Drug target deconvolution by chemical proteomics

Available online at www.sciencedirect.com

Drug target deconvolution by chemical proteomicsManfred Raida

Drug target deconvolution is a process where the action of a

drug, a small molecule, is characterised by identifying the

proteins binding the drug and initiating the biological effect. The

biological relevant target has to be extracted, or deconvoluted,

from a list of proteins identified in such an approach. Beside the

medically desired action of the drug, the identification of other

proteins binding the drug can help to identify side effects and

toxicity at a very early stage of drug development. The current

approach to identify the proteins binding to the drug is an

affinity-enrichment based approach, where the drug molecule

is immobilised to a matrix through a linker and the proteins

binding to the drug are identified by proteomics.

Address

Experimental Therapeutics Centre, A*STAR, 31 Biopolis Way, Nanos L3-

01, Singapore, 138669, Singapore

Corresponding author: Raida, Manfred ([email protected])

Current Opinion in Chemical Biology 2011, 15:570–575

This review comes from a themed issue on

Next Generation Therapeutics

Edited by Alex Matter and Thomas H. Keller

Available online 18th July 2011

1367-5931/$ – see front matter

# 2011 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.cbpa.2011.06.016

IntroductionAffinity chromatography using small molecules immobil-

ised through a linker to a solid matrix has been used over

several decades to enrich and purify proteins. Back in

1968 and later in 1985 this approach was used to identify

the protein binding to Isoquinoline H9 [1,2]. While in

these early years the identification of the protein was the

major bottleneck, today state-of-the-art mass spectrom-

etry and available protein and genome databases enable a

fast and sensitive identification of the proteins. This

makes the method relevant for the identification of

proteins that bind to drug candidates and other com-

pounds of interest. In the past few years, the major focus

in drug target deconvolution was on kinase inhibitors.

Through the identification of the protein targets of a drug

or a drug candidate, it becomes possible to understand side

effects and toxicity in an early state of development, which

should help to reduce the attrition rate in development.

Even after 50 years, the teratology of Thalidomide


(Contergan) [3,4] was explained by chemical proteomics

[5�].

By analysing kinase inhibitors in clinical use, the targets

of these drugs could be identified by chemical proteomics

and side effects and toxicity rationalised [6–17,18��].Besides the kinase inhibitors, other examples are

cGTP-binding proteins [19,20], transcription factors

and other proteins involved in nucleic acid processing

[21], serine hydrolases [22], cysteine proteases [23,24]

aspartyl proteases [25], metalloproteases [26], glycosi-

dases [27,28], histone deacetylases [29,30], nitrilases

[31,32] and oxireductases [33,34] and epigenetic enzymes

as potential targets for cancer treatment [35��].

There are some limitations to the identification of the

relevant targets out of a large number of identified

proteins, such as unspecific binding of background

proteins, the risk of missing the interesting target owing

to low abundance, and the risk of missing the target owing

to sample processing steps. In any case, the identified

target has to be validated by independent biological

experiments, as shown for a tankyrase inhibitor [36�].

Another important aspect that is crucial for a chemical

proteomics approach is the orientation of the drug in the

binding pocket of the target and the off-target protein.

Parkinson drugs, for example, bind in different manner to

the target protein and to the off-target or the liver-toxicity

causing protein [37��].

In the past years some methods for chemical proteomics

have been developed that will be introduced here.

Direct deconvolution of drug targets byaffinity probesThe direct way to identify a molecular target of a drug is the

use of a chemical derivative of the drug to be immobilised

on a solid matrix, as agarose, sepharose or magnetic

beads [6,9,12,15��,18��,38,39�,40–42]. Incubating complex

protein mixtures, as cell, tissue or organ lysates with the

matrix-bound drug captures the target proteins. There are

some restrictions to this approach, first of all, the drug

molecule must retain the biological activity, a linker may

interfere with the structure-activity relationship (SAR) of

the native molecule. In many cases the attachment of a

linker requires the complete new synthesis of the molecule

and an attachment of linkers to different positions to avoid

destruction of the SAR. The use of a linker attached

to different sites of the molecule can give further

insight into the drug actions as one compound can have

different orientations in binding pockets in different

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Drug target deconvolution Raida 571

Figure 1

Current Opinion in Chemical Biology

Principle of direct chemical proteomics: The drug bound to a solid matrix through a linker is incubated with the lysate and target proteins identified

after removal of unbound proteins by extensive washing.

proteins, resulting in side effects and toxicity beside the

desired biological action [37��]. Matrix and linker have to

be selected to have little or no unspecific binding of

proteins, a control with beads with linker but no drug

molecule has to be done for every experiment to identify

unspecific binders. This direct approach works generally

for kD’s below 1 mM. Another problem is the less specific

binding of a high abundant protein that also interferes with

the binding to the real, often low abundant target. The lysis

conditions are also of high importance, if the target is a

membrane bound protein, a nuclear or a cytosolic protein;

different conditions have to be applied. The chemical

proteomics approach has been shown applicable also to

membrane proteins [43] (Figure 1).

