U.S. patent application number 11/569457 was filed with the patent office on 2011-12-29 for identification of compounds modifying a cellular response.
This patent application is currently assigned to Carlsberg A/S. Invention is credited to Frederik Diness, Grith Hagel, Dorte W. Kaznelson, Morten Meldal, Thomas E. Nielsen, Ole Thastrup.
Application Number | 20110319274 11/569457 |
Document ID | / |
Family ID | 34968405 |
Filed Date | 2011-12-29 |
![](/patent/app/20110319274/US20110319274A1-20111229-C00001.png)
![](/patent/app/20110319274/US20110319274A1-20111229-C00002.png)
![](/patent/app/20110319274/US20110319274A1-20111229-C00003.png)
![](/patent/app/20110319274/US20110319274A1-20111229-C00004.png)
![](/patent/app/20110319274/US20110319274A1-20111229-C00005.png)
![](/patent/app/20110319274/US20110319274A1-20111229-C00006.png)
![](/patent/app/20110319274/US20110319274A1-20111229-C00007.png)
![](/patent/app/20110319274/US20110319274A1-20111229-C00008.png)
![](/patent/app/20110319274/US20110319274A1-20111229-C00009.png)
![](/patent/app/20110319274/US20110319274A1-20111229-C00010.png)
![](/patent/app/20110319274/US20110319274A1-20111229-C00011.png)
View All Diagrams
United States Patent
Application |
20110319274 |
Kind Code |
A1 |
Hagel; Grith ; et
al. |
December 29, 2011 |
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. Test compounds modulating a cellular
response, for example via a cell surface molecule may be identified
by selecting solid supports comprising cells, wherein the cellular
response of interest has been modulated. The cellular response may
for example be changes in signal transduction pathways modulated by
a cell surface molecule.
Inventors: |
Hagel; Grith; (Dragor,
DK) ; Meldal; Morten; (Kobenhavn Nv, DK) ;
Kaznelson; Dorte W.; (Kobenhaven N, DK) ; Thastrup;
Ole; (Birkerod, DK) ; Nielsen; Thomas E.;
(Kobenhavn V, DK) ; Diness; Frederik; (Kobenhavn
O, DK) |
Assignee: |
Carlsberg A/S
Valby
DK
|
Family ID: |
34968405 |
Appl. No.: |
11/569457 |
Filed: |
May 25, 2005 |
PCT Filed: |
May 25, 2005 |
PCT NO: |
PCT/DK05/00348 |
371 Date: |
March 31, 2007 |
Current U.S.
Class: |
506/3 ; 435/375;
506/18; 506/23; 523/449; 530/300 |
Current CPC
Class: |
G01N 33/6845 20130101;
G01N 2035/00158 20130101; C07K 7/08 20130101; C40B 30/06 20130101;
C07K 5/1008 20130101; C12Q 1/025 20130101; C07K 1/047 20130101;
G01N 33/5023 20130101; G01N 33/54313 20130101; C07K 7/06 20130101;
G01N 2500/10 20130101 |
Class at
Publication: |
506/3 ; 435/375;
506/18; 506/23; 523/449; 530/300 |
International
Class: |
C07K 16/00 20060101
C07K016/00; C40B 50/00 20060101 C40B050/00; C40B 20/02 20060101
C40B020/02; C40B 40/10 20060101 C40B040/10; C08L 33/00 20060101
C08L033/00; C12N 5/02 20060101 C12N005/02 |
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 covalently linked to one member of a
library of test compounds and wherein at least two solid supports
comprise different library members; and (b) Attaching cells
comprising said reporter system(s) onto said solid support; wherein
cells are directly attached to the solid support, and/or at least
10% of the solid supports comprise cell adhesion molecules as well
as said library member, and cells adhere to said cell adhesion
molecules; and (c) 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 (d) Selecting solid
supports comprising cells meeting said at least one selection
criterion; and (e) 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
support, 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 1, wherein the solid supports are
spots or regions on a surface or a plated gel or a membrane.
6. The method according to claim 2, wherein said cell adhesion
compound is a peptide with an overall positive netcharge.
7. (canceled)
8. The method according to claim 6, 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 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.
