U.S. patent application number 11/569597 was filed with the patent office on 2009-09-24 for identification of compounds modifying a cellular response.
This patent application is currently assigned to 2cureX. Invention is credited to Jens Chr. Norrild, Grith Hagel, Morten Hentzer, Morten Meldal, Ole Thastrup.
Application Number | 20090239755 11/569597 |
Document ID | / |
Family ID | 34968405 |
Filed Date | 2009-09-24 |
United States Patent
Application |
20090239755 |
Kind Code |
A1 |
Thastrup; Ole ; et
al. |
September 24, 2009 |
Identification of Compounds Modifying A Cellular Response
Abstract
The present invention relates to methods for identifying
compounds capable of modulating a cellular response. The methods
involve attaching living cells to solid supports comprising a
library of test compounds. The test compounds are linked to the
solid support via cleavable linkers and may thus be released from
the solid supports. Solid supports comprising cells, wherein the
cellular response of interest has been modulated are selected and
the test compound of the solid support can then be identified. The
cellular response may for example be changes in complex formation
between proteins.
Inventors: |
Thastrup; Ole; (Birkerod,
DK) ; Meldal; Morten; (Kobenhavn, DK) ; Hagel;
Grith; (Dragor, DK) ; Chr. Norrild; Jens;
(Birkerod, DK) ; Hentzer; Morten; (Holbaek,
DK) |
Correspondence
Address: |
GIFFORD, KRASS, SPRINKLE,ANDERSON & CITKOWSKI, P.C
PO BOX 7021
TROY
MI
48007-7021
US
|
Assignee: |
2cureX
Birkerod
DK
|
Family ID: |
34968405 |
Appl. No.: |
11/569597 |
Filed: |
May 25, 2005 |
PCT Filed: |
May 25, 2005 |
PCT NO: |
PCT/DK05/00347 |
371 Date: |
November 27, 2006 |
Current U.S.
Class: |
506/7 ; 506/13;
546/121 |
Current CPC
Class: |
C12Q 1/025 20130101;
C07K 5/1008 20130101; G01N 33/5023 20130101; C07K 7/08 20130101;
G01N 33/6845 20130101; C07K 1/047 20130101; G01N 33/54313 20130101;
C07K 7/06 20130101; G01N 2035/00158 20130101; G01N 2500/10
20130101; C40B 30/06 20130101 |
Class at
Publication: |
506/7 ; 506/13;
546/121 |
International
Class: |
C40B 30/00 20060101
C40B030/00; C40B 40/00 20060101 C40B040/00; C07D 471/04 20060101
C07D471/04 |
Foreign Application Data
Date |
Code |
Application Number |
May 25, 2004 |
DK |
PA 2004 00821 |
May 25, 2004 |
DK |
PA 2004 00822 |
Claims
1. A method of identifying a compound modifying at least one
cellular response, wherein each cellular response is linked to
different reporter systems generating detectable outputs, said
method comprising the steps of: (a) Providing multiple solid
supports capable of supporting adherence and growth of cells,
wherein each solid support is linked to multiple copies of a member
of a library of test compounds via cleavable linkers and wherein at
least two solid supports comprise different library members; and
(b) Attaching cells comprising said reporter system(s) onto said
solid support; and (c) Releasing a proportion of said library
member from the solid support; and (d) Screening said solid
supports for solid supports comprising cells meeting at least one
predetermined selection criterion, wherein said selection criterion
is linked directly or indirectly to said detectable output; and (e)
Selecting solid supports comprising cells meeting said at least one
selection criterion; and (f) Identifying said library member,
thereby identifying a compound modifying said at least one cellular
response.
2. The method according to claim 1, wherein said adherence is
mediated through a cell adhesion compound coupled to said solid
supports, wherein said cell adhesion compound enables said solid
support to support growth of cells.
3. The method according to claim 1, wherein the solid supports are
resin beads.
4. (canceled)
5. The method according to claim 3, wherein the resin bead
comprises or consists of polyethylene glycol
6. (canceled)
7. The method according to claim 2, wherein said cell adhesion
compound is a peptide with an overall positive netcharge.
8. (canceled)
9. The method according to claim 2, wherein said cell adhesion
compound is selected from the group consisting of SEQ ID 1, SEQ ID
2, SEQ ID 3, SEQ ID 4, SEQ ID 5, SEQ ID 6, SEQ ID 7, SEQ ID 8, SEQ
ID 9, SEQ ID 10, SEQ ID 11, SEQ ID 12, SEQ ID 13, SEQ ID 14, SEQ ID
15, SEQ ID 16, SEQ ID 17, SEQ ID 18, SEQ ID 19, SEQ ID 20, SEQ ID
21, SEQ ID 22, SEQ ID 23, SEQ ID 24, SEQ ID 25, SEQ ID 26, SEQ ID
27, SEQ ID 28, SEQ ID 29, SEQ ID 30, SEQ ID 31, SEQ ID 32, SEQ ID
33, SEQ ID 34, SEQ ID 35, SEQ ID 46, SEQ ID 47, SEQ ID 48, SEQ ID
49, SEQ ID 50, SEQ ID 51, SEQ ID 52, SEQ ID 53, SEQ ID 54, SEQ ID
55, SEQ ID 56, SEQ ID 57, SEQ ED 58, SEQ ID 59, SEQ ID 60, SEQ ID
61, SEQ ID 62, SEQ ID 63, SEQ ED 64, SEQ ID 65, SEQ ID 66, SEQ ID
67, SEQ ID 68, SEQ ID 69 and SEQ ED 70.
10. The method according to claim 2, wherein the adhesion compound
is linked to the resin bead via a cleavable linker.
11. The method according to claim 1, wherein the cellular response
is a change in interaction between two or more cellular
molecules.
12. (canceled)
13. (canceled)
14. The method according to claim 11, wherein the cellular response
is a change in interaction between proteins involved in regulation
of apoptosis.
15. The method according to claim 1, wherein said cellular response
is a modulation of a signal transduction pathway.
16. The method according to claim 1, wherein said cellular response
is a modulation of signal transduction pathway, wherein said
modulation is selected from the group consisting of Upregulation or
downregulation of the level of a member of the pathway;
Relocalisation of a member of the pathway; Complex formation
between members of the pathway or between members of the pathway
with other cellular compounds; Enhanced or reduced transcription
from genes regulated by the pathway; Modification by
phosphorylation of a member of the pathway; Activation or
inhibition of an enzyme of the pathway; Degradation of a cellular
compounds due to upregulation or downregulation of the pathway;
Altered secretion of a compound; Change in ion-flux; Morphological
changes; and Change in viability
17. (canceled)
18. (canceled)
19. (canceled)
20. (canceled)
21. (canceled)
22. (canceled)
23. (canceled)
24. (canceled)
25. (canceled)
26. (canceled)
27. The method according to claim 1, wherein the cellular response
is apoptosis.
28. The method according to claim 1, wherein the reporter system is
a system endogenous to said cells.
29. (canceled)
30. (canceled)
31. (canceled)
32. (canceled)
33. (canceled)
34. The method according to claim 1, wherein the reporter system
comprises a bioluminescent moiety.
35. (canceled)
36. (canceled)
37. (canceled)
38. The method according to claim 1, wherein the reporter system is
a proximity ligation reporter system.
39. (canceled)
40. The method according to claim 1, wherein one predetermined
selection criterium is a quantitative level of said bioluminescence
above or below a specific threshold.
41. (canceled)
42. (canceled)
43. (canceled)
44. The method according to claim 1, wherein said cells are
selected from the group consisting of mammalian cells.
45. (canceled)
46. (canceled)
47. The method according to claim 3, wherein at least 100 resin
beads comprising different library members are provided.
48. (canceled)
49. The method according to claim 1, wherein the library is
selected from the group consisting of peptides, glycopeptides,
lipopeptides, nucleic acids (DNA or RNA), oligosaccharides;
chemically modified peptides, oligomers of amino acids,
glycopeptides, and small organic molecules.
50. (canceled)
51. (canceled)
52. (canceled)
53. The method according to claim 1, wherein the library comprises
polymers of in the range of 3 to 6 amino acids selected from the
group of amino acids mentioned in table 3, table 4, table 5 and
table 6.
54. The method according to claim 1, wherein compounds modifying at
least two cellular responses are identified, wherein step c)
involves screening said resin beads for beads comprising cells
meeting at least two predetermined selection criterion, wherein
each selection criterion is related to a different detectable
output.
55. The method according to claim 1, wherein at least one cleavable
linker is selected from the group consisting of acid-labile,
base-labile, fluoride-labile and photo-labile linkers.
56. (canceled)
57. (canceled)
58. The method according to claim 1, wherein step c) comprises
releasing in the range of 5 to 95% of the copies of the library
member.
59. A library useful in the method according to claim 1, comprising
multiple solid supports linked to multiple copies of a member of a
library of test compounds via cleavable linkers and wherein at
least two solid supports comprise different library members; and
furthermore wherein the solid supports are coupled to a cell
adhesion compound, wherein said cell adhesion compound is selected
from either: i) the group consisting of peptides of: SEQ ID 1, SEQ
ID 2, SEQ ID 3, SEQ ID 4, SEQ ID 5, SEQ ID 6, SEQ ID 7, SEQ ID 8,
SEQ ID 9, SEQ ID 10, SEQ ID 11, SEQ ID 12, SEQ ID 13, SEQ ID 14,
SEQ ID 15, SEQ ID 16, SEQ ID 17, SEQ ID 18, SEQ ID 19, SEQ ID 20,
SEQ ID 21, SEQ ID 22, SEQ ID 23, SEQ ID 26, SEQ ID 27, SEQ ID 28,
SEQ ID 29, SEQ ID 30, SEQ ID 31, SEQ ID 32, SEQ ID 33, SEQ ID 34,
SEQ ID 35, SEQ ID 46, SEQ ID 47, SEQ ID 48, SEQ ID 49, SEQ ID 50,
SEQ ID 51, SEQ ID 52, SEQ ID 53, SEQ ID 54, SEQ ID 55, SEQ ID 56,
SEQ ID 57, SEQ ID 58, SEQ ID 59, SEQ ID 60, SEQ ID 61, SEQ ID 62,
SEQ ID 63, SEQ ID 64, SEQ ID 65, SEQ ID 66, SEQ ID 67, SEQ ID 68,
SEQ ID 69 and SEQ ID 70 or: ii) a peptide comprising at least one
D-form amino acid, said peptide being selected from the group
consisting of: SEQ ID 1, SEQ ID 2, SEQ ID 3, SEQ ID 4, SEQ ID 5,
SEQ ID 6, SEQ ID 7, SEQ ID 8, SEQ ID 9, SEQ ID 10, SEQ ID 11, SEQ
ID 12, SEQ ID 13, SEQ ID 14, SEQ ID 15, SEQ ID 16, SEQ ID 17, SEQ
ID 18, SEQ ID 19, SEQ ID 20, SEQ ID 21, SEQ ID 22, SEQ ID 23, SEQ
ID 24, SEQ ID 25, SEQ ID 26, SEQ ID 27, SEQ ID 28, SEQ ID 29, SEQ
ID 30, SEQ ID 31, SEQ ID 32, SEQ ID 33, SEQ ID 34, SEQ ID 35, SEQ
ID 46, SEQ ID 47, SEQ ID 48, SEQ ID 49, SEQ ID 50, SEQ ID 51, SEQ
ID 52, SEQ ID 53, SEQ ID 54, SEQ ID 55, SEQ ID 56, SEQ ID 57, SEQ
ID 58, SEQ ID 59, SEQ ID 60, SEQ ID 61, SEQ ID 62, SEQ ID 63, SEQ
ID 64, SEQ ID 65, SEQ ID 66, SEQ ID 67, SEQ ID 68, SEQ ID 69 and
SEQ ID 70.
60. A method of manufacturing a compound modifying at least one
cellular response, wherein said method comprises the steps of: a)
Identifying said compound by the method according to claim 1 b)
Preparing said compound by chemical synthesis c) Thereby
manufacturing said compound
61. A method of modulating the interaction between two or more
cellular molecules comprising the steps of a) Identifying a
compound by the method according to claim 11 b) Incubating said
compound together with cells expressing said two cellular molecules
c) Thereby modulating the interaction between the two cellular
molecules
62. Compound identified by the method according to claim 1.
63. The compound according to claim 62, wherein the compound is an
oligomer of in the range of 3 to 6 amino acids selected from the
group of amino acids mentioned in table 3, table 4, table 5 and
table 6.
64. A library comprising at least two oligomers of in the range of
3 to 6 amino acids selected from the group of amino acids mentioned
in table 3, table 4, table 5 and table 6.
Description
[0001] All patent and non-patent references cited in the
application are hereby incorporated by reference in their
entirety.
FIELD OF INVENTION
[0002] The present invention relates to a method and tools for
extracting information relating to an influence, for example on
intracellular molecule(s), in particular an influence caused by
contacting a cellular molecule with a substance, which has been
released from a solid support to which a cell expressing the
cellular component is attached. In particular the method related to
a solid support that allow chemical synthesis of individual
substances on beads of the solid support
[0003] The method of the invention may be used as a very efficient
procedure for testing or discovering the influence of a library of
substances on a physiological process, for example in connection
with screening for new drugs, testing of substances for toxicity,
identifying drug targets for known or novel drugs. Other valuable
uses of the method and technology of the invention will be apparent
to the skilled person on the basis of the following disclosure
BACKGROUND OF INVENTION
[0004] Combinatorial synthesis of peptide as well as small-molecule
libraries has proven very useful as a method for generating vast
numbers of highly diverse compounds (see for example Comprehensive
Survey of Combinatorial Library Synthesis: 2002 Roland E. Dolle J.
Comb. Chem., 2003, pp 693-753). To fully exploit this high capacity
of combinatorial chemistry to produce huge numbers of compounds
several technologies have been developed that allow screening
directly on the solid support (M. Meldal, 1994, METHODS: A
companion to methods of enzymology 6:417-424). In the field of drug
discovery such methods have successfully been applied for example
for the identification of enzyme modulators. The library can be
synthesized on resin beads that each carry one specific compound,
and these "one-bead-one compound" libraries are then screened
against the purified biological component of interest (e.g.
cellular proteins or peptides),
[0005] Before progressing active compounds, identified though such
procedure, further in the drug discovery process, the compound will
have to be re-synthesized and tested for efficacy in a cell-based
or in-vivo test system.
[0006] Novel ways to screen combinatorial libraries in a
physiological more correct way are assumed to greatly accelerate
the drug discovery process, and show importance in areas like
chemo-genomics and chemo-proteomics.
[0007] Screening of combinatorial libraries in intact cells have
been done by capturing mammalian or yeast cells together with a
limited number of resin-beads in a "nanodroplet" (Borchart et al.
Chem Biol 1997 4:961). Compounds immobilized on the resin are
released through disruption of a photo-cleavable linker and the
compound-associated effects on the intact cells are monitored.
[0008] In an alternative method the compounds are released through
acidolysis resin-beads carrying the library members area are spread
out on a lawn of mammalian cells, and the spatial localization of a
cellular response is monitored and beads in that region is
isolated, and the remaining compound is structure elucidated
Jayawickreme et al, 1998, Combinatorial peptide Library Protocols,
Ed. Shmuel Cabilly, Humana Press, p. 107-128).
[0009] WO03/038431 describes methods for screening combinatorial
bead libraries by capturing cells from body fluids. Beads
comprising a compound enabling cells to adhere to said bead may be
selected.
[0010] US2003/0059764 describes multiplexed cell analysis systems
using non-positional or positional arrays of coded carriers.
SUMMARY OF INVENTION
[0011] It is of great importance to provide new and efficient
methods for identification of compounds influencing specific
cellular processes. In particular, such methods wherein a very
large quantity of candidate compounds may be tested for a specific
effect on a cell within a relatively short period of time.
[0012] It is therefore an object of the present invention to
provide very efficient procedures for testing or discovering the
influence of compounds of a library on a physiological process in a
cell. In particular, the methods provides means for testing very
large numbers of different compounds for one or more physiological
effects within a rather short time period. This may be obtained by
attaching living cells to resin beads coupled to a test compound.
The test compounds may be released from the resin beads and thus
influence physiological processes in said cells. Said influence(s)
may be detected and beads containing cells displaying the desired
influence(s) may be selected. Once selected the compounds coupled
to the selected beads may be identified. These methods may for
example be very useful in connection with screening for new drugs,
testing of substances for toxicity, identifying drug targets for
known or novel drugs.
[0013] Accordingly, it is a first objective of the invention to
provide methods of identifying a compound modifying at least one
cellular response, wherein each cellular response is linked to
different reporter systems generating detectable outputs, said
method comprising the steps of: [0014] (a) Providing multiple resin
beads capable of supporting growth of cells, wherein each resin
bead is linked to multiple copies of a member of a library of test
compounds via a cleavable linker and wherein at least two beads
comprise different library members; and [0015] (b) Attaching cells
comprising said reporter system(s) onto said resin beads; and
[0016] (c) Releasing a proportion of said library member from the
resin bead; and [0017] (d) Screening said resin beads for beads
comprising cells meeting at least one predetermined selection
criterion, wherein said selection criterion is linked directly or
indirectly to said detectable output; and [0018] (e) Selecting
beads comprising cells meeting said at least one selection
criterion; and [0019] (f) Identifying the library member remaining
linked to the selected resin bead, thereby identifying a compound
modifying said at least one cellular response.
[0020] The method involves release of a proportion of library
member. The released library member may enter into cells in the
immediate surroundings and thus influence cellular responses within
said cells. In practical terms, the cells present in the immediate
surroundings, will be the cells attached to the resin bead, from
which the library member is released. Thus resin beads comprising
cells, wherein the particular cellular response has been modified
may be selected and the library member remaining bound to said
resin beads can be identified.
[0021] The invention furthermore relates to methods of
manufacturing a compound modifying at least one cellular response,
wherein said method comprises the steps of: [0022] a) Identifying
said a compound modifying a cellular response according to the
methods described herein [0023] b) Preparing said compound by
chemical synthesis [0024] c) Thereby manufacturing said
compound
[0025] The invention also relates to methods of modulating the
interaction between two cellular molecules comprising the steps of
[0026] a) Identifying a modulating interaction between two cellular
molecules according to the methods described herein [0027] b)
Incubating said compound together with cells expressing said two
cellular molecules [0028] c) Thereby modulating the interaction
between the two cellular molecules
[0029] Depending on the nature of the two cellular molecules,
specific cellular responses may be inhibited/activated. This may in
particular be interesting for use of the compounds in therapy.
[0030] The invention furthermore relates to compounds identified by
the methods disclosed herein.
DESCRIPTION OF DRAWINGS
[0031] FIG. 1A illustrates a method of identifying a resin bead
comprising a compound influencing a cellular response linked to a
reporter system generating a fluorescent output. The method
involves cultivating cells on resin beads, fixing cells, FABS
calibration using a positive and a negative control, identification
and isolation of positive hits.
[0032] FIG. 1B illustrates a method of identifying a resin bead
comprising a compound influencing a cellular response linked to a
reporter system generating a fluorescent output detectably using a
plate reader or image acquisition analysis. The method involves 1)
Grow cells on beads for 24 hrs and Fix cells in EtOH, 2) Add app.
20 beads to each well and Identify hit wells using plate reader or
image acquisition/analysis and 3) Transfer beads from hit wells to
a new 384 well plate--one bead/well and identify hit wells using
plate reader or image acquisition. If for example 500.000 beads are
screened with 20 beads/well, approx. 25.000 wells, i.e. approx. 68
plates must be screened. With a 0.1% hit rate, there will be
approx. 500 hit wells comprising approx. 10.000 beads, which
amounts to analysis of approx. 27 plates in the second round.
Alternatively, positive beads may be picked directly (preferably
without fixation) after the first identification using image
acquisition analysis. The method may for example be used for
analysis of expression of a Cre-YFP construct.
[0033] FIG. 2A illustrates a multiplexed screen involving FABS and
microscopy. The screen involves I) identification of positive hits
by FABS as displayed in FIG. 1, followed by II) a step of
microscopy identifying resin beads comprising cells with an
internal fluorescent signal.
[0034] FIG. 2B illustrates a multiplexed screen involving two FABS
analysis. The screen involves I) identification of positive hits by
FABS as displayed in FIG. 1, followed by II) a second FABS
analysis.
[0035] FIG. 3 illustrates a plasmid map of pCRE-d2EGFP
[0036] FIG. 4 illustrates examples of cleavable linkers useful with
the present invention.
[0037] FIG. 5 illustrates spectra and structure determination by
accurate mass differences from single beads
[0038] FIG. 6 illustrates structure determination by accurate mass
differences from single beads
[0039] FIG. 7 illustrates a fragmentation pathway
[0040] FIG. 8 illustrates examples of an adhesion peptide
displaying bead covered with cells (U2OS).
DEFINITIONS
[0041] Naturally occurring amino acids are named herein using
either their 1-letter or 3-letter code. If nothing else is
specified amino acids may be of D or L-form. In the description
(but not in the sequence listing) 3-letter codes starting with a
capital letter indicate amino acids of L-form, whereas 3-letter
codes in small letters indicate amino acids of D-form. Three- and
one-letter abbreviations for amino acids are used according to the
recommendations from IUPAC, see for example
http://www.chem.qmw.ac.uk/iupac.
[0042] The term "a" as used herein, can mean one or more, depending
on the context in which it is used.
[0043] In the present context, the term "green fluorescent protein"
or (GFP) is intended to indicate a protein which, when expressed by
a cell, emits fluorescence upon exposure to light of the correct
excitation wavelength (cf. [(Chalfie et al. 1994)]). "GFP" as used
herein means any protein or fragment thereof capable of fluorescing
when excited with appropriate radiation. This includes fluorescent
proteins that are either naturally occurring or engineered and
proteins that have been modified to be fluorescent. Naturally
occurring fluorescent proteins have been isolated from the
jellyfish, Aequorea vistoria, the sea pansy, Renilla reniformis,
Phialidium gregarium and Discosoma coral (W. W. Ward et al. (1982)
Photochem. Photobiol, 35:803-808; Levine et al. (1982) Biochem.
Physiol., 72B:77-85; Fradkov et al. (2000), FEBS Lett.
479:127-130). GFPs have also been engineered to emit different
colors and to fluoresce more intensely in mammalian organisms (U.S.
Pat. No. 5,625,048; WO 97/28261; WO 96/23810; EP0851874; U.S. Pat.
No. 6,172,188; WO01/98338).
[0044] A variety of Aequorea-related fluorescent proteins have been
engineered to have different excitation and emission spectra by
modifying the naturallt occurring amino acid sequence (D. C.
Prasher et al. (1992) Gene 111:229-233; Heim et al. (1994) Proc.
Natl. Acad. Sci. USA 91: 12501-12504; U.S. Pat. No. 5,625,048; WO
96/23810 and PCT/US97/14593).
[0045] The term "living cell" is used to indicate a cell which is
considered living according to standard criteria for that
particular type of cell such as maintenance of normal membrane
potential, cell membrane integrity and energy metabolism
[0046] The terms "image processing" and "image analysis" are used
to describe a large family of digital data analysis techniques or
combination of such techniques which reduce ordered arrays of
numbers (images) to quantitative information describing those
ordered arrays of numbers. When said ordered arrays of numbers
represent measured values from a physical process, the quantitative
information derived is therefore a measure of the physical
process.
[0047] The term "fluorescent probe" is used to indicate a
fluorescent fusion polypeptide comprising a GFP or any functional
part thereof which is N- or C-terminally fused to a biologically
active polypeptide as defined herein, optionally via a peptide
linker consisting of one or more amino acid residues, where the
size of the linker peptide in itself is not critical as long as the
desired functionality of the fluorescent probe is maintained. A
fluorescent probe according to the invention is expressed in a cell
and basically mimics the physiological behaviour of the
biologically active polypeptide moiety of the fusion
polypeptide.
[0048] The term "determining the fluorescence" is used to describe
the process used to monitor a change in fluorescence
properties.
[0049] The term "bioluminescence" is used to describe a process
where light is produced through a chemical reaction that natively
is occurring in a biological system. For the reaction to occur at
least two chemicals are required: the one that produces the light
(called "luciferin") and the other (called "luciferase") that
catalyzes the reaction. Sometimes the luciferin and luciferase are
brought together in one single unit (called "photoprotein" an
example of the last group is aequorin.
[0050] The term "FRET" is used to describe the occurrence of
Fluorescence resonance energy transfer between a fluorophore donor
and an acceptor fluorophore. It is a distance-dependent interaction
between the electronic excited states of two fluorophores in which
excitation is transferred from a donor fluorophore to an acceptor
fluorophore without emission of a photon. The efficiency of FRET is
dependent on the inverse sixth power of the intermolecular
separation, making it useful over distances comparable with the
dimensions of biological macromolecules. Thus, FRET is an important
technique for investigating interactions between cellular molecules
for example complex formation.
[0051] The term "BRET" is used to describe a process that is
related to FRET, but differs from FRET in that donor is a
bioluminescent protein like luciferase that generates its own
luminescence emission in the presence of a substrate, and that can
pass the energy to an acceptor fluorophore. For either BRET or FRET
to work, the donor's emission spectrum must overlap the acceptor's
absorption spectrum, their transition dipoles must be in an
appropriate orientation, and the donor and acceptor must be in
close proximity (usually within 30-80 .ANG. of each other,
depending on the degree of spectral overlap).
[0052] The term "Scintillation Proximity Assay" is used to describe
an assay determining the distance between two compounds, wherein
one compound (bound to a bead) will emit light when radiation from
an isotope occurs in close proximity and the other compound is
containing a radioactive isotope.
[0053] The term "Proximity ligation" is used to describe an assay
determining the presence of a target molecule through the
convergence of two different protein-binding reagents that
specifically recognise said target molecule. Attached to each
protein-binding reagent are nucleic acid sequences that when broad
into close proximity will create a DNA reporter sequence through a
ligation reaction (see Gullberg et al. Curr Opinion Biotechnology
2003, 14:82)
[0054] The term "mammalian cell" is intended to indicate any cell
of mammalian origin. The cell may be an established cell line, many
of which are available from The American Type Culture Collection
(ATCC, Virginia, USA) or a primary cell with a limited life span
derived from a mammalian tissue, including tissues derived from a
transgenic animal, or a newly established immortal cell line
derived from a mammalian tissue including transgenic tissues, or a
hybrid cell or cell line derived by fusing different celltypes of
mammalian origin e.g. hybridoma cell lines. The cells may
optionally express one or more non-native gene products, e.g.
receptors.
