectivity for the receptor and for bioavailability and other

in vivo pharmacological and physiological properties, which

may eventually result in a drug candidate.

NMR can assist the above process at different stages,

namely, in hit finding, hit validation, and lead optimization.13

These three areas of application will be discussed in detail in

the following sections. The drug discovery process is summar-

ized in Figure 1, in which shaded boxes indicate where

incorporation of NMR techniques is possible.

NMR offers some unique features that make it an attractive

alternative for applications in drug research. The primary,

intrinsic advantage, however, is the ability to detect weak

intermolecular interactions, e.g. between a ligand and a target,

with unmatched sensitivity. This ability makes NMR ideal for

fragment-based screening.1,14–18 In a fragment-based

approach, comparably small and simple molecules are

screened for binding to a target. These compounds often

reveal only a weak affinity. However, when several fragments

for different binding sites of the target are identified, they can

be linked to form higher affinity ligands. Although the linkage

of fragments is an additional difficulty, this approach accesses

a larger structural space because each fragment can be

optimized separately.

In contrast, structural space is significantly restricted in

conventional high throughput screening (HTS), in which the

identified hits often are hydrophobic and possess relatively

high molecular masses already, and it is difficult to improve

their activity without further increasing either their hydro-

phobicity or molecular weight, or both. Lipinski’s ‘‘rule of

five’’19 (Table 1) requires a certain window of lipophilicity, a

maximum of five hydrogen bond donors and ,10 hydrogen

bond acceptors, and in addition a molecular weight below

500 Da. Clearly, the fragment-based approach carries a higher

potential for designing efficient drug candidates within the

limits set by the ‘‘rule of five’’. The advantage of the fragment-

based approach over other techniques like HTS is illustrated in

Figure 2.

Additionally, high throughput screens usually work as

functional or competitive binding assays with labelled ligands

aDepartment Chemie, TU Munchen, Lichtenbergstr. 4, 85747 Garching,GermanybDepartment of Protein Evolution, Max-Planck-Institute forDevelopmental Biology, Spemannstrasse 35, 72076 Tubingen, Germany

{ This is Chapter 12 taken from the book Exploiting ChemicalDiversity for Drug Discovery (Edited by M. Entzeroth and P. A.Bartlett) which is part of the RSC Biomolecular Sciences series.

Fig. 1 A flowchart outlining the drug discovery process; steps

allowing the application of NMR techniques are shaded.

BOOK CHAPTER www.rsc.org/molecularbiosystems | Molecular BioSystems

This journal is � The Royal Society of Chemistry 2006 Mol. BioSyst., 2006, 2, 318–332 | 319

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that are often derived from the natural binding molecules.

Establishing a functional assay can be tedious, whereas

binding can be measured easily. An advantage of NMR is

that it is applicable even when no functional assay or known

binders are available; moreover, NMR can be used for

validation of hits from functional HTS screening.

A further key advantage of NMR is its ability to provide

additional structural information, which is welcome even at

the earliest stages of the drug development process. Structural

information supports rational lead design or the design of

biased libraries based on known structure activity relationships

(SARs).

The number of compounds that can be screened by NMR is

limited to about 103–104depending on the technology used –

much lower than for HTS and in silico screening methods (105–

106compounds). Hence, it is not the number of compounds

that makes NMR an attractive method, but rather the

possibility to use fragment-based screening. In combination

with the structural information that can be extracted

simultaneously, NMR experiments can open up new,

unexplored regions of chemical diversity in the search for

drugs.

2 NMR screening: general aspects

It is clear that to carry out ligand screening by NMR, the

library compounds (ligands) must be distinguished from the

target. One of the most important methods of achieving this is

through their vastly different molecular masses. Several NMR

parameters are dependent on the rotational and translational

diffusion rates of the subject molecule, and therefore on

molecular mass. Notably, the transverse (entropic) relaxation

of the NMR signal is dependent on the rotational diffusion

rate. Large, slowly tumbling target molecules relax much faster

than rapidly tumbling small molecules. As the transverse

relaxation rate is directly related to linewidth, small molecules

can be distinguished by their narrow lines. Also, filters

suppressing broad signals can be used to minimize signals

from the target. Other relaxation-related parameters, such as

the nuclear Overhauser effect (NOE), are also related to

rotational diffusion rates; small molecules show a positive

enhancement and large molecules a negative enhancement. In

contrast to rotational diffusion, translational diffusion can be

measured directly by NMR. This technique allows rapidly

diffusing small molecules to be distinguished from the slowly

diffusing target by suppressing the signal from the former.

These differences in NMR properties between small and

large molecules form the basis of many NMR screening

experiments. When a small ligand binds to a large target, it

adopts the properties of the target to an extent dependent on

the residence time of the binding event (Figure 3). NMR

screening experiments can therefore be designed to detect

molecules with intermediate properties. It is clear that

distinction on the basis of molecular mass would fail if the

ligands were not considerably smaller than the target. It should

also become clear in the following sections that the use of

relaxation or translational diffusion filters to suppress ligand

and/or target signals places considerable affinity limits on the

techniques involved.

