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II: Hill' Mill I 

(id EP 1 464 960 A1 



(1 2) EUROPEAN PATENT APPLICATION 



(43) Date of publication: 


(51) Intel* G01N 33/68 


06.10.2004 Bulletin 2004/41 


(21) Application number: 03007690.5 




(22) Date of filing: 03.04.2003 




(84) Designated Contracting States: 


• Grandl, Paola 


AT BE BG CH CY CZ DE DK EE ES Fl FR GB GR 


69117 Heidelberg (DE) 


HU IE IT LI LU MC NL PT RO SE SI SK TR 


• Kruse, Ulrich 


Designated Extension States: 


69221 Dossenhelm (DE) 


AL LT LV MK 






(74) Representative: Krauss, Jan B. } Dr. 


(71) Applicant: CELLZOME AG 


Forrester & Boehmert, 


^ 69117 Heidelberg (DE) 


Petten koferstrasse 20-22 




80336 Munchen (DE) 


(72) Inventors: 




• Gavin, Anne-Claude 




69181 Leimen (DE) 





(54) Screening Method for the Identification of new Proteome-interacting Compounds 



(57) The present invention relates to the search for proteome-interacting compound. Furthermore, new and 
new drugs and in particular to a method for screening a yet unidentified interactions between the proteome and 
library of potentially proteome-interacting candidate compounds can be identified using the method accord- 
compounds for identifying a protein/protein-complex in- ing to the present invention, 
teracting compound and thereby further identifying a 



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Description 

[0001] The present invention relates to the search for 
new drugs and in particular to a method for screening a 
library of potentially proteome-interacting candidate 
compounds for identifying a protein and/or protein com- 
plex interacting compound and thereby further identify- 
ing a proteome-interacting compound. Furthermore, 
new and yet unidentified interactions between the pro- 
teome and compounds can be identified using the meth- 
od according to the present invention. 
[0002] The search for new drugs in essentially all ther- 
apeutic fields of pharmaceutical industry has been lim- 
ited so far by the identification of so-called "suitable drug 
targets". These targets are mostly present as proteins 
and/or enzymes that constitute targets by which, for ex- 
ample, the processes underlying a specific disease can 
be modulated in order to achieve a beneficial therapeu- 
tic effect. In addition, these targets must be "druggable", 
that is, must be available for the interaction with a drug 
to be applied. 

[0003] Currently, the concept of "drugability" (availa- 
bility) is limited to a rather small number of proteins with 
enzymatic activity and some cell surface/nuclear recep- 
tors. Around 1 0 % of the human genome can be targeted 
for the development of new drugs (i.e. are expected to 
be "druggable"), according to top pharmaceutical indus- 
try scientists (BA Festival of Science at the U niversity of 
Glasgow, 3rd September 2001; see also, for example, 
Johnson J A. Harris S, Foord SM. Drug target pharma- 
cogenomics: an overview. Am J Pharmacogenomics 
2001;1(4):271-81). 

[0004] "Genomics is already beginning to reshapethe 
way drugs are discovered, with around 3,500 of human 
genes representing potential druggable targets. So far, 
the complexities of the drug discovery process have 
meant that the pharmaceutical industry has exploited 
only 450 targets." (Mark Fidock, Pfizer Ltd. "From genes 
and cells to healthcare forum", organised by the Bio- 
technology and Biological Sciences Research Council). 
[0005] A greater understanding of the molecular path- 
ways that lead to disease will provide better targets and 
more selective and safer compounds. This will help re- 
duce the rate of attrition-drugs that fail to make it to mar- 
ket due to poor clinical efficacy, or safety - and improve 
the overall efficiency of the process. A deeper under- 
standing of cellular processes is needed, particularly in 
disease. Novel therapeutic targets can be found from 
understanding how proteins interact with each other and 
how they work co-operatively in cells as part of larger 
complexes. 

[0006] From a pharmaceutical perspective, not all 
genes are equal and the industry is heavily investing in 
technologies required to identify those genes that are 
both highly druggable and disease relevant. 
[0007] Oneway out of the dilemma of the lack of drug- 
gable would be to increase the number of drugs that are 
available for the druggable targets. The recent develop- 



ments of new drug classes, like anti-sense RNA (and 
probably also RNAi) and antibodies could definitively 
help to bypass this limitation. These approaches remain 
however at relatively early stages and their very general 
5 use as future therapeutic tools to cure diseases is still 
under question. 

[0008] Therefore, the therapeutic tools of choice for 
curing diseases remain small compounds. The broad- 
ening of the "druggable space" for small compounds 
io and the possibility to target new enzyme classes will rep- 
resent a clear advantage. 

[0009] In case of traditional drug discovery, in one ap- 
proach so-called "small molecule libraries" are 
screened against a single pre-defined target. Little is yet 

is known about selectivity of such a assay in a crude pro- 
teome (the specific protein composition of a cell or tis- 
sue); for example, the ability to bind related enzymes 
(coming from similar or identical protein families) or oth- 
er enzymes/protein classes including, but not limited to, 

20 drug transporters and/or drug modifying enzymes. 
[0010] The current methods that are developed in or- 
derto tacklethe above mentioned issues usually involve 
proteins that are expressed in heterologous systems, 
like bacteria or phages. A very commonly used system 

25 employs the phage display system. 

[0011] One example for this system is the so-called 
"Proteome scan™", which screens for targets and lead 
structures (i.e. structures forming the chemical and 
structural basis ("core") of a future drug) at the proteome 

30 level. The method comprises a screening format in 
which one compound/peptide is tested against a phage 
display library that usually contains approx. 10000 hu- 
man proteins (in case of a high quality library). The drugs 
themselves are compounds with unknown modes of ac- 

35 tion and can constitute a library as well. Currently, the 
introduction of robotics leads to a "through-put" of 200 
scans per month, a number which is said to be improved 
up to an expected through-put of 4000 screens per 
months. 

40 [0012] WO 02/092118 relates to proteome chips com- 
prising arrays having a large proportion of all proteins 
expressed in a single species and to methods for using 
proteome chips to systematically assay all protein inter- 
actions in a species in a high-throughput manner. Fur- 

45 thermore, also methods for making protein arrays by at- 
taching double-tagged fusion proteins to a solid support 
are described. 

[0013] Evans DM et al. (in: Evans DM, Williams KP, 
McGuinness B, Tarr G, Regnier F, Afeyan N, Jindal S. 

50 Affinity-based screening of combinatorial libraries using 
automated, serial-column chromatography. Nat Bio- 
technol 1996 Apr;14(4):504-7) describe an automated 
serial chromatographic technique for screening a library 
of compounds based upon their relative affinity for a tar- 

55 get molecule. A "target" column containing the immobi- 
lised target molecule is set in tandem with a reversed- 
phase column. A combinatorial peptide library is inject- 
ed onto the target column. The target-bound peptides 



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are eluted from the first column and transferred auto- 
matically to the reversed-phase column . The target-spe- 
cific peptide peaks from the reversed-phase column are 
identified and sequenced. Using a monoclonal antibody 
(3E-7) against beta-endorphin as a target, a single pep- 
tide with the sequence YGGFL from approximately 5800 
peptides present in a combinatorial library was selected. 
The technique is described as having broad applications 
for high throughout screening of chemical libraries or 
natural product extracts. 

[0014] Although powerful, the above methods have 
limitations, since most proteins require laborious tech- 
niques, folding, post-translational modifications and as- 
sociation in complexes with regulatory/modulatory com- 
ponents. Although important, these post-translational 
events are not always sustained in heterologous sys- 
tems. 

[001 5] Thus, there is an ongoing need in the pharma- 
ceutical industry for efficient screening systems that are 
suitable for high-throughput screening and allow for the 
identification of new and more "physiological" targets, i. 
e. methods that more efficiently mimic the situation in 
the proteome in vivo. 