[0055] The phrase "fluorescence properties" means absorption
properties, such as wavelength and extension, and spectral
properties of the emitted light, such as wavelength, fluorescence
lifetime, intensity or polarisation, or the intracellular
localisation of the fluorophore. It may thus be localised to a
specific cellular component (e.g. organelle, membrane,
cytoskeleton, molecular structure) or it may be evenly distributed
throughout the cell or parts of the cell.
[0056] The term "fixed cells" is meant to cover cells treated with
a cytological fixative such as glutaraldehyde, methanol, acetone or
formaldehyde, treatments which serve to chemically cross-link
and/or stabilize soluble and insoluble proteins within the
structure of the cell or to dehydrate cells. Once in this state,
such proteins cannot be lost from the structure of the now-dead
cell.
[0057] The term "cell line" is meant to cover a group of cells,
wherein the cells of that group are essentially genetically
indistinguishable from each other. The cells of a cell line are
thus all progeny of the same cell.
[0058] The term "comprising" should be understood in an inclusive
manner. Hence, by way of example, a composition comprising compound
X, may comprise compound X and optionally additional compounds.
[0059] The term "multiple" should be understood as "at least
two".
[0060] The term "library of test compounds" should be understood as
a collection of test compounds comprising at least 2 different test
compounds.
[0061] The term "small organic molecules or compounds" refers
herein to non-oligomeric, carbon containing compounds producible by
chemical synthesis and generally having a size of less than 600
mass units.
[0062] The term split/mix refer herein to the process of i)
dividing a bead assembly into m portions and reacting each portions
with a different building block followed by mixing the resin into
one portion providing an even distribution throughout the assembly
of beads containing said building blocks, ii) preparing
(activating) the resin for attachment of the next building block
and repeating the process n times of dividing, reacting, mixing and
activating, thus providing an exponential growth of the number
(m.sup.n) of distinct molecular entities of complexity n each
attached to separate beads.
[0063] The term "one bead-one compound library" refers to libraries
immobilised on resin beads, wherein each individual resin bead does
not comprise more than one library member in one or multiple
copies. In a particular form of such libraries each member is
represented by multiple fragments of said member obtained by ladder
synthesis encoding.
[0064] The term "one bead-two compound library" refers to libraries
immobilised on resin beads, wherein each individual resin bead does
not comprise more than one library member in one or multiple copies
and wherein each individual resin bead in addition to said library
member also comprises an adhesion compound. All beads may comprise
identical adhesion compounds.
[0065] The term "cleavable linker" is used to describe any chemical
moiety which may be used to attach any molecule to a solid support
either covalently or via complex formation and thereafter release
said molecule by the action of either acid, base, electrophiles,
nucleophiles, oxidative agents, reductive agents, metals, heat or
light.
DETAILED DESCRIPTION OF THE INVENTION
Library of Test Compounds
[0066] In the present invention, libraries of compounds are used to
screen for compounds having a desired physiological influence on a
living cell. As used herein, the term "library" means a collection
of molecular entities or test compounds, herein also designated
"library members" obtained after a series of chemical
transformation.
[0067] In preferred embodiments of the present invention the
library is a combinatorial library. Non-limiting examples of
combinatorial libraries that may be used with the present invention
and methods of producing such libraries are given in: Comprehensive
Survey of Combinatorial Library Synthesis: 1998 Roland E. Dolle and
Kingsley H. Nelson, Jr. J. Comb. Chem., 1999, pp 235-282;
Comprehensive Survey of Combinatorial Library Synthesis: 1999
Roland E. Dolle J. Comb. Chem., 2000, pp 383-433; Comprehensive
Survey of Combinatorial Library Synthesis: 2000 Roland E. Dolle J.
Comb. Chem., 2001, pp 477-517; Comprehensive Survey of
Combinatorial Library Synthesis: 2001 Roland E. Dolle J. Comb.
Chem., 2002, pp 369-418 and Comprehensive Survey of Combinatorial
Library Synthesis: 2002 Roland E. Dolle J. Comb. Chem., 2003, pp
693-753. The skilled person will appreciate that these protocols
may be easily be adapted to specific need of a particular
embodiment of the present invention.
[0068] In one embodiment, these molecular entities can be natural
oligomers (oligomers of building blocks occurring in Nature) such
as peptides, glycopeptides, lipopeptides, nucleic acids (DNA or
RNA), or oligosaccharides. By way of example, a natural oligomer
may be any peptide consisting of naturally occurring amino acid,
even if said peptide comprises a sequence not present in nature.
The libraries may comprise different natural oligomers or the
libraries may comprise only one kind of natural oligomer, for
example the library may be a peptide library. In another
embodiment, they can be unnatural oligomers (oligomers comprising
one or more building blocks not occurring in Nature) such as
chemically modified peptides, glycopeptides, nucleic acids (DNA or
RNA), or, oligosaccharides, and the like. Said chemical
modification may for example be the use of unnatural building
blocks connected by the natural bond linking the units (for
example, a peptide amide linkage), the use of natural building
blocks with modified linking units (for example, oligoureas as
discussed in Boeijen et al, 2001, J. Org. Chem., 66: 8454-8462;
oligosulfonamides as discussed in Monnee et al, 2000, Tetrahedron
Lett., 41: 7991-95), or combinations of these (for example, statine
amides as discussed in Dolle et al, 2000, J. Comb. Chem., 2:
716-31.). Preferred unnatural oligomers include oligomers
comprising unnatural building blocks connected to each other by a
naturally occurring bond linking. Said oligomers may thus comprise
a mixture of naturally occurring and unnatural building blocks
linked to each other by naturally occurring bonds. By way of
example, the oligomer may comprise naturally occurring amino acids
and unnatural building blocks linked by peptide bonds f.x. PNA or
LNA. Thus, in one embodiment of the invention preferred oligomers
comprise modified amino acids or amino acid (mimics). Other
preferred unnatural oligomers include, for example oligoureas, poly
azatides, aromatic C--C linked oligomers and aromatic C--N linked
oligomers. Still other preferred oligomers comprise a mixture of
natural and unnatural building blocks and natural and unnatural
linking bonds. For example, the unnatural oligomer may be any of
the oligomers mentioned in recent reviews see: Graven et al., 2001,
J. Comb. Chem., 3: 441-52; St. Hilaire et al., 2000, Angew. Chem.
Int. Ed. Engl., 39: 1162-79; James, 2001, Curr. Opin. Pharmacol.,
1: 540-6; Marcaurelle et al., 2002, Curr. Opin. Chem. Biol., 6:
289-96; Breinbauer et al., 2002, Angew. Chem. Int. Ed. Engl., 41:
2879-90. The libraries of the invention may also comprise cyclic
oligomers, for example cyclic natural oligomers, such as cyclic
peptides or cyclic unnatural oligomers. In certain embodiments of
the invention, libraries of cyclic oligomers may be advantageous to
use due to the rigid structure. This may result in higher
selectively and affinity.
[0069] In yet another embodiment, the molecular entities may
comprise non-oligomeric molecules such as peptidomimetics or other
small organic molecules. Peptidomimetics are compounds that mimic
the action of a peptidic messenger, such as bicyclic thiazolidine
lactam peptidomimetics of L-proplyl-L-leucyl-glycinamide (Khalil et
al, 1999, J. Med. Chem., 42: 2977-87). In a preferred embodiment of
the invention, the library comprises or even more preferably
consists of small organic molecules. Small organic molecules are
non-oligomeric compounds of less than about 600 mass units
containing any of a variety of possible functional groups and are
the product of chemical synthesis, or isolated from nature, or
isolated from nature and then chemically modified, and include, for
example, Bayer's urea-based kinase inhibitors (Smith et al., 2001,
Bioorg. Med. Chem. Lett., 11: 2775-78). Small organic compounds may
for example be selected from the group consisting of alcohols,
ethers, carboxylic acids, aryloxy, acyloxy, thiol, alkylthio,
arylthio, heteroarylthio, sulphonyl, sulphoxy, amino, alkylamino,
dialkylamino, acylamino, diacylamino, alkoxycarbonylamino, amides,
alkyl, branched alkyl, aryl, heteroaryl, nitro, cyano, halogeno,
silyloxy, keto, heterocycles, fused ring systems, fused
heterocycles and mixtures thereof, wherein each of the
aforementioned may be substituted independently on each position
with one or more groups selected from the group consisting of --H,
--OH, --SH, halogen, carboxyl, carbonyl, alkoxy, aryloxy, acyloxy,
alkylthio, arylthio, heteroarylthio, sulphonyl, sulphoxy, amino,
alkylamino, dialkylamino, acylamino, diacylamino,
alkoxycarbonylamino, amides, alkyl, aryl, heteroaryl, nitro, cyano,
halogeno, silyloxy, keto, heterocycles, fused ring systems, and
fused heterocycles.
[0070] Non-limiting examples of small organic molecule libraries
that may be used with the present invention and methods of
producing them may for example be found in the reviews Thompson et
al., 1996, Chem. Rev., 96: 555-600; Al-Obeidi et al., 1998, Mol.
Biotechnol., 9: 205-23; Nefzi et al., 2001, Biopolymers, 60: 212-9;
Dolle, 2002, J. Comb. Chem., 4: 369-418.
[0071] The libraries according to the invention may comprise at
least 20, such as at least 100, for example at least 1000, such as
at least 10,000, for example at least 100,000, such as at least
1,000,000 different test compounds. Preferably, the libraries
comprises in the range of 20 to 107, more preferably 50 to
7,000,000, even more preferably 100 to 5,000,000, yet more
preferably 250 to 2,000,000 different compounds. In a very
preferred embodiment of the present invention the libraries
comprises in the range of 1000 to 20,000 or for example in the
range of 20,000 to 200,000 different test compounds.
[0072] In preferred embodiments of the invention the library
comprises in the range of 10,000 to 1,000,000 different test
compounds.
[0073] Preferably, the libraries to be used with the present
invention are immobilised on resin beads. Said resin beads may be
any of the beads described herein below. At least 2, preferably at
least 20, more preferably at least 100, even more preferably at
least 1000, yet more preferably at least 10,000, for example at
least 100,000, such as at least 1,000,000 resin beads comprising
different library members, i.e. different test compounds may be
used with the methods according to the invention. Preferably, the
in the range of 20 to 107, more preferably 100 to 7,000,000, even
more preferably 1000 to 5,000,000, yet more preferably 5000 to
2,000,000, even more preferably 10,000 to 1,000,000 resin beads
comprising different library members, are used with the methods
according to the invention.
[0074] In one very preferred embodiment of the invention, each
resin bead does not comprise more than one library member in one or
more copies, i.e. each resin bead only comprises one kind of test
compound, however said test compound may be present on the resin
bead in multiple copies. Such libraries may also be designated
one-bead-one-compound libraries. Preferably, each resin beads
comprises sufficient copies of said library member in order to
exert the desired influence of cells attached to said resin bead
and in order to analyse the chemical structure of the compound.
Such libraries may be prepared by different methods, for example by
a split/mix method or by coupling individually a specific compound
to a bead. One-bead-one compound libraries offer the advantage that
once a resin bead has been selected according to the methods
described herein, the desired compound may easily be identified
(see useful methods herein below).
[0075] The libraries may in one preferred embodiment be synthesized
directly on resin beads using a split/mix method (vide infra) which
gives rise to one-bead-one-compound libraries. Split/mix methods in
general comprise the steps of: [0076] 1. Providing several pools of
resin beads [0077] 2. Performing one or more different chemical
synthesis steps on each pool of resin beads [0078] 3. Mixing pools
of resin beads, thereby obtaining a mixed pool. [0079] 4. Splitting
the mixed pool of resin beads thereby obtaining new pools. [0080]
5. Optionally repeating step 1 and 4
[0081] Alternatively steps 3 and 4 may be as follows: [0082] 3.
Splitting said pools to obtain fractions [0083] 4. Mixing fractions
from different pools, thereby obtaining new pools
[0084] One-bead-one-compound libraries may for example be prepared
as described in M. Meldal, Multiple column synthesis of quenched
solid-phase bound fluorogenic substrates for characterization of
endoprotease specificity in Methods: A Companion to Methods in
Enzymology 6:417-424, 1994 or in M. Meldal, The One-bead
Two-Compound Assay for Solid Phase Screening of Combinatorial
Libraries in Biopolymers, Peptide Science 66:93-100, 2002; or in
Combinatorial peptide library protocols, Ed. by Shmuel Cabilly,
Humana Press, 1998, p. 1-24 and 51 to 82.
[0085] In one embodiment of the invention the library may be a
library of oligomers of amino acids immobilised on resin beads,
wherein said amino acids may be naturally occurring amino acids or
any other kind of amino acid or a mixture of both. In general,
oligomers of amino acids are referred to as peptides, regardless
whether they comprise naturally occurring or not naturally
occurring amino acids or a mixture, unless it is clear from the
context that the term only cover oligomers of naturally occurring
amino acids. For example, the oligomers may comprise or consist of
at least 3, such as at least 4, for example at least 5, such as at
least 6, for example at least 7, such as at least 10, for example
at least 20, such as in the range of 3 to 5, for example in the
range of 4 to 10, such as in the range of 5 to 15, for example in
the range of 10 to 20 amino acids, such as in the range of 15 to
30, for example in the range of 3 to 50, such as in the range of 3
to 25, for example in the range of 4 to 15 amino acids. Such
libraries may for example be prepared as described in example 1
herein below.
[0086] The library of oligomers of amino acids may in one
embodiment comprise an appended sequence. Thus, every library
member may share a common sequence, i.e. the appended sequence. In
addition every library member comprises an individual sequence. The
appended sequence may be a sequence of amino acids, for example in
the range of 3 to 5, for example in the range of 4 to 10, such as
in the range of 5 to 15, for example in the range of 10 to 20 amino
acids, such as in the range of 15 to 30, for example in the range
of 3 to 50, such as in the range of 3 to 25, for example in the
range of 4 to 15 amino acids.
[0087] In another embodiment of the invention the library may be a
one-bead-two-compounds library. Each individual resin bead of such
a library comprises only one library member in one or more copies.
In addition each individual resin bead comprises a second compound,
such as a cell adhesion compound. The cell adhesion compound could
for example be any of the cell adhesion compounds mentioned herein
below. It is comprised within the invention that several library
resin beads, such as all library resin beads comprises identical
adhesion compound(s) in one or more copies. One-bead-two-compound
libraries may for example be prepared by a method involving the
steps of: [0088] 1. Providing resin beads comprising a plurality of
reactive groups [0089] 2. Reacting said reactive groups with two
chemical moeities comprising different and preferably orthogonal
protective groups [0090] 3. Deprotecting a subset of the reactive
groups by removal of one kind of protective groups, preferably
selective removal of one kind of protective group, [0091] 4.
Attaching or synthesizing a split/mix library of test compounds to
the deprotected reactive group [0092] 5. Deprotecting the remaining
reactive groups by removal the other kind of protective group
[0093] 6. Attaching the second compound to the deprotected reactive
groups
[0094] The method may also be performed by first attaching the
second compound and then synthezising the library. Accordingly, the
steps of the method may be performed in the following order: 1, 2,
3, 6, 5 and 4. The library of test compounds may be first
synthesized and then attached to the resin beads or it may be
synthesized directly into the resin bead. Similarly, the second
compound may be first synthesized and then attached to the resin
beads or it may be synthesized directly into the resin bead.
[0095] Preferred resin beads are described in the section "resin
beads" herein below. The reactive group may be any suitable
reactive group, preferably however, the reactive group is either a
hydroxyl group, a thiol or a primary amino group. The reactive
group may also preferably be an azido or a secondary amino group.
The protective group may be any suitable protective group known to
the person skilled in the art, such as acid labile, alkaline
labile, fluoride labile, oxidation labile, reduction labile or
photolabile protective groups, preferably the protective group is
selected from the group consisting of Fmoc, Boc, Alloc and N.sub.3.
It is preferred that the different protective groups may be removed
by different treatment, for example that if one protective group is
acid labile, then the other is not acid labile, but instead for
example alkaline labile or photo labile. In an preferred embodiment
one protective group is Fmoc and the other protective group is
Alloc or N.sub.3. Step 3 may for example be performed by a
split/mix method as described herein above, thereby generating a
one-bead-one-compound library. The second compound is preferably a
cell adhesion compound.
[0096] In yet another embodiment of the invention the library may
be a mixed compound library, wherein each individual resin bead
comprises a plurality of library members.
[0097] Selection of an appropriate library is dependent upon the
specific embodiment of the invention. For example, a totally random
library designed to contain interesting and greatly diverse
compounds may be used with the invention. An advantage of this
approach is that the outcome of the screening is not prejudiced in
any specific manner or at least less prejudiced. Since the
invention permits screening of millions of diverse compounds, for
example, immobilized on resin beads, a large number, for example in
the range of 3 to 5 million, of random molecules can be used in the
ligand library.
[0098] Alternatively, a smaller, targeted library (hundreds to
thousands of compounds) can be used, for example, starting with a
known compound or compounds, and providing numerous variations of
these known compounds for targeted screening. For example, in
embodiments of the invention wherein compounds modulating the
activity of a specific cell surface molecule, a compound known to
modulate said specific cell surface molecule may be used as
starting compound for the preparation of a targeted library.
Alternatively, a smaller targeted library of compounds mimicking a
compound known to modulate the activity of said cell surface
molecule may be prepared, for example using computer aided
modelling followed by chemical synthesis. The smaller, targeted
library can also comprise random molecules. Examples of libraries
and methods of preparing such libraries, which may useful in
embodiments of the invention, wherein the cellular response is
mediated through an intracellular signalling pathway are known to
the skilled person. The library may contain a parallel array of
random modifications of one or more test compounds. In one
embodiment, the library may be formed as a parallel array of random
modifications to a known compound or compounds. The term "parallel
array" is meant to cover synthesis of a library by subjecting a
given compound to a known set of reactions in an isolated vessel or
well. Thus, the nature of a compound in a given container or well
is known. The array of test compounds is preferably prepared
directly on resin beads using techniques known by those skilled in
the art. Briefly, the resin may be portioned into a number of
vessels or wells, usually less than 500 and the reagents added.
There is in general no mixing step and after the appropriate
washing steps, subsequent reactions are carried out by addition of
additional reagents to the wells. There is no exponential increase
in the number of compounds generated and that is equal to the
number of vessels used. The compound can be easily identified by
keeping track of the reagent added to each well.
[0099] The library may also have been prepared by parallel
synthesis using a tag to enable identification of, what chemical
synthesis steps the individual resin bead has been submitted to.
This may for example be done by IROR1 or radiofrequency tag.
Alternatively, chemical synthesis steps may be performed in
parallel to preparing a polymeric tag. Identification of the tag
will thus provide knowledge of the compound.
[0100] Attachment of a label to a compound may alter the properties
of said compound. Hence, in one embodiment of the present
invention, the compounds of the library are not labelled, i.e. the
compounds are not connected to a detectable label, such as a
fluorescent component, a nucleic acid or a nucleic acid homologue
such as PNA, a dye, a probe comprising a reactive moiety or the
like. In particular it is preferred that all compounds are not
connected to the same detectable label.
[0101] The peptides used for preparation of any of the libraries
mentioned above may be oligomers of naturally occurring or not
naturally occurring amino acids or a mixture of both, preferably
they are oligomers of the 20 amino acids naturally present in
proteins, wherein said amino acids may be in either D- or L-form.
It is preferred that each peptide (or peptide mimetic) is
immobilised on a solid support, such as any of the solid supports
mentioned herein below. More preferably the solid support is resin
beads and it is preferred that each resin bead comprises only one
library member in one or more copies.
[0102] Preferably at least 2, such as at least 10, for example at
least 100, such as at least 1000, for example at least 10.000
different peptides and/or peptide mimetics are provided. Each
peptide may comprise in the range of 2 to 100 amino acids, such as
in the range of 2 to 50 amino acids, for example 2 to 25 amino
acids, such as in the range of 2 to 15 amino acids, for example 2
to 10 amino acids, such as in the range of 3 to 8 amino acids, for
example 4 to 6 amino acids,
[0103] The invention also relates to libraries prepared by any of
the methods described above.
[0104] Libraries of heterocyclic compounds obtained by cyclisation
of a peptide aldehyde through an intramolecular Pictet-Spengler
reaction may also be used with the present invention. Such
libraries may for example be any of the libraries described in
Danish patent application PA 2003 00967, which is hereby
incorporated by reference.
[0105] In one embodiment of the invention the library comprises
oligomers of amino acids, such as oligomers of 3 to 10 amino acids,
such as oligomers of 3 to 6 amino acids, for example oligomers of 3
to 4 amino acids, wherein the oligomer optionally may comprise
additional moieties, such as 1 to 4, for example 1 to 3, such as 1
to 2, for example 1 additional moiety, which is not an amino acid.
The additional moiety may be an acyl moiety, such as an acyl
halide. The additional moiety is preferably situated at one end of
the oligomer, preferably at the C-terminus. The amino acids may be
either naturally occurring or not naturally occurring amino acids
or a mixture of both. They are preferably linked via peptide bonds.
In one embodiment the library comprising compounds consisting of 3
amino acids linked via peptide bonds and an additional moeity which
may be an amino acid or for example an acyl halid. Such libraries
may for example be useful for identifying compounds capable of
interacting with inhibitors of apotosis, such as ML-IAP or XIAP.
Examples of such libraries and methods of preparing such libraries
are described herein below in Examples 4, 5a, 5b and 5c. In one
embodiment libraries prepared essentially as described in examples
5b or 5c are preferred.
[0106] In one embodiment the library may comprise or essentially
consist of oligomers of amino acids, such as oligomers of in the
range of 3 to 6 amino acids, for example oligomers of 4 amino
acids, wherein the amino acids are selected from the group of amino
acids mentioned in table 3, table 4, table 5 and table 6. A
preferred example of such a library is given in example 4.
Resin Beads
[0107] The library members of this invention are preferably bound
to a solid support. Preferred solid supports to be used with the
present invention are resin beads (see herein below).
[0108] The solid support may however also be a spot or region on a
surface or a plated gel or a membrane. A spot or a region is a
defined area on said surface, to which the library member is bound
via a cleavable linker. One can therefore envisage one surface
comprising a plurality of spots or regions, wherein each such spot
or region is covalently attached to only one library member in one
or more copies. Said surface could for example be a silicium wafer,
a glass surface, a plastic surface or a gel. Plastic surface may
for example be prepared from polystyrene, polycarbonate
polypropylene, ethylene and/or teflon. Gels could be prepared from
for example poly acrylamid or PEGA.
[0109] In this invention however, the compounds of the library are
preferably bound to a resin bead, conferring the advantage of
compartmentalized "mini-reaction vessels" for attachment of
cells.
[0110] In general more compounds may be screened and several of the
steps in the procedure may be performed on one bead with sufficient
material. Hence, preferably, the library is bound to resin beads.
Each member of the library is a unique compound and is physically
separated in space from the other compounds in the library,
preferably, by immobilizing the library on resin beads, wherein
each bead at the most comprises one member of the library.
Depending on the mode of library synthesis, each library member may
contain, in addition, fragments of the library member. Since ease
and speed are important features of this process invention, it is
preferred that the screening step take place on the same solid
support used for synthesis of the library, and also that
identification of the members of the binding pair can take place on
the same support, such as on a single resin bead. Thus, preferred
solid supports useful in the process invention satisfy the criteria
of not only being suitable for organic synthesis, but are also
suitable for screening procedures, such as "on-bead" screening as
well as suitable for attachment of cells. It is furthermore
preferred that the resin bead is suitable for "on-bead"
identification of library members as described herein below. The
resin bead may be prepared from any suitable material such as
polystyrene, polyethylene, polyacrylamide, controlled pore glass or
PEG. The resin bead could thus for example be selected from the
group consisting of Toyopearl, sepharose, sephadex, CPG, silica,
POPOP, PEGA, SPOCC, Expansin, Tentagel, Argogel, Polystyrene,
Jandagel, polydimethylacrylamide resin, Polyacrylamide resin,
kieselghur supported resins and polystyrene supported resins.
Hydrophilic supports are preferred. Examples of preferred
hydrophilic resin beads includes TentaGel (commercially available
from Rapp polymere, Tubingen, Germany), ArgoGel (commercially
available from Argonaut Technologies Inc., San Carlos, Calif.),
PEGA (commercially available from VersaMatrix, Copenhagen), POEPOP
(Renil et al., 1996, Tetrahedron Lett., 37: 6185-88; available from
Versamatrix, Copenhagen, Denmark) and SPOCC (Rademann et al, 1999,
J. Am. Chem. Soc., 121: 5459-66; available from Versamatrix,
Copenhagen, Denmark). Examples of on-bead screening attempts are
described in the following references: Chen et al., 1996, Methods
Enzymol., 267: 211-19; Leon et al., 1998, Bioorg. Med. Chem. Lett.,
8: 2997-3002; St. Hilaire et al., 1999, J. Comb. Chem., 1: 509-23;
Smith et al., 1999, J. Comb. Chem., 1: 326-32; Graven et al., 2001,
J. Comb. Chem. 3: 441-52; Park et al., 2002, Lett. Peptide Sci., 8:
171-78). TentaGel and ArgoGel are made up of polyethylene glycol
chains grafted on to a polystyrene core. However, use of these
supports in biological screening is limited by a size restriction,
and by denaturation of certain proteins, particularly enzymes.
[0111] Preferred resin beads according to the present invention are
resin beads, useful for on-bead library synthesis, screening and
identification of ligand/protein. Hence, preferred resins according
to the present invention are resin comprising polyethylene glycol.
More preferably, the resin is PolyEthyleneGlycol Acrylamide
copolymer (PEGA), Super Permeable Organic Combinatorial Chemistry
(SPOCC) resin or PolyOxyEthylene-PolyOxyPropylene (POEPOP) resin.
Another preferred resin comprises a crosslinked polyacrylamide
resin.