A second major method of distinguishing ligand and target

signals does not rely on molecular mass, but on the much

greater dispersion of chemical shifts usually observed for the

target. This property can be exploited to create non-

equilibrium magnetization specifically on the target, which

can be transferred to binding ligands and subsequently

detected. By far, the most robust method of distinguishing

ligands and target, however, takes this a step further, using

specific isotope labelling of one or the other component to

create a unique chemical shift range. This strategy allows some

of the simplest NMR screening techniques to be used, relying

on the change in chemical environment induced by the binding

ligand. This change in environment affects the chemical shifts

of both the target and a binding ligand, predominantly at the

binding site. Thus, chemical shift changes observed in a (15N-

or 13C-labelled) target can localize and partially characterize

the binding site. Distinction via isotope labelling avoids the use

of relaxation- and/or diffusion-based filters to suppress the

Fig. 2 Schematic representation of the advantage of the fragment-

based approach over conventional HTS screening.14 The range of

affinities and molecular weights of average HTS hits and fragments are

illustrated. The thickly dashed line indicates the size limit set by

Lipinski’s ‘‘rule of five’’. Whereas HTS hits tend to have high

molecular masses, fragments show much lower molecular weights but

only slightly lower affinities, which conveys higher lead-like character.

Table 1 Properties of orally available drug-like compounds

‘‘Rule of five’’ criteria19

N Molecular weight ¡500 DaN Log P ¡ 5N Hydrogen bond donors (OH and NH) ¡ 5N Hydrogen bond acceptors (lone-pairs of hetero-atoms, like O andN) ¡10Other criteria(Verber-rules77)N Number of heavy atoms 10–70N Rotatable bonds 2–8N Number of rings 1–6, aromatic ¡3N Molar refractivity 40–130N Size of hydrophobic area

320 | Mol. BioSyst., 2006, 2, 318–332 This journal is � The Royal Society of Chemistry 2006

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unwanted component, giving these techniques a much wider

affinity range. These techniques are also ideally suited to

competitive screening applications, which represent some of

the most promising new developments in the field.

3 Ligand-vs. target-detected methods

NMR screening can be divided into ligand- and target-detected

methods. In the first class, changes induced in the ligand’s

NMR signals by binding to a large target are observed,

whereas in the second class, the influences of ligands on the

spectra of the target are detected. Both techniques have their

intrinsic advantages for different applications, making them

largely complementary.

Ligand-based methods predominantly make use of one-

dimensional (1D) NMR spectra and therefore are comparably

fast, allowing higher throughput in screening. The detection of

the ligand signals offers the opportunity to screen mixtures of

ligands without the need for deconvolution, as long as the

signals of the ligands do not overlap. Moreover, the amount of

target required for the screening process is smaller and there

are fewer restrictions on the properties of the target.

Commonly, the target is not isotopically labelled and its

molecular mass is practically unlimited; in some techniques

(e.g. saturation transfer difference (STD), see below), the

target can even be immobilized in membranes. Finally, some

crude information about the binding epitope and the binding

mode can be extracted by ligand-based methods.20

Most ligand-detected methods are limited to low and

medium affinities because of the need to suppress the signals

of the target molecule. Ligands that bind too tightly are

indistinguishable from the target and thus are suppressed as

well, resulting in false negatives. In contrast, non-specific

binding can result in the appearance of false positives.

Target-based methods can easily distinguish between non-

specific and specific binding. In addition, effects caused by

aggregation and pH-changes can be excluded. The detectable

affinity range is much higher than in ligand-detected techni-

ques, particularly at the high-affinity end. However, the most

valuable advantage is the possibility of extracting detailed

structural information about the ligand–target complex from

the spectra. For target-detected experiments comparably large

amounts of the protein target are needed, because the

observation of its NMR signals requires concentrations in

the range of 0.1–1 mM. These targets usually have to be

labelled with NMR active isotopes (15N and/or 13C) making

these techniques quite costly. In contrast to ligand-detected

screening methods, it is not possible to screen mixtures of

compounds without deconvolution. The standard implemen-

tation of these experiments includes two-dimensional (2D)

spectra, which require longer acquisition times. As the targets

usually have high molecular masses (>10 kDa), the signals are

subject to fast relaxation. This limitation restricts the size of

targets that can be observed to molecular weights ,100 kDa,

even if techniques like transverse relaxation-optimized spectro-

scopy (TROSY)21 or cross-relaxation-induced polarization

transfer (CRIPT)22 are included. Although this limit may be

overcome with specific methyl group labelling in the future,

interesting targets might still remain intractable with these

techniques.

3.1 Sample requirements

Because the interaction between the ligand and the target

usually takes place in aqueous solution, the ligands have to be

soluble in water to an appropriate level; otherwise, aggregation

effects lead to false positives during the screening process. For

standard ligand-based techniques, a concentration of 100 mM

is found to be satisfactory for a reasonable signal-to-noise

ratio. The target, which is usually unlabelled, is sufficiently

concentrated when it is within a range of 1–50 mM; for a 0.5 mL

solution, this corresponds to 0.1–5 mg of protein with a

molecular weight of 20 kDa. In target-detected experiments,

however, labelling is essential to avoid signal overlap.