[0016] This problem is solved by providing a method 
for screening a library of potentially proteome-interact- 
ing candidate compounds, comprising: a) providing a li- 
brary comprising non-labelled potentially proteome-in- 
teracting candidate compounds, b) providing a second 
library comprising a variety of proteomes, wherein each 
proteome contains at least one labelled polypeptide, c) 
contacting the library from a) with the library in b) in a 
manner as to allow for an interaction of the candidate 
compounds with the at least one labelled polypeptide, 
d) determining an interaction between said candidate 
compounds and said at least one labelled polypeptide, 
and thereby identifying a a proteome-interacting com- 
pound. 

[0017] A "library" according to the present invention 
relates to a (mostly large) collection of (numerous) dif- 
ferent chemical entities that are provided in a sorted 
manner that enables both a fast functional analysis 
(screening) of the different individual entities, and at the 
same time provide for a rapid identification of the indi- 
vidual entities that form the library. Examples are collec- 
tions of tubes or wells or spots on surfaces that contain 
chemical compounds that can be added into reactions 
with one or more defined potentially interacting partners 
in a high-throughput fashion. After the identification of a 
desired "positive" interaction of both partners, the re- 
spective compound can be rapidly identified due to the 
library construction. Libraries of synthetic and natural or- 
igins can either be purchased or designed by the skilled 
artisan. 

[0018] In the context of the present invention, two dif- 
ferent libraries are used. In the first "library of potentially 
proteome-interacting candidate compounds", a library 
of potentially proteome-interacting compounds is pro- 
vided, wherein a collection of compounds is provided 



that potentially interact with the proteome to be analysed 
(screened). Examples of compounds are synthetic and/ 
or naturally occurring chemical compounds, peptides, 
proteins, nucleic acids, antibodies, and the like. 

5 [0019] Examples of the construction of libraries are 
provided in, for example, Breinbauer R, Manger M, 
Scheck M, Waldmann H. Natural product guided com- 
pound library development. Curr Med Chem. 2002 Dec; 
9(23):21 29-45, wherein natural products are described 

10 that are biologically validated starting points for the de- 
sign of combinatorial libraries, as they have a proven 
record of biological relevance. This special role of nat- 
ural products in medicinal chemistry and chemical biol- 
ogy can be interpreted in the light of new insights about 

15 the domain architecture of proteins gained by structural 
biology and bioinformatics. In order to fulfil the specific 
requirements of the individual binding pocket within a 
domain family it is necessary to optimise the natural 
product structure by chemical variation. Solid-phase 

20 chemistry is said to become an efficient tool for this op- 
timisation process, and recent advances in this field are 
highlighted in this review article. Other related referenc- 
es include Edwards PJ, Morrell Al. Solid-phase com- 
pound library synthesis in drug design and develop- 

25 ment. Curr Opin Drug Discov Devel. 2002 Jul;5(4): 
594-605.; Merlot C, Domine D, Church DJ. Fragment 
analysis in small molecule discovery. Curr Opin Drug 
Discov Devel. 2002 May;5(3):391-9. Review; Goodnow 
RA Jr. Current practices in generation of small molecule 

30 new leads. J Cell Biochem Suppl. 2001 ;Suppl 37:13-21 ; 
which describes that the current drug discovery proc- 
esses in many pharmaceutical companies require large 
and growing collections of high quality lead structures 
for use in high throughput screening assays. Collections 

35 of small molecules with diverse structures and "drug- 
like" properties have, in the past, been acquired by sev- 
eral means: by archive of previous internal lead optimi- 
sation efforts, by purchase from compound vendors, 
and by union of separate collections following company 

40 mergers. Although high throughput/combinatorial 
chemistry is described as being an important compo- 
nent in the process of new lead generation , the selection 
of library designs for synthesis and the subsequent de- 
sign of library members has evolved to a new level of 

45 challenge and importance. The potential benefits of 
screening multiple small molecule compound library de- 
signs against multiple biological targets offers substan- 
tial opportunity to discover new lead structures. Subse- 
quent optimisation of such compounds is often acceler- 

50 ated because of the structure-activity relationship (SAR) 
information encoded in these lead generation libraries. 
Lead optimisation is often facilitated due to the ready 
applicability of high-throughput chemistry (HTC) meth- 
ods for follow-up synthesis. Some of the strategies, 

55 trends, and critical issues central to the success of lead 
generation processes are discussed. One use of such 
a library is finally described in, for example, Wakeling 
AE, Barker AJ, Davies DH, Brown DS, Green LR, 



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Cartlidge SA, Woodburn JR. Specific inhibition of epi- 
dermal growth factor receptor tyrosine kinase by 4-ani- 
linoquinazolines. Breast Cancer Res Treat. 1996;38(1): 
67-73. 

[0020] The proteome of a cell, tissue, and/or organism 
is largely sub-organised in protein-complexes, i.e. ag- 
gregations of proteins that form functional and/or struc- 
tural sub-divisions of a cell. Examples for these com- 
plexes and their functional relevance are described in, 
for example, Gavin AC, Superti-Furga G. Protein com- 
plexes and proteome organisation from yeast to man. 
Curr Opin Chem Biol 2003 Feb;7{1):21-7, wherein pro- 
tein complexes are described as well being the most rel- 
evant molecular units of cellular function. The activities 
of protein complexes have to be regulated both in time 
and space to integrate within the overall cell programs. 
The cell can be compared to a factory orchestrating in- 
dividual assembly lines into integrated networks fulfilling 
particular and superimposed tasks. 
[0021] The library of the potentially proteome-inter- 
acting compounds contains entities that are usually 
"non-labelled", i.e. that do not contain a marker that 
would be necessary for an identification of the library 
compound. Nevertheless, also libraries of labelled po- 
tentially proteome-interacting compounds can be 
screened without using the labels of the compounds. 
[0022] The present invention uses furthermore a sec- 
ond "library of a variety of proteomes". This library there- 
fore contains a collection of proteomes from different 
cells, organs, tissues or pool of cells. "Proteomes" as 
used in the context of the present invention are, for ex- 
ample, described in Gavin AC, Bosche M, Krause R, 
Grandi P, Marzioch M, Bauer A, Schultz J, Rick JM, Mi- 
chon AM, Cruciat CM, Remor M, Hofert C, Schelder M, 
Brajenovic M, Ruffner H, Merino A, Klein K, Hudak M, 
Dickson D, Rudi T, Gnau V, Bauch A, Bastuck S, Huhse 
B, Leutwein C, Heurtier MA, Copley RR, Edelmann A, 
Querfurth E, Rybin V, Drewes G, Raida M, Bouw- 
meesterT, Bork P, Seraphin B, Kuster B, Neubauer G, 
Superti-Furga G. Functional organization of the yeast 
proteome by systematic analysis of protein complexes. 
Nature. 2002 Jan 10;415(6868):141-7. Thus, a "pro- 
teome" according to the present invention is the specific 
protein composition of a cell or tissue or organism. De- 
pending on the individual cell or tissue, one culture of 
yeast cells could, theoretically, contain as many pro- 
teomes as there are cells in said culture. For conven- 
ience, the proteomes of one cell culture under the same 
growth conditions are regarded as identical, i.e. repre- 
senting one proteome. In other words, the proteomes of 
one type of organism differ depending from the status 
of the cells and the genomic background of the cells. 
[0023] An "interaction" according to the present in- 
vention does relate to both an interaction between dif- 
ferent components of the libraries that leads to function- 
al or non-functional effects. That is, a compound is at- 
tached to the other with or without modifying the function 
of the polypeptide, the complex and/or the proteome. 