The approach described above uses the immobilised drug

on a solid matrix; a biphasic system of the solubilised

proteins and the solid phase bound drug molecules. Pore

size of the matrix may prevent larger proteins to reach the

drug molecules and reduce the binding, but may also

capture proteins in an unspecific way. Another approach is

the use of the drug molecule connected to a sorting

function with a defined specificity, like a biotin group,

through a linker. In this case the drug and the protein

mixture are incubated in solution, only after binding the

target molecules to the drug, they are affinity captured

through the biotin residue [44,45].

The deconvolution of the potential drug targets [46��] is

done in consecutive steps, experiments are repeated at

least two times and only proteins identified in both

experiments are considered to be valid. Next the frequent

hitters, proteins seen repeatedly in independent exper-

iments using unrelated drugs or with matrices without

immobilised drugs, are removed from the list. Then

highly abundant proteins that are present in many cell


lines, the so-called ‘core-proteome’ proteins [47], are

removed from the list. These two steps always carry

the risk that the target is removed because it falls into

one of the exclusion criteria. The last step is the com-

petition, an independent experiment to identify the

relevant protine. The lysate is pre-incubated with the

molecule of interest in solution, and then the binding to

the immobilised compound is carried out. Proteins com-

peted from binding to the immobilised compound are

then probably the most relevant targets.

Usually the proteins purified by the affinity step are then

extracted from the matrix under denaturing conditions

and separated by gel-electrophoresis. The bands are

submitted to in-gel tryptic digestion and the proteins

identified by consecutive liquid-chromatography tan-

dem-mass spectrometry (LC–MS/MS) followed by data-

base searches. Questions of transient phosphorylation in

relation of a drug action on pathways can be addressed by

using specific enrichments of the phosphorylated pep-

tides [15��,46��].

In the past years this approach has been shown to be

applicable for a number of drugs and drug like molecules.

The major targets have been kinases, as their involve-

ment in cancer has been described for many cases. But

even for successful drugs with a well-defined target

profile, as Imatinib or Dasatinib, off-targets have been

identified [17,48–51]. The use of a single immobilised

kinase inhibitor [52��,53] allows the capturing of specific

target proteins while a combination of kinase inhibitors,

for example ‘Kinobeads’, allows the affinity capturing of

over 300 kinases from cell lysates [15��,16,18��]. While in

the first approach the specific targets of the inhibitor are

identified, the second approach is required for the com-

petition approach described below.


572 Next Generation Therapeutics

Figure 2


Principle of competition chemical proteomics: The lysate is pre-incubated with the drug or with molecules in development that block the binding

pocket and prevent the target protein from binding to the immobilised compound.

Figure 3S

tand

ard

No

com

petit

ion

Com

petit

ion


Virtual 1D SDS-gel of proteins affinity purified by the direct and the

competition approach. The bands marked in red are not detected in the

competition approach, therefore suggested as target candidates, while

the other proteins are unspecific bound or background.

Chemical proteomics has not only been applied to lysates

but also to living cells [54�].

A further development of this approach is the use of a

photo-reactive group close to the molecule [37��,55,56].

This allows for the identification of enzymes that

modify the compound and releases it, or use the com-

pound through intermediate binding to modify other

proteins, for examples as substrates as in the case of

proteases, epigenetic enzymes or kinases. The cell

lysate is incubated with a construct containing the

selective group, the drug moiety, a photo-reactive

group, and a sorting group or function, in most cases

a biotin. After binding to the drug moiety, the proteins

are covalently captured by the photo-reactive after UV

irradiation and the drug-protein construct is affinity

purified by the biotin group and identified as described

above.

The competition approachAffinity capturing of proteins using immobilised com-

pounds usually results in a long list of proteins identified

by mass spectrometry. Easily several hundreds of proteins

are identified [36�] in one experiment, selecting the real

target candidate from this list requires a deconvolution

process as described above [40]. After removing frequent

hitters, the core proteome, and proteins found only once

in repeated experiments the next step is to repeat the

experiment with the lysate pre-incubated with the

soluble form of the immobilised compound or with a

known competitor. Competing for the binding pocket of

the target protein, the soluble compound will prevent or

reduce the binding to the immobilised compound

[36�,57]. Only proteins competed by the compound in

solution are considered as real target candidates, further

verification has to be done by biological experiments

(Figure 2).


But beside the target verification, the competition

approach offers more possibilities in the drug develop-

ment process (Figure 3).

Often it is not easily possible to attach a linker to the

molecule without destroying the SAR. For many natural



compounds, the chemical re-synthesis itself alone and

with linker is impossible or is very time consuming and

man-power consuming. In many cases the linker has to

be attached to the molecule on different locations to

ensure capturing all potential targets. This has only

been shown for the Parkinson drugs [37��]. Very few

natural compounds have been used in chemical proteo-

mics; only Staurosporine, a fungal pan-kinase inhibitor,

has been immobilised to capture kinases [18��,58]. But

some of the natural compounds may also become future

drugs or leads and their target protein have to be

identified. The unbiased approach cannot be done in

competition experiments; the analysis has to be limited

to a class of potential target proteins. For these classes,

relative unspecific binders have to be immobilised to

cover as many as possible members of the enzyme class.

