efficiency of hit generation and structural characterization in fragment-based ligand discovery

Available online at www.sciencedirect.com

Efficiency of hit generation and structural characterization infragment-based ligand discoveryAndreas Larsson1, Anna Jansson1, Anders Aberg2,3 and Par Nordlund1,2

Fragment-based ligand discovery constitutes a useful strategy

for the generation of high affinity ligands with suitable

physico-chemical properties to serve as drug leads. There is an

increasing number of generic biophysical screening strategies

established with the potential for accelerating the generation of

useful fragment hits. Crystal structures of these hits can

subsequently be used as starting points for fragment evolution

to high affinity ligands. Emerging understanding of the

efficiency and operative aspects of hit generation and

structural characterization in FBLD suggests that this method

should be well suited for academic ligand development of

chemical tools and experimental therapeutics.

Addresses1 School of Biological Sciences, Nanyang Technological University,

61 Nanyang Drive, Singapore 639798, Singapore2 Department of Medical Biochemistry and Biophysics, Karolinska

Institutet, Scheeles vag 2, SE-171 11 Stockholm, Sweden3 Current address: Sprint Bioscience, Box 23052, S-104 35 Stockholm,

Sweden.

Corresponding author: Nordlund, Par ([email protected])

Current Opinion in Chemical Biology 2011, 15:482–488

This review comes from a themed issue on

Next Generation Therapeutics

Edited by Alex Matter and Thomas H. Keller

Available online 1st July 2011

1367-5931/$ – see front matter

# 2011 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.cbpa.2011.06.008

IntroductionFragment-based ligand discovery (FBLD) has emerged as

an efficient strategy for the pharmaceutical industry to

generate high affinity ligands with suitable chemical prop-

erties to serve as leads for further development into clinical

candidates [1]. Small size and low hydrophobicity are

valuable qualities of clinical candidates generated with

FBLD, as these properties correlate with success rates

in subsequent clinical trials [2]. FBLD also constitute an

efficient strategy to sample chemical space, allowing

ligands for more challenging targets to be developed [1,3�].

A critical stage in a typical FBLD projects is the revel-

ation of bound fragments in an appropriate ligand-binding

site in crystal structures, when this, in addition to confirm-

ing that the target is druggable; gives support for that the

available protein samples, screening tools, and crystal


systems are of sufficient quality to pursue a full FBLD

project. In the present review we will summarize recent

trends in the FBLD literature with an emphasis on

strategies used for efficient hit generation and structural

characterization of hits. Efficiency up to this critical stage

of the process is useful not only for drug design projects in

industry but also for the academic sector where ligand

development is starting to play an increasingly important

role for generating selective high affinity ligands as

chemical tools and experimental therapeutics [4].

Until recently, the literature on FBLD has been domi-

nated by success stories for individual protein targets,

primarily from industry, with relatively little details on

attrition and problems with the methods presented.

Therefore it has been hard to estimate the efficiency

of the methodologies applied. Recently, however, more

extensive information has been revealed from some

industrial programs and at the same time the academic

sector starts to make significant contributions to this

literature [1,5].

Detection of fragment hits —noncrystallographic primary screeningAn overall scheme of possible paths through the FBLD

process is seen in Figure 1. Although structural infor-

mation on protein–fragment complexes can be obtained

with NMR [6��,30], this is still the exception, and our

emphasis in this review is on crystallographic structures.

Generic noncrystallographic primary screening based on

biophysical methods can be used to identify hits, but to be

really useful these hits should correlate with bound frag-

ments confirmed in crystal structures. The comparison of

different screening methodologies based on the current

literature is complicated when practice for what is con-

sidered a hit, a ‘positive’, a validated hit, etc., varies

significantly between individual researchers, labs, and

technologies (Table 1). Direct head-to-head benchmark-

ing of technologies is also rare. Early efforts in FBLD

were dominated by primary screening by NMR and

crystallography, two generic methods that are still widely

employed [1]. Lately, thermal shift assays (TSA) [6��,7]

and in particular surface plasmon resonance (SPR) [8��]have become popular for primary screening.

There are two principal ways to screen using NMR [1,9].

Hits can be detected either by observing changes in protein

signals (protein-observed) or changes in ligand signals

(ligand-observed) upon ligand binding. Protein-observed

NMR (typically via 15N/1H HSQC) is very information rich

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http://dx.doi.org/10.1016/j.cbpa.2011.06.008

The hit generation and structural characterization in FBLD Larsson et al. 483

Figure 1

Current Opinion in Chemical Biology

Fragment libraryTypically 500-2000 small (Mw less than 250)

highly soluble and diverse compounds

High throughput protein production

High throughput X-rayCrystallography screening .