9. The method according to claim 1, wherein said cellular response
is modulation of a signal transduction pathway mediated by a cell
surface molecule.
10. The method according to claim 9, wherein said cell surface
molecule is a G-protein coupled receptor (GPCR).
11. The method according to claim 10, wherein said GPCR is selected
from the group consisting of GPCR of table 3.
12. (canceled)
13. (canceled)
14. (canceled)
15. (canceled)
16. (canceled)
17. The method according to claim 1, wherein the cellular response
is modulation of transcriptional activity.
18. (canceled)
19. The method according to claim 1, wherein the cellular response
is change in the intracellular level of a compound
20. (canceled)
21. (canceled)
22. The method according to claim 1, wherein the cellular response
is relocalisation of a compound.
23. (canceled)
24. (canceled)
25. (canceled)
26. (canceled)
27. (canceled)
28. (canceled)
29. The method according to claim 1, wherein the reporter system is
a system endogenous to said cells.
30. (canceled)
31. (canceled)
32. (canceled)
33. The method according to claim 1, wherein the reporter 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.
34. (canceled)
35. The method according to claim 9, wherein the reporter system
comprises 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).
36. The method according to claim 9, wherein the reporter system
comprises a nucleic acid comprising a nucleotide sequence encoding
a detectable polypeptide operably linked to transcriptional
response element (TRE).
37. (canceled)
38. (canceled)
39. The method according to claim 1, wherein said detectable
polypeptide is selected from the group consisting of fluorescent
proteins and enzymes.
40. (canceled)
41. The method according to claim 1, wherein the reporter system
comprises a bioluminescent moiety.
42. (canceled)
43. (canceled)
44. The method according to claim 1, wherein one predetermined
selection criteria is a quantitative level of said bioluminescence
above or below a specific threshold.
45. The method according to claim 1, wherein the predetermined
selection criteria is specific localisation of a fluorescent
signal.
46. The method according to claim 1, wherein said cells are
selected from the group consisting of mammalian cells.
47. (canceled)
48. (canceled)
49. The method according to claim 9, wherein the cells attached to
the resin beads comprise a nucleic acid comprising a first
nucleotide sequence encoding said cell surface molecule operably
linked to a second nucleotide sequence not naturally associated
therewith directing expression of said first sequence.
50. The method according to claim 1, wherein at least 100 resin
beads comprising different library members are provided.
51. (canceled)
52. 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, glycopeptides, nucleic acids (DNA or
RNA) [oligosaccharides,] and small organic molecules.
53. The method according to claim 1, wherein the library is a
library of small organic molecules.
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 the resin bead
comprises or consists of polyethylene glycol
56. (canceled)
57. A cell adhesion compound selected from either: i) the group
consisting of peptides of: SEQ ID 1, SEQ ID 2, SEQ ID 3, SEQ ED 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.
58. A resin bead comprising a cell adhesion compound 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.
59. The resin bead according to claim 58, wherein said resin bead
comprises polyethylene glycol.
60. (canceled)
61. 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
62. A method of modulating the activity of a GPCR receptor
comprising the steps of a) Providing a compound identified by the
method according to claim 10 b) Incubating said compound together
with cells expressing said GPCR c) Thereby modulating the activity
of said GPCR
63. Compound identified by the method according to claim 1
64. A method of synthesising a cyclic peptide or peptide mimetic
library, comprising the steps i) Providing a plurality of peptides
or peptide mimetics covalently linked to an azide moiety and an
acetylene moeity; and ii) cyclizing said peptide or peptide mimetic
through a Cu(I) catalysed reaction between said azide- and said
acetylene moiety; and iii) thereby obtaining a library of cyclic
peptides or peptide mimetics.
65. The method according to claim 64, wherein each peptide or
peptide mimetic are immobilised on a solid support.
66. The method according to claim 64, wherein the solid support is
resin beads and each resin bead comprises only one library member
in one or more copies.
67. A library prepared by the method according to claim 64.
68. The method according to claim 1, wherein the library of test
compounds is a cyclic peptide or peptide mimetic library prepared
by a method comprising the steps i) providing a plurality of
peptides or peptide mimetics covalently linked to an azide moiety
and an acetylene moeity; and ii) cyclizing said peptide or peptide
mimetic through a Cu(I) catalysed reaction between said azide- and
said acetylene moiety; and iii) thereby obtaining a library of
cyclic peptides or peptide mimetics.