The attachment can be either covalent or non-covalent. 
[0024] In general, the method of the present invention 
determines the interaction of a protein with a compound. 
Usually, a "protein-complex interacting compound" is a 

5 compound that interacts with at least one protein of a 
cell. Consequently, a "protein-complex interacting com- 
pound" is a compound that interacts with at least one 
protein of the cell as a protein-complex or as a subunit 
of a protein-complex. A "protein-complex" according to 

10 the present invention therefore can be formed by only 
one polypeptide. A "protein-complex interacting com- 
pound" therefore is a compound from one library that 
interacts with a complex or a protein as subunits of a 
proteome. Consequently, a "proteome-interacting corn- 
's pound" is a protein-complex interacting compound that 
interacts with a complex as part of a proteome. 
[0025] In one embodiment of the present invention, a 
method is provided, wherein the potentially proteome- 
interacting candidate compounds are selected from en- 

20 zymes, polypeptides, peptides, antibodies and frag- 
ments thereof, nucleic acids or derivatives thereof, and 
chemical entities having a molecular mass of less than 
1 000 kDa ("small molecules"). Thus, in one library of the 
present invention potentially proteome-interacting com- 

25 pounds are provided that potentially interact with the 
proteome to be analysed (screened). Examples of such 
compounds are synthetic and/or naturally occurring 
chemical compounds, peptides, proteins, antibodies, 
and the like. Since the library is related to "small" com- 

30 pounds, i.e. other that complete enzymes, antibodies or 
other proteins, the molecular weight of these com- 
pounds is preferably below 1000 kDa, more preferably 
below 500 kDa. Of course, these compounds can be 
much smaller, e.g. smaller than 1000 Da. Such com- 

35 pounds can be suitable as "leads" for further optimisa- 
tion. One example of a peptide library is, for example, 
described in Sachpatzidis A, et al. Identification of allos- 
teric peptide agonists of CXCR4. J Biol Chem 2003 Jan 
10;278(2):896-907, wherein a synthetic cDNA library 

40 coding for 160,000 different SDF-based peptides was 
screened for small molecule CXCR4 agonist activity in 
a yeast strain. 

[0026] In another embodiment of the method accord- 
ing to the present invention, said potentially proteome- 

45 interacting candidate compounds is a nucleic acid, such 
as a DNA, RNA and/or PNA. Such nucleic acids can be 
present in the form of oligonucleotides or polynucle- 
tides, covering specific binding-specific nucleotide spe- 
cific nucleic acid sequences and/or motifs. Hybrid nu- 

50 cleic acids between the different forms might also be 
employed. Furthermore, the library can be present on a 
chip for high-throughput screening purposes. 
[0027] In the context of the present invention, libraries 
comprising synthetic and/or naturally occurring "small" 

55 chemical compounds (e.g. drugs, metabolites, prod- 
rugs, potential drugs, potential metabolites, potential 
prodrugs and the like) are most preferred. 
[0028] As mentioned above, the library of potentially 



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proteome-interacting candidate compounds can be 
present in different formats, such as in liquid solution, 
such as in tubes, microtiter-plates, or on a solid support, 
such as on filters, glass slides, silicon surfaces, beads 
or a customised chemical microarray. Microarrays are 
preferred due to their easy handling and their uses in 
high-throughput formats. 

[0029] In one preferred embodiment of the method 
according to the present invention, the potentially pro- 
teome-interacting candidate compounds are bound to 
beads, such as sepharose (e.g. NHS-activated sepha- 
rose) or agarose beads. In contrast to Evans DM et al. 
(1996, see above), the present invention uses immobi- 
lised compound libraries that are screened with pro- 
teomes, which allows for a much more flexible screening 
procedure than using columns with immobilised binding 
partners. Furthermore, on columns the "/nvivo-like con- 
ditions" of the present method is easily lost, which 
renders some interactions unspecific and leads to im- 
precise results. Preferably, said potentially proteome-in- 
teracting candidate compounds are bound to said beads 
via an amino-group orcarboxy-group. 
[0030] The use of proteomes (and protein-complex- 
es) allows for an increased flexibility during the screen- 
ing procedure. In contrast to other methods according 
to the art, the present method can also be used in an in 
vivo-environment. Thus, in performing the method ac- 
cording to the present invention, a variety of proteomes 
can be used that is present in or derived from one single 
cell or cell culture or from a mixture of cells, such as a 
tissue, organ or organism. In an even more preferred 
embodiment of the present invention the variety of pro- 
teomes is present in or derived from one single cell or 
cell culture or from a mixture of cells, such as a tissue, 
organ or organism, wherein said single cell or cell cul- 
ture or mixture of cells, such as a tissue, organ or or- 
ganism was exposed to certain conditions, such as heat, 
stress, starvation, drugs, radioactivity, chemical agents, 
toxins, viral infection, antibiotics, and ageing. These 
conditions lead to different "sets" of proteomes that can 
be used for the screening procedure. This approach re- 
sembles a situation that is as identical to the "real" situ- 
ation in vivo, as possible. 

[0031] The method according to the present invention 
can be employed on a large variety of proteomes that 
are present in or derived from a cell selected from 
prokaryotic or eukaryotic cells, such as a bacterial cell, 
a pathogenic micro-organism, a fungal cell, a yeast cell, 
a plant cell, a mammalian cell, a fish cell, a nematode 
cell, an insect cell, and, in particular, a non-human stem- 
cell. Furthermore, the method according to the present 
invention can be employed on a large variety of pro- 
teomes that are present in or derived from a tissue or 
organ, such as connective tissue, endothelial tissue, 
brain, bone, liver, heart, skeletal muscles, prostate, co- 
lon, kidney, glands, lymph nodes, pancreas, roots, 
leaves, and flowers. Finally the variety of proteomes can 
be present in a non-human organism or derived from an 



organism, such as E. coli, Drosophila melanogaster, 
Caenorhabditis elegans, zebrafish, rat, hamster, 
mouse, goat, sheep, monkey, human, jellyfish, rice, po- 
tato, Arabidopsis, wheat, oat, and tobacco. A human in- 
5 vivo use of the present invention is explicitly excluded 
from the scope of the present invention. 
[0032] In the case of a preferred in-vitro method ac- 
cording to the present invention, said variety of pro- 
teomes is present in or derived from a lysate of one sin- 
to gle cell or cell culture or from a lysate of a mixture of 
cells, such as a tissue, organ or organism, identical to 
those described above. 

[0033] As already pointed out, the method according 
to the present invention in general makes no use of 

15 screening compound libraries that contain labelled po- 
tentially proteome-interacting candidate compounds. 
Although labelled libraries could also be used for 
screening, according to the preferred embodiment of the 
present invention, each proteome used for screening 

20 contains only one proteome-complex comprising one la- 
belled polypeptide. Even more preferred is a method ac- 
cording to the present invention, wherein each member 
of the library in b) above contains only one labelled 
polypeptide that is different from the other members of 

25 said library. 

[0034] One example for such a library would be the 
production of an essentially complete collection of cells 
in which a different gene (or polypeptide) is labelled 
(tagged), respectively. One particular example is TAP- 

30 tagging in yeast which recently was used in order to 
identify the yeast proteome (Gavin et al. Nature 
415,141-7 (2002)). 