[0112] PEGA (PolyEthyleneGlycol Acrylamide copolymer; Meldal M.,
1992, Tetrahedron Lett., 33: 3077-80), POEPOP
(PolyOxyEthylene-PolyOxyPropylene resin; Renil et al., 1996,
Tetrahedron Lett., 37: 6185-88) and SPOCC (Super Permeable Organic
Combinatorial Chemistry resin; Rademann et al, 1999, J. Am. Chem.
Soc., 121: 5459-66) resins are made primarily of polyethylene
glycol and swell well in organic as well as aqueous solvents.
Because they have very reduced or no non-specific binding, PEGA and
SPOCC resins have been effectively used in the screening of myriad
proteins including enzymes of different classes. Furthermore, these
resins are available in different pore sizes and can allow large
proteins to enter while retaining activity. For example, PEGA6000
resins allow proteins up to 600 kDa to enter. In the Examples
below, PEGA4000 and PEGA1900 resin with a molecular weight cut off
of 200 and 90 kDa, respectively, were used for screening. In
principle, any hydrophilic support that is useful for
compartmentalized synthesis, retains the activity of the proteins,
and has minimal non-specific binding, may be used in this process
invention.
[0113] One aspect of the invention relates to a method comprising
the step of providing multiple resin beads capable of supporting
growth of cells. Preferably, all resin beads provided are capable
of supporting growth of cells. In one preferred embodiment all
resin beads are similar and each is capable of supporting growth of
cells, wherein the resin beads only differs by comprising different
library members. In embodiments of the invention wherein the resin
beads comprise a cell adhesion molecule, it is preferred that at
least 10%, more preferably at least 20%, even more preferably at
least 30%, yet more preferably at least 40%, even more preferably
at least 50%, yet more preferably at least 60%, %, even more
preferably at least 70%, yet more preferably at least 90%, even
more preferably essentially all, yet more preferably all resin
beads comprise the cell adhesion molecule as well as a library
member.
Release of Library Compounds or of Adhesion Compound
[0114] The library of test compounds is preferably linked to the
resin beads or solid supports by a cleavable linker. Hence in one
embodiment of the invention, a proportion of the library members
may be released from the resin beads, preferably by cleaving the
cleavable linker. The thus released library members may then
interact with cells in the immediate proximity, i.e. normally with
cells attached to the same bead, and it is even possible that the
library member may enter the cells and interact with intracellular
compounds. Later selection of a single bead allows elucidation of
the structure of the specific library member remaining attached to
said bead.
[0115] Preferably, "releasing a proportion of a library member"
means releasing one or more copies of the library member attached
to a solid support or resin bead. Preferably, said copies of the
library member are released by cleaving the cleavable linker.
Preferably, in the range of 5 to 95% of all copies of a library
member attached to a resin bead are released, more preferably in
the range of 10 to 90%, even more preferably in the range of 20 to
80%, such as in the range of 30 to 70%, for example in the range of
40 to 60%, such as at least 5%, for example at least 10%, such as
at least 20%, such as at least 30%, for example at least 40%, such
as at least 50%, for example at least 60%, such as at least 70%,
for example at least 80%, such as at least 90%, for example at
least 95%, such as at the most 5%, for example at the most 10%,
such as at the most 20%, such as at the most 30%, for example at
the most 40%, such as at the most 50%, for example at the most 60%,
such as at the most 70%, for example at the most 80%, such as at
the most 90%, for example at the most 95% of all copies of a
library member attached to a resin bead are released.
[0116] It is also comprised within the invention that the adhesion
compound may be attached to the resin bead via a cleavable linker.
Cleavage of said cleavable linker may release the adhesion compound
as well as cells attached to said adhesion compound. When the
cleavable linkers linking the library compound and the adhesion
compound, respectively, are differentially cleavable, then
selective release of either library compound or adhesion compound
may be achieved.
[0117] The cleavable linker may be any chemical moiety which may be
used to attach any molecule to a solid support either covalently or
via complex formation, and thereafter release said molecule by the
action of either acid, base, electrophiles, nucleophiles, oxidative
agents, reductive agents, metals or light. Preferably, the
cleavable linker attaches the library member to the solid support
covalently. Depending on the nature of the cleavable linker, a
person skilled in the art will be capable of controlling cleavage
of the cleavable linker, so only a proportion of the copies of a
library member are released. A comprehensive review describing
state of the art for "cleavable linkers" is "Linkers and Cleavage
Strategies in Solid-Phase Organic Synthesis and Combinatorial
Chemistry", F. Guillier, D. Orain, and M. Bradley, Chem. Rev. 2000,
100, 2091-2157. Any of the cleavable linkers described therein may
be used with the present invention.
[0118] Examples of useful acid labile linkers include the most
commonly used linkers for acidic detachment from a solid support,
the Wang and Rink linkers (FIGS. 4A and B). Detachment of peptide
esters from Wang linkers require up to 95% TFA in DCM whereas
detachment of Rink esters (FIG. 4B, X.dbd.OH) may be cleaved under
milder conditions (AcOH-DCM 1:9) which does not cleave the normal
protection groups on the peptides. The Rink amides (FIG. 4A,
X.dbd.NH.sub.2) require 95% TFA (aq). Partial detachment of the
compounds attached to the resin may also be achieved using gaseous
acids such as HCL or TFA vapour in a sealed container. The use of
gases allow rigorous control of the degree of cleavage obtained
with concentration of acid and time of exposure. The skilled person
may readily establish a suitable concentration of acid and time of
exposure to obtain a desried degree of cleavage.
[0119] Examples of useful base-labile linkers includes Wang and
HMBA linkers, which may be cleaved under alkaline conditions.
Saponification with 0.1 M NaOH may be applied but even milder
conditions such as potassium carbonate in MeOH are applicable. The
HMBA linker is stable to TFA under normal conditions.
[0120] In a preferred embodiment the cleavable linker is a light
sensitive cleavable linker which, upon the action of light with a
given wave length and intensity, may release any active compound
from the solid support.
[0121] Photo-labile linkers provide a tool for solid phase
synthesis which enables the detachment of the synthesized molecules
in the prescence of acid or base-sensitive functionalities within
the molecules. In 1973 Rich proposed the use of o-nitrobenzyl type
of linkers (nitrated analogs of the Wang linker). Irradiation with
UV-light gave detachment of the free acids or amides although only
in moderate yields. Detachment yields could be improved by applying
the NBA type linkers (see FIG. 4E). Even better result have been
obtained with the Holmes-type linkers (FIG. 4F). Detachment from
photolabile linkers is performed by illuminating the resins with
ultraviolet light, preferable at 365 nm. The wave length and
intensity of the light and the time of exposure might need
optimization for the individual case. A person skilled in the art
can readily establish conditions wherein a desired proportion of
copies of a library member are released. Detachment yields may be
over 90% under ideal conditions.
[0122] Example of a preferred photo sensitive cleavable linker:
##STR00001##
[0123] Depending on the nature of the cleavable linker, the library
member may be released using different methods. For example, if the
linker is photo labile, the library member may be released by
illumination. The release should preferably be partial, so that
only a proportion of the library member is released. The person
skilled in the art will readily be able to establish the conditions
required for partial release using a specific cleavable linker. An
example of how to achieve partial release is given in example 6
herein below.
[0124] It is also comprised within the present invention that a
library member may be linked to a resin bead via different
cleavable linker. For example some copies of a library member may
be linked to a resin bead via a first kind of cleavable linker,
whereas other copies of the same library member may be linked to
the resin bead via a second kind of cleavable linker. Preferably,
the first kind of cleavable linker is cleavable by another method
than the second kind of cleavable linker. By way of example, the
first cleavable linker could be acid or base labile, whereas the
second kind of cleavable linker could be photolabile. Thus, some
copies of the library member could be released during the screening
procedure of the invention, for example during step c) in the
method outlined in the "Summary" herein above, whereas other copies
could be retained on the resin beads and released during the
identification, for example during step f) in the method outlined
in the "Summary" herein above. Thus, releasing a proportion of a
library member could be controlled by using different cleavable
linkers.
Cells
[0125] The cells to be used with the present invention may be any
useful cells available or prepared for the purpose. Preferably, the
cells are selected from the group consisting of mammalian cells.
For example the cells may be human cells. The cells may be cells
capable of growing in suspension or they may be adherent cells.
Adherent cells may preferably be cultivated directly on the resin
beads used with the invention (see also herein below). It is
preferred that the cells are adherent cells. Cells with a better
adherence are preferred over cells with a poorer adherence. Cells
which adhere well to resin beads comprising an adhesion compound as
described herein above are very preferred.
[0126] Cells could for example be primary cells or established cell
lines. Preferred cell lines include but are not limited to those
mentioned in table 1.
TABLE-US-00001 TABLE 1 Cell line Species Tissue Morphology 3T3-L1
Mouse Embryonic fibroblast Fibroblast 3T3-Swiss albino (CCL-92)
Mouse Embryo Fibroblast A10 Rat thoracic aorta Myoblast Att 20
Mouse Pituitary Small round cells BAE Cow Aorta Endothelial Balb/c
Mouse Embryonic fibroblast Fibroblast BHK:R P.1#4aa PTP1B fl BHK-21
Hamster Kidney Fibroblast BHK467 Hamster Kidney BHK570 Hamster
Kidney Fibroblast BJ Human Foreskin Fibroblast C2C12 Mouse Muscle
Myoblast Caki-1 Human Kidney Epithelial CAL-54 Human Kidney
Epithelial CHOhIR Chinese hamster Ovary Fibroblast CHO-K1 Hamster
Ovary Epithelial COS 1 Monkey Kidney Fibroblast COS 7 Monkey Kidney
Fibroblast G-8 Mouse Muscle Myoblast GT1-7 HCT 116 Human Colorectal
Epithelial HEK293 Human Embryonic kidney Epithelial Hela Human
Cervix adenocarcinoma Epithelial HEP-G2 Human Liver Epithelial
HT-1080 Human Fibrosarcoma Epithelial HT-29 Human Colon Epithelial
HUVEC Human umbilical vein Endothelial Ins-1 Jurkat clone E6-1
Human T lymphocyte Lymphoblastoid K-562 Human Bone marrov
Lymphoblastoid L-6 Rat Muscle Myoblast MCF 7 Human Mammary Gland
Epithelial MDA-MB-231 Human Adenocarcinoma Epithelial MDA-MB-468
Human Mammary Gland Epithelial MDCK Canine Kidney Epithelial Min6
Mv 1 Lu (NBL-7) Mink Lung Epithelial NIH-3T3 Mouse Embryo
Fibroblast PAE Pig Aorta PC 12 Rat Adrenal gland PC-3 Human
Prostate Epithelial RAT2 Rat Normal Fibroblast RAW 264.7 Mouse
Monocyte RIN Rat Epithelial SK-ML-28 Human Melanoma SK-N-AS Human
Neuroblastoma Epithelial SK-N-DZ Human Neuroblastoma Epithelial
SK-N-F1 Human Brain Epithelial SK-NM-C Human Neuroepithelioma
Epithelial SK-N-SH Human Caucasian neuroblastoma Epithelial SW480
Human Colorectal Epithelial U-2 OS Human Bone, osteosarcoma
Epithelial U-87 MG Human Brain Epithelial U937 Human Lymphoma
Monocyte VERO Monkey Kidney Fibroblast-like WI-38 Human Lung
Fibroblast WM-266-4 Human Skin Epithelial WEHI Human
[0127] In one embodiment of the invention the cells have been
genetically or otherwise modified in order to enhance their
usability with the present invention. The modification may be
stable or only transient or a mixture of both. For example, the
cells may have been modified to contain one or more of the reporter
systems described herein below. Depending on the nature of the
reporter system this may be achieved by a number of different
methods. For example, if the reporter system comprises a nucleic
acid, said nucleic acid may be inserted into said cell by
conventional recombinant techniques (see below).
[0128] In another preferred example the cell comprises nucleic acid
comprising first nucleotide sequences encoding cellular proteins or
polypeptides being part of an intracellular signal transduction
pathway operationally linked to a reporter system detecting the
enzymatic activity or subcellular localization of said first
sequences, or detecting direct interactions between these first
sequences.
[0129] Useful second sequences includes for example promoters
active in the particular cells, for example mammalian promoters,
viral promoters or synthetic promoters. A large number of useful
eukaryotic promoters are known to the person skilled in the art,
useful promoters are for example described in "Mechanism of
Transcription" (1998) Cold Spring Harbor Symposia on Quantitative
Biology Vol. LXIII; Cold Spring Harbor Laboratory Press
[0130] Such promoters may be constitutively active or they may be
active only temporarily. In one example the promoter may be
regulated by an external signal, for example the promoter may be
inducible or repressable.
[0131] The nucleic acid may be inserted into the cells by any
useful method, for example by conventional recombinant techniques,
such as any of the techniques described in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 1989, Cold Spring Harbor
Laboratory, New York, USA
[0132] In another embodiment the cells are primary cells. Primary
cells are cells with a limited life span that preferably are
derived from a mammalian tissue. Preferred primary cells are cells
which are adherent. The mammalian tissue may for example be a human
tissue, such as healthy or diseased tissue. In one embodiment the
tissue is or comprises a neoplastic tissue, for example tissue
removed from a cancer patient by surgery, for example from a
patient suffering from melanoma, breast cancer or colon cancer. The
tissue may also be hypertrophic cells, such as cardiac myocytes.
Preferably said cancer patient has not been subjected to
radiotherapy prior to surgery. In embodiments of the invention
wherein the cells are primary cells it is preferred that the
reporter system is endogenous to said primary cells.
Cell Attachment to Resin Beads and Cell Cultivation
[0133] The present invention relates to methods comprising the step
of attaching cells comprising a reporter system(s) to resin beads.
The cells may for example attach to said resin beads directly or by
attaching a second compound conferring adhesion to the resin
bead.
[0134] The resin beads useful for the present invention should
preferably be able to support cell growth. The resin beads may per
se be able to support cell growth, however frequently the resin
beads will comprise a cell adhesion compound that enables the resin
beads to support growth of cells. Said cell adhesion compound may
be coupled to said resin beads by any useful means known to the
person skilled in the art depending on the nature of the cell
adhesion compound.
[0135] Any cell adhesion compound known to the person skilled in
the art may be used with the present invention. It is frequently an
advantage if the cell adhesion compound comprises at least one
positively charged moiety at neutral pH, more preferably the cell
adhesion compound has a positive overall netcharge at neutral
pH.
[0136] In one preferred embodiment of the invention the cell
adhesion compound comprises a peptide or a polypeptide, more
preferably the cell adhesion compound consists of a peptide. Such
peptides are herein also designated "adhesion peptides".
[0137] Said peptide preferably consists of in the range of 3 to
100, preferably in the range of 3 to 75, more preferably in the
range of 3 to 50, even more preferably in the range of 3 to 30, yet
more preferably in the range of 3 to 25, even more preferably in
the range of 3 to 20, yet more preferably in the range of 3 to 15,
such as in the range of 3 to 10, for example in the range of 3 to
8, for example in the range of 6 to 7 amino acids. In general, it
is sufficient if the peptide comprises at least 3 amino acids.
[0138] It is preferred that the peptide comprises at least one
amino acid selected from the group consisting of arginine and
lysine, more preferably the peptide comprises at least 2 basic
amino acids, such as 3 basic amino acids selected from the group
consisting of Arg and Lys, even more preferably the peptide has an
overall positive netcharge. In one preferred embodiment the peptide
comprises the following sequence of 4 amino acids:
basic-basic-lipophilic-basic. Basic amino acids may for example be
selected from the group consisting of arginine and lysine, whereas
the lipophilic amino acid may be selected from the group consisting
of Gly, Ala, Val, Leu, Ile, Phe, Trp, Pro and Met of either D or
L-form. Preferably, the peptide comprise at least 1, preferably at
least 2, more preferably at least 3, even more preferably at least
4 amino acid on the D-form, yet more preferably all amino acids are
on the D-form. Preferably .beta.-amino acids are used to enhance
the metabolic stability but also L-amino acids may be used.
[0139] Preferred examples of peptides useful as cell adhesion
compounds are given in table 2 herein below:
TABLE-US-00002 TABLE 2 No 1 2 3 4 5 6 7 SEQ ID NO 1 ala arg ile arg
ile gln his SEQ ID: 1 2 ala lys cys arg trp cys met SEQ ID: 2 3 ala
lys ala arg cys lys ser SEQ ID: 3 4 ala lys tyr trp ser tyr lys SEQ
ID: 4 5 ala his leu tyr arg asn lys SEQ ID: 5 6 ala arg arg cys phe
arg asp SEQ ID: 6 7 ala ala arg his cys tyr tyr SEQ ID: 7 8 ala tyr
tyr cys gln gln arg SEQ ID: 8 9 ala asp leu lys arg pro met SEQ ID:
9 10 ala gly gly lys arg lys phe SEQ ID: 10 11 ala pro arg lys arg
cys gly SEQ ID: 11 12 ala thr arg arg val ala arg SEQ ID: 12 13 ala
gly lys lys asn lys asn SEQ ID: 13 14 ala ala lys arg trp lys phe
SEQ ID: 14 15 ala arg trp pro tyr arg gly SEQ ID: 15 16 ala leu tyr
trp thr trp arg SEQ ID: 16 17 ala ala tyr arg trp tyr arg SEQ ID:
17 18 ala arg cys ile arg gly asp SEQ ID: 18 19 ala thr lys cys lys
gly arg SEQ ID: 19 20 ala val tyr met arg asn ile SEQ ID: 20 21 ala
arg lys arg ile arg gln SEQ ID: 21 22 ala lys ile arg glu lys arg
SEQ ID: 22 23 ala arg arg phe lys met tyr SEQ ID. 23 24 arg arg phe
lys SEQ ID: 24 25 arg arg ile arg SEQ ID: 25 26 leu arg his arg leu
lys SEQ ID: 26 27 lys phe gly gln lys (cys) SEQ ID: 27 28 lys val
tyr met his lys SEQ ID: 28 29 ile arg tyr arg leu arg SEQ ID: 29 30
ala gln arg pro arg trp SEQ ID: 30 trp tyr ala lys arg arg SEQ ID:
31 lys arg ile arg gln arg leu arg SEQ ID: 32 lys arg ile arg gln
arg lys SEQ ID: 33 arg ile arg gln arg SEQ ID: 34 arg gln arg ile
arg SEQ ID: 35 lys phe gly gln lys cys SEQ ID: 36 arg arg leu leu
pro ile SEQ ID: 37 pro phe arg lys lys cys SEQ ID: 38 tyr arg trp
arg ile Ala SEQ ID: 39 arg ser lys arg ile Asn SEQ ID: 40 arg ser
ala lys arg cys SEQ ID: 41 lys lys gln phe trp Phe SEQ ID: 42 arg
met lys leu his lys SEQ ID: 43 arg his trp gly arg ile SEQ ID: 44
thr lys arg leu lys thr SEQ ID: 45 thr lys gly lys ala lys SEQ ID:
46 ala lys thr arg his arg SEQ ID: 47 asn arg pro arg val arg SEQ
ID: 48 val pro arg lys val gln SEQ ID: 49 lys met arg tyr cys gln
SEQ ID: 50 ile arg lys his leu ile SEQ ID: 51 pro arg arg val val
ile SEQ ID: 52 lys arg glu ser lys arg SEQ ID: 53 ser arg lys asp
arg lys SEQ ID: 54 arg cys lys lys leu ile SEQ ID: 55 arg lys leu
arg val asn SEQ ID: 56 val arg thr val arg val SEQ ID: 57 arg ala
phe lys tyr tyr SEQ ID: 58 ile thr arg arg thr gln SEQ ID: 59 lys
met pro lys lys asn SEQ ID: 60 lys pro lys met met cys SEQ ID: 61
lys lys met arg phe trp SEQ ID: 62 lys lys lys phe tyr tyr SEQ ID:
63 lys ser asn lys val arg SEQ ID: 64 lys trp pro his his arg SEQ
ID: 65 arg his ile gln trp tyr SEQ ID: 66 leu arg Ieu lys pro lys
SEQ ID: 67 glu arg lys arg cys thr SEQ ID: 68 arg arg ala arg gln
asp SEQ ID: 69 arg glu lys gly ala arg SEQ ID: 70
[0140] Furthermore, preferred peptide may be any of the peptides
identified by any of SEQ ID: 1 to 70, preferably any of SEQ ID: 1
to 23 and 26 to 35, such as SEQ ID: 1 to 23, for example SEQ ID: 25
to 35, wherein 3 amino acids, preferably 2 amino acids, more
preferably 1 amino acid have been substituted for another amino
acid. Preferably, said substitution is a conservative substitution,
i.e. substitution for an amino acid with similar characteristics.
Said characteristic could for example be acidic/basic properties,
polarity or lipophilicity. It is also comprised within the
invention that the peptide may be a peptide of above mentioned size
comprising any of the peptides identified by SEQ ID: 1 to 70. In
particular, in order to immobilised the peptide on a resin bead it
may be useful to synthesise the adhesion peptide on an amino acid
immobilized on the resin bead, for example a Gly.
[0141] In one embodiment the peptide is preferably selected from
the group consisting of peptides identified by SEQ ID: 21 to 23 and
36 to 35, more preferably from the group consisting of 26 to 35,
even more preferably SEQ ID:35. In another embodiment the peptide
defined by SEQ ID:21 is preferred.
[0142] In one embodiment of the invention it is preferred that the
peptide has low or essentially no fluorescent properties. It is
particularly preferred that the peptide has low or essentially no
fluorescent properties when attached to a solid support, such as a
resin bead. By "essentially no fluorescent properties" is meant
that the peptide does not emit any detectable fluorescence. This is
in particularly relevant for embodiments of the invention wherein
the detectable output is fluorescence (see herein below). Preferred
peptides to use with this embodiment of the invention may be
selected from the group consisting of SEQ ID:26 to 35.
[0143] Peptides useful as cell adhesion compounds may be identified
using any suitable method. Said method may for example include the
steps of [0144] i) synthesizing or coupling a test peptide on to a
resin bead; [0145] ii) incubating said resin bead with cells under
cell cultivation conditions; [0146] iii) testing whether said cells
attach to said resin bead [0147] iv) identification of the peptide
sequence wherein the test peptide is useful as cell adhesion
compound If more cells attach to said resin bead in the presence,
than in the absence of said test peptide. Preferably, the test
peptide is useful as cell adhesion compound If at least 200, more
preferably at least 500, even more preferably at least 1000 cells
attach to said resin bead after incubation. This is in particular
the case in embodiments of the invention, wherein the resin beads
are PEGA beads. For example useful test peptides may be identified
as described in example 1 herein below.
[0148] In embodiments of the invention wherein it is preferred that
the peptide has no or low fluorescence it is preferred that the
method comprises an additional step performed at any point
subsequent to step i), such as immediately subsequent to step i)
prior to step ii). Said additional step comprises testing whether
said peptide has fluorescent properties. This may for example be
performed by sorting resin beads in a FABS or manually with the aid
of a fluorescence microscope. If this is done prior to step ii)
then only resin beads with no or low fluorescence properties are
incubeted with cells, A non-limiting example of a useful method is
described in example 2b.
[0149] The peptide may be coupled to the resin bead by any useful
method, for example by synthesising the peptide directly onto an
amino functionalised resin bead using a standard Fmoc-protocol for
peptide synthesis. Other protective groups may be used instead of
Fmoc, for example Boc, N.sub.3 or Alloc. In one embodiment Alloc is
the preferred protective group. It is preferred that different
protecting groups are used for synthesis of the adhesion peptide or
for library synthesis. The peptide may also be synthesised by
anchoring an Fmoc amino acid to a hydroxyl functionalised resin
bead, such as a hydroxymethylbenzoic acid (HMBA) derivatised PEGA
resin followed by peptide assembly using standard Fmoc technology
as described in B. Blankemeyer-Menge, M. Nimtz, and R. Frank, An
Efficient method for ancoring Fmoc-amino acids to
hydroxyl-functionalised solid supports. Tetrahedron Lett.
31:1701-1704, 1990 and A. Dryland and R. C. Sheppard. Peptide
synthesis. Part 11. A system for continuous flow solid phase
peptide synthesis using fluorenylmethoxycarbonyl-amino acid
pentafluorophenyl esters. Tetrahedron 44(3):859-876, 1988
Sidechains may be protected with acid labile protecting groups such
as t-Bu, Trt, Pmc, Boc etc. The protected peptide may for example
be cleaved off the resin using alkaline conditions or hydrazine and
the structure may be determined e.g. by on bead Edman Degradtion.
The HMBA-linked peptide may also be cleaved under mild alkaline
condition.
[0150] In one embodiment the peptide may be linked to the resin
bead via a linker, which may be a cleavable linker. This may for
example be achieved by synthesizing the linker directly on resin
beads or coupling the linker to the resin beads and subsequently
coupling or synthesizing the library onto the resin beads. Thus,
before coupling of the library the linker preferably comprises a
protective group as described herein above. The cleavable linker
may be any of the cleavable linkers described herein above. If the
resin beads are coupled to the library via a cleavable linker it is
preferred that the cleavable linker linking the adhesion compound
is differentially cleavable.
[0151] In embodiments wherein cells adhere to the resin bead via
the adhesion compound and the adhesion compound is attached to the
resin bead via a cleavable linker, cells may be at least partially
or even essentially fully released from the resin bead by cleavage
of the cleavable linker.
[0152] Testing whether cells attach to resin beads may be done by
any conventional methods, such as by manual inspection with the aid
of a light microscope. If the cells have fluorescent properties,
for example if the cells express a fluorescent protein, then resin
beads with attached cells may be identified using a fluorescent
microscope or a FABS, preferably a fluorescent microscope.
[0153] In one preferred embodiment of the invention, the cells may
be cultivated directly on the resin beads. In general, a method of
cultivating cells on resin beads may comprise the steps of [0154]
Providing resin beads capable of supporting growth of cells [0155]
Seeding cells onto said resin bead [0156] Incubating said resin
beads comprising said cells in a cell culture medium under cell
cultivation conditions [0157] Optionally allowing said cells to
divide on said resin bead [0158] Thereby cultivating cells on resin
beads
[0159] The cells may adhere actively to the resin beads and will
then generally be referred to as adherent cells.
[0160] Cells cultivation conditions depends on the specific cells.