Moreover, higher concentrations are necessary, raising the

amount of target needed to 100–300 mM. Depending on the

size and the relaxation properties of the target, different

labelling schemes are necessary. For relatively small proteins,

uniform 15N-labelling is sufficient. For larger proteins,13C-labelling and/or deuteration may be required. Site- or

amino acid-selective labelling23 can extend the size of the

proteins, which can be studied as overlap is reduced. Selective13C-labelling of the methyl groups of valine, leucine, and

isoleucine24–27 has proved to be especially advantageous

because these residues are at the surface of hydrophobic areas,

which are often involved in binding events. In addition, methyl

Fig. 3 Alteration of the physicochemical properties during the process of binding. On binding, the ligand adopts the properties of the large target

molecule due to drastic increase in the effective molecular mass.


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group signals are usually narrower and therefore better

resolved.

4 Incorporation of NMR into the drug discoveryprocess

In the following detailed discussion of the application of NMR

techniques to drug discovery, a short description for hit

finding, hit validation, and hit (lead) optimization is given. In

addition, the corresponding requirements for the NMR

experiments and the resulting implementations are described.

Only typical experiments are given explicitly, but other

techniques are summarized in Table 2.

The classification of drug discovery into different phases is

to a certain extent artificial – the different phases overlap or

can be applied simultaneously – but the general outline

indicated here shows that more complex NMR techniques

are needed when the drug discovery process is more advanced.

4.1 Hit finding

In hit finding, a large number of compounds, the so-called

library, is screened vs. a target to identify components that

bind. Libraries can meet different types of requirements, and

efforts are made to optimize them with respect to diversity,

solubility, drug-like character (see Table 1), and the synthetic

accessibility of the compounds they contain.28,29 Substance

libraries can contain small organic fragments, synthetic

compounds of diverse chemical structures, or natural

products.30

Experiments for hit finding by NMR spectroscopy should

allow high throughput, since substance libraries can easily

contain up to 104 compounds. Hence, time-optimized techni-

ques are required that not only reduce experimental time, but

also the time for sample preparation and data analysis.

Consequently, lengthy 2D or three-dimensional (3D) techni-

ques are unfavourable, as are complex experiments that

require fine tuning for each sample. For an industrial

application, the ability to identify binding components from

large mixtures without deconvolution is nearly indispensable.

Moreover, labelled samples and target-consuming techniques

should be avoided for cost reduction. These requirements

make 1D ligand-based experiments the most favourable

alternative.

4.1.1 STD and WaterLOGSY. STD31,32 and

WaterLOGSY33,34 both take advantage of the fact that the

intermolecular NOE transfer is strongly negative. A non-

equilibrium magnetization is created on the receptor–ligand

–complex, and the subsequent alteration of the free ligand

signal intensities is monitored. Two principle implementations

are imaginable. Either all resonances (target, binding, and non-

binding ligands) are perturbed simultaneously, followed by

selective suppression of some signals, or the excitation itself is

already selective. Selective suppression for instance can be

implemented by a diffusion filter,35 eliminating signals of small

molecules or by a relaxation element,36 filtering out target

resonances.

STD is one of the most useful experiments for NMR

screening. Here, non-equilibrium magnetization is achieved by

selective excitation (on-resonance) of target resonances. The

magnetization is transferred within the target via spin diffusion

and eventually to a bound ligand (Figure 4). When this ligand

dissociates from the target into solution, the magnetization

change transferred in the bound state is retained in the free

ligand. The difference from a reference spectrum taken without

on-resonant irradiation thus yields a spectrum containing only

those ligands that have been perturbed by binding to the

target. The reference spectrum is obtained by off-resonance

irradiation in a spectral window where no target signals appear

(Figure 5).

Table 2 Overview of common screening techniques and their application10

Method Application Limits and requirements Identification of

Target MWlimit

Affinitylimit

Labelledtarget req.

Binding siteon target

Binding epitopeon ligand

Binding comp.in mixtures

Diffusion filtering Hit finding Lower U/L No No No YesRelaxation filtering Hit finding Lower U/L No No No YesTrNOE Hit finding Lower U/L No No No YesNOE pumping Hit finding Lower U/L No No No YesRev. NOE pumping Hit finding Lower U/L No No Yes YesWaterLOGSY Hit finding Lower U/L No No Yes YesSTD Hit finding Lower U/L No No Yes Yes19F-screening Hit finding None None 19F ligand No No YesCSM Hit validation Upper None 15N or 13C Yes No NoComp. screening Hit optimization None None No Yes No NoComp. 19F-screening Hit optimization None None 19F ligand Yes No No

Fig. 4 Illustration of the effect of on-resonant irradiation of protein

signals. Due to spin diffusion, the resulting non-equilibrium magne-

tization spreads out across the target molecule and is transferred to the

ligand. The magnetization of the bound ligand decays rapidly with a

rate of R1,BL, which is of the same order as the rate for the target

molecule R1,R. On dissociation into solution, the relaxation properties

of the ligand change and the acquired non-equilibrium magnetization

now decays with a rate of R1,FL for the free ligand.12


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STD spectroscopy offers several advantages. First, there is

almost no upper limit for the size of the target, even membrane

proteins or immobilized targets are applicable.32 On the

contrary, since the correlation time increases with increasing

molecular mass, spin diffusion becomes more and more

effective, which in turn leads to stronger STD effects. For

this technique, the amount of target and ligand needed to give

reasonable results is relatively small. In larger ligands, the part

of the molecule that is in direct contact with the target is most

strongly affected. Hence, structural information of the

complex from the ligand side is provided. Estimations of the

binding constants can also be drawn.20 STD spectra are

usually recorded as 1D spectra, but they can be easily extended

to 2D or 3D versions if necessary.37 This option offers a

diverse tool for a variety of different tasks in screening. STD

fundamentally depends on an effective spin-diffusion mechan-

ism and therefore on proton density. Hence, the observed STD

effect might not be large enough to be detected, especially

when small targets are used. As it is the free fraction of the

ligand that is observed, one normally sees a dramatic

amplification effect if the ligand concentration is higher than

that of the target. However, similar to other ligand-based

techniques, STD is not applicable to high-affinity binders.