ITC

Prim

ary

scre

en

KD

LE

Chemical tractabilityDiversity

Binding mode

Hit

char

acte

rizat

ion

Medicinal chemistry - Fragment optimization and expansion

TSA SPR

Structural characterization

Time (min)

Molar Ratio

Schematic view of the FBLD process with key experimental screening and characterization steps. High-throughput protein production with

multiconstruct design approach enhances the likelihood for obtaining soluble expression of protein as well as crystals. The fragment library used

consists of small, highly soluble compounds. Primary screening for fragment hits can be done directly by crystallography or with generic

noncrystallographic biophysical methods such as surface plasmon resonance (SPR), thermal shift assay (TSA) (using differential scanning fluorimetry

(DFS) or differential static light scattering (DLSL)), and other methods like NMR and electrospray-masspectrometry (ESI-MS). For hit validation and

characterization, isothermal titration calorimetry (ITC) allows measurements of thermodynamic properties of ligand interactions and concurrently

crystallography with the aim to solve the structure of the fragment binding to the protein. Together these methods will give a range of information about

Kd, thermodynamics, kinetics, ligand efficiency (LE), binding mode, etc. LE is generally defined as the binding free energy for a ligand divided by its

molecular size [41]. LE allows ligands of different sizes to be compared and helps to focus on efficiency, in addition to affinity, when selecting which

fragments to progress. This structural and biophysical information can subsequently be used to guide the iterations of evolution and optimization of the

fragments using medicinal chemistry.

www.sciencedirect.com Current Opinion in Chemical Biology 2011, 15:482–488

484 Next Generation Therapeutics

Table 1

Overview of commonly used biophysical screening methods for detection of fragments.

Screening method Speed Protein

consumption

Low affinity

sensitivity

Pros Cons Ref

Ligand-observed NMR Medium Medium–high High - High sensitivity - High rate of false

positive using only

one experiment

-Expensive equipment

[1,5,29]

Protein-observed NMR Very low High Medium - Provides structural

information

- Needs isotope labeled

protein of large quantities

- Expensive equipment

- Need expert knowledge

[1,5,29]

X-ray crystallograhy Low–medium Medium–high High - Provides detailed

structural information

- Requires robust and

well diffracting crystals

- Need expert knowledge

[28–31]

Surface plasmon

resonance

Medium Low Medium–high - High screening

capacity

- Provides kinetic data

- Protein needs to sustain

immobilization

- Expensive equipment

[5,7]

Thermal shift assay

with DSLS

High Medium–low Medium - Fast

- Easy to acquire data

- Attrition, give lower

completeness

- High rate of false negative

for some proteins

[15�], Larsson,

unpublished

Thermal shift assay

with DSF

High Medium–low Medium - Fast

- Easy to acquire data

- Inexpensive and

robust equipment

- High rate of false negative

for some proteins

- Incompatible with

hydrophobic proteins

[14,5]

Native MS Medium Low Medium - Low protein

consumption

- Expensive equipment [18,19]

but requires isotopically labeled protein and relatively long

experiment times and is thereby rarely used for primary

screening.

Ligand-observed NMR (for example using water-

LOGSY, STD, T1rho) [10,11] is a very versatile and

sensitive screening strategy and likely the fragment

screening technique with the widest range of detection.

Because of relatively high protein consumption and long

experimental time, fragments are typically screened in

cocktails grouped to minimize spectral overlap of com-

pounds. Strategies involving recording of several different

types of experiments with and without a competitive

inhibitor are commonly employed [6��] to confirm bind-

ing in active site and reduce false positives. Filtering

initial screening data through multiple types of ligand-

observed NMR experiments can give high correlation

with visible hits in crystal structures [6��].

The use of SPR for both primary screening and hit

characterization is growing fast. These are likely driven

in part by development of new, better hardware but also

improved knowledge to guide experimental aspects of

SPR-based fragment screening and hit characterization

[8��,12]. Biacore are most commonly used, and still most

sensitive, but instruments from other manufacturers also

work well for fragment screening [13,14].

In addition to Kd determination, SPR also allows the

determination of stoichiometry of binding as well as


kinetics and thermodynamic parameters [15�]. In spite

of its usefulness, the linking of the protein or reference

ligand onto the SPR chip could still be a significant hurdle

for some targets. TSA measure the influence of ligands on

the thermostability of the target protein, where increased

stability indicates binding. Thermal shift-based methods

offer a relatively robust, inexpensive, and rapid way to

screen for binding fragments, and in our hands do corre-

late well with success in X-ray crystallography character-

ization (Larsson, unpublished). On the negative side, the

response is highly phenomenological and can be hard to

interpret, and several investigators have reported that

generally fewer (albeit also unique) hits were found using

TSA compared to ligand-observed NMR screening

[6��,16�].