For a large number of mammalian cells, such conditions comprise
high humidity, preferably close to 100%, approximately 5% CO.sub.2
and around 37.degree. C. It is often desirable to keep the resin
beads immersed in a suitable cultivation medium and frequently it
is also desirable that the resin beads can be circulated within
said medium, for example by stirring or rotation. Said stirring or
rotation may be continuous or in intervals. It is also possible
that the container comprising the resin beads is simply rocked
gently a few times every now and then.
[0161] In one embodiment of the invention more than one cell line
or type of primary cell is attached to or cultivated on the beads.
Hence for example 2, such as 3, for example 4, such as 5, for
example 6, such as 7, for example 8, such as 9, for example 10,
such as in the range of 10 to 20, for example in the range of 20 to
50, such as more than 50 different cell lines may be attached to or
cultivated on said beads. Also different specific primary cells may
be attached to the cultivated beads.
[0162] It is possible that a subgroup of resin beads only comprise
one cell line or a specific kind of primary cells and another
subgroup of resin beads comprises another cell line or another
specific kind of primary cell and so forth. However, it is also
possible that in principle every resin beads comprises all the
different cell lines and/or different primary cells.
[0163] Intermediates between these two extremes may also be
envisaged. Preferably, said different cell lines and/or primary
cells comprise different reporter systems, hence it is possible
that the different cell lines are derived from the same parent cell
lined by insertion of different reporter systems. However, the
different cell lines and/or primary cells may also be
unrelated.
Cellular Molecules
[0164] In one particularly preferred embodiment of the invention
the methods of the invention involve identification of compounds
modulating a cellular response, which is mediated through an
interaction between cellular molecules, more preferably through an
interaction between intracellular molecules. Cellular molecules may
be any cellular molecule, such as proteins, polypeptides, DNA, RNA,
molecules of non-protein nature or metal-ions. In preferred
embodiment, the cellular molecule is a protein or polypeptide.
Intracellular molecules are molecules that are not accessible from
the extracellular surface of intact cells. Intracellular molecules
may for example be proteins, polypeptides, DNA, RNA, molecules of
non-protein nature or metalions. In one preferred embodiment,
intracellular molecules are proteins or polypeptides not accessible
from the extracellular surface of intact cells.
[0165] In one embodiment, the cellular response is mediated through
an interaction between cellular molecules of a cellular signal
transduction pathway. Hence, the invention, for example may be
useful for identifying compounds modulating the activity of a
signal transduction pathway. Such compounds could for example
activate or repress a signal transduction pathways by [0166]
modulating the interaction between different or the same cellular
molecules, [0167] modulating the catalytic activity of enzymes,
[0168] modulating the synthesis or degradation of cellular
molecules, [0169] modulating transcriptional activity, [0170]
modulating the localization or movement of cellular molecules.
[0171] modulating the level of cellular molecules (i.e. in a
specific cellular compartment or on average throughout the whole
cell)
[0172] Within the context of the present invention the term "signal
transduction pathway" should be understood in its common cell
biological meaning, i.e. modulation of an intracellular event
triggered by a cell surface receptor.
[0173] Signal transduction pathways may for example involve steps
of changed catalytic activity of enzymes, phosphorylation, cleavage
of proteins, synthesis of cAMP, activation of transcription,
inhibition of transcription, change i intracellular Ca.sup.2+
concentration, change in membrane potential, subcellular
relocalisation of cellular components, complex formation of
cellular components, degradation of cellular components and/or
change in energy metabolism
[0174] The signal transduction pathway could for example be a
pathway activated/repressed by a cell surface receptor selected
from the group consisting of Gprotein coupled receptors (GPCR),
protein kinase coupled receptors, receptor kinases with intrinsic
kinase activity, orphan receptors or transmembrane channels. The
signal transduction pathway may also be a pathway resulting in
modulation of transcription, for example modulation of
transcription regulated by a response element, for example a
response element selected from the group consisting of CRE, SRE,
TRE and AP-1 In one embodiment of the invention the signal
transduction pathway is a pathway resulting in apoptosis.
[0175] Preferably the cellular molecules are proteins or parts
thereof or derivatives thereof, more preferably the cellular
molecules are proteins. Even more preferably the cellular molecules
belong to the classes of: serine/threonine protein kinases;
tyrosine protein kinases, protein phosphatases, phospholipid
dependent serine/threonine protein kinases, calmodulin dependent
serine/threonine protein kinases, mitogenactivated serine/threonine
protein kinases, cycline dependent serine/threonine protein
kinases, transcription factors, structural proteins, protein
scaffolds, proteases, such as caspases, metallo-matrix-proteases,
rennin, cathepsins, viral proteases, secretases or ADAM family
proteases, or hydrolases, nucleases, synthases, isomerases,
polymerises, oxido-reductases, ATPases or GTPases.
[0176] The cellular molecules are more preferably proteins that are
known to participate in protein-protein interactions or complex
formations. Such proteins can be selected from proteins listed in
databases like BIND (Biomolecular Interaction Network Database)
http://bind.ca.
[0177] In one embodiment of the invention the cellular molecules
are involved in regulation of apoptosis. Thus the cellular
molecules may be proteins or functional fragments thereof involved
in regulation of apoptosis. Proteins involved in apoptosis includes
for example caspases, inhibitors of apoptosis (IAPs) or Smac.
Inhibitors of apoptosis may for example be XIAP, cIAP1/BIRC2,
ML-IAP/BIRC7, DIAP1, DIAP2, OPIAP3, cIAP2, NAIP, Apollon or
Survivin (see also Vaux and Silke, 2005, Nature Reviews,
6:287-297). Thus, in one example proteins involved in
protein-protein interaction may be Smac and XIAP or ML-IAP or a
Smac binding fragment thereof. Smac binding fragments of XIAP
preferably comprises the BIR3 domain of XIAP, whereas Smac binding
fragments of ML-IAP preferably comprises the BIR domain. The
domaine structure of IAPs is well described, see for example Wu,
G., Chai, J., Suber, T. L., Wu, J.-W., Du, C., Wang, X., and Shi,
Y. (2000) Nature 408, 1008-1012; Matthew C. Franklin et al.,
Biochemistry 2003, 42, 8223-8231; and Liston et al. Oncogene. 2003
Nov. 24; 22(53):8568-80
[0178] A cell surface receptor within the meaning of the present
invention is preferably a protein, more preferably a protein that
is accessible from the extracellular surface. Yet more preferably,
the cell surface molecule is a cell surface protein receptor
(herein also merely designated "receptor"). A "receptor" within the
meaning of the present invention, is a molecule, which at least
sometimes is localised at the cell surface and which is capable or
associating with at least one ligand. The ligand binding site is
accessible from the extracellular surface. Frequently, association
with said ligand may alter the activity of the receptor.
Cellular Response
[0179] The invention relates to methods of identifying compounds
modulating, such as activating or inhibiting, a cellular response
linked to a reporter system. The reporter system may be any of the
reporter systems described herein below. The methods disclosed by
the present invention may be used to identify compounds modifying
any cellular response, which is or may be linked to a reporter
system generating a detectable output. The person skilled in the
art will appreciate that the specific methods disclosed herein may
be adapted to any such cellular response. Below, non-limiting
examples of cellular responses are described.
[0180] In a particularly preferred embodiment of the invention, the
cellular response is mediated through interaction between cellular
molecules, such as intracellular molecules. The cellular molecules
may for example be components of a signal transduction pathway, and
thus the cellular response may be activation or repression of a
signal transduction pathway. Hence, the cellular response may for
example be modulation of a signal transduction pathway within a
cell, such as modulation of a signal transduction pathway mediated
by a cell surface molecule. By "activation of a receptor" is meant
that the receptor is influenced in a manner that it activates
downstream signalling events. Accordingly, the methods according to
the present invention may be employed to identify activators or
inhibitors of signal transduction pathways.
Examples of modulations of signal transduction pathway includes
[0181] Upregulation or downregulation of the level of a member of
the pathway [0182] Relocalisation of a member of the pathway [0183]
Complex formation between members of the pathway or between members
of the pathway with other cellular compounds [0184] Enhanced or
reduced transcription from genes regulated by the pathway [0185]
Modification by for example phosphorylation of a member of the
pathway [0186] Activation or inhibition of an enzyme of the pathway
[0187] Degradation of a cellular compounds due to upregulation or
downregulation of the pathway [0188] Altered secretion of a
compound [0189] Change in ion-flux [0190] Morphological changes
[0191] Change in viability
[0192] In a preferred embodiment the modulation of a signal
transduction pathway can for example be monitored by measuring:
[0193] the enzymatic activity of an enzyme being part of said
signal transduction pathway [0194] the level of cyclic nucleotides,
i.e. cAMP or cGMP [0195] the activity of transcription factors
[0196] the level of specific proteins as quantified through
standard proteomics techniques [0197] the level of inositol or
lipid phosphates [0198] the level of phosphorylation of specific
proteins as quantified through standard proteomics techniques
[0199] the binding between two or more proteins or polypeptides
[0200] the cellular localization of proteins or polypeptides
[0201] The enzymatic activity could for example be the enzymatic
activity of serine/threonine protein kinases or of tyrosine protein
kinases or of protein phosphatases or of phospholipid dependent
serine/threonine protein kinases or of calmodulin dependent
serine/threonine protein kinases or of mitogenactivated
serine/threonine protein kinases or of cycline dependent
serine/threonine protein kinases or of proteases or of hydrolases
or of nucleases or of synthases or of isomerases or of polymerises
or of oxido-reductases or of ATPases or of GTPases.
[0202] The cellular response may in one embodiment be modulation of
transcriptional activity, such as activation or reduction of
transcription of one or more genes. In particular, activation or
reduction of transcription of genes regulated by a response
element. Said response element could for example be selected from
the group consisting of CRE, SRE, TRE and AP-1.
[0203] Hence, the cellular response may also be an increased or
decreased level of a particular mRNA within a cell.
[0204] By the term "regulated by a response element" is meant that
transcription is modulated by said response element, however other
elements may also modulate transcription of said gene. By the term
"activation of response element" is meant increased transcription
of genes regulated by said response element.
[0205] In another embodiment of the invention the cellular response
is: [0206] change in the intracellular level of a compound; or
[0207] change in the level of a compound within a specific cellular
compartment, for example within the cytoplasm, in the golgi, in the
endoplasmatic reticulum, in lysosomes, in endosomes or in the
nucleus
[0208] The compound may be any compound, preferably a naturally
occurring compound. Frequently, the compound is a compound
endogenous to the cell. The compound may thus for example be a
salt, an ion, a nucleotide or a derivative thereof, a peptide, a
saccharide, a lipid or a biomacromolecule. Biomacromolecules
includes for example RNA such as mRNA, polypeptides and proteins.
An example of an ion is Ca.sup.2+ and an example of a nucleotide
derivative is cAMP.
[0209] In yet another embodiment of the invention the cellular
response is relocalisation of a compound. Relocalisation may for
example be [0210] concentration of a compound otherwise dispersed
in one or more specific locations [0211] relocalisation from one
cellular compartment to another, for example relocalisation from
the cellular membrane to the cytoplasma. [0212] relocalisation from
one location within a compartment to another location within the
same compartment [0213] internalisation of an extracellular
compound
[0214] The compound may be any compound, such as any of the
compounds mentioned in the section above. In one preferred
embodiment the compound, which is relocalised is a
biomacromolecule, such as RNA, polypeptides or proteins. For
example, the compound may be a cell surface receptor (receptor).
The cellular response may thus be internalisation of said receptor
or relocalisation of said receptor from the cellular membrane to
the cytoplasma.
[0215] In one embodiment of the invention the cellular response is
change in the activity of a compound, such as an increase or a
decrease in the activity of a compound. Said compound may for
example be an enzyme. The cellular response may for example be
induction of the activity of a caspase. Preferred caspases are
Caspase 3 or 7.
[0216] In another embodiment of the invention the cellular response
is change in phosphorylation of a compound.
[0217] In another embodiment of the invention the cellular response
is formation or disruption of a complex between compounds.
[0218] In another embodiment of the invention the cellular response
is change in the concentration of a compound.
[0219] The cellular response may also be altered secretion of a
compound, such as increased or decreased secretion of a compound.
Said compound could for example be a biomacromolecule, such as a
protein, a polypeptide, a peptide, a hormone, a cytokine, or the
like.
[0220] In another embodiment of the invention the cellular response
is change in pH in an intracellular compartment, for example in the
cytoplasm.
[0221] In yet another embodiment the cellular response is a change
in a membrane potential, for example a change in membrane potential
over the cell membrane or over the mitochondria membrane.
[0222] In an even further embodiment of the invention the cellular
response is change in morphology, such as change in size or shape.
The cellular response may also be change in viability (e.g.
apoptosis or necrosis), such as change in viability under specific
conditions.
[0223] In a preferred embodiment of the present invention the
cellular response is change in interaction between two or more
cellular molecules, preferably between two cellular molecules, such
as establishment of an interaction between two or more cellular
molecules or disruption of an interaction between two or more
cellular molecules. Thus the cellular response may be formation of
a complex or disruption of a complex. The cellular molecules may be
any of the cellular molecules mentioned above, however, preferably
the cellular molecules are proteins or fragments thereof.
[0224] In one embodiment of the present invention the cellular
response is induction or facilitation of apoptosis in living cells,
such as induction or facilitation of apoptosis in tumour cells,
preferably induction of apoptosis. The cellular response may also
be induction or facilitation of apoptosis in cells that have
undergone an apoptosis promoting treatment. The cellular response
may also be induction or facilitation of apoptosis in cells that
have undergone an apoptosis inhibiting treatment, In another
embodiment of the invention the cellular response is inhibition or
reduction of apoptosis, for example reduction of apoptosis in cells
prone to undergo apoptosis or reduction in apoptosis in cells that
have undergone an apoptosis promoting treatment.
[0225] The apoptosis promoting treatment may be contacting cells
with an inducer of apoptosis. The inducer of apopotosis may be any
compound known to be capable of inducing apoptosis, for example the
compound may be staurosporine (STS). Alternatively, the apoptosis
promoting treatment may be illumination with radiation, such as
with UV-light with a predetermined wave length and intensity.
[0226] The methods according to the invention may also include
identification of compounds modulating more than one cellular
response, such as 2, for example 3, such as 4, for example 5, such
as more than 5 different cellular responses. Said cellular
responses may be any of the responses discussed above.
Reporter System
[0227] The reporter system to be used with the present invention
should be selected according to the particular cellular response.
The reporter system should be capable of generating a detectable
output.
[0228] In some embodiments of the invention the reporter system may
be identical to the cellular response. This is in particular true
when the cellular response may be detected without the aid of an
additional reporter system, for example when the cellular response
is an increase/decrease in the level of a compound, relocalisation
of a compound, change in membrane potential, change in pH, change
in morphology or the like.
[0229] Hence, the reporter system may be a system endogenous to
said cells. For example, the reporter system may comprise the
endogenous system regulating the intracellular level of an
endogenous compound. By way of example, the reporter system may be
the endogenous system of a cell regulating the intracellular
Ca.sup.2+ level.
[0230] In another example, the reporter system comprises the
intracellular localisation of an endogenous compound.
[0231] In yet another example, the reporter system may comprise the
activity of an enzyme. This may in particular be relevant when the
cellular response is modulation of an enzymatic activity or
modulation of a signal transduction pathway which modulates an
enzymatic activity. Then the reporter system could be direct
detection of for example the enzymatic activity of serine/threonine
protein kinases or of tyrosine protein kinases or of protein
phosphatases or of phospholipid dependent serine/threonine protein
kinases or of calmodulin dependent serine/threonine protein kinases
or of mitogenactivated serine/threonine protein kinases or of
cycline dependent serine/threonine protein kinases or of proteases,
such as caspases, metallo-matrix-proteases, rennin, cathepsins,
viral proteases, secretases or ADAM family proteases or of
hydrolases or of nucleases or of synthases or of isomerases or of
polymerises or of oxido-reductases or of ATPases or of GTPases.
[0232] It is preferred that said enzymatic activity may be detected
for example because the enzymatic activity leads to formation of a
coloured compound, a fluorescent compound, a radioactive compound
or the like. This may be achieve by the use of appropriate
substrates. If the enzyme is a protease, the enzymatic activity may
for example be detected by use of a substrate, which generates a
detectable output when cleaved by said protease. For example the
substrate could be a peptide or a polypeptide comprising a
fluorescent moeity and a quencher, wherein cleavage would lead to
formation of two peptides/polypeptides, wherein one comprises the
fluorescent moeity and the other comprises the quencher.
Fluorescence would thus be detectable only in the presence of an
active protease.
[0233] In embodiments wherein the enzyme is a caspase, the reporter
system may be a substrate for said caspase. Useful caspase
substrates are known to the skilled person and several caspase
substrates are commercially available, for example from Beckman
Coulter Inc. Examples of Caspase substrates are Caspase 3 and/or 7
substrates. It is preferred that cleavage of the substrates is
readily detectable. Thus fluorogenic substrates comprising a
quenching group which may be cleaved of by caspases may be useful.
Cleavage of such substrate can simply be detected by determining
fluorescence. Non-limiting examples of fluorogenic caspase
substrates are the CellProbe HT Caspase 3/7 Whole Cell Assay,
Beckman Coulter, Inc, or any of the substrates described in U.S.
Pat. No. 6,342,611. It is not required that such a reporter system
is introduced permanently into living cells. Thus the substrate may
be added directly to the cell culture medium or to an assay buffer
comprising resin beads with cells. Thus such a reporter system may
also be useful in embodiments of the invention wherein primary
cells are employed. A non-limiting example of a useful Caspase
assay is given in example 12 herein below.
[0234] However, the reporter system may also be heterologous to the
cell, i.e. a reporter system which has been inserted into the cell
for example by recombinant techniques.
[0235] In several embodiments of the invention the reporter system
comprises a fusion protein comprising a first protein and
detectable polypeptide. The detectable polypeptide may for example
be an enzyme or part thereof, a transcription factor or part
thereof or a bioluminiscent protein, such as a fluorescent protein.
Preferred detectable polypeptides are luciferase or fluorescent
proteins. The first protein may be selected according to the
cellular response. If the cellular response is change in level or
location of a given protein, the first protein could be that
particular protein. If the cellular response is change in
interaction between two or more proteins, the first protein could
be one of the proteins taking part in that interaction.
[0236] In embodiments of the invention, wherein the cellular
response is modulation of transcription from gene(s) regulated by a
response element, it is preferred that the report system comprises
a nucleic acid comprising a nucleotide sequence encoding a
detectable polypeptide operably linked to a response element, the
activity of which is modulated by the cellular response.
[0237] In embodiments of the invention, wherein the cellular
response is modulation of a signal transduction pathway, the
reporter system may comprises a nucleic acid comprising a
nucleotide sequence encoding a detectable polypeptide operably
linked to a response element, the activity of which is modulated by
said signal transduction pathway.
[0238] For example, if the cellular response is modulation of a
signal transduction pathway influencing the activity of CRE and/or
SRE, then the reporter system may comprise a nucleic acid
comprising a nucleotide sequence encoding a detectable polypeptide
operably linked to a response element selected from the group
consisting of cAMP response element (CRE) and serum response
element (SRE). Examples of such signal transduction pathways
include the signal transduction pathways modulated by GPCR of the
rhodopsin family or secretin family and by protein kinase receptors
and receptors belonging to the family of receptor kinases.
[0239] By way of example: 1) If the cellular response is activation
of a signal transduction pathway activated by a GPCR coupled to a
G.sub.S (see herein above) that stimulates adenylate cyclase, then
the reporter system may be a nucleic acid comprising a nucleotide
sequence encoding a detectable polypeptide operably linked to CRE.
Activation of said GPCR may then be detected by detection of
increased levels of said detectable polypeptide. 2) If the cellular
response is activation of signal trans-duction pathway activated by
a GPCR coupled to a G.sub.I (see herein above) that inhibits
adenylate cyclase, then the reporter system may be a nucleic acid
comprising a nucleotide sequence encoding a detectable polypeptide
operably linked to CRE. Activation of said GPCR may then be
detected by detection of decreased levels of said detectable
polypeptide.
[0240] Similarly, if the cellular response is modulation of a
signal transduction pathway that influences the activity of TRE,
then the reporter system may comprise a nucleic acid comprising a
nucleotide sequence encoding a detectable polypeptide operably
linked to TPA response element (TRE). Examples are GPCRs that are
linked to activation of Protein Kinase C such as Gq coupled
receptors (see herein above).
[0241] Similarly, if the cellular response is modulation of a
signal transduction pathway that influences the activity of SRE,
then the reporter system may comprise a nucleic acid comprising a
nucleotide sequence encoding a detectable polypeptide operably
linked to SRE. Examples of such signal transduction pathways
include the signal transduction pathways modulated by growth
hormones or cytokines through protein kinase receptors and
receptors belonging to the family of receptor kinases.
[0242] Similarly, if the cellular response is modulation of a
signal transduction pathway that influences the activity of AP-1,
then the reporter system may comprise a nucleic acid comprising a
nucleotide sequence encoding a detectable polypeptide operably
linked to AP-1. Examples of such signal transduction pathways
include the signal transduction pathways modulated by cytokines or
growth factors cytokines through protein kinase receptors and
receptors belonging to the family of receptor kinases
[0243] The detectable polypeptide may be any detectable
polypeptide, however preferably the detectable polypeptide is
selected from the group consisting of fluorescent proteins and
enzymes.
[0244] Fluorescent proteins may for example be green fluorescent
protein (GFP) and fluorescent mutants thereof, such as yellow
fluorescent protein (YFP) or cyan fluorescent protein (CFP). The
fluorescent protein can also be a protein complex, e.g. a di- or
tetramer of a fluorescent protein, such as dsRed. Enzymes may for
example be selected from the group consisting of luciferase, CAT,
galactosidase, alkaline phosphatase and beta-lactamase.
[0245] In one embodiment of the invention the reporter system may
comprise a bioluminescent moiety. For example, if the cellular
response is relocalisation of a compound, then the reporter system
may for example be said compound linked to a luminescent moiety,
such as a fluorescent moeity. Hence, for example if the cellular
response is relocalisation of a polypeptide the reporter system may
be a chimeric protein made up of said polypeptide and a fluorescent
protein, such as GFP, YFP or CFP. In one preferred embodiment said
polypeptide may be receptor.
[0246] In one embodiment of the invention the reporter system may
detect the level of a cellular molecule, such as a protein. This
may for example be achieved by quantifying the amount of a compound
i.e. an antibody that specifically binds to the cellular molecule.
The quantification can for example be achieved by covalently
coupling a fluorescent, bioluminescent or coloured moiety to said
compound. The quantification could be confined to a specific
cellular compartment.
[0247] In one embodiment of the invention the reporter system may
detect the level of modification of a cellular molecule for example
but not limited to phosphorylation, glycosylation or
ubiguitination. This may for example be achieved by quantifying the
amount of a compound i.e. an antibody that specifically binds to
the modified cellular molecule. The quantification can for example
be achieved by covalently coupling a fluorescent, bioluminescent or
coloured moiety to said compound. The quantification could be
confined to a specific cellular compartment.
[0248] In one embodiment of the invention the reporter system may
detect complex formation or disruption between two cellular
proteins (designated first protein and second protein in this
paragraph). This is in particular relevant when the cellular
response is change in interaction between two cellular proteins.
Several different reporter systems may be used to detect
interaction between a first and a second protein. Below preferred
reporter systems are described, however, the invention is not
limited to these specific reporter systems.
[0249] The reporter system may for example comprise the first
protein linked to a bioluminescent moiety, such as luciferase and
the other protein linked to a fluorescent moiety, such as a
fluorescent protein. Such reporter systems are referred to as "BRET
reporter systems" herein. The bioluminiscent moeity should
preferably be able to directly or indirectly generate light of a
wavelength capable of exciting the fluorescent moiety. The skilled
person will readily be able to select useful bioluminiscent
moeities and fluorescent moeities. Preferably, the BRET reporter
system comprises a first chimeric protein comprising the first
protein linked to a bioluminescent protein, preferably luciferase
and a second chimeric protein comprising the second protein linked
to a fluorescent protein. Such a reporter system may be introduced
into a cell by introducing nucleic acids encoding the first and the
second chimeric proteins under control of suitable promoters into
said cell. Direct interaction between the proteins can after
expression of the two chimeric proteins be detected through
occurrence of BRET (Bioluminescence Resonance Energy Transfer). In
one embodiment, BRET2 technology may be used which is based on
energy transfer between a bioluminescent donor (a Renilla
luciferase (Rluc) fusion protein) and a fluorescent acceptor (a
Green Fluorescent Protein (GFP2) fusion protein). In presence of
its substrate DeepBlueC.TM. (a coelenterazine derivative), Rluc
emits blue light (.about.395 nm). Thus the reporter system may
comprise a first chimeric protein comprising the first protein and
Rluc and a second chimeric protein comprising the second protein
and GFP2. A protein-protein interaction between Rluc and GFP2
chimeric proteins allows energy transfer to GFP2, which reemits
green light (510 nm). Expression of Rluc alone, in the presence of
the substrate DeepBlueC.TM., gives an emission spectrum with a peak
at .about.395 nm, whereas when the Rluc and GFP2 chimeric proteins
interact, there is efficient energy transfer between Rluc and GFP2
and the 510 nm signal represents a major peak.
[0250] In another similar reporter system the first protein and the
second protein are linked to different fluorescent moieties,
preferably a fluorescent proteins. Such reporter systems are
referred to as "FRET reporter systems" herein. Preferably, one
fluorescent moiety is capable of emitting light of a wavelength
capable of exciting the other fluorescent moeity. FRET reporter
systems preferably comprise a first chimeric protein comprising the
first protein and a fluorescent protein and a second chimeric
protein comprising the second protein and another flourescent
protein. It is then possible to detect the complex formation
through the occurrence of FRET (Fluorescence Resonance Energy
Transfer). BRET or FRET according to the present invention may for
example be performed as described in (Nicolas B, R Jockers, and T
Issad Trends in Pharmacological Sciences 23 (8):351-354, 2002;
and/or A. Roda, M. Guardigli, P. Pasini, and M. Mirasoli. Anal.
Bioanal. Chem 377 (5):826-833, 2003)
[0251] Complex formation may also be detected by proximity
ligation. In such an embodiment the reporter system comprises two
affinity probes raised against the first and the second protein.