If the target does not provide a large enough proton density,

as is the case with nucleic acids,12 the WaterLOGSY (water-

ligand observed via gradient spectroscopy) technique might be

preferred. The large bulk water magnetization is used for an

effective transfer via the ligand–target complex to the free

ligand in solution. By selectively inverting or saturating the

water resonance, the magnetization is transferred to the target

and then finally to the ligand. The exact details of the transfer

are more complicated and will not be discussed in this review.

The important point is the difference in the cross-relaxation

properties of the water with non-binding ligands and binding

ligands. In the first case, the relaxation includes small

correlation times leading to positive cross-relaxation rates, in

contrast to the second case in which positive cross-relaxation

rates are observed. As a consequence, non-binding and binding

ligands show different signs in the spectrum. This combination

can lead to an erroneous interpretation if positive and negative

peaks cancel each other out.

4.1.2 Libraries of 19F-containing ligands. A comparably new

technique is the screening of ligands that contain 19F-labels,

for example, in the form of fluorinated aromatic or trifluoro-

methyl groups.38–40 The 19F-nucleus has some unique features

that make it an attractive probe for screening. It has a high

gyromagnetic ratio (cF y 0.94 cH) and occurs at 100% natural

abundance making it a very sensitive NMR nucleus. It has

broad chemical shift dispersion, allowing the use of large

mixtures without signal overlap. The target, additives, and

solvents generally do not contain fluorine, meaning only ligand

signals are observed in the spectra. Upon binding, the chemical

shift and linewidth of the ligand 19F-signal is strongly affected

(as a result of the large chemical shift anisotropy (CSA) of the19F-nucleus). Hence, the acquisition of 1D spectra with and

without the target molecule is sufficient to screen for binding.

Of course, the need for 19F-labels seems to be a profound

drawback. However, about 10% of all drugs on the market

already contain fluorine10 mainly for modification of meta-

bolic stability. Additionally, the small fluorine atom can be

often replaced by hydrogen without loss of binding affinity.

4.2 Hit validation

For all compounds found in the primary screen, the binding

has to be validated to remove false positives, which is

especially important for hits of functional assays. False

positives might originate from changes in the pH, aggregation,

or non-specific binding. By monitoring binding on a molecular

level, interactions with the wrong binding site and chemical

reactions with the drug target can also be excluded. The

number of compounds investigated during the validation

process is much lower than for the hit finding process.

Therefore, the problems of deconvolution, concentration,

and experimental time can be less rigorous, whereas a large

window of affinities is still essential. Even more important is

the lack of ambiguity of the experiment with respect to the

binding of the ligands. To extract true binders from the hits, it

is advisable to apply several experiments to confirm the mode

of interaction.

4.2.1 Chemical shift mapping. A comparably simple

approach uses perturbations of the target’s chemical shifts to

confirm binding. Multidimensional spectra are usually used, as

signal overlap obscures the analysis in 1D spectra. In these

spectra, the displacement of peaks due to the binding event is

monitored. Obviously, the use of 2D- or 3D spectra results in

Fig. 5 Schematic representation of the procedure for the measure-

ment of STD experiments.12 Polygons and stars represent binding and

non-binding components, respectively. During the off-resonant experi-

ment (top panel), no magnetization is transferred from the binding

ligand to the target molecule. If the irradiation changed to ‘‘on-

resonant frequencies’’ (middle panel), magnetization transfer from the

bound ligand to the target occurs, which lowers the signal intensity of

the ligand. The difference between the two spectra only contains the

signals of binding components.


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an increased experimental time. As an additional feature of

this technique, the dissociation constant KD may be extracted

from the experiment. This analysis is possible if the displace-

ment is related to the concentration of the ligand via a

titration. Of course, these experiments require a reference

spectrum without the ligand.

Chemical shift perturbations are most commonly tracked in13C- and 15N-HSQC spectra, although HNCO spectra have

also been used.15 15N-labelling is relatively cheap if an

appropriate overexpression system has been established, while

in contrast, uniformly 13C-labelling is expensive. Nowadays,

new labelling schemes enable the selective incorporation of13C-labels in the methyl groups of valine, leucine, and

isoleucine.27 Isotopic enrichment with 13C at specific locations

forms a convenient alternative to 15N-labelling, especially

considering that the apparent signal intensity of methyl

groups is three times that of amide protons. The chemical

shift dispersion of the methyl moieties is merely somewhat

smaller than that of the amide protons. The amide protons

visible in the 15N-HSQC spectrum are hydrophilic, whereas

the methyl groups of the above-mentioned residues are

hydrophobic. Therefore, both techniques are partially

complementary.

4.3 Hit optimization

For hit optimization, the confirmed hits from the validation

process are first ranked and clustered. Specific properties, e.g.