Typically, TSA data are collected as differential scanning

fluorimetry (DSF) [17], but differential static light scat-

tering (DSLS) is also very useful [18]. In contrast to DSF,

a DSLS experiment does not rely on a reporter dye,

commonly Sypro Orange [19,20]. Instead the increase

in scattered light from the aggregates that forms during

denaturation of the protein is measured. This allows

proteins having hydrophobic pockets or surface patches

to be screened, proteins for which DSF typically fail.

Head-to-head benchmarking of SPR, NMR, and DSF

using a small fragment library on HSP90 shows similar

number of hits for the three methods, respectively [6��].Around half of the hits for each target could be seen in

crystal structures, and around half of the total hits were



common to all three methods. This indicates that the

three methods could be similarly useful but also comp-

lementary, and therefore that screening and hit confir-

mation with multiple methods are useful.

The toolbox of generic biophysical technologies being

adapted for fragment screening is rapidly growing. Native

ESI-MS has emerged as a highly sensitive method for

screening compound binding [21�,22] even in the mM Kd

affinity range. Novel optical biosensors related to SPR

have potential for being adapted for fragment screening,

back-scatter interferometry [23], biolayer interferometry

[24], and SPR with colloidal gold in solution [25]. Weak

affinity chromatography (WAC) is another method that

has capability for detection of fragment binding [26]. In

some cases methods have been tailored to address pro-

blems with specific targets, as in a recent example where

equilibrium dialysis with a 3H-labeled reporter com-

pound was used to identify fragments interacting with

the Escherichia coli TPP thiM riboswitch [27]. All the

above methods differ in their sensitivity, rates of false

positive or negative measurements, and protein require-

ments. In practise this means that a particular method

might be more suitable for specific types of targets, and

that all methods will not work for all proteins. Also, there

are large differences in operative aspects such as exper-

imental costs, time of data collection, and required exper-

tise for data collection and analysis. A nongeneric

approach to fragment screening is high-concentration

activity assays. Although feasible, recent benchmarking

against biophysical data suggests that they are prone

to high levels of false positive and false negative hits

[6��].

Crystallographic fragment screening andstructural characterizationPrimary screening using X-ray crystallography [31] has

typical used cocktails of ligands in each soaking exper-

iment, to minimize the experimental effort of screening

an entire library [32]. The cocktails have often been

composed by mixing structurally diverse fragments to

allow them to be distinguished in the resulting electron

density. Direct crystallographic library screening has for

example been extensively used by Astex, Structural

GenomiX (SGX), and Vertex. SGX reports hit rates of

1–5%, but generally it is anticipated that hit rate will

depend strongly on the target [1,32]. As these examples

are likely to be high priority drug targets, it can be

assumed that very robust crystal systems had been devel-

oped before initiating the screening.

Recently, academic groups have reported more detailed

experiences of primary screening with X-ray crystallogra-

phy. Verlinde et al. used an in-house library pooled into

68 cocktails of 10 compounds each [33]. Twenty-six target

proteins, which were structurally characterized in their

protozoa structural genomic program, were screened with


this library. They report that seven proteins yielded clear

evidence for bound fragments in electron density. The

attrition of targets appears to have been mainly because of

the crystal deterioration induced by compound soaks and

DMSO sensitivity, but also some proteins did not yield

fragment hits. The high attrition of this project illustrates

the strong requirement for robust crystals for successful

FBLD.

Martins and colleagues [21�] describe an X-ray crystal-

lography-based screening of phenylethanolamine

N-methyltransferase (PNMT) using a commercial frag-

ment library composed of 96 cocktails of 4 structurally

diverse compounds each. They identify 12 hits out of

which 9 could be confirmed with Isothermal titration

calorimetry (ITC) to bind with and affinity <700 mM.

After the initial screening of the 96 cocktails, the decon-

volution of compounds from electron densities requires

an additional 50 data sets soaked with individual com-

pounds to be collected. This indicates a relatively large

deconvolution effort when using this X-ray crystallogra-

phy-based screening strategy with structurally diverse

libraries.

The structural chemistry team at Johnsson & Johnsson

has recently presented an interesting approach to crystal-

lographic fragment screening [34�] where they instead of

using cocktails of structurally diverse compounds, use

cocktails of 5 structurally similar compounds to screen a

900 compound library. The presence of different com-

pounds in the mixture can often be directly deconvoluted

based on crystallographic corefinement, and no follow-up

data sets are collected for deconvolution. They argue

instead that the electron density maps of similar frag-

ments can directly give a good representation of the ‘hot

spots’ for interactions to the protein. After screening of

the initial library a subset of hits (structures and informa-

tive electron density) are used to direct the synthesis of a

secondary library, which is screened in a similar way. No

quantitative binding data are generated before the second

library generation. The strategy appears to minimize

operative time and requirement of deconvolution of hit

cocktails. Fragment occupancy will partly serve as an

affinity measurement. However, the overall strategy

requires the synthesis of a relatively large secondary

library and is therefore expensive. It is possible that there

are less negative effects of cocktails on diffraction and

compound behavior when they are composed of

similar fragments, as compared to cocktails with diverged

fragments. In the later case it is more likely that

single problematic fragment scaffolds can poise multiple

cocktails.