Such reporter systems are designated "proximity ligation reporter
systems" herein. When the two proteins come in close proximity a
ligation reaction creates a DNA reporter sequence that can be
amplified. The amplified sequence can be detected by any useful
method, for example it may be detected through photolabelling.
Preferably, the DNA reporter sequence is amplified by PCR, rolling
circle replication or ligation chain reaction. In order to detect
the amplified sequence, the sequence may for example be amplified
using primers labelled with a detectable label, such as a
fluorescent label or the sequence may be detected using a
detectably labelled probe, such as a fluorescently labelled probe.
The affinity probes in general comprise or consist of a binding
moeity and a nucleic acid moeity. The binding moiety of the
affinity probes can be any molecule that binds either the first or
the second protein with high affinity. Preferably, the binding
moeity is capable of specifically recognising and binding either
the first or the second protein. Examples of useful binding
moieties of affinity probes are monoclonal- or polyclonal
anti-bodies or antigen binding fragments thereof, chimeric
antibodies, recombinant antibodies, single chain antibodies or
aptamers. Antibodies may be prepared using any conventional method
known to the person skilled in the art. Aptamers may be prepared by
any method known to the skilled person, for example by iterative
cycles of screening nucleic acid libraries for compounds capable of
binding a tare nucleic acid molecules selected for their ability to
specifically bind a target. Aptamers may for example be produced
using a SELEX process (Sun S Curr Opin Mol Ther 2000 February
2:100-5; Jayasena S D Clin Chem 1999 September 45:1628-50). The
nucleic acid moeity may comprise or consist of any nucleic acid
sequence, preferably a sequence, which when ligated to another
nucleic acid moeity creates a DNA reporter sequence, which can be
amplified by PCR, rolling cycle amplification or ligase chain
reaction using appropriate primers. A person skilled in the art can
design useful nucleic acid moeity sequences and corresponding
primers. The affinity probes can be introduced into cells by a
number of different methods, for example they may be introduced
into the cells after said cells have been fixed and permeabilized
or they can be introduced by using traditional cDNA transfection
methods, for example by using standard procedure for Fugene6
transfection. Proximity ligation may for example be carried out as
described in Frederiksson et al. Nature Biotechnology 2002, 20:
473; Gullberg et al. Curr Opinion Biotechnology 2003, 14: 82. The
reporter system may also be a "two-hybrid reporter system".
Two-hybrid reporter systems comprises two chimeric proteins,
wherein the first chimeric protein comprises the first protein
fused to a DNA binding domain and the second chimeric protein
comprises the second protein fused to a transactivating domain.
Furthermore, the two hybrid reporter system comprises a reporter
construct comprising a nucleic acid sequence encoding a detectable
polypeptide the expression of which is controlled by the
transactivating/DNA binding domain. Thus if the first protein
interacts with the second protein the DNA binding domain and the
transactivating domain are brought into close proximity and may
activate transcription from the reporter construct. Interaction can
then be determined by detection of the detectable polypeptide. The
detectable polypeptide may be any of the detectable polypeptides
mentioned herein above. Two-hybrid reporter systems are well
described in the art, see for example U.S. Pat. No. 5,283,173.
[0252] The reporter system may also be an enzyme complementation
reporter system. Enzyme complementation reporter systems comprises
two chimeric proteins, wherein the first chimeric protein comprises
the first protein fused to a first part of an enzyme and the second
chimeric protein comprises the second protein fused to a second
part of an enzyme. The first and the second part of an enzyme
should together constitute a functional enzyme. Thus, when the
first protein interacts with the second protein the first part and
the second part of an enzyme will form a functional enzyme, the
activity of which may be determined. One example of an enzyme
useful for enzyme complementation system is DHFR (dihydrofolate
reductase), where the activity of the reconstituted enzyme is
monitored as a fluorescent read-out based on stoichiometric binding
of fluorescein-methotrexate to reconstituted DHFR (Remy I, Michnick
S W Proc Natl Acad Sci USA 1999 May 96:5394-9).
[0253] Hence, if the cellular response is relocalisation of a cell
surface molecule, then the reporter system may comprise a
fluorescent moiety covalently coupled to said cell surface
molecule.
[0254] In some embodiments of the invention the cellular response
is modulation of a signal transduction pathway involving activation
of phospholipase C. Phospholipase C may for example be activated by
GPCRs coupled to G.sub.Q (see herein above). Activation of
phospholipase C in general leads to increase in the intracellular
level of Ca.sup.2+ and thus in such embodiments the reporter system
may be the intracellular Ca.sup.2+ level. This reporter system may
thus be endogenous to the cell.
[0255] When the cellular response is induction/facilitation of
apoptosis a number of reporter systems may be employed.
Induction/facilitation of apoptosis may be determined by
determining caspase activity as described herein above.
Induction/facilitation of apoptosis may also be determined by
determining cell growth/number of cells, for example cell
growth/number of cells after cultivation of for example normal
cells or tumour cells, such as cells expressing high levels of XIAP
or ML-IAP. Other methods of determining apoptosis are well known to
the skilled person.
Detectable Output
[0256] The detectable output may be any output, which is detectable
directly or indirectly. For example the detectable output may be
the concentration of a compound within a cell, localisation of a
compound within a cell, luminiscence, activity of an enzyme or the
like.
[0257] In preferred embodiments of the invention the detectable
output is luminiscence, fluorescence, bioluminescence, FRET or
BRET. Bioluminiscence may be detected by any conventional methods,
for example with the aid of a Plate reader. BRET or FRET may for
example be detected using FABS, a plate reader, a fluorescence
microscope or the like.
[0258] Alternatively, the detectable output may preferably be
linked (directly or indirectly) to a bioluminiscent signal.
[0259] However, the detectable output could also be radioactivity,
a coloured compound or a colour signal, a heavy metal, an
electrical potential, a redox potential, a temperature or the
detectable output may be linked to a radioactive signal, a coloured
compound or a colour signal or a heavy metal or an electrical
potential, or a redox potential or a temperature. Said radioactive
signal could for example be .sup.35S, .sup.32P, .sup.3H. The
coloured compound could for example be the product of any of the
enzymatic reaction described herein elsewhere. The heavy metal
could for example be gold.
[0260] In embodiments of the invention, wherein the cellular
response is change in the intracellular level of a compound or
change in the level of a compound within a specific cellular
compartment, then the detectable output may be said level of said
compound. Depending on the nature of the compound, said level may
be detected directly or indirectly.
[0261] If the compound for example is a fluorescent compound, the
level of said compound may be determined by determining the
fluorescence properties. This may be done by any suitable means,
for example by the aid of a fluorescence microscope, a FACS
(Fluorescence Activated Cell Sorter), a FABS (Fluorescence
Activated Bead Sorter), fluorescence plate-reader or a fluorescence
spectrometer,
[0262] If the compound for example is an enzyme then the level of
said compound may be determined by determining the activity of said
enzyme. By way of example, if the enzyme catalyses a reaction
leading to a product, which is directly detectable, for example by
colorimetric or chemiluminescent detection techniques, the activity
of said enzyme may be detected by detecting said compound. For
example, if the enzyme is luciferase, the activity of said enzyme
may be detected by detecting emmission of light upon oxidation of
the added substrate, luciferin.
[0263] Several other enzymes such as CAT, .beta.-galactosidase,
alkaline phosphatase, horseradish peroxidase and beta-lactamase
are, when provided with suitable substrates, capable of catalysing
reactions leading to coloured or chemiluminescent products, which
may be detected using any colorimetric or chemiluminescent
detection technique.
[0264] If the compound for example is Ca.sup.2+, then the
intracellular concentration of said ion can be measured by using
any suitable method, for example by inserting into the cells
Ca.sup.2+ binding fluorescent compounds like Fura-2, Fluo-3 or
Fluo-4 (Molecular Probes), which change fluorescent properties
according to a changed Ca.sup.2+ concentration. A non-limiting
example of a method of determining cytosolic free Ca.sup.2+ is
given in example 13 herein below. Other ion concentrations can be
monitored using suitable fluorescent compounds, which for example
are available from Molecular Probes Inc.
[0265] If the compound for example is a protein, then it may for
example be detected using a first specific binding partner. Said
first specific binding partner could be a second protein capable of
specifically interacting with said protein, such as a specific
anti-body or said first specific binding partner could be an
aptamer. Said first specific binding partner could be conjugated to
a directly detectable compound, such as a fluorescent compound, a
radioactive compound or a heavy metal or to an indirectly
detectable compound, such as an enzyme, which for example could be
any of the enzymes mentioned herein above. It is also possible that
the first specific binding partner may be detected with a second
specific binding partner, capable of interacting specifically with
the first specific binding partner. Said second specific binding
partner may be conjugated to a directly or indirectly detectable
compound similarly to the first specific binding partner.
Additional specific binding partners may be used.
[0266] In embodiments of the invention wherein the cellular
response is relocalisation of a compound the detectable output
could be a detectable label conjugated to said compound. In
particular, the compound may be conjugated to a directly detectable
label, such as a fluorescent label or a heavy metal. Thus the
localisation of the compound may be directly detected, for example
using a fluorescence microscope, Fluorescent plate-reader,
fluorescence spectrometer, a FACS or a FABS instrument In one
preferred embodiment the compound is a fusion protein comprising a
protein of interest and a fluorescent protein, such as GFP. The
compound may thus be a fluorescent probe. Thus the detectable
output may be localisation of a fluorescent signal. Alternatively,
the compound is a fusion protein comprising the protein of interest
and a tag. Said tag could be a tag specifically interacting with a
specific binding partner, for example the tag could be an HA-tag or
a flash domain. Alternatively, localisation of a compound may be
determined with the aid of a specific binding partner as outlined
above. Intracellular localisation may also be detected using
methods capable of detecting distance between two compounds, for
example BRET or FRET.
[0267] In embodiments of the invention wherein the cellular
response is change of activity of a compound, the detectable output
may be a product of said activity. I.e. when said compound is an
enzyme the detectable output could be a product of a reaction
catalysed by said enzyme. Said product could thus be a coloured
product or a chemiluminescent product as discussed herein
above.
[0268] In embodiments of the invention wherein the cellular
response is enhanced or reduced transcription from one or more
genes, then the cellular response could be mRNA transcribed from
said gene, a protein encoded by said gene or in case the protein is
an enzyme, the detectable output could be a product of a reaction
catalysed by said enzyme. The enzyme and the products could be any
of the enzymes or products discussed herein above.
[0269] mRNA may be detected by any useful means, for example with
the aid of a probe capable of hybridising specifically with said
mRNA. Said probe could be labelled with a directly detectable
label, for example a radioactive compound, a fluorescent compound
or a heavy metal or an indirectly detectable label such as an
enzyme or a specific binding partner.
[0270] Said protein may be detected with the aid of specific
binding partners as outlined herein above. However, in a preferred
embodiment the protein is a fluorescent protein and may thus be
detected directly. Hence, the detectable output could be
bioluminescence, such as fluorescence.
[0271] In embodiments of the invention wherein the cellular
response is modification by for example phosphorylation of a
compound this can be detected through binding of an antibody that
specifically bind the phosphorylated protein said antibody can then
be quantified by specific fluorescence labelling.
[0272] In embodiments of the invention wherein the cellular
response is change in pH in an intracellular compartment, the
detectable output will in general be said pH. The pH may be
determined using any suitable method, for example using a pH
indicator or a pH-meter. For example the pH may be determined using
a fluorescent indicator for intracellular pH. Suitable compounds
are compounds with a fluorescent excitation profile which is
pH-dependent, such as BCECF (available from Molecular Probes). In
embodiments of the invention wherein the cellular response is a
change in a membrane potential, the detectable output will in
general be said membrane potential. The membrane potential may be
determined using any suitable method such as applying a fluorescent
molecule to cells that distribute over the membrane dependent upon
the membrane potential. Examples of such compounds are DiBAC,
various ANEP dyes, JC-1 and JC-9 (Molecular Probes). For example,
JC-1 and JC-9 are cationic dyes that exhibit potential-dependent
accumulation in mitochondria leading to a shift in fluorescence
emission from green to red. Thus mitochondrial depolarization may
for example be determined by decrease in red/green fluorescence
intensity ratio (see also product information from Molecular
Probes). ANEP dyes are in particularly useful for detection of
changes in membrane potential. The fluorescence can be read for
instance by a fluorescence microscope, a fluorescence plate-reader,
a FACS, or a FABS instrument.
[0273] In embodiment of the invention wherein the cellular response
is change in morphology, the detectable output will in general be
the morphology of the cell. The morphology may be observed using
any suitable method for example by the aid of a microscope, using a
FACS or FABS.
[0274] In embodiments of the invention where the cellular response
is change in an interaction between two cellular proteins, the
detectable outout may for example be BRET or FRET, which is
detectable by determining the occurrence of fluorescence of a given
wavelength. BRET or FRET may for example be detected using a FABS,
FACS, fluorescent microscope or any other equipment useful for
detection of fluorescence.
[0275] In embodiments of the invention wherein the reporter system
is proximity ligation the detectable output is dependent on the
detectable label used to label the amplified DNA reporter sequence.
In embodiments, wherein the DNA reporter sequence is amplified
using fluorescently labelled primers, the detectable output will be
said fluorescent label, which may be detected using a FABS, FACS,
fluorescent microscope or any other equipment useful for detection
of fluorescence.
[0276] Depending on the detectable output, it will frequently be an
advantage to fix cells prior to detecting said detectable output.
However, in some embodiments of the invention it is preferred that
the cells are not fixed. Cells may be fixed according to any useful
protocol (see also definitions herein above).
Selection
[0277] The methods according to the invention involves screening
resin beads for beads comprising cells meeting at least one
predetermined selection criterion, wherein said selection criterion
is linked directly or indirectly to said detectable output. Hence,
the selection criterion will be dependent on the detectable
output.
[0278] For example the predetermined selection criterium may be a
quantitative criterium, such as a quantitative level of
bioluminescence above or below a specific threshold value.
[0279] In embodiments of the invention, wherein the detectable
output is fluorescence or the detectable output may be linked to a
fluorescent signal, then the predetermined selection criterion
could be any fluorescence property. For example, the selection
criterion could be intensity of said fluorescence above or below a
predetermined threshold value or emission of light of a specific
wavelength or absorption of light of a specific wavelength or
intensity of emitted light of a specific wavelength above or below
a predetermined threshold value. The selection criterion could also
be based on Fluorescence lifetime and/or fluorescence polarization
The selection criterion could also be a specific localisation of
the fluorescent signal, such as intensity of a fluorescent signal
in a specific cellular compartment above or below a predetermined
threshold value. The selection criterion could also be a
predetermined change in fluorescence lifetime or in fluorescence
polarization. Fluorescence intensity and/or localisation may for
example be determined using image processing and/or image analysis,
a fluorescence microscope, FACS, FABS or fluorescence plate
reader.
[0280] In one embodiment of the invention the selection criterion
is high fluorescence intensity. This may for example be the case,
when the cellular response is activation of a signal transduction
pathway and the reporter system comprises a gene encoding a
fluorescent protein, where activation of the signal transduction
pathway leads to increased expression of said gene. This may also
be the case when the cellular response is establishment of
interaction between two cellular proteins, wherein the reporter
system is a FRET or BRET reporter system. After release of a
proportion of the library members to be tested, resin beads may be
selected using a method comprising the steps of: [0281] 1.
Determining the fluorescence intensity of positive control resin
beads and setting this fluorescence intensity to 100% [0282] 2.
Determining the fluorescence intensity of negative control resin
beads and setting this fluorescence intensity to 0% [0283] 3.
Selecting resin beads having a fluorescence intensity corresponding
to at least 5%, preferably at least 10%, more preferably at least
20%, even more preferably at least 30%, such as at least 40%, for
example at least 50%, such as at least 60%, for example at least
70&, such as at least 80%, for example at least 90%, such as in
the range of 5 to 100%, for example in the range of 10 to 100%,
such as in the range of 20 to 100%, for example in the range of 30
to 100%, such as in the range of 40 to 100%, for example in the
range of 50 to 100%.
[0284] The positive control may for example be a resin bead (or
optionally several resin beads kept in a separate container or
well) comprising a compound known to influence the cellular
response. By way of example, if the cellular response is activation
of an intracellular signal transduction pathway, then the positive
control may be a resin bead comprising a known compound that
stimulate one or multiple components of said intracellular signal
transduction pathway. The positive control signal is then obtained
after release from the resin of the compound. Alternatively the
positive control may be a resin bead comprising a known ligand of a
receptor activating the signal transduction pathway, for example a
naturally occurring ligand. The negative control may be a resin
bead (or optionally several resin beads kept in a separate
container or well) optionally comprising a cell adhesion compound,
but otherwise comprising no library member or other test
compound.
[0285] In another embodiment of the selection criterion is low
fluorescence. This may for example be the case, when the cellular
response is inhibition of a signal transduction pathway and the
reporter system comprises a gene encoding a fluorescent protein,
where an active signal transduction pathway leads to expression of
said gene. This may also be the case when the cellular response is
disruption of interaction between two cellular proteins, wherein
the reporter system is a FRET or BRET reporter system. After
release of a proportion of the library members to be tested, resin
beads may be selected using a method comprising the steps of:
[0286] 1. Determining the fluorescence intensity of positive
control resin beads and setting this fluorescence intensity to 0%
[0287] 2. Determining the fluorescence intensity of negative
control resin beads and setting this fluorescence intensity to 100%
[0288] 3. Selecting resin beads having a fluorescence intensity
corresponding to at least 5%, preferably at least 10%, more
preferably at least 20%, even more preferably at least 30%, such as
at least 40%, for example at least 50%, such as at least 60%, for
example at least 70&, such as at least 80%, for example at
least 90%, such as in the range of 5 to 100%, for example in the
range of 10 to 100%, such as in the range of 20 to 100%, for
example in the range of 30 to 100%, such as in the range of 40 to
100%, for example in the range of 50 to 100%.
[0289] The positive control may for example be a resin bead(s)
comprising a compound known to influence the cellular response. By
way of example, if the cellular response is inhibition of an
intracellular signal transduction pathway, then the positive
control may be a resin bead(s) comprising a compound known to
inhibit one or multiple components of signal transduction pathway.
The control signal is then obtained after release from the resin of
the compound. Alternatively the positive control may be a resin
bead comprising a known antagonist of a receptor known to activate
said signal transduction pathway. If the cellular response is
induction of apoptosis, then the positive control may be a resin
bead comprising a compound known to induce apoptosis The negative
control may be a resin bead optionally comprising a cell adhesion
compound, but otherwise comprising no library member or other test
compound.
[0290] One method of selecting resin beads using FABS is
illustrated in FIG. 1A.
[0291] In one preferred embodiment selection is performed manually
with the aid of a fluorescence microscope. In this embodiment the
fluorescence intensity or other fluorescence properties are judged
manually.
[0292] When the selection criterion is fluorescence intensity of
localisation, the resin beads may also be analysed using a plate
reader or image acquisition. An example of such an analysis is
given in FIG. 1B.
[0293] If the selection criterion is localisation, then resin beads
are generally analysed by a fluorescence or imaging microscope.
Said microscope may optionally be equipped with a micromanipulator
capable of picking out single beads. Resin beads are scanned for
cells where the fluorescence signal is located at the desired
intracellular location and these resin beads are selected. The
selection may be manually or it may be automated.
[0294] In embodiments of the invention, wherein the detectable
output is light emission or the detectable output may be linked to
a light signal, then the predetermined selection criterion could be
any property of the light. For example the selection criteria could
be light intensity above or below a predetermined threshold value.
Light can be detected for example by the eye, in a microscope, and
if the light is emitted via bioluminescence it can be measured by a
luminometer
[0295] In embodiments of the invention, wherein the detectable
output is a radioactive signal or the detectable output may be
linked to a radioactive signal, then the selection criterion could
be any property of said radioactive signal, such as intensity above
or below a predetermined threshold value or localisation of the
radioactive signal.
[0296] In embodiments of the invention, wherein the detectable
output is a colour signal or the detectable output may be linked to
a colour signal, then the selection criterion could be any property
or said colour signal. For example the predetermined selection
criterion could be a colour intensity above or below a specific
threshold value or it could be a specific colour. The colour signal
could be detected using any suitable calorimetric method, such as a
spectrophotometer or a microscope.
[0297] Resin beads comprising cells meeting at least one selection
criterion, such as any of the selection criteria mentioned herein
above are selected. In certain embodiments of the invention resin
beads comprising cells meeting at least two, for example 2, such as
3, for example 4, such as in the range of 5 to 10, for example of
in the range of 10 to 25 selection criteria are selected.
[0298] It is also possible within the present invention to select
resin beads comprising cells meeting one or more predetermined
selection criteria and subsequently to subject said beads to one or
more additional selection rounds, wherein resin beads comprising
cells meeting one or more additional selection criteria are
selected.
[0299] Resin beads meeting said at least one predetermined
selection criteria may be selected by manually sorting for example
with the aid of a microscope, or for example by sorting by
fluorescence or by colour or by morphology depending on the
detectable output and the selection criterion. Positive beads may
be picked directly under the microscope, such as under a
fluorescence microscope for example manually or with the aid of a
micromanipulator. Frequently, in the range of 100 to 1,000,000, for
example in the range of 1000 to 100,000, such as in the range of
5000 to 50,000 resin beads may be placed on a suitable surface,
such as in a dish or on a coverglass and subsequently examined by
microscopy. Alternatively, the sorting process may be automated
with the use of specially designed, commercially available bead
sorters (Union Biometrica, Sommerville, Mass.) and detecting for
example fluorescence intensity (Meldal, 2002, Biopolymers, 66:
93-100). In general, resin beads can be sorted at a rate of up to
100 beads per second, or even faster depending on the equipment
used and its reading capacity. A range of about 5-30 beads per
second is generally used with known instruments. Slower rates may
be used to increase accuracy, however any suitable rate may be used
with the present invention, such as much higher rates. Preferred,
is a rate where only one resin bead passes through the detector at
a time. It is also comprised within the present invention to select
resin beads using a plate reader. In general in the range of 1 to
1000, such as 10 to 500, for example 50 to 100 resin beads are
placed in each well of a multiter plate and analysed. Beads from
positive wells may then be further examined.
[0300] In one embodiment of the invention resin beads may be
selected by comparing the detectable output, with the detectable
output generated by control resin beads, for example positive
and/or negative control resin beads. Positive control resin beads
are beads comprising a compound capable of inducing the desired
cellular response, whereas negative control resin beads comprises
no such compound. By way of example, if the cellular response is
activation of an intracellular signal trans-duction pathway with a
known natural ligand, the positive control resin bead may comprise
said ligand, whereas the negative control resin bead comprises no
compound except optionally a cell adhesion compound.
[0301] If the detectable output is a quantifiable signal, then
resin beads may be selected, comprising cells where the detectable
output is higher or lower than the detectable output from cells
attached to the positive or negative control resin bead. By way of
example, if the detectable output is fluorescence intensity, then
resin beads comprising cells displaying a fluorescence intensity
which is higher than the negative control and lower than the
positive control could for example be selected.
[0302] Non-limiting examples of methods of selecting resin beads
are illustrates in FIGS. 1 and 2.
Identification of Compound
[0303] Once a resin bead has been selected, the compound that is
remaining after partial release on said bead may be identified.
Preferably, only one resin bead is used at a time. Thus if said
resin bead only comprises one library member in one or more copies,
then only one compound is identified at a time.
[0304] The process for identification of the library member depends
on the type of library used. For a library of primarily oligomeric
compounds, the library member can be analysed by Mass Spectroscopy
(MS), particularly if the library was synthesized in such a way
that the synthetic history of the compound is captured, for
example, using a capping procedure to generate fragments of the
compound that differ in mass by one building block (see, for
example, Youngquist et al., 1995, J. Am. Chem. Soc., 117: 3900-06).
This capping procedure is most efficient when the cap and the
building block are reacted at the same time. The capping agent can
be any class of compound that has at least one functional group in
common with the building block used to generate the oligomer, so
that both the capping agent and the building block can react when
added to the resin in an appropriate ratio. Alternatively, the
capping agent can have two functional groups in common with the
building block where one of the groups in common, such as the group
in the building block that is used for the elongation of the
oligomer, is orthogonally protected. For example, in a synthesis of
a peptide using the Fmoc strategy, the capping agent could be the
same as the building block but with a Boc group protecting the
reactive amine instead of the Fmoc group (see St. Hilaire et al.,
1998, J. Am. Chem. Soc., 120: 13312-13320). In another example, if
the building block is a protected haloamine, the capping agent
could be the corresponding alkylhalide.
[0305] Where the library is synthesized by parallel synthesis (a
parallel array), the compound can be identified simply by the
knowledge of what specific reaction components were reacted in a
particular compartment. The structure can be confirmed by cleavage
of a small portion of compound from the solid support and analyzed
using routine analytical chemistry methods such as infrared (IR),
nuclear magnetic resonance (NMR), mass spectroscopy (MS), and
elemental analysis. For a description of various analytical methods
useful in combinatorial chemistry, see: Fitch, 1998-99, Mol.
Divers., 4: 39-45; and Analytical Techniques in Combinatorial
Chemistry, M. E. Swartz (Ed), 2000, Marcel Dekker: New York.
[0306] In a preferred embodiment however the library has been
synthesised by a split-mix approach where the precise structure of
the compound of a specific bead is unknown. In this embodiment, the
library member can be identified using a variety of methods. The
compound may be cleaved off the resin bead, for example by cleaving
the cleavable linker and then analyzed using IR.sup.X, MS, or NMR.
For NMR analysis, larger beads containing approximately 5 nmoles of
material are preferably used for the acquisition of 1-dimensional
(1-D) and 2-dimensional (2-D) NMR spectra. Furthermore, these
spectra can be attained using high-resolution MAS NMR (magic angle
spinning nuclear magnetic resonance) techniques. Alternatively,
high resolution-MAS NMR spectra can be acquired while the ligand is
still bound to the solid support, as described for example, in
Gotfredsen et al., 2000, J. Chem. Soc., Perkin Trans., 1: 1167-71.
The compound may also be identified by release of the compound and
fragmentation by MS-MS in MALDI or electrospray mode.