[0035] EP 1 1 05 508 B1 as well as Puig O, et al. ('The 
tandem affinity purification (TAP) method: a general pro- 

35 cedure of protein complex purification." Methods. 2001 
Jul;24(3):21 8-29.; and Rigaut G, et al. A generic protein 
purification method for protein complex characterization 
and proteome exploration. Nat Biotechnol 1999 Oct;17 
(1 0):1 030-2) describe the general principle of the TAP- 

40 method. The tandem affinity purification (TAP) method 
is described as a tool that allows rapid purification under 
native conditions of complexes, even when expressed 
at their natural level. Prior knowledge of complex com- 
position or function is not required. The TAP method re- 

45 quires fusion of the TAP tag, either N- or C-terminalfy, 
to the target protein of interest. The TAP method was 
initially developed in yeast but can be successfully 
adapted to various organisms, such as mammalian cells 
(Cox DM, DuM, Guo X, Siu KW, McDermott JC. Tandem 

50 affinity purification of protein complexes from mamma- 
lian cells. Biotechniques. 2002 Aug;33(2):267-8, 270.) 
[0036] Preferably, the label of the labelled polypeptide 
is selected from the group of radiolabels, such 

as 32p t 35 S> 3 Hj 125| t 99m TC( 111| n> and the |j kej dye la- 

55 bels, labels that can be detected with antibodies, en- 
zyme labels, and labels having a detectable mass. Pre- 
ferred are labels that can be specifically detected with 
antibodies. Examples for the use of mass spectroscopy 



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EP 1 464 960 A1 



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in proteome analysis is described in Ho Y, et al. (without 
labels) ("Systematic identification of protein complexes 
in Saccharomyces cerevisiae by mass spectrometry" 
Nature. 2002 Jan 1 0;41 5(6868):1 80-3.); and Gu S, et al 
("Precise peptide sequencing and protein quantification 
in the human proteome through in vivo ly sine-specific 
mass tagging". J Am Soc Mass Spectrom 2003 Jan; 14 
(1 ):1 -7) and Williams C and Addona TA ("The integration 
of SPR biosensors with mass spectrometry: possible 
applications for proteome analysis." Trends Biotechnol. 
2000 Feb;18(2):45-8). Preferably, the label of the la- 
belled polypeptide is selected from phosphorescent 
markers, fluorescent markers, chemiluninescent mark- 
ers, phosphatases, streptavidin, biotin, TAP-method 
markers, and peroxidases. A wide variety of labels can 
be used when performing the method of the present in- 
vention. In addition to TAG, other markers are Arg-tag, 
calmodulin-binding peptide, cellulose-binding domain, 
DsbA, c-myc-tag, glutathione S-transferase, FLAG-tag, 
HAT-tag, His-tag, maltose-binding protein, NusA, S-tag, 
SBP-tag, Strep-tag, and thioredoxin. The uses of these 
tags are extensively described in the literature (for ex- 
ample, in Terpe K. Overview of tag protein fusions: from 
molecular and biochemical fundamentals to commercial 
systems. Appl Microbiol Biotechnol 2003 Jan;60(5): 
523-33). 

[0037] In another aspect of the method according to 
the present invention, each member of the library in b) 
above comprises a mixture or collection (pool) of differ- 
ent proteomes. This embodiment is used in particular in 
an initial screening step that shall result into the forma- 
tion of library-subgroups in order to accelerate the 
throughput of all library samples. These subgroups 
could, for example, each contain approximately 1000 
proteomes in the initial screening, then 100 in the sec- 
ond screening of positive initial pools and finally are 
screened based on the individual proteomes. In accord- 
ance with this embodiment, different labels can be used 
in order to further discriminate between the subsets and/ 
or proteomes in the pools. 

[0038] Most preferred is a method according to the 
present invention, wherein contacting the library from a) 
above with the library in b) above is performed essen- 
tially under physiological conditions. This approach re- 
sembles a situation that is as identical to the "real" situ- 
ation in vivo as possible, an elementary advantage of 
the method of the present invention, in contrast to other 
methods according to the state of the art that easily re- 
sult in false positive results. For this contacting the li- 
brary from a) above with the library in b) above is pref- 
erably performed using a suitable buffer and, optionally, 
a cofactor, such as calcium, magnesium, potassium, 
ATP, ADP, CAMP, and the like. Cofactors can significant- 
ly improve the formation of both complexes and pro- 
teomes and at the same time the interaction of the pro- 
teome and/or the complexes with the potentially inter- 
acting compound. As used herein the term "physiologi- 
cal conditions" refers to temperature, pH, ionic strength, 



viscosity, and like biochemical parameters which are 
compatible with a viable organism, and/or which typical- 
ly exist intracellular^ in a viable cultured yeast cell or 
mammalian cell. For example, the intracellular condi- 

5 tions in a yeast cell grown under typical laboratory cul- 
ture conditions are physiological conditions. Suitable in 
vitro reaction conditions for in vitro transcription cock- 
tails are generally physiological conditions. In general, 
in vitro physiological conditions comprise 50-200 mM 

10 NaCI or KCI, pH 6.5-8.5, 20-45.degree. C. and 0.001 -1 0 
mM divalent cation (e.g., Mg ++ , Ca ++ ); preferably about 
150 mM NaCI or KCI, pH 7.2-7.6, 5 mM divalent cation, 
and often include 0.01-1.0 percent nonspecific protein 
(e.g., BSA). A non-ionic detergent (Tween, NP-40, Tri- 

15 ton X-1 00) can often be present, usually at about 0.001 
to 2%, typically 0.05-0.2% (v/v). Particular aqueous con- 
ditions may be selected by the practitioner according to 
conventional methods. For general guidance, the fol- 
lowing buffered aqueous conditions may be applicable: 

20 10-250 mM NaCI, 5-50 mM Tris HCI, pH 5-8, with op- 
tional addition of divalent cation(s) and/or metal chela- 
tors and/or non-ionic detergents and/or membrane frac- 
tions and/or anti-foam agents and/or scintillants. 
[0039] In a most preferred embodiment of the method 

25 according to the present invention, determining an in- 
teraction between said candidate compounds and said 
at least one labelled polypeptide comprises isolation of 
the labelled polypeptide and/or the complexes. Such 
isolation can be performed using common separation 

30 and/or purification techniques, such as chromatogra- 
phy, gel filtration, precipitation, immune absorption, gel 
electrophoresis, centrifugation, and the like. Further- 
more preferred is method according to the present in- 
vention, wherein said determining an interaction be- 

35 tween said candidate compounds and said at least one 
labelled polypeptide comprises a detection of the bound 
labelled polypeptide using antibodies, radioactivity de- 
tection methods, dye detection methods, enzymatic de- 
tection methods and mass spectroscopy. Such detec- 

40 tion depends on the type of label that is used. Since the 
proteome-complex interacting compound interacts with 
a complex as a subunit of a proteome, it is possible, not 
only to analyse the direct interaction of the actually la- 
belled polypeptide and the proteome-complex interact- 

45 ing compound, but also to obtain information regarding 
the context of the binding by analysis of the other part- 
ners in the proteome-complex, whereby a first informa- 
tion of the "mode of action" of a compound can be ob- 
tained. Furthermore, if the compound is a known phar- 

50 maceutical, the indirectly interacting protein-partners of 
said compound can be identified via the binding/inter- 
action with a specific proteome-complex. Finally, differ- 
ent compound and proteome-complex interactions in 
different proteomes can identify different modes of ac- 

55 tions between different organisms, cellular states and 
different diseases, both of known and/or unknown com- 
pounds. Consequently, the polypeptide-interacting 
compound as a proteome-complex interacting com- 



11 



EP 1 464 960 A1 



12 



pound also reveals information about the proteome(s). 
This can not be achieved using other methods as 
present in the art. 

[0040] As mentioned above, in a preferred method ac- 
cording to the present invention screening is performed 
in vivo or in vitro. Preferred are uses in vitro, as de- 
scribed above. 

[0041] In yet another embodiment of the method ac- 
cording to the present invention, the steps a) above to 
d) above are repeated, wherein the interacting com- 
pounds identified in d) above are used to provide an im- 
proved candidate library for step a) above. These 
rounds can be regarded as "pre-screening" rounds and/ 
or be performed as control screens. In addition, the com- 
position of the proteome library and or compound library 
can be modified between the screens. Furthermore, 
pools can be generated, as indicated above. Finally, an 
additional round of screening can be performed as end- 
screening, in order to improve the reliability of the 
present method. 