69. A method of synthesising a library of heterocyclic ureas,
comprising the steps of i) Providing a plurality of urea containing
peptide aldehydes; and ii) Subjecting said urea containing peptides
to an intramolecular Pictet-Spengler reaction; and iii) Thereby
obtaining a library of heterocyclic ureas
70. The method according to claim 69, wherein said urea containing
peptide aldehydes are immobilised on a solid support.
71. A library obtained by the method according to claim 69.
72. The method according to claim 1, wherein the library of test
compounds is a library of heterocyclic ureas prepared by a method
comprising the steps of i) providing a plurality of urea containing
peptide aldehydes; and ii) subjecting said urea containing peptides
to an intramolecular Pictet-Spengler reaction; and iii) thereby
obtaining a library of heterocyclic ureas
73. The method according to claim 1, wherein the library of test
compounds is a library of heterocyclic compounds obtained by
cyclisation of a peptide aldehyde through an intramolecular
Pictet-Spengler reaction.
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 a
surface receptor, in particular an influence caused by contacting a
receptor with a substance linked to a solid support to which a cell
expressing the surface receptor 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 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 comprises one member of a library of test compounds 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) 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 a detectable output; and [0017] (d) Selecting beads
comprising cells meeting said at least one selection criterion; and
[0018] (e) Identifying said the library member, thereby identifying
a compound modifying said at least one cellular response.
DESCRIPTION OF DRAWINGS
[0019] 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.
[0020] 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 detectable 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.
[0021] 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. The screen could for example be a
screen for Cre-YFP and MC4R-GFP or HA-MC4R internalisation, wherein
I) Cre-YFP reporter hits are identified and isolated by FABS and
II) MC4R-GFP or HA-GFP internalisation positive hits are
picked.
[0022] 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. The screen could for example be a screen for Cre-YFP and
HA-MC4R internalisation, wherein I) Cre-YFP reporter hits are
identified and isolated by FABS into a 10 ml. tube (see FIG. 1) and
II) HA-MC4R internalisation hits are isolated (=low
fluorescence).
[0023] FIG. 3 illustrates a plasmid map of pCRE-d2EGFP
[0024] FIG. 4A illustrates synthesis of
Ac-His-(D)phe-Arg-Trp-NH.sub.2.
[0025] FIG. 4B illustrates synthesis of
Ac-His-(D)phe-Arg-Trp-Gly-PEGA.sub.1900
[0026] FIG. 4C illustrates synthesis of Fmoc-Dap(N.sub.3)
[0027] FIG. 5 illustrates synthesis of the cyclic peptide of
example 3
[0028] FIG. 6a illustrates synthesis of a combinatorial library
(6a) via an intramolecular N-acyliminium Pictet-Spengler
reaction
[0029] FIG. 6b illustrates synthesis of a combinatorial library
(6b) via an intramolecular N-acyliminium Pictet-Spengler
reaction
[0030] FIG. 7 illustrates spectra and structure determination by
accurate mass differences from single beads
[0031] FIG. 8 illustrates structure determination by accurate mass
differences from single beads
[0032] FIG. 9 illustrates a fragmentation pathway
[0033] FIG. 10 illustrates examples of an adhesion peptide
displaying bead covered with cells (U2OS).
[0034] FIG. 11 illustrates quantification of MC4R-GFP
internalization on beads
[0035] FIG. 12 illustrates intracellular Ca.sup.2+ mobilization as
visualised using the Flou4 probe.
[0036] FIG. 13 illustrates the aMSH induced CRE-YFP transcription
in HEK293 and U2OS cells, respectively, expressing MC4.
[0037] FIG. 14 illustrates signal obtained from a subfraction of
identified hits after functional screening (CRE-YFP) of a
library.
[0038] FIG. 15a is a picture of a bead with cells screened as
described in Example 14a comprising the compound designated
TEN-636-36-36.
[0039] FIG. 15b illustrates QTOF MSMS analysis of the compound
designated TEN-636-33-26.