[0307] Frequently, resin beads used for library synthesis contain
about 100 to 500 pmoles of material, which is generally
insufficient for direct analysis using NMR techniques. In such
situations, the libraries can be synthesised with special encoding
to facilitate identification of the library member. For a review of
encoding strategies employed in combinatorial chemistry see: Barnes
et al., 2000, Curr. Opin. Chem. Biol., 4: 346-50. Most coding
strategies include the parallel synthesis of the encoding molecule
(for example, DNA, PNA, or peptide) along with the library
compounds. This strategy requires a well-planned, time consuming,
orthogonal protecting group scheme. Furthermore, the encoding
molecule itself can sometimes influence the cell leading to false
positives. Alternatively, the library members can be encoded using
radiofrequency tags or using optical encoding, such as quantum dot
encoding, spherical encoding or distance encoding. These methods
alleviates the problem of false positives stemming from the coding
tags, but is generally only useful for small libraries in a
one-bead-one-compound system due to the sheer bulk of the
radiofrequency tag. Alternatively, single beads can be analyzed in
a non-destructive manner using infrared imaging. This method gives
limited information and while useful for prescreening, is not
recommended for conclusive structural determination.
[0308] In a preferred embodiment of the invention the library
member(s) comprised within selected resin beads are identified
using mass spectrometry (MS). MS can be used alone to identify the
library member. The library member can be cleaved from the resin
bead, the molecular mass determined, and subsequently fragmented
into subspecies to conclusively determine the structure by
combination with knowledge of contained structures in the library.
MS-based methods of compound identification are useful in this
invention, as they require very little material, and can utilise
pico- to femtomole amounts of compound.
[0309] After identification of the compound it may be desirable to
confirm the activity of said compounds by further in vitro and/or
in vivo assays. For example, resin beads comprising the identified
compound and optionally an adhesion compound may be synthesized and
the cellular response confirmed. It is also possible to test
identified compounds in in vitro assays in the absence of beads.
Cells may for example be grown directly in a tissue culture dish,
flask or coverglass and the identified compound can be added
directly to the medium of said cells. If several reporter systems
are available for the particular cellular response then preferably
several different reporter assays may be tested in vitro, in order
to identify very useful compounds. By way of example, if the
cellular response is induction of apoptosis, then for example
activity of caspases, cell growth/cell death and/or interaction
between Smac and inhibitors of apoptosis may be tested.
Multiplexing
[0310] The methods disclosed by the present invention may also be
used in multiplexing methods.
[0311] For example, the methods may be used to identify compounds
modifying at least two cellular responses, such as 2, for example
3, such as 4, for example in the range of 5 to 10, such as in the
range of 10 to 25 cellular responses.
[0312] In such methods step c) of the method outlined above (see
the section "Summary of the invention") preferably involves
screening resin beads for beads comprising cells meeting at least
two, such as 2, for example 3, such as 4, for example in the range
of 5 to 10, such as in the range of 10 to 25 predetermined
selection criteria, wherein each selection criterion is preferably
related to a different detectable output.
[0313] In such a method more than one kind of cell may be attached
to each resin bead and the different cellular responses may be
detected in different kinds of cells. For example, a first cell
line comprising a first reporter system linked to a first cellular
response and a second cell line comprising a second reporter system
linked to a second cellular response and optionally additional cell
line(s) comprising additional reporter system(s) linked to
additional cellular response(s) may all be attached to a single
bead. Resin beads comprising cells meeting selection criteria
linked to all the different reporter systems may then be
selected.
[0314] Depending on the detectable outputs, said detectable output
may be determined using any of the methods described herein above.
In one preferred embodiment at least two detectable outputs are
fluorescent outputs, preferably of different excitation and/or
emmision. Thus resin beads meeting said at least two selection
criteria may be selected in one step using a FABS with at least 2
channels in both excitation and emmision. Similarly, more than two
different fluorescent properties may be selected for in an suitable
FABS. The at least two detectable outputs may be in the same cell
line or they may be in different cell lines.
[0315] Examples of multiplexing methods are illustrated in FIGS. 2A
and 2B.
EXAMPLES
Example 1
General Methods for Solid Phase Peptide Synthesis (SPPS)
General for Chemical Synthesis:
[0316] All chemicals described, commercially available and used
without further purification. All solvents were HPLC-grade.
PEGA-resins were purchased from VersaMatrix A/S, Copenhagen. Each
washing step lasted 2 min unless otherwise stated. Purifications
were performed on a standard reverse phase HPLC using gradients of
acetonitrile-Water with various amounts of TFA.
Coupling of HMBA Linker to PEGA-Resin:
[0317] Dry PEGA-resin was swelled in DCM and washed with DMF
(3.times.). 3.0 eq. HMBA, 2.9 eq. TBTU and 3.0 eq. NEM were mixed
in appropriate DMF and allowed to react for 10 min. The mixture was
added to resin and after 2 h the resin was washed with DMF
(6.times.), DCM (6.times.) and lyophilised.
General Procedure for Coupling of Amino Acid to HMBA-Linker:
[0318] Dry PEGA-resin with HMBA-linker was swelled in DCM. 3.0 eq.
Fmoc-protected amino acid, 2.25 eq. Melm and 3.0 eq. MSNT were
mixed in appropriate amount of DCM and added to resin. After 1 h
the resin was washed with DCM (3.times.) and the coupling was
repeated as above once. After coupling for 1 h the resin was washed
with DCM (6.times.), DMF (6.times.), DCM (6.times.) and
lyophilised.
General SPPS Coupling Procedure:
[0319] The terminal amino acid on the resin was Fmoc-deprotected by
treatment with 20% piperidine in DMF (1.times.2 min+1.times.18 min)
followed by washing with DMF (6.times.). 3.0 eq. Fmoc-protected
amino acid, 2.9 eq. TBTU and 3.0 eq. NEM were mixed in appropriate
amount of DMF and allowed to react for 10 min. The mixture was
added to the resin and after 2 h the resin was washed with DMF
(6.times.).
I. General Side Chain Deprotection Procedure:
[0320] Dry PEGA-resin with linker, usually acid stable linker(s)
and peptide or optionally compound was swelled in H.sub.2O and the
side chains was deprotected with 95% TFA (aq) (2.times.15 min). (If
Pmc groups were present cleavage time was 6 h). The resin was
washed with H.sub.2O until washing water had pH=5-7. The resin was
then washed with DMF (6.times.), DCM (6.times.) and
lyophilised.
General HMBA Cleavage Procedure:
[0321] Dry PEGA-resin with HMBA linker and attached compound was
swelled in water and NaOH (aq.) 0.1 M was added. After 2 h HCl
(aq.) 0.1 M was used for neutralisation and then AcN was added
until the H.sub.2O/AcN ratio was 1:1 by volume. The resin was
filtered off and the liquid was used direct for RP-HPLC or/and
Q-TOF MS analysis.
[0322] The above general procedures are used for solid phase
peptide synthesis in the following examples unless otherwise
specified.
Example 2a
Screening of Adhesion Peptide Library
[0323] Approx. 100 adhesion peptide library beads were mixed with
1.times.10E6 cells (BHK, CHO, U2OS, Hek) in each well of a Falcon
12 well plate using 2 ml growth medium. The adhesion peptide
library was prepared according to the general methods for solid
phase peptide synthesis outlined above. The library consisted of
heptamers of D-amino acids. The peptide library beads were PEGA
beads each coupled to a potential adhesion peptide. The cells and
beads were mixed gently every 15 min for 2 hrs. Supernatant with
nonattached cells were removed and new growth medium added.
Cells/beads were incubating for another 16 hrs. (37.degree. C., 5%
CO.sub.2). Cell adhesive beads were identified using a microscope
with 10.times. objective and positive beads were transferred to a
filter paper (to suck off medium). Peptides were identified by
amino acid sequencing. Examples of useful peptides are given in
table 2.
Example 2b
Identification of an Adhesion Peptide with Low Absorption of
Fluorescent Components from Growth Medium and High Adhesion
Properties
[0324] An adhesion D-amino peptide library was synthesized (500.000
members) as described above in Example 2a and screened for low
fluorescence/high adherence properties. This was done in 4
steps:
1) Selection of low fluorescent beads by Fluorescence Activated
Bead Sorting (FABS).
[0325] The 500.000 member adhesion peptide library was FABSorted
and 150.000 low fluorescent beads were isolated.
2) Selection of beads with good cell adhesion properties.
[0326] The 150.000 low fluorescent beads were incubated with GFP
expressing U2OS cells followed by FABS sorting for high
fluorescence (high cell adhesion). 536 beads were isolated.
3) Identification and isolation of beads with high Hek293 cell
adherence properties.
[0327] The 536 beads were cleared for U2OS cells and incubated with
GFP expressing Hek293 cells. 47 beads with high cell adhesion
properties were isolated using a fluorescence microscope.
4) Sequence elucidation and re-synthesis of selected peptides.
[0328] 22 peptides were sequenced and six of them were
re-synthesized. Based on Structure-Activity of the six peptides,
four additional ones were synthesized. The peptide defined by SEQ
ID 35 showed the best overall performance.
Example 3
Synthesis of Peptides Binding to the ML-IAP BIR Domain
[0329] This example describes the preparation of 6 peptides known
to interact with the BIR domain of ML-IAP (livin). The syntheses
were performed according to the "general SPPS coupling Procedure"
above.
Synthesis of H-Ala-Val-Pro-Ill-Ala-Gln-Lys-Ser-Glu-OH (X),
H-Ala-Val-Pro-Phe-Ala-Val-Lys-Ser-Glu-OH (X), and
H-Ala-Glu-Ala-Val-Pro-Trp-Lys-Ser-Glu-OH (X)
[0330] Coupling 1-3: HMBA modified PEGA 800 beads (600 mg,
Versabeads A-800, 315-500 .mu.m, 0.32 mmol NH.sub.2/g) were coupled
under standard MSNT coupling conditions (Example 1) with
Fmoc-Glu(O.sup.tBu)OH followed by standard TBTU coupling of
Fmoc-Ser(O.sup.tBu)OH and FmocLys(Boc)OH.
[0331] Coupling 4-8: The resin was separated into three 200 mg
portions and three parallel couplings were performed with the
respective amino acids according to the "general SPPS coupling
procedure in Example 1 (Trp, Glu and Gln were side chain protected
as the N-Boc, O-.sup.tBu and N-trityl derivatives respectively).
After the last coupling side chain deprotection was performed
according to the general procedure (Example 1). The peptides were
detached according to the general HMBA cleavage procedure (Example
1) and the peptides purified with preparative HPLC.
[0332] H-Ala-Val-Pro-Ill-Ala-Gln-Lys-Ser-Glu-OH (X): Yield after
purification: 30 mg (>98% pure according to HPLC). MALDI-MS
Calcd. (M+H).sup.+ 942.5; Found 942.7. NMR data were in accordance
with the predicted structure.
[0333] H-Ala-Val-Pro-Phe-Ala-Val-Lys-Ser-Glu-OH (X): Yield after
purification: 32 mg (>98% pure according to HPLC). MALDI-MS.
Calcd. (M+H).sup.+ 947.5; Found 948.0. NMR data were in accordance
with the predicted structure.
[0334] H-Ala-Glu-Ala-Val-Pro-Trp-Lys-Ser-Glu-OH (X): Yield after
purification: 38 mg (>98% pure according to HPLC). MALDI-MS.
Calcd. (M+H).sup.+ 1016.5; Found 1016.5. NMR data were in
accordance with the predicted structure.
Synthesis of H-Ala-Val-Pro-Ile-OH (X)
[0335] Synthesis performed according to the general SPPS procedure
on 200 mg of HMBA modified PEGA 800 beads. Yield after HMBA
cleavage and purification: 17 mg. (>98% pure according to HPLC).
ES-MS. Calcd. (M+H).sup.+ 399.2. Found 399.2. NMR data were in
accordance with the predicted structure.
Synthesis of H-Ala-Glu-(1-amino-cyclopentanecarboxyl)-Phe-OH
(X)
[0336] Synthesis performed according to the general SPPS procedure
on 200 mg of HMBA modified PEGA 800 beads. Yield after HMBA
cleavage and purification: 22 mg. (>98% pure according to HPLC).
ES-MS. Calcd. (M+H).sup.+ 477.2. Found 477.2. NMR data were in
accordance with the predicted structure.
Synthesis of H-Ala-Glu-(azetidine-1-carboxyl)-(homo-Phe)-OH (X)
[0337] Synthesis performed according to the general SPPS procedure
on 200 mg of HMBA modified PEGA 800 beads. Yield after HMBA
cleavage and purification: 21 mg. (>98% pure according to HPLC).
ES-MS. Calcd. (M+H).sup.+ 463.2. Found 463.2. NMR data were in
accordance with the predicted structure.
[0338] The structures of the 6 synthesized peptides are given
below.
##STR00002## ##STR00003##
Example 4
Synthesis of a 44.000 Member Tetrapeptide Library on Beads with
Fixed Adhesion Peptide
[0339] This example describes the preparation of a library of
tetrapeptides (44.000 members) on resin beads containing a "cell
adhesive peptide". This "two compound one bead" (TCOB) library may
be used for in vivo screening of the tetrapeptides for e.g.
antagonists action on the ML-IAP receptor.
FmocLys(Fmoc)/BocVal (.about.1:1) Modification of Pega 1900
Beads
[0340] Dry Versabeads A-1900 (315-500 .mu.m, 0.2 mmol NH2/g)(1.20
g) were dissolved in dry DMF (10 mL) and coupled with
FmocLys(Fmoc)OH (0.24 mmol, 1 eq) and BocValOH (0.48 mmol, 2 eq)
using the general SPPS coupling procedure.
Fmoc to Alloc Transformation of Lys Protecting Groups
[0341] The beads were Fmoc deprotected using the standard
procedure. The beads were washed several times with DCM
(-10.degree. C.) and finally added DCM (10 mL) with the same
temperature. DIPEA (0.9 mL) was added followed by dropwise addition
of AllocCl (allyloxycarbonylchloride) (0.26 mL). The reaction
mixture was kept under N.sub.2 and stirred at RT for 2 h (Kaiser
test was negative). The resin was washed with DCM (6.times.), DMF
(6.times.) and DCM (6.times.) and lyophilised overnight.
Attachment of Photolabile Linker (PLL) to Val
[0342] The dried beads (.about.1.2 g) were Boc deprotected with 30%
TFA in DCM (2+ 45 min). The resin was washed with DCM (3.times.),
20% pip in DMF (2.times.) and DMF (6.times.). The photo cleavable
linker (4-[(1-Fmoc-aminoethyl)-2-methoxy-5-nitrophenoxy]butanoic
acid) (187 mg, 0.36 mmol, 3 eq) was dissolved in DMF followed by
NEM (100 .mu.L, 0.72 mmol, 6 eq) and TBTU (111 mg, 0.35 mmol, 2.9
eq). After stirring for 5 minutes the mixture was added to the
beads and left with occasional stirring for 3.5 h. (Kaiser test
neg). Wash with DMF (10.times.), DCM (6.times.) and lyophilised
overnight in the dark.
Preparation of Tetrapeptide Library with 44.000 Members
[0343] The beads containing the Fmoc protected photo linker (1.00
g) were placed in a custom made 20 well synthesizer. Four couplings
using the general SPPS TBTU coupling procedure were performed using
a split and mix protocol. Amino acids
(20.times.20.times.10.times.10) used for couplings are given in
Tables 3 to 6. For the 3.sup.rd and 4.sup.th coupling each amino
acid is added to 2 wells. Protection groups are left on the
peptides after end couplings.
TABLE-US-00003 TABLE 3 Amino acids used for the 1st coupling.
Member number 1 2 3 4 Structure ##STR00004## ##STR00005##
##STR00006## ##STR00007## Mw Mol. Wt.: 405.42 Mol. Wt.: 405.42 Mol.
Wt.: 455.43 Mol. Wt.: 412.44 micromol 10 10 10 10 Weight (mg) 4.05
4.05 4.55 4.12 Member number 5 6 7 8 Structure ##STR00008##
##STR00009## ##STR00010## ##STR00011## Mw Mol. Wt.: 412.44 Mol.
Wt.: 443.51 Mol Wt.: 393.46 Mol Wt.: 447.48 micromol 10 10 10 10
Weight (mg) 4.12 4.43 3.93 4.47 Member number 9 10 11 12 Structure
##STR00012## ##STR00013## ##STR00014## ##STR00015## Mw Mol. Wt.:
359.37 Mol. Wt.: 351.4 Mol. Wt.: 365.42 Mol. Wt.: 610.7 micromol 10
10 10 10 Weight (mg) 3.59 3.51 3.65 6.11 Member number 13 14 15 16
Structure ##STR00016## ##STR00017## ##STR00018## ##STR00019## Mw
Mol. Wt.: 411.45 Mol. Wt.: 387.43 Mol. Wt.: 417.45 Mol. Wt.: 421.87
micromol 10 10 10 10 Weight (mg) 4.11 3.87 4.17 4.22 Member number
17 18 19 20 Structure ##STR00020## ##STR00021## ##STR00022##
##STR00023## Mw Mol. Wt.: 413.47 Mol. Wt.: 427.49 Mol. Wt.: 648.77
Mol. Wt.: 355.41 micromol 10 10 10 10 Weight (mg) 4.13 4.27 6.49
3.55
TABLE-US-00004 TABLE 4 Amino acids used for the 2nd coupling.
Member number 1 2 3 4 Structure ##STR00024## ##STR00025##
##STR00026## ##STR00027## Mw Mol. Wt.: 337.37 Mol. Wt.: 337.37 Mol.
Wt.: 355.41 Mol. Wt.: 323.34 micromol 10 10 10 10 Weight (mg) 3.37
3.37 3.55 3.23 Member number 5 6 7 8 Structure ##STR00028##
##STR00029## ##STR00030## ##STR00031## Mw Mol. Wt.: 351.4 Mol. Wt.:
365.42 Mol. Wt.: 351.4 Mol. Wt.: 413.47 micromol 10 10 10 10 Weight
(mg) 3.51 3.65 3.51 4.13 Member number 9 10 11 12 Structure
##STR00032## ##STR00033## ##STR00034## ##STR00035## Mw Mol. Wt.:
405.42 Mol. Wt.: 387.43 Mol. Wt.: 393.46 Mol. Wt.: 610.7 micromol
10 10 10 10 Weight (mg) 4.05 3.87 3.93 6.11 Member number 13 14 15
16 Structure ##STR00036## ##STR00037## ##STR00038## ##STR00039## Mw
Mol. Wt.: 339.39 Mol. Wt.: 353.41 Mol. Wt.: 411.45 Mol. Wt.: 427.49
micromol 10 10 10 10 Weight (mg) 3.39 3.53 4.11 4.27 Member number
17 18 19 20 Structure ##STR00040## ##STR00041## ##STR00042##
##STR00043## Mw Mol. Wt.: 648.77 Mol. Wt.: 397.46 Mol. Wt.: 477.51
Mol. Wt.: 311.33 micromol 10 10 10 10 Weight (mg) 6.48 3.97 4.77
3.11
TABLE-US-00005 TABLE 5 Amino used for the 3rd coupling. Member
number 1 2 3 4 Structure ##STR00044## ##STR00045## ##STR00046##
##STR00047## Mw Mol. Wt.: 339.39 Mol. Wt.: 353.41 Mol. Wt.: 353.41
Mol. Wt.: 397.46 micromol 10 10 10 10 Weight (mg) 3.39 3.53 3.53
3.97 Member number 5 6 7 8 Structure ##STR00048## ##STR00049##
##STR00050## ##STR00051## Mw Mol. Wt.: 411.45 Mol. Wt.: 596.67 Mol.
Wt.: 425.47 Mol. Wt.: 610.7 micromol 10 10 10 10 Weight (mg) 4.11
5.97 4.25 6.11 Member number 9 10 11 12 Structure ##STR00052##
##STR00053## Mw Mol. Wt.: 477.51 Mol. Wt.: 440.49 micromol 10 10
Weight (mg) 4.78 4.40
TABLE-US-00006 TABLE 6 Amino acids used for the 4th coupling.
Member number 1 2 3 4 Structure ##STR00054## ##STR00055##
##STR00056## ##STR00057## Mw Mol. Wt.: 175.18 Mol. Wt.: 189.21 Mol.
Wt.: 217.26 Mol. Wt.: 261.31 micromol 10 10 10 10 Weight (mg) 3.50
3.78 4.35 5.22 Member number 5 6 7 8 Structure ##STR00058##
##STR00059## ##STR00060## ##STR00061## Mw Mol. Wt.: 275.34 Mol.
Wt.: 203.24 Mol. Wt.: 485.66 micromol 10 10 10 10 Weight (mg) 5.51
4.02 4.06 9.71 Member number 9 10 11 12 Structure ##STR00062##
##STR00063## Mw Mol. Wt.: 215.25 Mol. Wt.: 535.7 micromol 10 10
Weight (mg) 4.30 10.71 indicates data missing or illegible when
filed
Attachment of "Adhesion Peptide":
[0344] Adhesion peptides were synthesized on the alloc-protected
lysine. One batch of the library was attached adhesion peptide A,
and a second batch with adhesion peptide B.
Adhesion peptide A:
(Boc-D-Ala-D-Arg(Pmc)-D-Lys(Boc)-D-Arg(Pmc)-D-Ile-D-Arg(Pmc)-D-Gln(Trt)-G-
ly-)
[0345] Alloc deprotection of lysine residues: The library beads
were treated under N.sub.2 with 3 eq Pd(PPh.sub.3).sub.4 in 10 mL
degassed CHCl.sub.3 containing 5% HOAc and 2.5% NEM for 2 h at RT.
The resin was washed with CHCl.sub.3 (6.times.), DMF containing
0.5% sodium diethyldithiocarbamat and 0.5% DIPEA (2.times.), and
DMF (10.times.).
[0346] Coupling: The adhesive peptide was synthesized directly
(stepwise) on the library beads using the general SPPS coupling
procedure.
[0347] Alternative method: The purified peptide
(Boc-D-Ala-D-Arg(Pmc)-D-Lys(Boc)-D-Arg(Pmc)-D-Ile-D-Arg(Pmc)-D-Gln(Trt)-L-
-Gly-OH) (3 eq) was coupled to the lysine NH.sub.2 groups using the
general SPPS coupling procedure.
[0348] Adhesion peptide B:
(Fmoc-D-Arg(Pmc)-D-Ile-D-Arg(Pmc)-D-Gln(Trt)-D-Arg(Pmc)-: Analogous
to synthesis of adhesion peptide A.
Adhesion Peptide B:
(Fmoc-D-Arg(Pmc)-D-Gln(Trt)-D-Arg(Pmc)-D-Ile-D-Arg(Pmc)-
[0349] Analogous to synthesis of adhesion peptide A.
Deprotection of Adhesion and Library Peptides
[0350] Final deprotection of protecting groups was performed
according to the general procedure described above yielding the
TCOB library ready for testing
Example 5a
Preparation of TAT Appended "Tetrapeptide" Library
[0351] The peptide library of example 4 is modified to include a
TAT sequence appended to the C-terminal end of the peptide library
in order to enhance the cell permeation properties of the
tetrapeptides.
Synthesisis of TAT Sequence (GGYGRKKRRQRRR) on Val Residues.
[0352] Method A: Beads (1.00 g) containing FmocPLL-Val and
AllocLys(Alloc) (as prepared in example 3) are subjected to
stepwise SPPS using the general TBTU protocol giving the sequence
FmocGGYGRKKRRQRRR attached to the photo labile linker (Arg is side
chain protected as Pmc, Gln protected as Trt, Lys protected as Boc,
and Tyr as .sup.tBu).
[0353] Method B: Beads (1.00 g) containing FmocPLL-Val and
AllocLys(Alloc) (as prepared in example 3) are coupled to the side
chain protected and purified TAT sequence FmocGGYGRKKRRQRRR using
the general SPPS coupling procedure. Protection of side chains as
in method A.
Formation of TAT Appended Tetrapeptide Library
[0354] Using the FmocTAT-Val derivatized beads a tetrapeptide
library is build according to the description in Example 3. The
resulting library is derivatized with the cell adhesive peptide and
followingly all protection groups are removed as described in
example 4.
Example 5b
Preparation of Tetrapeptide Library with 44.000 Members with Base
Labile Adhesion Peptide
[0355] In certain preferred embodiments it might be advantageous to
be able to clear selected beads from both cells and adhesion
peptide before either analysis of active compound or further
processing. This example describes the synthesis of the above
described library on beads where the adhesion peptide is linked to
the bead via an HMBA linker.
Synthesis of FmocGly/AllocGly (Ratio .about.1:1) Modified PEGA 1900
Beads
[0356] PEGA 1900 beads (300-500 .mu.m, 0.24 mmol NH.sub.2/g) (4 g,
0.96 mmol.about.1 eq)) were treated with a 1:1 TBTU coupling
mixture of FmocGlyOH (2 eq) and AllocGlyOH (2 eq), NEM (17 eq) and
TBTU (3.8 eq). Coupling time 3.5 h. Beads washed 10.times. with
DMF.
Coupling of Holmes Photolinker to the "Core" Fmoc-Glycine
[0357] FmocGly/AllocGly beads were standard Fmoc deprotected with
20% piperidine in DMF leaving the Alloc glycine untouched. Then
standard TBTU coupling of the Holmes photolinker
(4-(1-aminoethyl-2-methoxy-5-nitrophenoxy)butanoic acid) to the
deprotected glycine (1 eq=0.48 mmol). After coupling, the beads
were washed with DMF and DCM and lyophilized.
[0358] Tetrapeptide Library Synthesis on the Photo Linker
[0359] Performed analogous to example 4
Alloc Deprotection of Second "Core" Glycine:
[0360] The beads from the 20 wells were all combined in a 50 mL
syringe and washed with CHCl.sub.3 (5.times.) and with Ar-degassed
CHCl.sub.3 containing 5% HOAc and 2.5% NEM (5.times.). A solution
of Pd(PPh.sub.3).sub.4 (3 eq, 0.72 mmol) in Ar-degassed CHCl.sub.3
containing 5% HOAc and 2.5% NEM (10 mL) was added to the beads and
after a few minutes bobling with Ar the syringe was sealed with
parafilm and left for 2 h. The beads were washed with CHCl.sub.3
(10.times.), DMF (10.times.), MeOH (10.times.), DMF (20% pip)
(2.times.), DMF (10.times.). Kaiser test was positive.
Attachment of HMBA Linker
[0361] According to standard procedure above.