Lipinski’s ‘‘rule of five’’, have to be considered to develop a

promising lead structure (Table 1).13,41 This assessment

includes the solubility, molecular weight, chemical accessibil-

ity, and affinities (binding constants). For a further improve-

ment of the binders, structural information is indispensable.15

The exact binding site as well as the precise binding mode has

to be extracted from the experiments. For the design of new

ligands, analogs of the lead structure are explored, often

assisted by combinatorial chemistry. The resulting compounds

are subjected to a screening comparable to the hit finding

process, but with more thorough evaluation. As targets often

have more than one binding site, these have to be identified

and screened too. If ligands for adjacent binding sites can be

linked, large increases in their affinities can be expected.

Depending on the stage of the hit optimization process, the

experiments should provide structural information of different

qualities. Whereas at the beginning, even crude SARs are

sufficient, more detailed information is required at an

advanced stage of drug development. Therefore, the techni-

ques change from ligand- to target-based methods and more

complex pulse sequences are applied. Target-based techniques

have the additional advantage of offering the possibility of

monitoring high-affinity ligands, as low-affinity binders are of

minor interest at this stage. Of course, the target needs to be

NMR accessible, which sets some limitations in size.

4.3.1 Chemical shift mapping. The above-mentioned imple-

mentations for chemical shift mapping (see hit validation)

usually allow the structural characterization of the binding

epitope as well, if a sequential assignment is available. A

simple mapping of the displaced signals identifies the binding

site. This method is of particular interest if two fragments are

to be combined as proposed by the SAR-by-NMR technique.1

In this approach, two fragments are fine tuned separately to

give higher affinity ligands. Both molecules bind to the same

target but at different binding sites. The subsequent linkage of

the two results in a high-affinity ligand (Figure 6). SAR-by-

NMR is a prominent example for structure- or fragment-based

drug design. It is also possible to draw conclusions about the

orientation of the ligand within the binding pocket from the

experiments.42,43 The ligand is docked into the binding site of

the target by computational techniques. A theoretical spec-

trum is calculated and compared to the experimental spectrum.

If several spectra are calculated for different conformations of

the ligand, information about the binding mode can be

generated.

Another interesting approach is the observation of binding

within a living cell.44–46 15N-HSQC spectra are recorded

directly of the bacterial slurry, resulting in spectra of practical

resolution. This result is of particular interest because many

compounds have high affinity in vitro but show much weaker

effects in vivo.

2D spectra sometimes are not sufficient to visualize the

binding process because of remaining signal overlaps. If the

binding site is already known, site-selective labelling might

help to avoid this problem. Single types of amino acids are

selectively labelled to simplify the spectra and focus it on the

binding pocket.

The labelling schemes described are of an immense value for

protein targets; however, there are still no comparable

labelling techniques that make target-based experiments

appropriate for nucleic acid targets.

Fig. 6 Illustration of the SAR-by-NMR technique. High-affinity

binders are identified on the basis of two medium-affinity binders of

different but adjacent binding sites.


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4.3.2 Competition-based screening. The major drawback of

ligand-based experiments is the restricted ability to screen

higher affinity ligands. Because strong binders possess a long

residence time in the binding pocket of the target, their

exchange rate is very low. This slow exchange results in the loss

of the information about the bound state as the non-

equilibrium magnetization that is created on the ligand decays

before it can be detected via the free form. Tighter binders can

be investigated to a certain extent if the concentration of the

ligand is lowered. Of course, this approach is limited by the

sensitivity of the technique used. Thus, high-affinity binders

usually cannot be detected by simple ligand-based techniques

and lead to false negatives.

An alternative approach is competition-based screen-

ing.47–49 A known medium-affinity binder (reporter ligand) is

added to the solution of the target. If other ligands (screening

ligands) are present in this solution as well, the reporter ligand

will be displaced according to the affinity of the screening

ligands (Figure 7). The prerequisite of fast exchange is only

valid for the reporter ligand and therefore the range of

affinities for competing ligands is not limited. For the

detection of weaker binders, the concentration of the reporter

ligand has to be decreased. During the experiment the focus is

on the reporter ligand, since only signals of this species are

detected. This limitation raises the problem of deconvoluting

mixtures of ligands if binders are discovered. With competi-

tion-based experiments, only ligands that bind to the same site

as the reporter ligand are detectable, also making this

application relevant to the hit validation process. All

competition experiments can be used to estimate the dissocia-

tion constant of the screened ligands if the KD of the reporter

ligand is known. An NMR titration has to be performed

with the ligand of interest while the concentration of the

target and the reporter ligand are kept fixed. The change of

the intensity of the reporter ligands signal and subsequent

fitting to corresponding equations12 gives the required

information.

In principle, all ligand-based techniques can be extended to a

competition type of experiment. However, experiments using

simple 1D spectra are especially worth considering, e.g.

WaterLOGSY,47 STD, and 19F-screening,40 which rank

among the most powerful ligand-based screening techniques.

Their advantage that they only require a small amount of

target and their high sensitivity is adopted by the competition

experiment. Competition-based fluorine screening has to be

highlighted in this context as it combines the advantages of

most techniques. Fluorine chemical shift anisotropy and

exchange for screening (FAXS) is one implementation of this

concept. The relaxation parameters of the reporter ligand

change dramatically on the displacement by other ligands.

Clean spectra are easily obtained without suppression of target

or solvent signals making it a highly effective tool for

screening, even in hit finding.