[0040] FIG. 16 illustrates MSMS analysis of material cleaved from a
single bead of a library prepared as described in Examples 6a or
6b. Structure elucidation is given by [M+H].sup.+,
[M-Gly-AA1].sup.+, and [M-Gly-AA.sub.1AA.sub.2].sup.+.
DEFINITIONS
[0041] Naturally occurring amino acids are named herein using
either their 1-letter or 3-letter code according to the
recommendations from IUPAC, see for example
http://www.chem.qmw.ac.uk/iupac. 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.
[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 naturally 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 chromophore. 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
chromophore 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 "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.
[0054] The phrase "fluorescence properties" means absorption
properties, such as wavelength and extension, or 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.
[0055] 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.
[0056] 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.
[0057] 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.
[0058] The term "multiple" should be understood as "at least
two".
[0059] The term "library of test compounds" should be understood as
a collection of test compounds comprising at least 2 different test
compounds.
[0060] 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.
[0061] 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.
[0062] 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.
DETAILED DESCRIPTION OF THE INVENTION
Library of Test Compounds
[0063] 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.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] 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.
[0068] 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 10.sup.7, 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, such as in the range of
20,000 to 200,000 different test compounds. In preferred
embodiments of the invention the library comprises in the range of
10,000 to 1,000,000 different test compounds.
[0069] 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 10.sup.7, 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.
[0070] 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 on 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).
[0071] 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: [0072] 1. Providing several pools of
resin beads [0073] 2. Performing one or more different chemical
synthesis steps on each pool of resin beads, [0074] 3. Splitting
said pools to obtain fractions [0075] 4. Mixing fractions from
different pools, thereby obtaining new pools [0076] 5. Optionally
repeating step 1 and 4
[0077] Alternatively steps 3 and 4 may be as follows: [0078] 3.
Mixing all pools of resin beads, thereby obtaining a mixed pool
[0079] 4. Splitting the mixed pool of resin beads into reaction
containers thereby obtaining new pools.
[0080] 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.
[0081] 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: [0082] 1. Providing resin beads comprising a plurality of
reactive groups [0083] 2. Reacting said reactive groups with two
chemical moeities comprising different and preferably orthogonal
protective groups [0084] 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, [0085] 4.
Attaching or synthezising a split/mix library of test compounds to
the deprotected reactive group [0086] 5. Deprotecting the remaining
reactive groups by removal the other kind of protective group
[0087] 6. Attaching the second compound to the deprotected reactive
groups
[0088] 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.
[0089] 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 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 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.
[0090] In one embodiment the library 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 below. If the
resin beads are coupled to an adhesion compound via a cleavable
linker it is preferred that the cleavable linker linking the
library is different to the cleavable linker linking the adhesion
compound. It is in particularly preferred that the linker are not
cleavable by the same mechanism. Thereby, the library may be
specifically released from the resin beads, without release of
adhesion compounds.
[0091] 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.
[0092] 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. 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.
[0093] 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 a G-protein coupled receptor are described in C.
Haskell-Luevano, A. Rosenquist, A. Souers, K. C. Khong, J. A.
Ellman, and R. D. Cone, 1999, J. Med. Chem. 42:4380-4387. Compounds
that activate the mouse melanocortin-1 receptor identified by
screening a small molecule library based upon the b-turn. J. Med.
Chem. 42:4380-4387, 1999; A. J. Souers, A. A. Virgilio, A.
Rosenquist, W. Fenuik, and J. A. Ellman. Identification of a potent
heterocyclic ligand to somatostatin receptor subtype 5 by the
synthesis and screening of b-turn mimetic libraries. J. Am. Chem.
Soc. 121 (9):1817-1825, 1999; J. Bondebjerg, Z. Xiang, R. M. Bauzo,
C. Haskell-Luevano, and M. Meldal. A solid phase approach to mouse
melanocortin receptor agonists derived from a novel thio-ether
cyclized peptidomimetic scaffold. J. Am. Chem. Soc.
124:11046-11055, 2002; B. A. Harrison, G. W. Pasternak, and G. L.
Verdine. 2,6-dimethyltyrosine analogues of a stereodiversified
ligand library: highly potent, selective, non-peptidic m opioid
receptor agonists. J. Med. Chem. 46:677-680, 2003; G. R. Marshall.