[0042] In another preferred embodiment according to 
the method according to the present invention, the 
screening is performed at least in part in a high through- 
put manner. High throughput techniques are usually em- 
ployed in strategies that involve large libraries of com- 
pounds, i.e. potentially proteome-interacting com- 
pounds and/or proteome-libraries. In general, in the 
course of the method according to the present invention , 
one single potentially proteome-interacting compound 
is screened (brought in contact) with a multitude of dif- 
ferently tagged proteomes, coming from one or several 
organisms to be screened. ) 
[0043] Examples of protein related high-throughput 
technologies are described in, for example, Sreekumar 
A and Chinnaiyan AM ("Protein microarrays: a powerful 
tool to study cancer". Curr Opin Mol Ther 2002 Dec;4 
(6):587-93), in which protein microarrays for examining 
the cellular proteome are described. Further described 
is the use of antigen and antibody arrays. Templin MF, 
et al. ("Protein microarray technology" Drug Discov To- 
day 2002 Aug 1 ;7(15):815-22) describe the use of 
microarray technology in the form of microspots of cap- 
ture molecules that are immobilised in rows and col- 
umns onto a solid support and exposed to samples con- 
taining the corresponding binding molecules. Readout 
systems based on fluorescence, chemiluminescence, 
mass spectrometry, radioactivity or electrochemistry 
can be used to detect complex formation within each 
microspot. Furthermore, arrays containing immobilised 
DNA probes that are exposed to complementary targets 
and their degree of hybridisation are described. Finally, 
US 6,197,599 relates to a device that comprises a solid 
support and multiple immobilised agents for protein de- 
tection. The immobilised agents are mainly proteins, 
such as antibodies and recombinant proteins. The im- 
mobilised agents can be synthesised peptides or other 
small chemicals. Agents are individually deposited in a 
predetermined order, so that each of the agents can be 



identified by the specific position it occupies on the sup- 
port. The immobilised agents on the solid support retain 
their protein binding capability and specificity. Methods 
employing the device are described as being extremely 

5 powerful in screening protein expression patterns, pro- 
tein post-translational modifications and protein-protein 
interactions. All these methods and devices can be eas- 
ily adapted for the use in a method according to the 
present invention. 

10 [0044] According to yet another embodiment of the 
method according to the present invention, the identify- 
ing of the proteome-complex interacting compound and 
said further identifying said proteome-interacting com- 
pound is performed, at least in part, by a computer sys- 

15 tern, 

[0045] The use of these systems is preferred, due to 
the enormous amount of data that is generated and/or 
has to be handled in the high-throughput environment. 
Such handling is described, for example, in Jansen R et 

20 al. ("Relating whole-genome expression data with pro- 
tein-protein interactions". Genome Res. 2002 Jan;12 
(1):37-46.),andStummG,et al . ("Deductive genomics: 
afunctional approach to identify innovative drug targets 
in the post-genome era." Am J Pharmacogenomics 

25 2002;2(4):263-71). Similarly, US 6,064,754 (hereby in- 
corporated by reference in its entirety) relates to com- 
puter-assisted methods and apparatus for identifying, 
selecting and characterising biomolecules in a biologi- 
cal sample. A two-dimensional array is generated by 

30 separating biomolecules present in a complex mixture 
and a computer-readable profile is constructed repre- 
senting the identity and relative abundance of a plurality 
of biomolecules detected by imaging the two dimension- 
al array. Computer-mediated comparison of profiles 

35 from multiple samples permits automated identification 
of subsets of biomolecules that satisfy pre-ordained cri- 
teria. Identified biomolecules can be automatically iso- 
lated from the two dimensional array by a robotic device 
in accordance with computer-generated instructions. A 

40 supported gel suitable for electrophoresis is provided 
that is bonded to a solid support such that the gel has 
two-dimensional spatial stability and the solid support is 
substantially non-interfering with respect to detection of 
a label, such as a fluorescent label, associated with one 

45 or more biomolecules in the gel. Finally, US 6,146,830 
(hereby incorporated by reference in its entirety) relates 
to methods and systems for characterising the actions 
of drugs in cells. In particular, described are methods for 
determining the presence of a number of primary targets 

50 through which a drug, drug candidate, or other com- 
pound of interest acts on a cell. Furthermore, also meth- 
ods for drug development based on the disclosed meth- 
ods for determining the presence of a number of primary 
targets of a drug are disclosed which involve: (i) meas- 

55 uring responses of cellular constituents to graded expo- 
sures of the cell to a drug of interest; (ii) identifying an 
"inflection concentration" of the drug for each cellular 
constituent measured; and (iii) identifying "expression 



13 



EP 1 464 960 A1 



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sets" of cellular constituents from the distribution of the 
inflection drug concentrations. Each expression set cor- 
responds to a particular primary target of the drug. Fi- 
nally computer systems are described which determine 
the presence of a number of targets of a drug by exe- 
cuting the disclosed methods. All these methods can be 
easily adapted for the use in a method according to the 
present invention. 

[0046] According to yet another aspect of the present 
invention, a proteome-interacting compound or its phar- 
maceutical^ acceptable salts is provided that has been 
identified based on the method according to the present 
invention as described above. These compounds can 
be used in order to provide new pharmaceutical compo- 
sitions that include a proteome-interacting compound 
according to the present invention, together with a suit- 
able carrier and/or diluent. The compounds that are 
found to be interactive with a labelled polypeptide that 
is part of a complex that, in turn, belongs to a specific 
proteome can be of varying nature. Depending on the 
library for screening, these compounds are selected 
from enzymes, polypeptides, peptides, antibodies and 
fragments thereof, nucleic acids or derivatives thereof, 
and chemical entities having a molecular mass of less 
than 1000 kDa ("small molecules"). Thus, in one library 
of the present invention potentially proteome-interacting 
compounds are provided that potentially interact with 
the proteome to be analysed (screened). Examples of 
such compounds are synthetic and/or naturally occur- 
ring chemical compounds, peptides, proteins, antibod- 
ies, and the like. Since the library is related to "small" 
compounds as indicated above, i.e. other that complete 
enzymes, antibodies or other proteins, the molecular 
weight of these compounds is preferably below 1000 
kDa, more preferably below 500 kDa. Such compounds 
can be suitable as "leads" for further optimisation. Fur- 
thermore, the proteome-interacting compound can be a 
nucleic acid, such as a DNA, RNA and/or PNA. Such 
nucleic acids can be present in the form of oligonucle- 
otides or polynucletides, covering binding-specific nu- 
cleic acid sequences and/or motifs, and can be suitable 
for, e.g., gene therapy or antisense-technology. 
[0047] The method according to the present invention 
can be employed in a variety of medical and pharma- 
ceutical uses, such as for further lead optimisation (e.g. 
in the cases of pre-screened compounds and/or already 
used pharmaceutical compounds), elucidating the 
mode of action of a compound (e.g. for scientific pur- 
poses and the finding of additional druggable targets), 
finding of further medical uses, the identification of po- 
tential side effects of the compound of interest in cases 
where the identified proteome-interacting compound is 
known to elicit side effects, and for the identification of 
diagnostic agents for a specific disease or condition. In 
yet another aspect, the method according to the present 
invention can be used for the identification of new lead 
compounds for established protein target classes, new 
protein target classes for known lead compounds and/ 



or new lead compounds for new protein target classes. 
Finally, according to yet another aspect of the present 
invention, the inventive method can be used for the de- 
velopment of new tools for the functional assessment of 
5 new targets (e.g. chemical knock-outs) or the develop- 
ment of prediction/modellisation-tools for the binding of 
compounds to targets, drug transporters, and drug mod- 
ifying enzymes (for example, by using computer-mod- 
elling techniques, see above), and as an improved data 
source for bioinformatic purposes. 
[0048] Many of the above uses can be accomplished 
by comparison with known protein-complex data with 
the newly identified labelled polypeptide-compound in- 
teraction. Since the proteome-complex interacting com- 
pound interacts with a complex as a subunit of a pro- 
teome, it is possible based on the data as present, not 
only to analyse the direct interaction of the actually la- 
belled polypeptide and the proteome-complex interact- 
ing compound, but also to obtain information regarding 
the context of the binding by analysis of the other part- 
ners in the proteome-complex, whereby a first informa- 
tion of the "mode of action" of a compound can be ob- 
tained. Furthermore, if the compound is a known phar- 
maceutical, the indirectly interacting protein-partners of 
said compound can be identified via the binding/inter- 
action with a specific proteome-complex. Finally, differ- 
ent compound and proteome-complex interactions in 
different proteomes can identify different modes of ac- 
tions between different organisms, cellular states and 
different diseases, both of known and/or unknown com- 
pounds. Consequently, the polypeptide-interacting 
compound as a proteome-complex interacting com- 
pound also reveals information about the proteome(s). 
This can not be achieved using other methods as 
present in the art and allows for the generation of impor- 
tant information with respect to the mode of action, fur- 
ther medical uses and/or potential side effects. One ex- 
ample for an analysis of complex protein-protein inter- 
actions can be found in Drewes G and Bouwmeester T 
("Global approaches to protein-protein interactions." 
Curr Opin Cell Biol 2003 Apr;1 5(2): 199-205) which de- 
scribe more global, systematic strategies that analyse 
genes or proteins on a genomeand proteome-wide 
scale and several large-scale proteomics technologies 
that have been developed to generate comprehensive, 
cellular protein-protein interaction maps. This analyses 
can also be used in the analyses of protein-complex and 
proteome interactions. 