4.3.3 Paramagnetic spin labels. Paramagnetic spin labels

offer the opportunity to screen for binding sites remote from a

known binding site.50 The introduction of these labels

increases the T2-relaxation rate of binding components and

therefore results in an observable broadening of their signals

(Figure 8). The origin of this modified T2-time is the increased

relaxation via the electron–proton dipole–dipole interaction.

Because of the vastly larger gyromagnetic ratio of the electron,

the amount of target and ligand needed is reduced. Also,

shorter contact times and comparably large distances (y20 s)

between the two binding sites are practicable. Obviously, the

general handicap is the introduction of a spin label that does

not alter the binding properties of the binding site. Therefore,

a detailed knowledge of the 3D structure of the target is

essential.

5 Representative case studies

In the following section, examples of NMR-based screening

are presented. The first two examples include the screening of

libraries containing 400 and 10,000 compounds, respectively.

One involves fluorine techniques as a method for primary

screening, while the other uses target-based methods for

detection. In the second example, the fragment-based

approach is described where NMR methods for hit finding,

hit validation, and hit optimization are included. The last

example deals with a new technique (saturation transfer double

difference (STDD)) that might have a considerable impact on

drug development in future. The examples reflect the

techniques we regard as the most promising alternatives for

the drug discovery process.

5.1 Fluorine screening

Recently, we screened riboflavin synthase (RiSy) against a

small library of 19F-containing ligands. The library encom-

passes about 400 ligands containing either an aromatic

fluorine or a trifluoromethyl moiety.

RiSy catalyzes the final step of riboflavin (vitamin B2)

biosynthesis. During the last step, two lumazine units

Fig. 7 Depiction of competition-based screening. Low- to medium-

affinity binders are displaced from their binding site by higher affinity

binders. In this experiment, only the signals of these low- to medium-

affinity binders (reporter ligands) are monitored.

Fig. 8 Introduction of paramagnetic spin labels for second site

screening. Paramagnetic spin labels drastically alter the relaxation

properties of binding components. The large gyromagnetic ratio of

unpaired electrons amplifies the relaxation via dipole–dipole interac-

tion, which is represented by the arrows. Stars at the edges of the

binding ligand illustrate the increased transverse relaxation.


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(1, DMRL) are fused to form riboflavin (2) and 5-amino-6-

ribitylamino-2,4-(1H,3H)-pyrimidinedione (3, ARP) (see

Figure 9). ARP is recycled afterwards in the biosynthetic

pathway via lumazine synthase. The disproportionation

reaction that results in the formation of vitamin B2 involves

the transfer of a C4-fragment between the two DMRL

molecules. The xylene ring system of riboflavin is obtained

by a head-to-tail assembly of the two substrate molecules.

RiSy consists of three identical subunits denoted a.51,52 They

form a complex of pseudo-D3 symmetry with a total

molecular mass of about 75 kDa. The a-subunit (23 kDa)

shows an internal sequence similarity resulting in a 26%

identity of the C- and N-terminus. The N-terminal domain can

be expressed separately and forms a homo-dimer that binds

the substrate with a similar affinity to the complete trimeric

protein.53–55

This enzyme forms an attractive target for medical chemistry

to create an anti-infective drug. Selective suppression of this

enzyme will result in a deficiency of vitamin B2. While

mammals have the ability to absorb riboflavin from their

nutrition, gram-negative bacteria and various yeasts lack an

equivalent mechanism56–59 and hence depend completely on

the endogenous biosynthesis of this vitamin. This requirement

facilitates the development of a specific drug, as the target is

present in bacteria and fungi but not in the host.

Several inhibitors mimicking the scaffold of lumazine (1)

have been identified,60 but none of them consisted of new

structural features. To overcome this drawback, a screening of

fluorinated ligands was performed.

The 19F-chemical shift was measured in advance for each

member of the library. These reference experiments were

carried out in D6-DMSO solutions with trifluoroethanol

(TFE) as an internal standard. The 400 ligands were

divided into groups of about 10–30 compounds, resulting in

15 mixtures. The combinations were chosen such as to avoid

reactions and overlap of 19F-chemical shifts. For the composi-

tion of these mixtures, stock solutions in D6-DMSO were set

up and added to buffer solutions (50 mM KH2PO4, 10 mM

EDTA, 10 mM Na2SO3, pH 7.1). To test for binding, a

portion of the stock solution was added to a solution of RiSy

(0.1 mM) in the same buffer. For both samples 1D 19F-spectra

were acquired at 14.09 T (564 MHz 19F-frequency).

Compounds binding to RiSy experienced a significant line

broadening (Figure 10); this effect was observed for 11 out of

the 400 ligands (Table 3). The six best (approximated from the

amount of broadening) were combined in a new mixture,

which was added in excess to a small amount of enzyme. In the

case of competitive binding it is assumed that the binder with

the highest affinity displaces all the other ligands. From this

experiment it was possible to identify the two strongest

binders, compounds 4 and 5 (Figure 11).

An enzymatic assay of RiSy revealed a Ki value of about

20–30 mM for compound 4, which could serve as a starting

point for further optimizations to generate an appropriate lead

structure.