MSNT Coupling of FmocGlyOH
[0362] According to standard procedure above.
TBTU Coupling of FmocLys(Fmoc)OH
[0363] According to standard procedure above.
Adhesion Peptide Synthesis
[0364] After deprotection of the lysine Fmoc groups performed
analogous to example 4.
Example 5c
Formation of a "Tetrapeptide" Library Linked Via an Internal Amide
Nitrogen
Synthesis of FmocGly/AllocGly (Ratio .about.1:1) Modified PEGA 1900
Beads
[0365] PEGA 1900 beads (300-500 .quadrature.m, 0.24 mmol
NH.sub.2/g) (4 g, 0.96 mmol .about.1 eq)) were treated with a 1:1
TBTU coupling mixture of FmocGlyOH (2 eq) and AllocGlyOH (2 eq),
NEM (17 eq) and TBTU (3.8 eq). Coupling time was 3.5 h. Beads were
washed 10.times. with DMF.
Coupling of Aldehyde Photolinker to the "Core" Fmoc-Glycine.
[0366] FmocGly/AllocGly beads were standard Fmoc deprotected with
20% piperidine in DMF leaving the Alloc glycine untouched. Then
standard TBTU coupling of the aldehyde photolinker
(4-(4-formyl-2-methoxy-5-nitrophenoxy)butanoic acid) to the
deprotected glycine (1 eq=0.48 mmol). After coupling, the beads
were washed with DMF and DCM and lyophilized.
TABLE-US-00007 TABLE 7 5 Phenylethylamines used for reductive
amination of aldehyde linker 1 2 3 4 5 ##STR00064## ##STR00065##
##STR00066## ##STR00067## ##STR00068## 121.18 g/mol 139.17 g/mol
139.17 g/mol 155.63 g/mol 151.21 g/mol d: 0.965 d: 1.061 d: 1.066
d: 1.119 d: 1.033
TABLE-US-00008 TABLE 8 Compounds used for BTC coupling Well number
1 2 3 4 Structure ##STR00069## ##STR00070## ##STR00071##
##STR00072## Mw (g/mol) 452.50 452.50 452.50 452.50 Mol .times.
10.sup.6 36 36 36 36 Weight (mg) 16.3 16.3 16.3 16.3 Well number 5
6 7 8 Structure ##STR00073## ##STR00074## ##STR00075## ##STR00076##
Mw (g/mol) 452.50 452.50 452.50 452.50 Mol .times. 10.sup.6 36 36
36 36 Weight (mg) 16.3 16.3 16.3 16.3 Well number 9 10 11 12
Structure ##STR00077## ##STR00078## ##STR00079## ##STR00080## Mw
(g/mol) 452.50 452.50 337.67 337.67 Mol .times. 10.sup.6 36 36 36
36 Weight (mg) 16.3 16.3 12.1 12.1 Well number 13 14 15 16
Structure ##STR00081## ##STR00082## ##STR00083## ##STR00084## Mw
(g/mol) 323.34 351.40 399.44 351.40 Mol .times. 10.sup.6 36 36 36
36 Weight (mg) 11.6 12.7 14.4 12.7 Well number 17 18 19 20
Structure ##STR00085## ##STR00086## ##STR00087## ##STR00088## Mw
(g/mol) 427.49 413.47 379.46 565.72 Mol .times. 10.sup.6 36 36 36
36 Weight (mg) 15.4 14.9 13.7 20.4
TABLE-US-00009 TABLE 9 Compounds used for acylation in wells 1-10.
1 2 3 4 5 ##STR00089## ##STR00090## ##STR00091## ##STR00092##
##STR00093## Mw: 132.59 Mw: 130.53 Mw: 154.60 Mw: 158.56 Mw: 146.62
d: 1.091 d: 1.324 d: 1.167 d: 1.342 d: 1.096 6 7 8 9 10
##STR00094## ##STR00095## ##STR00096## ##STR00097## ##STR00098##
Mw: 120.58 Mw: 134.61 Mw: 106.55 Mw: 104.54 Mw: 78.50 d: 0.989 d:
0.969 d: 1.017 d: 1.152 d: 1.104
TABLE-US-00010 TABLE 10 Compounds used for TBTU coupling Member
number 1 2 3 4 Structure ##STR00099## ##STR00100## ##STR00101##
##STR00102## Mw (g/mol) 353.41 379.46 393.48 411.45 Mol .times.
10.sup.6 36 36 36 36 Weight (mg) 12.7 13.7 14.1 14.8 Member number
5 6 7 8 Structure ##STR00103## ##STR00104## ##STR00105##
##STR00106## Mw (g/mol) 405.42 387.43 397.46 351.40 Mol .times.
10.sup.6 36 36 36 36 Weight (mg) 14.6 13.9 14.3 12.6 Member number
9 10 11 12 Structure ##STR00107## ##STR00108## ##STR00109##
##STR00110## Mw (g/mol) 353.41 477.51 425.47 596.67 Mol .times.
10.sup.6 36 36 36 36 Weight (mg) 12.7 17.2 15.3 21.5 Member number
13 14 15 16 Structure ##STR00111## ##STR00112## ##STR00113##
##STR00114## Mw (g/mol) 565.42 323.34 610.70 447.49 Mol .times.
10.sup.6 36 36 36 36 Weight (mg) 20.4 11.6 22.0 16.1 Member number
17 18 19 20 Structure ##STR00115## ##STR00116## ##STR00117##
##STR00118## Mw (g/mol) 325.36 383.44 393.46 311.33 Mol .times.
10.sup.6 36 36 36 36 Weight (mg) 11.7 13.8 14.2 11.2
TABLE-US-00011 TABLE 11 Compounds used for TBTU coupling of amino
acids and acylation with acyl chlorides Member number 1 2 3 4
Structure ##STR00119## ##STR00120## ##STR00121## ##STR00122## Mw
203.24 201.22 189.21 189.21 Mol .times. 10.sup.6 36 36 36 36 Weight
(mg) 7.3 7.2 6.8 6.8 Member number 5 6 7 8 Structure ##STR00123##
##STR00124## ##STR00125## ##STR00126## Mw (g/mol) 203.24 203.24
442.63 442.63 Mol .times. 10.sup.6 36 36 36 36 Weight (mg) 7.3 7.3
15.9 15.9 Member number 9 10 11 12 Structure ##STR00127##
##STR00128## ##STR00129## ##STR00130## Mw (g/mol) 175.18 485.66
231.29 231.29 Mol .times. 10.sup.6 36 36 36 36 Weight (mg) 6.3 17.5
8.3 8.3 Member number 13 14 15 16 Structure ##STR00131##
##STR00132## ##STR00133## ##STR00134## Mw (g/mol) 217.26 215.25
275.34 134.60 Mol .times. 10.sup.6 36 36 36 120 Weight (mg) 7.8 7.7
9.9 16.2 (0.017 mL) Member number 17 18 19 20 Structure
##STR00135## ##STR00136## ##STR00137## ##STR00138## Mw (g/mol)
120.58 104.53 106.55 78.50 Mol .times. 10.sup.6 120 120 120 120
Weight (mg) 14.4 (0.015 mL) 12.6 (0.011 mL) 12.8 (0.013mL) 9.4
(0.009 mL)
Library Synthesis:
Step 1, Reductive Amination of Photo Linker Aldehyde:
[0367] 5 portions of the above beads (0.8 g dry beads) were placed
in 5 syringes of 5 mL. The beads were swollen in DMF and
phenylethylamines (see Table 7) were coupled to the free aldehyde
on the photolinker by reductive amination as follows: 0.8 g dry
beads is 96 .quadrature.mols.about.1 eq were pre-treated with the
reaction solvent (DMF/HOAc/TEOF/EtOH, 1:1:1:1). Then to each
syringe was added one of the five phenylethylamines (20 eq)
dissolved in the reaction solvent (400 .quadrature.L), and the same
solvent was added so the beads were covered. After 0.5 h
NaBH.sub.3CN (20 eq) was added and the beads stirred cautiously
until all dissolved. After 1 h another portion of NaBH.sub.3CN (20
eq) was added and the mixture left for additional 2 h. The beads
were washed with DMF (10.times.), DCM (10.times.), MeOH (10% HOAc)
(2.times.), MeOH (10.times.), DMF (20% pip) (2.times.), and DMF
(10.times.), DCM (10.times.). The 5 samples were lyophilized over
night.
Step 2, Mix and Split of Phenylethyl Amines:
[0368] 0.4 g of each of the 5 bead samples from above were swelled
in DMF, mixed and transferred to a custom made 20-well library
synthesizer such that each well contained approximate equal amounts
of beads.
Step 3, BTC Coupling of First Amino Acid:
[0369] Total amount of beads=2.0 g.about.0.24 mmol gives 12
.quadrature.mol NH/well.about.1 eq. 20 BTC-couplings from 11
different amino acids as shown in Table 8 was made as follows:
Beads were pre-treated with a 1:1 vol % THF/DIPEA solution for 5
minutes and drained. Of each amino acid in Table 2, 3 eq (36
.quadrature.mol) was dissolved in dry THF (200 .quadrature.L) and
BTC (1.67 eq) added as 200 .mu.L of a freshly made stock solution
in dry THF. Then 2,4,6-collidine (14 eq), as 200 .mu.L of a freshly
made stock solution in dry THF, was added and the resulting 20
suspensions left for 5 minutes. Each suspension was added to the
respective well and after short mixing the synthesizer was sealed
and left over night with gentle shaking. Next morning the beads
were washed with THF (10.times.) and DMF (10.times.) without mixing
the wells.
Step 4, Acylation of Well 1-10:
[0370] Beads in well 1-10 were washed with DCM (10.times.) and Boc
deprotected with 30% TFA in DCM followed by wash with DCM
(10.times.), DCM (5% DIPEA), DMF, and DCM (10.times.). From each of
the 10 acyl chlorides in Table 9 was made a solution of 10 eq acyl
chloride (120 .mu.mol) and DIPEA (20 eq) in dry DCM (400
.quadrature.L) containing catalytic amounts of DMAP. The resulting
solutions were added to well 1-10 and left with gentle shaking for
1 h. The reaction was repeated. After end reactions the beads were
washed with DCM (10.times.), and DMF (10.times.).
Step 5, Removal of Fmoc Protection Groups:
[0371] Well 1-20 were standard Fmoc deprotected, followed by wash
with DMF (10.times.).
Step 6, Mix and Split of the 20 Wells
[0372] The content of the 20 wells was thoroughly mixed and
re-distributed equally into the wells.
Step 7, Coupling of Second Amino Acid (20 Amino Acids):
[0373] To each well was coupled an amino acid according to Table 10
by a standard TBTU coupling. Coupling time 5 h. After end reaction
the beads were washed with DMF (10.times.).
Step 8, Coupling of Third Amino Acid/Acyl Chloride (15 Amino
Acids-5 Acyl Chlorides):
[0374] The beads in all wells were standard Fmoc deprotected and
for well 1-15 standard TBTU coupled with the Boc-protected amino
acids in Table 11. For wells 16-20 the beads were first washed with
DCM (10.times.) and the N-terminal amines acylated with the five
acyl chlorides listed in Table 11 (entry 16-20) analogous to step
4. After couplings all wells were washed with DMF (10.times.).
Step 9, Alloc Deprotection of Second "Core" Glycine:
[0375] The beads from the 20 wells were all combined in a 50 mL
syringe and washed with CHCl.sub.3 (5.times.) and with Ar-degassed
CHCl.sub.3 containing 5% HOAc and 2.5% NEM (5.times.). A solution
of Pd(PPh.sub.3).sub.4 (3 eq, 0.72 mmol) in Ar-degassed CHCl.sub.3
containing 5% HOAc and 2.5% NEM (10 mL) was added to the beads and
after bobling a few minutes with Ar the syringe was sealed with
parafilm and left for 2 h. The beads were washed with CHCl.sub.3
(10.times.), DMF (10.times.), DMF (5% DIPEA, 5% sodium
diethyldithiocarbamate)(5.times.), MeOH (10.times.), DMF DMF
(20.times.). Kaiser test was positive.
Step 10, Attachment of HMBA Linker:
[0376] According to the standard procedure above.
Step 11, MSNT Coupling of FmocGlyOH:
[0377] According to the standard procedure above.
Step 12, TBTU Coupling of FmocLys(Fmoc)OH
[0378] According to the standard procedure above.
Step 13, Attachment of Adhesion Peptides
[0379] Adhesion peptides were synthesized on the Fmoc-protected
lysine such that the final beads had two adhesion peptides pr.
library molecule. One batch of the library was attached adhesion
peptide A, and a second batch with adhesion peptide B. Adhesion
peptide A:
(Boc-D-Ala-D-Arg(Pmc)-D-Lys(Boc)-D-Arg(Pmc)-D-Ile-D-Arg(Pmc)-D-Gln(Trt)-G-
ly-:
[0380] Fmoc deprotection of lysine residues: According to the
standard procedure.
[0381] Coupling: The adhesive peptide was synthesized directly
(stepwise) on the library beads using the general SPPS coupling
procedure.
[0382] Alternative method: The purified peptide
(Boc-D-Ala-D-Arg(Pmc)-D-Lys(Boc)-D-Arg(Pmc)-D-Ile-D-Arg(Pmc)-D-Gln(Trt)-L-
-Gly-OH) (3 eq) was coupled to the lysine NH.sub.2 groups using the
general SPPS coupling procedure.
[0383] Adhesion peptide B:
(Fmoc-D-Arg(Pmc)-D-Gln(Trt)-D-Arg(Pmc)-D-Ile-D-Arg(Pmc)(SEQ ID:
35)
[0384] Analogous to synthesis of adhesion peptide A.
Deprotection of Adhesion and Library Peptides
[0385] Final deprotection of protecting groups was performed
according to the general procedure described above yielding the
TCOB libraries ready for testing.
Example 6
Partial Release of Library Compounds from Beads
[0386] The library beads of Examples 4 and 5 are swelled in the
assay medium and illuminated with an OMNILUX E-40 (400 W UV lamp,
365 nm, #89514005, Steinigke Showtechnic GmbH, Germany) for 15 min.
The beads could be placed in any environment (for example plates,
wells etc. used for the assays). This treatment releases
appropriate amounts of active peptide for assay use and the beads
withholds enough active peptide for subsequent identification.
Example 7
Clearance of Cells and Adhesion Peptide
[0387] A portion of the library beads with cells attached were
drained from medium and treated with a 0.1 M NaOH solution for 1 h.
Beads were gently spinned down and the supernatant removed. The
beads were washed with water (10.times.) to ensure complete removal
of cells and proteins.
Example 8
Functional Assay for Identification of Compounds that Stimulate a
Gs Coupled Signal Transduction Pathway (Cre-GFP Reporter Assay
Detected with a Fluorescence Activated Bead Sorter)
Cre-GFP:
[0388] Cre-GFP is commercially available from clontech
(pCre-d2eGFP) The vector contains three copies of Cre-binding
sequence fused to a TATA-like promoter. The vector is holding a
neomycin resistance gene. A map of the vector is shown in FIG.
3.
MC4R:
[0389] PCR amplified MC4R encoding DNA is introduced into the
gateway Entry Vector (pENTR) by topoisomarase-mediated ligation.
The DNA is subsequently recombined into Destination Vector
pDEST12.2. (pDEST12.2MC4R)
Cell Line Establishment:
[0390] HEK cells are transfected with pDEST1.2.2MC4R using standard
procedure for Fugene6 transfection. Cells are put under G418
selection for 4 weeks to obtain a cell line stably expressing
MC4R.
[0391] The U2OS cell line stably expressing the human MC4R
(melanocortin4 receptor) is further transfected with Cre-GFP the
day before culturing them on PEGA beads displaying adhesion peptide
and respectively 1) Negative control (PEGA beads with adhesion
peptide, but no library compound), 2) Positive control (PEGA beads
with adhesion peptide but no library compound) and 3) Library
compounds. The three cultures are handled separately in each their
culture flask.
Bead/Cell Preparation:
[0392] Cells are trypsinized and mixed with the PEGA beads in
growth medium (DMEM containing 10% FCS, in the proportion 4000
cells/bead and app. 50 ml growth medium/5000 beads.
[0393] 1) Positive control: 50 ml Growth medium+5000 positive
control beads+2.times.10E7 cells+10 .mu.M Forskolin.
[0394] 2) Negative control: 50 ml Growth medium+5000 negative
control beads+2.times.10E7 cells.
[0395] 3) Screening library (eg. 100.000 compounds): 1000 ml Growth
medium+100.000 library beads+4.times.10E8 cells was illuminated
with an OMNILUX E-40 UV light (365 nm) for 30 min for obtaining a
partial release of the library compounds (as described in example
6)
[0396] The three culture flasks are placed on a Magnetic stirring
platform (Techne) designed for cell culture in suspension and
incubated at 37.degree., 5% CO2 for 16-24 hrs using spinning
interval 30 rpm, 3 min stirring, 10 min pause.
[0397] Beads, now covered with cells, are allowed to sediment for
10 min (no centrifugation needed) and the growth medium is removed
using a 50 ml pipette. 10 ml 99% EtOH per 5000 beads is added,
mixed gently and left for 15 min. Beads are washed w. 10 ml
PBS/5000 beads .times.3 by allowing sedimentation for 10 min
between each wash. Cells are now preserved and fixed to the
beads
Bead Sorting:
[0398] A Fluorescence Activated Bead Sorter (FABS) equipped with a
multiline Argon laser 488 nm excitation line and 500-650 nm
emission filter and sorting capability into 96 well plate is used
to identify and isolate positive hit beads.
[0399] The FABS is calibrated to identify and isolate positive hit
beads (increased GFP fluorescence) by determining the dynamic range
of the assay using positive control beads prepared as described
above as Smax (maximum response) and negative control beads
comprising only cell adhesion peptide as 5 min (minimum response).
A cut off at 30% response compared to negative control beads is set
as threshold for a positive hit bead.
[0400] Positive hits are separated into each their well of a 96
well plate and are hereafter ready for compound elucidation,
re-synthesis and re-test as well as test for effects in other
assays.
Example 9
Functional Assay for Identification of Compounds that Inhibit a Gq
Coupled Receptor (Muscarinic M1) Signal Transduction Pathway (Ca++
Mobilization Using Fluo-4)
Ca++ Antagonist Assay:
[0401] This assay is designed to identify muscarinic M1 antagonist
compounds. The read out is changes in intracellular Ca++ conc.
detected using the Fluo-4 probe from Molecular probes (see
description elsewhere). Positive hits are compounds that inhibit
Carbacol (muscarinic M1 agonist) induced increase in intracellular
Ca++.
[0402] U2OS cells are transfected with Muscarinic M1 receptor using
standard procedure for Fugene6 transfection. Cells are put under
zeocin selection for 4 weeks to obtain a cell line stably
expressing the Muscarinic M1 receptor.
[0403] U2OS cells expressing the Muscarinic M1 receptor are
cultured on PEGA beads displaying adhesion peptide and respectively
1) Negative control (Beads comprising only cell adhesion compound),
2) Positive control (beads comprising cell adhesion compound and
Atropine) and 3) Library compounds. The three cultures are handled
separately in each their culture flask.
Bead/Cell Preparation:
[0404] Cells are trypsinized and mixed with beads in DMEM
containing 10% FCS in the proportion 4000 cells/bead and app. 50 ml
growth medium/5000 beads.
[0405] 1) Positive control: 50 ml Growth medium+5000 positive
control beads+2.times.10E7 cells.
[0406] 2) Negative control: 50 ml Growth medium+5000 negative
control beads+2.times.10E7 cells.
[0407] 3) Screening library (eg. 100.000 compounds): 1000 ml Growth
medium+100.000 library beads+4.times.10E8 cells was illuminated
with an OMNILUX E-40 UV light (365 nm) for 30 min for obtaining a
partial release of the library compounds (as described in example
6)
[0408] The three culture flasks are placed on a Magnetic stirring
platform (Techne) designed for cell culture in suspension and
incubated at 37.degree., 5% CO.sub.2 for 16-24 hrs using spinning
interval 30 rpm, 3 min stirring, 10 min pause.
[0409] Measurement of changes in the cytoplasmic free calcium
concentration [Ca.sup.2+].sub.i Beads, now covered with cells, are
allowed to sediment for 10 min (no centrifugation needed) and the
growth medium is removed using a 50 ml pipette. 10 ml Krebs Ringer
buffer (KRW; KrebsRingerWollheim, pH 7.4: NaCl 0.14 M, KCL 3.6 mM,
NaH.sub.2PO.sub.4, H.sub.2O 0.5 mM, MgSO.sub.4, 7H.sub.2O 0.5. mM,
NaHCO.sub.3,2H.sub.2O 1.5 mM, D-Glucose 6 mM, CaCl.sub.21.5 mM,
HEPES 10 mM) added 1 uM Fluo-4 (Molecular Probes F-14201)+0.02%
Pluronic (Molecular Probes F-127) per 5000 beads is added, mixed
gently and cells/beads are incubated at 37.degree. C. for 30 min.
Beads are hereafter washed w. 10 ml KRW/5000 beads .times.3 by
allowing sedimentation for 10 min between each wash. The Fluo-4
loaded cells are now ready for detection of changes in
[Ca.sup.2+].sub.i.
[0410] The fluorescence is monitored in either a Fluorescence
Activated Bead Sorter (FABS) (COPAS from Union Biometrica, US) that
is equipped with multiple laser excitation lines (476 nm, 483 nm,
488 nm, 496 nm, 514 nm, 520 nm, 568 nm, 647 nm, 676 nm) or a
fluorescence plate-reader (Polarstar Optima from BMG Labtech,
Germany) that is equipped with a flash Xenon blitz lamp. Fluo4
fluorescence is detected in FABS by exciting with 488 nm and
collecting the emitted light on to a PMT through a 530.+-.30 nm
emission filter, and on the plate-reader the cells were excited
through a 490.+-.5 nm excitation filter and the emission collected
through a 510.+-.5 nm emission filter. For calculation of the exact
[Ca.sup.2+].sub.i the fluorescence intensity was converted to
[Ca.sup.2+].sub.i by using the equation
[Ca.sup.2+].sub.i=K.sub.D[(F-F.sub.min)/(F.sub.max-F)] where the
dissociation constant K.sub.D is 345 nM, F is fluorescence
intensity, F.sub.min is total fluorescence in the absence of
Ca.sup.2+ and F.sub.max is total fluorescence when Fluo3 is
saturated with Ca.sup.2+. To obtain F.sub.min the cells were
pre-incubated in a calcium low buffer (pH 7.4: NaCl 0.14 M, KCL 3.6
mM, NaH.sub.2PO.sub.4, H.sub.2O 0.5 mM, MgSO.sub.4, 7H.sub.2O 0.5.
mM, NaHCO.sub.3, 2H.sub.2O 1.5 mM, D-Glucose 6 mM, EGTA 1.5 mM,
HEPES 10 mM) and was challenged with 1 uM ionomycin immediately
before the fluorescence detection. Similarly F.sub.max was obtained
by suspending the cells in a calcium saturated buffer (pH 7.4: NaCl
0.14 M, KCL 3.6 mM, NaH.sub.2PO.sub.4, H.sub.2O 0.5 mM, MgSO.sub.4,
7H.sub.2O 0.5. mM, NaHCO.sub.3,2H.sub.2O 1.5 mM, D-Glucose 6 mM,
CaCl.sub.2 1.5 mM, HEPES 10 mM) and challenged with 1 uM ionomycin
immediately before detection.
[0411] In several of our screening assay we do not use exact ion
[Ca.sup.2+].sub.I, but express the response of screening compounds
as relative to control compounds (see below).
Bead Sorting for Fluo-4 Signal:
[0412] A Fluorescence Activated Bead Sorter (FABS) equipped with
488 nm laser excitation line and 528-572 nm emission filter and
injection capability is used to identify and isolate positive hit
beads (=inhibition of Carbachol induced Ca++ response=decreased
fluorescence compared to negative control).
[0413] The FABS is calibrated to identify and isolate positive hit
beads by determining the dynamic range of the assay using positive
control beads as Smax (maximum inhibition=minimal fluorescence) and
negative control beads as 5 min (minimum inhibition=maximal
fluorescence). Carbacol 1 uM is injected into the flow stream
resulting in an increase in fluorescence for negative control beads
and an unchanged or minor increase in fluorescence for positive
control beads. A cut off at 30% inhibition compared to negative
control beads is set as threshold for a positive hit bead. Positive
hits are separated into each their well of a 96 well plate and are
hereafter ready for compound elucidation, re-synthesis and re-test
as well as test for effects in other assays.
Example 10
[0414] Functional Assay for Identification of Compounds that
Stimulate a Gs Coupled Signal Transduction Pathway (Cre-GFP
Reporter Assay Detected with Fluorescence Plate Reader or
Fluorescence Imaging Equipment)
Cre-GFP:
[0415] Cre-GFP is commercially available from clontech
(pCre-d2eGFP) The vector contains three copies of Cre-binding
sequence fused to a TATA-like promoter. The vector is holding a
neomycin resistance gene. A map of the vector is shown in FIG.
3.
MC4R:
[0416] PCR amplified MC4R encoding DNA is introduced into the
gateway Entry Vector (pENTR) by topoisomarase-mediated ligation.
The DNA was subsequently recombined into Destination Vector
pDEST12.2. (pDEST12.2MC4R)
Cell Line Establishment:
[0417] U2OS cells are transfected with pDEST1.2.2MC4R using
standard procedure for Fugene6 transfection. Cells were put under
G418 selection for 4 weeks to obtain a cell line stably expressing
the MC.sup.4R.sup.X.
[0418] The U2OS cell line stably expressing the human MC4R
(melanocortin4 receptor) is further transfected with Cre-GFP the
day before culturing them on PEGA beads displaying adhesion peptide
and respectively 1) Negative control (PEGA beads with adhesion
peptide, but no library compound), 2) Positive control (PEGA beads
of example 2) and 3) Library compounds. The three cultures are
handled separately in each their culture flask.
Bead/Cell Preparation:
[0419] Cells are trypsinized and mixed with the PEGA beads in
growth medium (DMEM containing 10% FCS, in the proportion 4000
cells/bead and app. 50 ml growth medium/5000 beads.
[0420] 1) Positive control: 50 ml Growth medium+5000 positive
control beads+2.times.10E7 cells.