[0049] All publications as cited herein are incorporat- 
ed herein by reference in their entirety. The present in- 
vention shall now be further described based on the fol- 
lowing examples, without being limited thereto. 

Example 1 : Production of a library of tagged 
(labelled) collection of proteomes 

[0050] The production of a library of a library of cells, 
in each of which a different gene is tagged (labelled), 



15 



20 



25 



30 



35 



40 



45 



50 



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EP 1 464 960 A1 



16 



respectively, has been described earlier, for example, in 
Gavin etal. Nature 41 5, 141 -7 (2002), and Rigaut et al. 
Nat. Biotechnol. 1 7,1 030-3 (1 999); and Puig et al, Meth- 
ods 24, 218-229 (2001); EP 1 105 508 B1 for the TAG 
approach. 5 

Example 2 Yeast drug pull-down protocol 

[0051 ] This example describes the use of a drug cou- 
pled to a sepharose matrix through an amine reaction w 
in order to pull down yeast TAP-tagged proteins that 
specifically interact with the drug. 

Materials: 

15 

Buffers: 

Yeast Lysis Buffer: 

[0052] 20 

50 mM Tris-HCI pH 7.5 
100mM NaCI 
0.15% Igepal 

1.5mMMgCI2 25 
0.5 mM DTT 

self-prepared protease inhibitors (1000x) + 1 mM 
PMSF (1 x stock of lysis buffer in cold room, DTT 
and protease inhibitors have to be added when 
making up 1x) so 

- TBST; TBS 1 x with 0,5% Tween 
lodoacetamide 200mg/ml 

Reagents: 35 

[0053] 

NHS-activated Sepharose 4 Fast Flow provided in 
isopropanol, Amersham Biosciences, 17-0906-01 40 
anhydrous Dimethylsulfoxide, Fluka, 41648 
Dimethylsulfoxide for washing, e.g. FLUKA 34869 
Ethanol (Merck, 1 .00983.1000, pro analysis) 
■ 2-Aminoethanol (Aldrich, 11 .016-7) 

Methanol, GR for analysis, Merck, 1 .060009 45 

Equipment: 

[0054] 

50 

End-over-end shaker (Roto Shake Genie, Scientific 
Industries Inc.) 

Materials: 

55 

[0055] 

50 mL Falcon tube 



15 mL Falcon tubes 
1 ,5 mL eppendorf tubes, siliconised 
NHS activated sepharose 4 fast flow, Amersham Bi- 
osciences, 17-0906-01 
UZ-polycarbonate tube, Beckmann, 355654 
Molbiol columns + filter 90 urn, MoBiTech, Angebot 
10055 

Glass beads (0.5 mm diameter) 

Poly-Prep Chromatography column, BIO-RAD, 

731-1550. 

Method: 

1) Coupling of the compounds to resins via primary 
amines (NHS-beads drug coupling protocol) 

[0056] Washing of beads: Use 1 ml (settled volume) 
activated NHS-beads for standard coupling reaction 
(NHS-activated Sepharose 4 Fast Row provided in iso- 
propanol, Amersham Biosciences, 17-0906-01); Insert 
10 ml chromatography column into 50 ml Falcon tube 
(Poly-Prep Chromatography column, BIO-RAD, 
731-1550); Pipet 2 ml of the re-suspended 50% slurry 
of NHS-beads into the column; Wash the beads with 1 0 
ml anhydrous DMSO (Di-methylsulfoxid, Fluka, 41648, 
H 2 0 <= 0.005%) by adding the solvent directly into the 
chromatography column; allow flow through by gravity; 
discard flow-through into non-halogenous waste 

2) Coupling reaction 

[0057] Dissolve the compound of interest in anhy- 
drous DMSO (final concentration = 100 nmol/mi); Add 
20 \i\ of the 100 iimol/mL compound solution onto the 
washed beads in a 2.0 mL Eppendorf tube; Add 14 jxL 
7.2 M Triethylamine (TEA) (final cone. = 100 jimol/mL) 
(50 x molar excess over compound) (SIGMA, T-0886, 
99% pure); Incubate at RT on an end-over-end shaker 
(Roto Shake Genie, Scientific Industries inc.) for 16 h. 

3) Blocking reaction: 

[0058] Add 50 ^L 1 6.56 M aminoethanol (2-Aminoeth- 
anol, Aldrich, 11.016-7) (final cone. = 830 ^mol/mL) (> 
40 fold excess over bead capacity) for blocking of non- 
reacted NHS-groups; Incubate at RT on the end-over- 
end shaker over night 

4) Washing 

[0059] Pipet this suspension back into a 15 ml Falcon 
tube (use cut off blue tip); Wash the beads first with 2 x 
10 ml DMSO (e.g. FLUKA, 34869 or equivalent, needs 
not to be anhydrous anymore), second with 2x1 0 ml eth- 
anol (Merck, 1.00983.1000, pro analysis); Resupend 
the beads with 1 mL ethanol to make a 50% slurry for 
storage at 4 °C (explosion proof refrigerator) 



25 



30 



17 



EP1 464 960 A1 



18 



5) Blocked beads (control) 

[0060] Washing of beads: Use 1 ml (settled volume) 
activated NHS-beads for standard coupling reaction 
(NHS-activated Sepharose 4 Fast Flow provided in iso- 5 
propanol, Amersham Biosciences, 17-0906-01); Insert 
10 ml chromatography column into 50 ml Falcon tube 
(Poly-Prep Chromatography column, BIO-RAD, 
731 -1 550); Pipet 2 ml of the resuspended 50% slurry of 
NHS-beads into the column; Wash the beads with 1 0 ml 
anhydrous DMSO (Pimethylsulfoxide, Fluka, 41648) by 
adding the solvent directly into the chromatography col- 
umn; allow flow through by gravity; discard flow-through 
into non-halogenous waste. 

6) Coupling reaction: 

[0061] 1 ml DMSO with 500 mM TEA (no drug) over 
night at room temperature 

7) Blocking reaction 

[0062] Pipet the suspension into a 2 ml Eppendorf 
tube (use cut off blue tip); Add 50 jil aminoethanol 
(2-aminoethanol, Aldrich, 11.016-7) for blocking of non- 
reacted NHS-groups; Incubate at RT on the end-over- 
end shaker over night washing; Pipet this suspension 
back into a 10 ml chromatography column (use cut off 
blue tip); Wash the beads first with 20 ml DMSO, second 
with 20 ml ethanol (Merck, 1 .00983.1 000, pro analysis); 
Store the coupled beads (50% slurry in ethanol) in col- 
umn at 4°C (explosion- proof refrigerator); Wash beads 
with 20 ml of the appropriate binding buffer before use. 