This example shows the ease of performing fluorine-based

screenings. Once the 19F-library is characterized (knowledge

about all 19F-chemical shifts) and standard mixtures can be

used for screening, it becomes a rather efficient technique. The

experimental time to acquire a spectrum of one mixture took

about 75 min in this case, but with higher concentrations,

experimental times can be reduced to 30 min or even less. This

estimate includes the sample set up, data acquisition, and the

change of the sample by the autosampler. Therefore, a

throughput of 400–1000 compounds per day can easily be

achieved if 20 components are assumed per mixture.

Fig. 9 Biosynthesis of riboflavine (2) by catalysis of RiSy. Two

molecules of lumazine (1) form one molecule of riboflavine and one

molecule of 5-amino-6-ribitylamino-2,4-(1H, 3H)-pyrimidinedione (3)

by disproportionation.

Fig. 10 Typical 19F spectrum of a screening mixture with (a) and without (b) protein. In the expansion, the change of the chemical shift as well as

the line broadening of the centre signal is visible.


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5.2 SAR-by-NMR

SAR-by-NMR1 represents a very intuitive way of rational,

fragment-based drug design. Two or more independently

optimized ligands of different binding sites are combined to

give a high-affinity binder. The example presented here deals

with the development of a selective inhibitor for the protein

tyrosine phosphatase 1B (PTP1B).61

PTP1B operates in the insulin-signalling pathway. When

insulin binds to its receptor, there is a conformational change

of intracellular region of the protein, which results in the

O-phosphorylation of three tyrosine residues as the first step in

the cascade of the insulin signalling.62 PTP1B is reckoned to be

responsible for the dephosphorylation of the insulin recep-

tor.63–67 Dephosphorylation results in the down regulation of

the insulin receptor and therefore selective inhibition of PTP1B

may enhance insulin activity.

The exogenous regulation of this enzyme is of general

interest as disturbed insulin signalling may lead to type II

diabetes. Currently, about 130 million people suffer from this

disease and it is expected that this number will rise during the

next years.68 Nowadays, the treatment encompasses the

application of insulin and hypoglycaemic drugs, but none of

them is sufficient to assuage the disease completely.69

Many processes in eukaryotic cells are regulated by

reversible phosphorylation. Hence, compounds interacting

with this class of proteins are regarded as promising candidates

for the therapy of many diseases. PTP1B belongs to one

subclass of these proteins, the tyrosine phosphatases.

Unfortunately, the catalytic domain is generally conserved

among these enzymes, complicating selective inhibition.

First, a primary screen of 10,000 compounds was performed

to find new scaffolds that bind to the active site of PTP1B.

This screen involved a shortened form of the protein (292

residues) that was either uniformly 15N-labelled or selectively

labelled with d-13CH3 isoleucine. Chemical shift perturbations

in the 15N-HSQC and 13C-HSQC spectra (Figure 12) were

used for the detection of binding ligands. From the screen,

compound 15 (Figure 13) could be identified as a ligand.

Comparison with the natural ligand phosphotyrosine (pTyr)

revealed that both ligands have equivalent binding modes. For

further improvement of the affinity, a derivative of the new

ligand with a bulkier group was synthesized (naphthyloxamic

acid, 16). This ligand showed an increase of the Ki value from

293 to 39 mM in a para-nitrophenyl phosphate (pNPP) assay.

Furthermore, covalent binding of 16 to PTP1B could be

excluded by enzyme kinetics. An X-ray analysis confirmed that

naphthyloxamic acid also binds in the same fashion as the

natural ligand.

Table 3 Hits found by 19F-screening

Fig. 11 19F spectrum of all compounds showing an interaction with the protein during the first screening with (a) and without (b) protein. It is

apparent that compounds 4 and 5 are the highest affinity binders among the ligands investigated.


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Apparent from the X-ray structure (Figure 14) is a groove in

the protein surface connecting the active site with a second,

inactive site. Further derivatization of the lead with the goal of

finding a linker for the two sites resulted in compound 17. A

diamido chain at 4-position gave a 40-fold boost in affinity

(Ki = 1.1 mM).

The second, non-active site is also a binding site for pTyr.

This non-catalytic site offers great potential to increase the

selectivity of the new ligand for PTP1B. Another NMR screen,

also involving 10,000 compounds, provided several hits,

mostly small fused-ring aromatics. This time, the screening

was performed with the truncated protein consisting of

selectively 13C-labelled methionine. Residue Met258 is present

in the second binding site and is therefore an excellent probe

for a binding event. The fusion of 3-hydroxy-2-naphtoic acid

and 17 resulted in a nanomolar binder (18) with a 50-fold

higher affinity (Ki = 22 nM).

Finally, this inhibitor was cross-checked against five other

phosphatases to determine the selectivity. Among those

analyzed, TCPTP is closely related to PTP1B on the basis of

sequence homology. Therefore, a difference in the affinity of

the evaluated compounds for these two enzymes would be a

remarkable achievement. The relative selectivities are summar-

ized in Table 4, indicating that compound 18 is indeed able to

‘‘distinguish’’ between PTP1B and TCPTP.

This example underscores the efficiency of SAR-by-NMR

and moreover illustrates the fragment-based approach.