Peptide interactions with G-protein coupled receptors. Peptide
Science 60:246-277, 2003; P. N. Arasasingham, C. Fotsch, X. Ouyang,
M. H. Norman, M. G. Kelly, K. L. Stark, B. Karbon, C. Hale, J. W.
Baumgartner, M. Zambrano, J. Cheetham, N. A. Tamayo, and
Structure-Activity relationship of (1-aryl-2-piperazinylethyl)
piperazines: Antagonists for the AGRP/Melanocortin receptor
binding. J. Med. Chem. 46:9-11, 2003. Further useful libraries are
described in examples 4, 5 and 6 herein below: The person skilled
in the art will appreciate that other libraries may be prepared by
adapting the protocols described in the aforementioned references.
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.
[0094] 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 IRORI 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.
[0095] 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.
[0096] In one aspect the present invention also relates to methods
of synthezising libraries of test compounds, wherein said libraries
are in particular useful for the screening methods of the
invention.
[0097] In one embodiment, the invention thus relates to methods of
synthesising a cyclic peptide or peptide mimetic library,
comprising the steps [0098] i) Providing a plurality of peptides or
peptide mimetics, (preferably peptides) covalently linked to an
azide moiety and an acetylene moeity; and [0099] ii) cyclizing said
peptide or peptide mimetic through a Cu(I) catalysed reaction
between said azide- and said acetylene moiety; and [0100] iii)
thereby obtaining a library of cyclic peptides or peptide
mimetics.
[0101] Each peptide preferably only comprises one azide moeity and
one acetylene moiety. An example of a method of preparing such a
library is given in example 4 herein below.
[0102] In another embodiment, the invention relates to methods of
synthesising a library of heterocyclic ureas, comprising the steps
of [0103] i) Providing a plurality of urea containing peptide
aldehydes; and [0104] ii) Subjecting said urea containing peptides
to an intramolecular Pictet-Spengler reaction; and [0105] iii)
Thereby obtaining a library of heterocyclic ureas
[0106] Said urea containing peptide aldehydes are preferably
peptides covalently linked to at least one urea moeity and one
aldehyde moeity. The intramolecular Pictet-Spengler reaction may
for example be performed as described in WO2004/113362 claiming
priority from Danish patent application PA 2003 00967, both are
hereby incorporated by reference.
[0107] An example of a method of preparing such libraries is given
in examples 5 and 5a herein below.
[0108] 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.
[0109] 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,
[0110] The invention also relates to libraries prepared by any of
the methods described above.
[0111] 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
WO2004/113362 claiming priority from Danish patent application PA
2003 00967, both are hereby incorporated by reference.
Resin Beads
[0112] 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).
[0113] 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
covalently bound. 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 poly-propylene,
ethylene and/or teflon. Gels could be prepared from for example
poly acrylamid or PEGA.
[0114] 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.
[0115] 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, Poly-acrylamide 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.
[0116] 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) or Poly-OxyEthylene-PolyOxyPropylene (POEPOP) resin.
Another preferred resin comprises a crosslinked polyacrylamide
resin.
[0117] PEGA (PolyEthyleneGlycol Acrylamide copolymer; Meldal M.,
1992, Tetrahedron Lett., 33: 3077-80), POEPOP
(PolyOxyEthylene-PolyOxyPropylene; Renil et al., 1996, Tetrahedron
Lett., 37: 6185-88) and SPOCC (Super Permeable Organic
Combinatorial Chemistry; 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, are 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.
[0118] 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.
Cells
[0119] 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.
[0120] 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 Mouse Embryo
Fibroblast albino (CCL-92) 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 Ovary Fibroblast hamster 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 Min 6
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
[0121] 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).
[0122] In another preferred example the cell comprises a nucleic
acid comprising a first nucleotide sequence encoding a cell surface
molecule operably linked to a second nucleotide sequence not
naturally associated therewith directing expression of said first
sequence. The cell surface molecule may be any of the cell surface
molecules described herein below. Such cells are in particular
useful for identification of compounds modulating the activity of
said cell surface molecule. Said nucleic acid may be introduced
transiently or stably into said cells.
[0123] 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
[0124] 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.
[0125] 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
[0126] 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
[0127] 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.
[0128] 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.
[0129] 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.