Example 3: Affinity capture of TAP-tagged proteins 
via immobiiised drugs 

* Method 

[0063] Grow a fresh TAP-expressing yeast cell line in 
YPD until OD 260 reaches 3,5; Wash cells in PBS and 
lyse in 1 ,5 volume of Yeast Lysis Buffer; (Vol lysis = 1 ,5 
x Vol pellet) using a planet mill beaker containing 25 mL 
of glass beads; Run 4times5min, 350 rpm; transfer su- 
pernatant (without glass beads) to a 50 ml_ Falcon tube; 
Wash glass beads with 1 0mL of Yeast Lysis Buffer; com- 
bine with the lysate; Centrifuge 1 0 min, 20.000 g; Trans- 
fer supernatant to a UZ-polycarbonate tube and centri- 
fuge 1 h 1 0 min, 1 0O.OOOg; Remove the lipid layer using 
a water pump, recover the supernatant and measure the 
protein concentration; Aliquot lysate by 100mg and 
freeze in liquid nitrogen; Store in a -80°C; Use 100 uL 
NHS-beads with coupled drug (2 p.mol/mL) or blocked 
NHS-beads as control; Wash the beads 3 times with 5 
mL of Yeast Lysis Buffer; invert tubes 3-5 times; centri- 
fuge 1 min, 400g, 4°C; Aspirate supernatant and dis- 
card; Thaw lysate quickly in 37°C water bath, then keep 
on ice; Dilute lysate with Yeast Lysis Buffer to a final con- 



centration of 10 mg/mL; Transfer supernatant to a UZ- 
polycarbonate tube (Beckmann, 355654); Spin lysate 
for 20 min. at 100.000 g at 4°C (33.500 rpm in T150.2, 
pre-cooled); Combine 1 00 mg of lysate with beads into 
a 1 5 mL Falcon tube; I ncubate for 2h at 4°C on a rotating 
wheel; Recover beads by centrifugation (1 min, 1000 
rpm , 4°C); take 40 \± as NBF; remove supernatant (> 
1 ml liquid should be left in the tube), Transfer beads to 
Mobi columns; Wash with 20 ml TBST; Centrifuge col- 
umn for 1 min, 400g in a table top centrifuge to remove 
excess buffer; Add 60 jiL of 2xSB, put column in a 1 .5 
mL siliconised 1 ,5 mL Eppendorf tube, heat for 5 min at 
95°C; Open column (first top, then bottom), put it back 
into the Eppendorf tube, centrifuge 1 min at 400g to re- 
cover the eluate; Load the eluate on a dot blot appara- 
tus; Detect the presence of TAP proteins using a perox- 
idase anti-peroxidase antibody. Develop with chemilu- 
minescence kit. 

[0064] Using the above strategy, as an example, the 
proteins Faa4, a long-chain fatty acid CoA ligase of the 
yeast Saccharomyces cerevisiae could be shown as in- 
teracting with the antifungal compound Nystatin. The in- 
teraction was specific, since Faa4 did not interact with 
an unrelated drug (Bisindolylmaleimide III) and Faa1 , a 
protein closely related to Faa4, did not interact with Nys- 
tatin. 

[0065] Using the above strategy, as a further exam- 
ple, the kinase PKC 1 of the yeast Saccharomyces cer- 
evisiae could be shown as interacting with the Bisin- 
dolylmaleimide HI kinase inactivator. The interaction 
was specific, since PKC1 did not bind to an unrelated 
drug Nystatin and the related kinase TPK1 did not inter- 
act with Bisindolylmaleimide III. 



Claims 

1 . A method for screening a library of potentially pro- 
teome-interacting candidate compounds, compris- 
ing: 

a) providing a library comprising, preferably 
non-labelled potentially proteome-interacting 
candidate compounds, 

b) providing a second library comprising a va- 
riety of proteomes, wherein each proteome 
comprises one proteome-complex comprising 
at least one labelled polypeptide, 

c) contacting the library from a) with the library 
in b) in a manner as to allow for an interaction 
of the candidate compounds with the at least 
one labelled polypeptide, 

d) determining an interaction between said can- 
didate compounds and said at least one la- 
belled polypeptide, and 

thereby identifying a proteome-complex inter- 
acting compound and thereby further identifying a 



15 



20 



25 



30 



35 



40 



45 



50 



19 



EP 1 464 960 A1 



20 



proteome-interacting compound. 

2. The method according to claim 1 , wherein said po- 
tentially proteome-interacting candidate com- 
pounds are selected from enzymes, polypeptides, 
peptides, antibodies and fragments thereof, nucleic 
acids or derivatives thereof, and chemical entities 
having a molecular mass of less than 1000 kDa 
("small molecules"). 

3. The method according to claim 2, wherein said po- 
tentially proteome-interacting candidate com- 
pounds is a nucleic acid, such as a DNA, RNA and/ 
or PNA or a small molecule, such as a drug, metab- 
olite, prodrug, and the like. 

4. The method according to any of claims 1 to 3, 
wherein said potentially proteome-interacting can- 
didate compounds are present in liquid solution, 
such as in tubes, microtiter-plates, or on a solid sup- 
port, such as on filters, glass slides, silicon surfac- 
es, beads or a customised chemical microarray. 

5. The method according to claim 4, wherein said po- 
tentially proteome-interacting candidate com- 
pounds are bound to beads, such as sepharose (e. 
g. NHS-activated sepharose) or agarose beads. 

6. The method according to claim 5, wherein said po- 
tentially proteome-interacting candidate com- 
pounds are bound to said beads via an amino-group 
orcarboxy-group. 

7. The method according to any of claims 1 to 6, 
wherein said variety of proteomes is present in or 
derived from one single cell or cell culture or from 
a mixture of cells, such as a tissue, organ or organ- 
ism. 

8. The method according to any of claims 1 to 7, 
wherein said variety of proteomes is present in or 
derived from one single cell or cell culture or from 
a mixture of cells, such as a tissue, organ or organ- 
ism, wherein said single cell or cell culture or mix- 
ture of cells, such as a tissue, organ or organism 
was exposed to certain conditions, such as heat, 
stress, starvation, drugs, radioactivity, chemical 
agents, toxins, viral infection, antibiotics, and age- 
ing. 

The method according to any of claims 1 to 7, 
wherein said variety of proteomes is present in or 
derived from a cell selected from prokaryotic or eu- 
karyotic cells, such as a bacterial cell, a pathogenic 
micro-organism, a fungal cell, a yeast cell, a plant 
cell, a mammalian cell, a fish cell, a nematode cell, 
an insect cell, and, in particular, a non-human stem- 
cell. 



10. The method according to any of claims 1 to 7, 
wherein said variety of proteomes is present in or 
derived from a tissue or organ, such as connective 
tissue, endothelial tissue, brain, bone, liver, heart, 

5 skeletal muscles, prostate, colon, kidney, glands, 
lymph nodes, pancreas, roots, leaves, and flowers. 

11. The method according to any of claims 1 to 7, 
wherein said variety of proteomes is present in or 

io derived from an organism, such as £ coli, Dro- 
sophila melanogaster, Caenorhabditis elegans, ze- 
brafish, rat, hamster, mouse, goat, sheep, jellyfish, 
rice, potato, Arabidopsis, wheat, oat, and tobacco. 

15 12. The method according to any of claims 1 to 11, 
wherein said variety of proteomes is present in or 
derived from a lysate of one single cell or cell culture 
or from a lysate of a mixture of cells, such as a tis- 
sue, organ or organism. 

20 

13. The method according to claim to any of claims 1 
to 1 2, wherein each proteome contains only one la- 
belled polypeptide. 

25 14. The method according to any of claims 1 to 13, 
wherein each member of the library in b) contains 
only one labelled polypeptide that is different from 
the other members of said library. 

30 15. The method according to any of claims 1 to 14, 
wherein said label of said labelled polypeptide is se- 
lected from the group of radiolabels, dye labels, la- 
bels that can be detected with antibodies, enzyme 
labels, and labels having a detectable mass. 

35 

16. The method according to claim 15, wherein said la- 
bel of said labelled polypeptide is selected from 
phosphorescent markers, fluorescent markers, 
chemiluninescent markers, phosphatases, strepta- 

40 vidin, biotin, TAP, and peroxidases. 