5.3 Saturation transfer double difference

STD spectroscopy9,31 is a frequently used tool to test ligands

for binding. The reason is the large variability and the high

sensitivity of this experiment and because it can also be applied

to the investigation of integral membrane proteins.32 These

proteins are especially difficult to study as they usually lose

their biological activity if removed from their natural

environment. Therefore, this class of proteins is reintegrated

into liposomes for NMR analysis to simulate the physiological

conditions. Nevertheless, most screening techniques are not

able to monitor the interaction between these targets and a

ligand.

Membrane-bound proteins are of highest interest for the

pharmaceutical industry, as about 30% of proteins in the

mammalian cells belong to this class. A subclass of these

proteins are the G-protein coupled receptors (GPCRs),70

which play an important role in signal transfer pathways.

One example is the aIibb3 integrin, which occurs on the surface

of platelets where it is the most prevalent component of the

exposed glycoproteins. The integrin is composed of two non-

covalently linked a and b subunits and is able to bind proteins

and peptides consisting of an RGD motif.71,72 Platelets are of

crucial importance in the blood clotting process by forming

platelet plugs. This process can be inhibited by RGD-

containing peptides and non-peptidic peptidomimetics. A

cyclic peptide that shows high activity (low nanomolar range)

for the avb3 integrin is cyclo(-RGDfV-).73,74 However, this

peptide shows the desired lower affinity (micromolar range)

for the aIibb3 integrin, which is required for the STD

experiment to achieve a sufficiently high STD amplification

by rapid exchange of bound and free ligand molecules.

For a proof of concept, Meinecke et al.75 reinvestigated this

interaction by STD spectroscopy on liposome, embedded

aIibb3 integrin. Recently, a further example that probably

represents a key step towards in-cell screening was published

by Claasen et al.76 They investigated the binding of the above-

mentioned system in vivo. Intact human platelets were mixed

with cyclo(-RGDfV-) and the resulting solution was analyzed

with a new technique called STDD spectroscopy (see below).

Suspensions of human platelets were prepared from

concentrated blood donations. The work up yielded two

samples, both containing the same composition of platelets in

a D2O-TBS (deuterated TRIS saline) buffer. The amount of

platelets was estimated to 7 6 109, representing an effective

Fig. 12 13C-HSQC of PTP1B with ligand (red) and without (black)

ligand. The chemical shift change for the isoleucine residue 219

indicates binding of the ligand to the corresponding amino acid.

Fig. 13 Structure evolution of a high-affinity binder (18) for PTP1B

via SAR-by-NMR screening.


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concentration of 100–600 pM if a number of 1–5 6 104

receptors is assumed per platelet. One sample (A) was treated

with the cyclic peptide at a concentration of 150 nM, while the

other one (B) remained unaltered. The normal STD analysis of

sample A containing a 250–1500-fold excess of the ligand did

not result in an interpretable spectrum. Even a T1r-filter

element integrated into the pulse sequence to suppress

resonances of large-size macromolecules did not lead to

significant improvement. The problem was primarily the result

of the fact that the suspension consisted of many small and

large molecules showing a lot of binding events. The resulting

STD spectrum obviously contained signals of these

compounds. To filter these contributions out of the spectrum,

a reference STD spectrum that included exactly these

ligands was recorded and subtracted afterwards. For this

reference, an STD spectrum of sample B was acquired.

Subtraction of the two resulted in the spectrum shown in

Figure 15D. This double difference method also has the

advantage that residual signals of macromolecules are

eliminated without the need for a relaxation filter element,

thus increasing the signal-to-noise ratio. The quality of the

resulting spectra is apparent by the comparison of the STDD

spectrum and the simple 1D spectrum of the pure cyclic

peptide (Figure 15E).

Compared to the STD spectrum of cyclo(-RGDfV-) in the

presence of integrin aIibb3 integrated into liposomes, the

STDD spectrum of the native integrin showed a fivefold higher

STD effect. Moreover, a difference in the STD response can be

observed, reflecting a slightly different binding mode. These

findings emphasize the value of this experiment, particularly if

all of the capabilities are considered.

Fig. 14 X-ray structure of compounds 15–18 with PTP1B (a–c) revealing the same binding mode. Also visible is the groove in the protein surface

that connects the catalytic and the non-catalytic binding sites.

Table 4 Relative sensitivities of the hits to different phosphatases

Compound

Phosphatase 15 16 17 18

PTP1B 1 1 1 1TCPTP 1 1 1 2LAR .3 1 6 36SHP-2 .3 .7 27 104CD45 .1 .4 380 2700Calicineurin .7 .270 .13000


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Of course, this technique is not useful for a primary screen,

as the ligands may interact with many targets and in addition

to the desired one. However, if ligands are found with a high

enough affinity and an accordingly high selectivity in vitro, this

experiment may serve as a control to distinguish scaffolds of

high activity in vivo from those with a low activity at an early

stage of the drug discovery process.

6 Conclusion

NMR spectroscopy has evolved into an important method for

screening ligand mixtures for binders to medicinal relevant

protein or nucleic acid targets. A number of new technologies

have been established for this purpose in the last decade; the

most efficient ones are STD, waterLOGSY, and the screening

of fluorine-containing libraries. Moreover, NMR spectroscopy

can assist the hit validation process and provide structural

information on the ligand–target complex.

To a large extent, NMR is, complementary to other

techniques. The ability to detect weak binders under quasi-

natural conditions is a particular advantage, making it ideally

suited for the fragment-based approach for developing lead

structures.

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NMR-based screening: a powerful tool in fragment-based drug discovery