[0421] 2) Negative control: 50 ml Growth medium+5000 negative
control beads+2.times.10E7 cells.
[0422] 3) Screening library (eg. 100.000 compounds): 1000 ml Growth
medium+100.000 library beads+4.times.10E8 cells was illuminated
with an OMNILUX E-40 UV light (365 nm) for 30 min for obtaining a
partial release of the library compounds (as described in example
6)
[0423] The three culture flasks are placed on a Magnetic stirring
platform (Techne) designed for cell culture in suspension and
incubated at 37.degree., 5% CO2 for 16-24 hrs using spinning
interval 30 rpm, 3 min stirring, 10 min pause.
[0424] Beads, now covered with cells, are allowed to sediment for
10 min (no centrifugation needed) and the growth medium is removed
using a 50 ml pipette. 10 ml 99% EtOH per 5000 beads is added,
mixed gently and left for 15 min. Beads are washed w. 10 ml
PBS/5000 beads .times.3 by allowing sedimentation for 10 min
between each wash. Cells are now preserved and fixed to the
beads.
Plate Reader Assay:
[0425] Control beads as well as library beads are seeded in 384
well black plates (eg. Nunc) with clear bottom app. 20 beads per
well. Positive and negative controls are placed in dedicated wells
in 2 times 4 replicates in each end of the plate. Negative
control=20 negative control beads, positive control=one positive
control bead+19 negative control beads. The plates are measured in
a fluorescence plate reader (PolarStar Optima from BMG) using 490
+-6 nm excitation filter and 510 +-5 nm emission filter. Positive
control wells are used to determine Smax (maximum response)=100%
activity and negative control wells to determine 5 min (minimum
response)=0% activity. Beads from wells showing activity >30%
are collected in a tube for re-seeding in a new 384 well plate,
this time having one bead per well. 5 min=one negative control bead
and Smax=one positive control bead. Read plates in plate reader and
identify hits beads using same procedure as described above.
Image Acquisition and Analysis:
[0426] Control beads as well as library beads are seeded in 384
well black plates (eg. Nunc) with clear bottom app. 20 beads per
well. Positive and negative control beads are placed in dedicated
wells in 2 times 4 replicates in each end of the plate. Negative
control=20 negative control beads, positive control=one positive
control bead+19 negative control beads. Plates are placed on a
microscope (Zeiss Axiovert 200M) equipped with filters allowing
fluorescence imaging of eGFP (excitation: 490 nm, emission: 510
nm), 10.times. objective and motorized stage. One image is acquired
for each well followed by image analysis (Metamorph) for
identification of hit beads (green). Beads from hit wells are
seeded in a new 384 well plate this time having one bead per well.
5 min=one negative control bead and Smax=one positive control bead.
Image acquisition and analysis described above is repeated and
final hit beads are identified.
[0427] Alternatively, approximately 5000 beads are seeded into
Lab-Tech Chambered Coverglass System (#155361; Nalge Nunc
INternational), imaging acquisition analysis is performed using the
fluorescence equipment described above, and individual beads that
display the required fluorescence properties are isolated using a
micromanipulator system (Eppendorf Injectman NK).
Example 11
Assay for Identification of Protein-Protein Interaction
(ML-IAP:Smac) Modulators
[0428] The present assay that is based on the principle:
Bioluminescence Resonance Energy Transfer (BRET.sup.2),
commercially available from Perkin Elmer. One of two interacting
proteins is fused to a bioluminescent donor (luciferase, Rluc) and
the other protein is fused to a fluorescent acceptor (GFP).
Melanoma Inihitor of Apoptosis Protein (ML-IAP) Probe:
[0429] The probe is constructed by PCR amplification of the ML-IAP
(GenBank Accession number: NM.sub.--139317 (alpha form);
NM.sub.--0221.61 (beta form) (the accession number refer to the
sequence available on 25 May 2005) from human cDNA libraries
(Marathon-Ready cDNA Library of human kidney cell line and a human
fetal brain cell line, Clontech Laboratories, Inc.) using the
primers livin-F1 (5'-CCA GTG TTC CCT CCA TGG GAC CTA A-3') (SEQ ID
71) and livin-R1 (5'-TAA GCC ATC CCC CAC GCC AAG-3') (SEQ ID 72).
The ML-IAP cDNA gene fragments are further modified to contain
restriction sites by PCR amplification using the primers Livin-F3
(5'-GAT AAG CTT CCA GTG TTC CCT CCA TGG GA-3') (SEQ ID 73),
Livin-R3 (5'-TAT GGA TCC AAG GTG CGC ACG CGG CT-3') (SEQ ID 74),
and Livin-F4 (5'-GAG AAT TCT CCT AAA GAC AGT GCC AAG TG-3') (SEQ ID
75) for amplification of full-length ML-IAP. The primers
Livin-BIR-F1 (5'-CAT GGTACC ATG ACA GAG GAG GAA GAG GAG-3') (SEQ ID
76) and Livin-BIR-R1 (5'-GC TGG ATC CGG GTC CCA GGA GCC CAG-3')
(SEQ ID 77) are used for amplification of the BIR domain of ML-IAP.
The restriction enzyme-treated amplificons are ligated in the BRET2
vectors (obtained from Perkin Elmer, Inc.) to produce N- and
C-terminal in-frame fusions to GFP and Rluc.
Smac Probe:
[0430] The probe is constructed by ligating restriction enzyme
treated PCR amplification products of the cDNA for human Smac
(GenBank Accession NM.sub.--019887 (variant 1); NM.sub.--138929
(variant 3)) in to the BRET2 vectors (obtained from Perkin Elmer,
Inc.) to produce C-terminal in-frame fusions to GFP and Rluc,
respectively. The following primers are used for PCR: Smac-F1
(5'-GCG CTG CAC AAT GGC GGC TCT-3') (SEQ ID 78), Smac-R1 (5'-GCA
CTC ACA GCT CAC AAA GGC GTC T-3') (SEQ ID 79), Smac-F3 (5'-GAT GGT
ACC CGC TGC ACA ATG GCG GCT CT-3') (SEQ ID 80), and Smac-R3 (5'-CGT
GGA TCC TCA CGC AGG TAG GCC TCC-3') (SEQ ID 81). Cytosolic
expression of biologically Smac may be achieved by constructing an
ubiquitin-smac fusion as described by Allison M. Hunter, Dan
Kottachchi, Jennifer Lewis, Colin S. Duckett, Robert G. Korneluk,
and P. Liston. A Novel Ubiquitin Fusion System Bypasses the
Mitochondria and Generates Biologically Active Smac/DIABLO. J.
Biol. Chem. 278 (9):7494-7499, 2003.
Cell Line Establishment:
[0431] Cells are co-transfected with the BRET fusions of ML-IAP and
Smac using standard procedure for Fugene6 transfection. Cells are
put under G418 and zeocin selection for 4 weeks to obtain a cell
line stably expressing the two genes.
Selection of Stable Cells:
[0432] The HeLa cell line stably expressing both probes are sorted
into a microtitre plate (one cell per well) for high GFP
fluorescence using a Fluorescence Activated Cell Sorter. The sorted
cells are grown for a week, and split into a plate that is
optimized for bio-luminescence. Final cell-clones are selected,
after addition of the luciferase substrate, DeepBlueC (Perkin
Elmer), for high lumiscence (detected with a PolarStar, BMG).
Bead/Cell Preparation:
[0433] HeLa/mammalian cells are cultured on PEGA beads displaying
adhesion peptide and respectively 1) Negative control (PEGA beads
with adhesion peptide, and a compound with no activity as the
library component), 2) Positive control (PEGA beads with adhesion
peptide, and a control compound, which disrupts the ML-IAP:Smac
interaction e.g. the compound of Example 20 of WO2004/005248, and
3) Library compounds (PEGA beads with adhesion peptide, and
screening compounds prepared as described in example 5b or 5c). The
three cultures are handled separately in each their culture
flask.
[0434] Cells are trypsinized and mixed with the PEGA beads in
growth medium (DMEM containing 10% FCS, in the proportion 4000
cells/bead and app. 50 ml growth medium/5000 beads. Culture flasks
are placed on a Magnetic stirring platform (Techne) designed for
cell culture in suspension and incubated at 37.degree., 5% CO.sub.2
for 16-24 hrs using spinning interval 30 rpm, 3 min stirring, 10
min pause. Alternatively, the culture flasks are gently rocked a
few times.
Cell-Coated Beads are Treated as Follows:
[0435] 1) Positive control (low BRET signal): 50 ml Growth
medium+approx. 5000 cell-coated control beads.
[0436] 2) Negative control (high BRET signal): 50 ml Growth
medium+approx. 5000 cell-coated control beads 3) Screening library
(eg. 100.000 compounds): 1000 ml Growth medium+approx. 100.000
cell-coated library beads.
[0437] The cell-coated beads are transferred to assay buffer
(either KRW (Krebs Ringer Wollheim) or PBS (phosphate buffered
saline) and illuminated with UV light (365 nm) for 2-20 min for
obtaining a partial release of the library compounds (as described
in example 6). The illuminated beads are further incubated at
37.degree., 5% CO.sub.2 for 15 min. Beads, now covered with cells,
are allowed to sediment for 10 min (no centrifugation needed) and
the growth medium is removed using a 50 ml pipette. Positive beads
may be identified by analysing individual beads in a
BRET-compatible microplate reader using 410-80 (370-450 nm) and
515-30 (500-530 nm) bandpass filters for dual emission measurements
of Rluc-luminescence and GFP-fluorescence, respectively. The
luciferase substrate, DeepBlueC, is added by an on-board injector
in the microplate reader: The BRET-signal for the protein-protein
interaction is calculated as the ratio of emission at 515 nm and
emission at 410 nm.
Alternatively Positive Beads may be Identified by Bead Sorting:
[0438] A Fluorescence Activated Bead Sorter (FABS) equipped with
filters for BRET detection and sorting capability into 96 well
plate is used to identify and isolate positive hit beads.
[0439] The luciferase substrate, DeepBlueC is injected into the
flow stream to emit luciferase-mediated luminescence.
[0440] The FABS is calibrated to identify and isolate positive hit
beads (decreased BRET signal) by determining the dynamic range of
the assay using positive control beads prepared as described in
above as Smax (maximum response) and negative control beads
comprising only cell adhesion peptide as 5 min (minimum response).
Positive hits are separated into each their well of a 96 well plate
and are hereafter ready for compound elucidation, re-synthesis and
re-test as well as test for effects in other assays.
Example 12
Caspase Activity Assay
[0441] Inhibitor of apoptosis proteins (IAPs) interact with
caspases to inhibit their activation and thereby cause a repression
of apoptosis. We seek to identify compounds, which can bind to IAP
with a higher affinity than caspase and, in turn, relieve
IAP-mediated caspase-inhibition. A screening assay is established
that measures caspase activation in response to an apoptosis
inducing signal (e.g. staurosporine, STS) and in the presence of
library compounds with putative IAP-binding activity.
[0442] The assay is based on measurements of caspase activity in
whole mammalian cells (HeLa/MCF-7/MeWo), preferably HeLa/SK-Mel28
cultured on PEGA beads displaying adhesion peptide and respectively
1) Negative control (PEGA beads with adhesion peptide, and a
compound with no activity as the library component), 2) Positive
control (PEGA beads with adhesion peptide, and a control peptide
that disrupts the ML-IAP:Smac interaction prepared as described in
example 3 and 3) Library compounds (PEGA beads with adhesion
peptide, and screening compounds prepared as described in example
5b or 5c). The three cultures are handled separately in each their
culture flask.
[0443] Caspase activity is measured in bead-attached whole cells by
incubating the cells with a fluorogenic caspase substrate with a
quenching leaving group, e.g. DEVD-Rh1 10-C8 or the CellProbe HT
Caspase 3/7 Whole Cell Assay, Beckman Coulter, Inc.). A fluorogenic
counter stain with different emission properties is applied to
correct for cellmass content on the individual beads.
[0444] Caspase substrate (DEVD-R-C8) is described in the following
publication: Sui Xiong Cai, Han Zhong Zhang, John Guastella, John
Drewe, Wu Yang, and Eckard Weber. Design and synthesis of Rhodamine
110 derivative and Caspase-3 substrate for enzyme and cell-based
fluorescent assay. Bioorganic & Medicinal Chemistry Letters 11
(1):39-42, 2001 and in the patent: U.S. Pat. No. 6,342,611 B1 (Jan.
29, 2002, Cytovia, Inc. San Diego, Calif.)
Bead/Cell Preparation:
[0445] Cells are trypsinized and mixed with the PEGA beads in
growth medium (DMEM containing 10% FCS, in the proportion 4000
cells/bead and app. 50 ml growth medium/5000 beads.
[0446] The beads/cells are placed in three culture flasks and
placed on a Magnetic stirring platform (Techne) designed for cell
culture in suspension incubated at 37.degree., 5% CO2 for 16-24
hours using spinning interval 30 rpm, 3 min stirring, 10 min
pause.
Cell-Coated Beads are Treated as Follows:
[0447] 1) Positive control: 50 ml Growth medium+approx. 5000
Positive control beads. 2) Negative control: 50 ml Growth
medium+approx. 5000 Negative control beads.
[0448] 3) Screening library (eg. 100.000 compounds): 1000 ml Growth
medium+approx. 100.000 cell-coated Library compound beads.
Screening Procedure:
[0449] The cell-coated beads are transferred to assay buffer (KRW
or PBS and illuminated with UV light (365 nm) for 30 min for
obtaining a partial release of the library compounds (as described
in example 6). The three culture flasks are placed on a Magnetic
stirring platform (Techne) designed for cell culture in suspension
and incubated at 37.degree., 5% CO.sub.2 for 3 to 6 hrs.
[0450] The beads are then allowed to sediment for 10 min (no
centrifugation needed) and the growth medium is removed using a 50
ml pipette. 1 ml medium containing the fluorogenic caspase
substrate and reagent buffer is added to the beads. The beads are
incubated without stirring at 37.degree., 5% CO.sub.2 for 0.5-5
hours. The beads are then fixed by treatment with 10 ml 99% EtOH
per 5000 beads, gentle mixing whereafter they are left for 15 min.
Beads are washed w. 10 ml PBS/5000 beads .times.3 by allowing
sedimentation for 10 min between each wash. Alternatively, beads
may be fixed with paraformaldehyde, acetone or zinc-based
fixatives. Cells are now preserved and fixed to the beads.
Bead Sorting:
[0451] The beads are analysed in a fashion similar to the procedure
described in example 11. The Fluorescence Activated Bead Sorter
(FABS) is equipped with 488 nm excitation filters and 500-550 nm
emission filter for rhodamine-110 measurements and sorting
capability into 96 well plate is used to identify and isolate
positive hit beads. The FABS is calibrated to identify and isolate
positive hit beads (increased caspase activity) by determining the
dynamic range of the assay using positive control beads with
apoptosis induced cells to determine the Smax (maximum response)
and negative control beads with uninduced cells as 5 min (minimum
response).
[0452] Positive hits are separated into each their well of a 96
well plate and are hereafter ready for compound elucidation,
re-synthesis and re-test as well as test for effects in other
assays.
Microscopy:
[0453] An alternative to FABS-based selection of positive hit beads
is epifluorescence microscopy using standard FITC filters
(excitation 480.+-.15 nm, emission 535.+-.20 nm) for visualization
of rhodamine 110 fluorescence. The microscope is equipped with
micromanipulators for extraction of positive hit beads.
Example 12b
Detection of Caspase Activity Using Proximity Ligation-Based
Assay
[0454] Caspase activity is detected with an assay based on the
proximity ligation technology described by S. Fredriksson, M.
Gullberg, J. Jarvius, C. Olsson, K. Pietras, S. M. Gustafsdottir,
A. Ostman, and U. Landegren. Protein detection using
proximity-dependent DNA ligation assays. Nat. Biotechnol. 20
(5):473-477, 2002. This assay allows direct detection of both
active caspases and cleaved caspase substrates for example PAPR.
Furthermore, the assay can be applied to fixed samples of primary
tissue.
[0455] We have developed the proximity ligation technique to
encompass a high volume discovery platform that allow
identification of primary hit compounds in a screening system that
because of the ability to conduct the screening in primary cells
closely resembles the conditions observed directly in the
patient.
[0456] The assay employs two antibodies, an antibody specific for
cleaved caspase-3 and an antibody for recognizing both the
pro-enzmye and the cleaved form of caspase-3. Both antibodies are
conjugated with an oligo nucleotide. Coordinated and proximal
binding of the two antibodies to an activated caspase allows
hybridisation and ligation of DNA probes which can be amplified by
rolling circle amplification using flourescently labelled
oligonucleotides or molecular beacons. The quantitative
incorporation of a fluorescence label may thus be done like in
quantitative real-time PCR. Detection and selection of beads with a
positive fluorescence signal is conducted either with the
Fluorescence-Activated-Bead-Sorter or a fluorescence microscope or
a plate reader.
Example 13
Identification of Compound
[0457] Once a resin bead is selected, the cells may be cleared off
the beads either by extensive washing or in case of HMBA-linked
adhesion peptides by treatment with 0.1 M NaOH followed by washing
(example 7). The library compound comprised within the bead may
then be identified. Selected bead(s) are washed and swelled in a
small drop of pure water and irradiated for 30 min. with an OMNILUX
E-40 (400 W UV lamp, 365 nm, #89514005, Steinigke Showtechnic GmbH,
Germany). The compound is identified with advanced mass
spectrometry combined with single bead and/or nano-scale NMR
techniques. For example advanced MS may be ES MS-MS analysis on a
MicroMass QTOF Global Ultima mass spectrometer (mobile phase 50%
CH.sub.3CN (aq), 0.1 .mu.L/min) employing a linear ramping of the
collision energy. The spectra are analyzed by generating the exact
mass differences between fragment ions and tabulated to provide the
fragmentation pathway and from that the structure of the compound
released from the selected bead is elucidated. Examples of spectra,
mass differences and fragmentation pathways are given in FIGS. 5 to
7.
ABBREVIATIONS
[0458] HGF: Hepatocyte Growth Factor [0459] NGF: Nerve Growth
Factor [0460] PDGF: Platelet Derived Growth Factor [0461] FGF:
Fibroblast Growth Factor [0462] EGF: epidermal Growth Factor [0463]
GH: Growth hormone [0464] TRE: TPA Response Element [0465] SRE:
serum response element [0466] CRE: cAMP response element [0467]
AcN: acetonitril; [0468] Boc: tert-butoxycarbonyl; .sup.tBu:
tert-butyl; [0469] DCM: dichloromethane; [0470] DMF:
dimethylformamide; [0471] Fmoc: 9-fluorenylmethoxycarbonyl; [0472]
HMBA: 4-hydroxymethylbenzoic acid; [0473] Q-TOF MS: quadrupole
time-of-flight mass spectrometry; [0474] Melm: N-methyl imidazole;
[0475] MS NT: 1-(mesitylene-2-sulphonyl)-3-nitro-1H-1,2,4-triazole;
[0476] NEM: N-ethyl morpholine; [0477] PEGA: polyethylene
glycol-polydimethyl acrylamide resin; [0478] Pfp:
pentafluorophenyl; [0479] Pmc:
2,2,5,7,8-pentamethylchroman-6-sulfonyl; [0480] RP-HPLC: reversed
phase high pressure liquid chromatography; [0481] SPPS: solid phase
peptide synthesis; [0482] TBTU:
O-(benzotriazol-1-yl-N,N,N',N'-tetramethyluronium
tetrafluoroborate; [0483] TCOB: Two-compound-one-bead [0484] TFA:
trifluoro acetic acid; [0485] Trt: Trityl.
Sequence CWU 1
1
8117PRTArtificial SequenceRandomly synthesized cell adhesion
peptide 1Ala Arg Ile Arg Ile Gln His1 527PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 2Ala Lys Cys Arg
Trp Cys Met1 537PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 3Ala Lys Ala Arg Cys Lys Ser1 547PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 4Ala Lys Tyr Trp
Ser Tyr Lys1 557PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 5Ala Tyr Tyr Cys Gln Gln Arg1 567PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 6Ala Arg Arg Cys
Phe Arg Asp1 577PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 7Ala Ala Arg His Cys Tyr Tyr1 587PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 8Ala Tyr Tyr Cys
Gln Gln Arg1 597PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 9Ala Asp Leu Lys Arg Pro Met1 5107PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 10Ala Gly Gly
Lys Arg Lys Phe1 5117PRTArtificial SequenceRandomly synthesized
cell adhesion peptide 11Ala Pro Arg Lys Arg Cys Gly1
5127PRTArtificial SequenceRandomly synthesized cell adhesion
peptide 12Ala Thr Arg Arg Val Ala Arg1 5137PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 13Ala Gly Lys
Lys Asn Lys Asn1 5147PRTArtificial SequenceRandomly synthesized
cell adhesion peptide 14Ala Ala Lys Arg Trp Lys Phe1
5157PRTArtificial SequenceRandomly synthesized cell adhesion
peptide 15Ala Arg Trp Pro Tyr Arg Gly1 5167PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 16Ala Leu Tyr
Trp Thr Trp Arg1 5177PRTArtificial SequenceRandomly synthesized
cell adhesion peptide 17Ala Ala Tyr Arg Trp Tyr Arg1
5187PRTArtificial SequenceRandomly synthesized cell adhesion
peptide 18Ala Arg Cys Ile Arg Gly Asp1 5197PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 19Ala Thr Lys
Cys Lys Gly Arg1 5207PRTArtificial SequenceRandomly synthesized
cell adhesion peptide 20Ala Val Tyr Met Arg Asn Ile1
5217PRTArtificial SequenceRandomly synthesized cell adhesion
peptide 21Ala Arg Lys Arg Ile Arg Gln1 5227PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 22Ala Lys Ile
Arg Glu Lys Arg1 5237PRTArtificial SequenceRandomly synthesized
cell adhesion peptide 23Ala Arg Arg Phe Lys Met Tyr1
5244PRTArtificial SequenceRandomly synthesized cell adhesion
peptide 24Arg Arg Phe Lys1254PRTArtificial SequenceRandomly
synthesized cell adhesion peptide 25Arg Arg Ile
Arg1266PRTArtificial SequenceRandomly synthesized cell adhesion
peptide 26Leu Arg His Arg Leu Lys1 5275PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 27Lys Phe Gly
Gln Lys1 5286PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 28Lys Val Tyr Met His Lys1 5296PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 29Ile Arg Tyr
Arg Leu Arg1 5306PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 30Ala Gln Arg Pro Arg Trp1 5316PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 31Trp Tyr Ala
Lys Arg Arg1 5328PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 32Lys Arg Ile Arg Gln Arg Leu Arg1
5337PRTArtificial SequenceRandomly synthesized cell adhesion
peptide 33Lys Arg Ile Arg Gln Arg Lys1 5345PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 34Arg Ile Arg
Gln Arg1 5355PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 35Arg Gln Arg Ile Arg1 5366PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 36Lys Phe Gly
Gln Lys Cys1 5376PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 37Arg Arg Leu Leu Pro Ile1 5386PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 38Pro Phe Arg
Lys Lys Cys1 5396PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 39Tyr Arg Trp Arg Ile Ala1 5406PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 40Arg Ser Lys
Arg Ile Asn1 5416PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 41Arg Ser Ala Lys Arg Cys1 5426PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 42Lys Lys Gln
Phe Trp Phe1 5436PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 43Arg Met Lys Leu His Lys1 5446PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 44Arg His Trp
Gly Arg Ile1 5456PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 45Thr Lys Arg Leu Lys Thr1 5466PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 46Thr Lys Gly
Lys Ala Lys1 5476PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 47Ala Lys Thr Arg His Arg1 5486PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 48Asn Arg Pro
Arg Val Arg1 5496PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 49Val Pro Arg Lys Val Gln1 5506PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 50Lys Met Arg
Tyr Cys Gln1 5516PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 51Ile Arg Lys His Leu Ile1 5526PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 52Pro Arg Arg
Val Val Ile1 5536PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 53Lys Arg Glu Ser Lys Arg1 5546PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 54Ser Arg Lys
Asp Arg Lys1 5556PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 55Arg Cys Lys Lys Leu Ile1 5566PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 56Arg Lys Leu
Arg Val Asn1 5576PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 57Val Arg Thr Val Arg Val1 5586PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 58Arg Ala Phe
Lys Tyr Tyr1 5596PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 59Ile Thr Arg Arg Thr Gln1 5606PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 60Lys Met Pro
Lys Lys Asn1 5616PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 61Lys Pro Lys Met Met Cys1 5626PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 62Lys Lys Met
Arg Phe Trp1 5636PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 63Lys Lys Lys Phe Tyr Tyr1 5646PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 64Lys Ser Asn
Lys Val Arg1 5656PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 65Lys Trp Pro His His Arg1 5666PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 66Arg His Ile
Gln Trp Tyr1 5676PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 67Leu Arg Leu Lys Pro Lys1 5686PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 68Glu Arg Lys
Arg Cys Thr1 5696PRTArtificial SequenceRandomly synthesized cell
adhesion peptide 69Arg Arg Ala Arg Gln Asp1 5706PRTArtificial
SequenceRandomly synthesized cell adhesion peptide 70Arg Glu Lys
Gly Ala Arg1 57125DNAArtificial SequencePrimer 71ccagtgttcc
ctccatggga cctaa 257221DNAArtificial SequencePrimer 72taagccatcc
cccacgccaa g 217329DNAArtificial SequencePrimer 73gataagcttc
cagtgttccc tccatggga 297426DNAArtificial SequencePrimer
74tatggatcca aggtgcgcac gcggct 267529DNAArtificial SequencePrimer
75gagaattctc ctaaagacag tgccaagtg 297630DNAArtificial
SequencePrimer 76catggtacca tgacagagga ggaagaggag
307726DNAArtificial SequencePrimer 77gctggatccg ggtcccagga gcccag
267821DNAArtificial SequencePrimer 78gcgctgcaca atggcggctc t
217925DNAArtificial SequencePrimer 79gcactcacag ctcacaaagg cgtct
258029DNAArtificial SequencePrimer 80gatggtaccc gctgcacaat
ggcggctct 298124DNAArtificial SequencePrimer 81ggatcctcac
gcaggtaggc ctcc 24
* * * * *
References