17. The method according to any of claims 1 to 16, 
wherein each member of the library in b) comprises 
a mixture or collection (pool) of different proteomes. 

45 

18. The method according to any of claims 1 to 17, 
wherein said contacting the library from a) with the 
library in b) is performed essentially under physio- 
logical conditions. 

50 

19. The method according to any of claims 1 to 18, 
wherein said contacting the library from a) with the 
library in b) is performed using a suitable buffer and, 
optionally, acofactor, such as calcium, magnesium, 

55 potassium, ATP, ADP, cAMP, and the like. 

20. The method according to any of claims 1 to 19, 
wherein said determining an interaction between 



21 



EP 1 464 960 A1 



22 



said candidate compounds and said at least one la- 
belled polypeptide comprises isolation of the la- 
belled polypeptide and/or the complexes. 

21. The method according to any of claims 1 to 20, 
wherein said determining an interaction between 
said candidate compounds and said at least one la- 
belled polypeptide comprises a detection of the 
bound labelled polypeptide using antibodies, radio- 
activity detection methods, dye detection methods, 
enzymatic detection methods and mass spectros- 
copy. 

22. The method according to any of claims 1 to 21, 
wherein said screening is performed in vivo or in 
vitro. 

23. The method according to any of claims 1 to 22, com- 
prising repeating steps a) to d), wherein the inter- 
acting compounds identified in d) are used to pro- 
vide an improved candidate library for step a). 

24. The method according to any of claims 1 to 23, 
wherein said screening is performed at least in part 
in a high throughput manner. 

25. The method according to any of claims 1 to 24, 
wherein said identifying of said proteome-complex 
interacting compound and said further identifying 
said proteome-interacting compound is performed, 
at least in part, by a computer system. 

26. A proteome-interacting compound, identified ac- 
cording to a method according to any of claims 1 to 
25, or its pharmaceutical^ acceptable salts. 

27. A pharmaceutical composition, comprising a pro- 
teome-interacting compound according to claim 26, 
together with a suitable carrier and/or diluent. 

28. Use of a method according to any of claims 1 to 27 
for further lead optimisation, elucidating the mode 
of action of a compound, finding of further medical 
uses, for the identification of potential side effects 
of the compound of interest when the identified pro- 
teome-interacting compound is known to elicit side 
effects, and for the identification of diagnostic 
agents for specific disease or condition. 

29. Use of a method according to any of claims 1 to 27 
for the identification of new lead compounds for es- 
tablished protein target classes, new protein target 
classes for known lead compounds or new lead 
compounds for new protein target classes. 

30. Use of a method according to any of claims 1 to 27 
for the development of new tools for the functional 
assessment of new targets (chemical knock-out) or 



the development of prediction/modellisation tools 
for the binding of compounds to targets, drug trans- 
porters, drug modifying enzymes, and as a data 
source for bioinformatic purposes. 

5 



25 



30 



35 



40 



45 



50 



EP 1 464 960 A1 



European Patent 
Office 



PARTIAL EUROPEAN SEARCH REPORT 



wtiich under Rule 45 of the European Patent Convention £p 03 00 
shall be considered, for the purposes of subsequent 
proceedings, as the European search report 



Application Number 

7690 



DOCUMENTS CONSIDERED TO BE RELEVANT 



Category 



Citation of document with indication, where appropriate, 
of relevant passages 



Relevant 
to claim 



CLASSIFICATION OF THE 
APPLICATION (fnt.CI.7) 



GOURI B S ET AL: "Interaction of SH3 
domain of Hck tyrosine kinase with 
cellular proteins containing proline-rich 
regions: evidence for modulation by unique 
domain." 

INDIAN JOURNAL OF BIOCHEMISTRY & 

BIOPHYSICS. INDIA 1997 FEB-APR, 

vol. 34, no. 1-2, February 1997 (1997-02), 

pages 29-39, XP009017954 

ISSN: 0301-1208 

* abstract * 

ZHU H ET AL: "Global analysis of protein 
activities using proteome chips" 
SCIENCE, AMERICAN ASSOCIATION FOR THE 
ADVANCEMENT OF SCIENCE,, US, 
vol. 293, no. 5537, 
14 September 2001 (2001-09-14), pages 
2101-2105, XP002201508 
ISSN: 0036-8075 

* abstract * 

* page 210, right-hand column, paragraph 2 
- page 211, middle column, paragraph 2; 
figures 1,2 * 

-/- 



1-25, 
28-30 



G01N33/68 



1-25, 
28-30 



TECHNICAL FIELDS 
SEARCHED (mtCL7) 



G01N 



INCOMPLETE SEARCH 



The Search Division considers that the present application, or one or more of Be claims, does/do 
not comply with the EPC to such an extent that a meaningful search into the state of the art cannot 
be carried out, or can only be carried out partially, for these claims. 

Claims searched completely : 



Claims searched Incompletely : 

Claims not searched : 

Reason for the limitation of the search: 

see sheet C 



3 



Place of search 

MUNICH 



Dale of completion of the eearch 

25 September 2003 



Luis Alves, D 



CATEGORY OF CITED DOCUMENTS 

X : particu tarty relevant if taken alone 

Y : particularly relevant if combined with another 

document of the same category 
A : technological background 
O : non-written disclosure 
P : intermediate document 



T : theory or principle underlying the invention 
£ : earlier patent document, but published on, or 

after the filing date 
D : document cited in the application 
L : document cited for other reasons 



& : member of the same patent family, corresponding 
document 



EP 1 464 960 A1 



J 



European Patent 
Office 



INCOMPLETE SEARCH 
SHEET C 



EP 03 00 7690 



Application Number 



Claim(s) searched completely: 
1-25, 28-30 



Claim(s) not searched: 
26, 27 



Reason for the limitation of the search: 

Independent claim 26 concerns compounds but does not characterise in any 
way said compounds. Said claim attempts to define the compounds merely by 
reference to a screening method. Thus, said claim lacks clarity (Article 
84 EPC) to such an extent that a meaningful search is not possible. 

Independent claim 27 concerns a composition comprising the compounds 
according to claim 26 and therefore lacks clarity (Article 84 EPC) for 
the same reasons as above. Consequently, a meaningful search is not 
possible for the subject-matter of claim 27. 



EP 1 464 960 A1 




Patent PARTIAL EUROPEAN SEARCH REPORT Application Number 

EP 03 00 7690 



DOCUMENTS CONSIDERED TO BE RELEVANT 


CLASSIFICATION OF THE 
APPLICATION (lnt.Ct7) 


Category 


Citation of document with indication, where appropriate, 
of relevant passages 


Relevant 
to claim 




X 

D,A 
A 


SHEVCHENKO ANNA ET AL: "Deciphering 
protein complexes and protein interaction 
networks by tandem affinity purification 
and mass spectrometry: Analytical 
perspective." 

MOLECULAR & CELLULAR PR0TEOMICS, 

vol, 1, no. 3 f March 20G2 (2002-03), pages 

204-212, XP002255660 

March, 2002 

ISSN: 1535-9476 

* the whole document * 

GAVIN A-C ET AL: "Functional organization 

of the yeast proteome by systematic 

analysis of protein complexes 0 

NATURE, MACMILLAN JOURNALS LTD. LONDON, 

GB, 

vol. 415, January 2002 (2002-01), pages 

141-14/, ArUUc^DOOOJ. 

ISSN: 0028-0836 

* abstract * 

KUKAR THOMAS ET AL: "Protein microarrays 
to detect protein-protein interactions 
using red and green fluorescent proteins." 
ANALYTICAL BIOCHEMISTRY, 
vol. 306, no. 1, 1 July 2002 (2002-07-01), 
pages 50-54, XP002255661 
July 1, 2002 
ISSN: 0003-2697 

* abstract * 


1-25, 
28-30 

1-25, 
28-30 

1-25, 
28-30 


TECHNICAL FIELDS 
SEARCHED (| nt .CI.7)