[0130] 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".
[0131] Said peptide preferably consists of in the range of 4 to
100, preferably in the range of 4 to 75, more preferably in the
range of 4 to 50, even more preferably in the range of 4 to 30, yet
more preferably in the range of 4 to 25, even more preferably in
the range of 4 to 20, yet more preferably in the range of 4 to 15,
such as in the range of 4 to 10, for example in the range of 4 to
8, for example in the range of 6 to 7 amino acids. In general, it
is sufficient if the peptide comprises at least 4 amino acids.
[0132] 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 D-amino acids are used to enhance the
metabolic stability but also L-amino acids may be used.
[0133] 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 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 leu 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
[0134] 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 immoblised 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.
[0135] 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.
[0136] 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.
[0137] Peptides useful as cell adhesion compounds may be identified
using any suitable method. Said method may for example include the
steps of [0138] i) coupling a test peptide to a resin bead; [0139]
ii) incubating said resin bead with cells under cell cultivation
conditions; [0140] iii) testing whether said cells attach to said
resin bead [0141] 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.
[0142] 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
incubated with cells, A non-limiting example of a useful method is
described in example 1a.
[0143] 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 N.sub.3 or Alloc. In one embodiment Alloc is the
preferred protective group. It is preferred that different
protecting group 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 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 anchoring Fmoc-amino acids to
hydroxyl-functionalised solid supports. Tetrahedron Lett.
31:1701-1704, 1990. 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. An non-limiting example of a method for
synthesizing an adhesion peptide is given in example 5a, "Synthesis
of adhesion peptide" herein below.
[0144] In one embodiment the adhesion compound 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 below. 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.
[0145] 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.
[0146] 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.
[0147] 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 [0148]
Providing resin beads capable of supporting growth of cells [0149]
Seeding cells onto said resin bead [0150] Incubating said resin
beads comprising said cells in a cell culture medium under cell
cultivation conditions [0151] Optionally allowing said cells to
divide on said resin bead [0152] Thereby cultivating cells on resin
beads
[0153] The cells may adhere actively to the resin beads and will
then generally be referred to as adherent cells.
[0154] 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 the
container comprising the resin beads is simply rocked gently a few
times every now and then.
[0155] In another embodiment of the invention cells may be attached
to resin beads, without active adherence. For example, this may be
the case for non-adherent cells, i.e. cells that may be cultivated
in suspension.
[0156] 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.
[0157] 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.
[0158] 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 may also be unrelated.
Cleavable Linkers
[0159] The library of test compounds and/or the adhesion compound
may in one embodiment be linked to the resin beads or solid
supports by a cleavable linker.
[0160] The cleavable linker may be any chemical moiety which may be
used to attach a molecule to a solid support either covalently or
via complex formation, and thereafter is capable of releasing 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/adhesion molecule to the solid support covalently. 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.
[0161] Examples of useful acid labile linkers include the most
commonly used linkers for acidic detachment from a solid support,
the Wang and Rink linkers. Examples of useful base-labile linkers
includes Wang and HMBA linkers, which may be cleaved under alkaline
conditions. Light sensitive cleavable linkers are linkers which,
upon the action of light with a given wave length and intensity,
may release the library member/adhesion compound from the solid
support. Photo-labile linkers cleavable by irradiation with
UV-light may be o-nitrobenzyl type of linkers (nitrated analogs of
the Wang linker), NBA type linkers or Holmes-type linkers. Paladium
linkers may also be used with the invention.
[0162] In one embodiment photolabile linkers are preferred
Cell Surface Molecules
[0163] 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 a cell
surface molecule. Hence, the invention, for example may be useful
for identifying compounds modulating the activity of a cell surface
molecule, preferably a cell surface molecule capable of
activating/repressing a signal transduction pathway. 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.
[0164] Signal transduction pathways may for example involve steps
of 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
[0165] The cell surface molecule 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.
[0166] In a preferred embodiment the cell surface molecule is a
G-protein coupled receptor (GPCR). GPCR is a family of receptors
coupled to a trimeric G-protein. GPCR to be used with the invention
preferably have 7 transmembrane domains. Examples of useful GPCR
are given in table 3.