Using a set of pan-kinase inhibitors immobilised to the

matrix and incubating cell lysates with the candidate

molecule, proteins are competed from binding to the

immobilised compounds. Pre-incubation of the lysate

with increasing concentrations of a compound leads to a

concentration-dependent competition, which allows the

determination of IC50 values and the selectivity and

specificity [15��]. The main limitation is the use of the

immobilised inhibitors, which do not cover the entire

human kinome. In case of kinase inhibitors, this

approach allowed determining the specificity of drugs

in development and in clinics and a binding constant

that roughly describes the KD of the soluble compound.

The competition approach has been described for

kinase inhibitors and for epigenetic enzyme inhibitors

[15��,35��].

LimitationsThe described process has several limitations. First of all,

the drug must maintain its SAR, the linker has to be

attached without interfering with the binding to the

protein target. To overcome this limitation, the linker

can be attached to different sites of the molecule, but

requiring often-difficult chemistry approaches. Another

limitation is the preparation of the cell or tissue lysate,

the protein target can be missed owing to the preparation

conditions, for example membrane-spanning protein

targets are not solubilised. The major problem is the

identification of the real target from the large number of

proteins identified in a drug-target deconvolution exper-

iment, often exceeding 500 proteins. Only competition

experiments can help to identify the correct protein

target. In case of a low-expressed protein it may be

missed owing to sensitivity reasons or may be masked

by a high abundant background protein. This case can

happen if 1-dimensional gel electrophoresis is used to

separate the proteins prior to mass spectrometric identi-

fication.

Another case is the binding of the drug to more than one

target with low affinity, leading to a synergistic effect. In


this case the binding may be too weak for an affinity

capturing of the protein targets.

Another major limitation is the still slow mass spectro-

metric identification of the proteins; an average exper-

iment can take up to several weeks for a full analysis. In

the current situation, no development is in sight to

increase the speed without losing analytical depth and

sensitivity.

Current developments and outlookSince the first publication of targeted affinity purification

of proteins using immobilised small molecule [2] the

process is still under development, the methods have

to be adapted for each molecule and cell or tissue source.

The number of drug-molecules for which the side effects

are not understood is large, chemical proteomics can help

to understand these.

The availability of faster and more sensitive mass spec-

trometers will help to identify low abundant proteins, but

also requires a cleaner sample preparation to avoid con-

taminations.

In combination with methods for protein structure

analysis, as NMR or X-ray crystallography of proteins

chemical proteomics will increase the knowledge of the

orientation of a small molecule in a binding pocket. This

increased knowledge can help to design better probes for

the identification of the protein targets.

SummaryDrug target deconvolution, chemical proteomics, chemo-

gentics, the identification of a drug target and the identi-

fication of the off-targets to explain side effects and

toxicity is becoming an essential step in early develop-

ment of a new drug and in understanding marketed drugs.

Currently the techniques are still under development, far

away from high-throughput approaches and can only be

applied to a limited number of molecules. But it will

become a major tool to support chemistry to develop more

targeted drugs in future and to identify potential side

effects and toxicity risks in early preclinical states.

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Chemical proteomics generally uses cell or tissue lysates to identify thetargets of the small molecule. This publication describes a construct of asmall molecule, the drug, and a cell-permeable peptide with a fluoro-phore. This allows direct visualization of the drug in the cell, but also thecapturing of the drug with the protein bound to it. This approach allows towork with living cells rather than cell lysate to study the action of a drugand the binding to the targets.

55. Koster H, Little DP, Luan P, Muller R, Siddiqi SM, Marappan S,Yip P: Capture compound mass spectrometry: a technologyfor the investigation of small molecule protein interactions.Assay Drug Dev Technol 2007, 5:381-390.

56. Luo Y, Fischer JJ, Baessler OY, Schrey AK, Ungewiss J, Glinski M,Sefkow M, Dreger M, Koester H: Gdp-capture compound – anovel tool for the profiling of gtpases in pro- and eukaryotes bycapture compound mass spectrometry (ccms). J Proteomics2010, 73:815-819.

57. Terstappen GC, Schlupen C, Raggiaschi R, Gaviraghi G: Targetdeconvolution strategies in drug discovery. Nat Rev DrugDiscov 2007, 6:891-903.

58. Fischer JJ, Graebner Baessler OY, Dalhoff C, Michaelis S,Schrey AK, Ungewiss J, Andrich K, Jeske D, Kroll F, Glinski Met al.: Comprehensive identification of staurosporine-bindingkinases in the hepatocyte cell line hepg2 using capturecompound mass spectrometry (ccms). J Proteome Res 2010,9:806-817.


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Drug target deconvolution by chemical proteomics