Normally, a number of structures of different bound

fragments are generated before deciding on synthetic

strategies. Fragment growing can initiate ligand-induced

conformational changes of the protein as described in



acteylcholin-binding protein (AChBP), where a tyrosine

flip occurs when the fragment is grown into the ligand

pocket [15�]. In a recent fragment screening study on

p53, a cysteine residue in the active site flips upon

fragment binding [16�]. b-Secretase (BACE1) is another

example showing larger conformational changes when

the flap region is moving with regards to binding of

different fragments [35,36]. Alternatively, substructures

of similar fragments have in some cases been shown to

bind in different ways [37]. However, for successful

FBLD projects presented in the literature, key sub-

structures and interactions of the initial fragment are

nearly always represented in the final lead compound,

where they bind the protein in a similar mode [1,3�].Therefore, the selection of fragment frameworks with

sufficient number of specific interactions, primarily

hydrogen bonds, is preferred to allow efficient fragment

evolution.

DiscussionThe repertoire of available fragment screening methods is

already playing a prominent and complementary role for

FBLD in industry. In light of the recent focus of the

academic sector to develop chemical tools for functional

studies of proteins, FBLD is emerging as an interesting

alternative for the academic sector to establish cost effi-

cient processes for high quality ligand generation. The

minimal requirements for a chemical tool are that it is cell

permeable and has sufficient affinity and selectivity for its

target. It does not, however, need all medical chemistry

optimizations required for a clinical candidate drug. So

far, many academic chemical biology programs for chemi-

cal tool generation have prioritized high-throughput

screening platforms, following the previous path of the

industrial sector. However several lines of evidence

suggest that FBLD will be a very suitable alternative

route for chemical tool generation. Operative aspects

suggest that the cost of FBLD platforms in the academic

sector is favorable; chemical library size is small, mini-

mizing purchasing and maintenance cost; biophysical

platforms for library screening and hit validation are often

already available in-house, crystallographic infrastructure

is in place and access to free synchrotron beam time is

often generous. Furthermore, costs for synthetic chem-

istry in FBLD are estimated to be much smaller than for

hit optimization after HTS [1]. The smaller size of FBLD

leads also correlates positively with cell permeability and

the structure-guided ligand optimization, which allow for

rational engineering of selectivity against homologous

proteins.

As discussed above, there are several working strategies

for generating useful fragments hits. Fragment screening

using SPR has emerged as an interesting alternative for

academic FBLD. It is suitable both for primary screening

and for detailed characterization of kinetic and thermo-

dynamic parameters during fragment hit selection and


fragment evolution. Optimization of thermodynamic

and kinetic parameters has recently been included for

fragment hit profiling and for prioritization of hits for

further synthetic chemistry in industry [6��]. ITC can

serve as a useful complement for fragment characteriz-

ation and thermodynamic analysis before prioritizing

fragments for synthetic chemistry.

From an operative point of view, screening with TSA

constitutes an interesting alternative to SPR and NMR

for academic groups because of the simplicity of the

methods. For example, a library of 500 fragments can

be screened in hours rather than days, which is the typical

data collection time for SPR and NMR. The rapid screen-

ing using TSA potentially allows multiple constructs

and multiple variants of the protein (e.g. with different

cosubstrates or effectors bound) to be screened to identify

druggable forms.

Eventually, screening using X-ray crystallography might

evolve as the preferred strategy for academic FBLD,

when sufficient synchrotron beam time is made avail-

able for even more rapid collection of data, and when the

data processing and analysis have been further stream-

lined. The output from the international structural

genomic programs has provided a large number of

structures of essential proteins from pathogens [38,39]

as well as human biomedically relevant pathways [40]

where, potentially, suitable crystal forms for FBLD have

already been established. With the establishment of

efficient FBLD platforms in the academic sector, these

data can potentially be harvested to support the

development of valuable ligands for understanding

biology and investigate the potentials of these proteins

as drug targets.

AcknowledgementsWe acknowledge the support from Swedish Research Council, the SwedishCancer Society, and a start-up grant from Nanayang TechnologicalUniversity, Singapore.

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� of special interest

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efficiency of hit generation and structural characterization in fragment-based ligand discovery

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