[0167] GPCR may be divided into subfamilies, accordingly the GPCR
may selected from the group consisting of GPCR belonging the
rhodopsin like family, the secretin family or the metabotropic
family, preferably from the group consisting of GPCR belonging the
rhodopsin like family or the secretin family.
[0168] Rhodopsin like GPCR are also referred to as Class I GPCR.
They are charaterised by a structurally similarity to the Rhodopsin
receptor. Preferred examples of members of this family includes
receptors for the following ligands: Acetylcholine (muscarinic
& nicotinic), Adrenoceptors, Alpha Adrenoceptors, Beta
Adrenoceptors, Dopamine, Histamine, Serotonin (5-HT), Angiotensin,
Bradykinin, C5a anaphylatoxin, Fmet-leu-phe, Interleukin-8,
ochernokine, Orexin, Nociceptin, CCK (Gastrin), Endothelin,
Melanocortin including any of melanocortin 1 to 5 receptors,
Neuropeptide Y, Neurotensin, Opioid, Somatostatin, Tachykinin
(Substance P, NKA.sub.1), Thrombin, vasopressin-like, Galanin,
Follicle stimulating hormone, Lutropinchoriogonadotropic,
Thyrotropin, Rhodopsin, Opsin, Prostaglandin, Lysophosphatidic
Acid, Sphingosine-1-phosphate, Leukotriene, Prostacyclin,
Thromboxane, Adenosine, Purinoceptors, Cannabis, Platelet
activating factor, Gonadotropin-releasing Hormone,
Thyrotropin-releasing hormone, Growth hormone-inhibiting factor or
Melatonin.
[0169] Secretin like GPCR are also referred to as Class II GPCR.
They are charaterised by a structurally similarity to the Secretin
receptor. (Accession No NM.sub.--002980) Preferred examples of
members of this family includes receptors for the following
ligands: Secretin, calcitonin, Corticotropin releasing
factor/urocortin, Gastric inhibitory peptide (GIP), Glucagon,
Glucagon-like Peptide 1 (GLP-1), Growth hormone-releasing hormone,
Parathyroid hormone, PACAP or Vasoactive intestinal polypeptide
(VIP).
[0170] Metabotropic GCPR are also referred to as class III GPCR.
Preferred examples of members of this group includes receptors for
the following ligands: Metabotropic Glutamate, GABA.sub.8, or
Extracellular Calcium Sensing.
[0171] In another preferred embodiment of the invention the GPCR is
coupled to a G-protein, such as G.sub.S, that stimulates adenylate
cyclase. In yet another preferred embodiment of the invention the
GPCR is coupled to a G-protein, such as G.sub.I, that inhibits
adenylate cyclase. Examples of GPCRs coupled to G.sub.S or G.sub.I
are given in table 3.
Gene Ontology Blast Serverfull
TABLE-US-00003 [0172] TABLE 3 Gene symbol* Gene Ontology Blast
Server Full name G-protein signaling, coupled to cyclic nucleotide
second messenger NEUY_HUMAN Neuropeptide Y precursor [Contains:
Neuropeptide Y ACM2_HUMAN Muscarinic acetylcholine receptor M2
SY02_HUMAN Small inducible cytokine A2 precursor B3AR_HUMAN Beta-3
adrenergic receptor TSHR_HUMAN Thyrotropin receptor precursor
CB1R_HUMAN Cannabinoid receptor 1 DADR_HUMAN D(1A) dopamine
receptor LSHR_HUMAN Lutropin-choriogonadotropic hormone receptor
precursor HH2R_HUMAN Histamine H2 receptor NY1R_HUMAN Neuropeptide
Y receptor type 1 5H1D_HUMAN 5-hydroxytryptamine 1D receptor
5H1B_HUMAN 5-hydroxytryptamine 1B receptor 5H1E_HUMAN
5-hydroxytryptamine 1E receptor SSR1_HUMAN Somatostatin receptor
type 1 SSR2_HUMAN Somatostatin receptor type 2 5H1F_HUMAN
5-hydroxytryptamine 1F receptor SSR4_HUMAN Somatostatin receptor
type 4 VIPR_HUMAN Vasoactive intestinal polypeptide receptor 1
precursor CKR1_HUMAN C-C chemokine receptor type 1 SSR3_HUMAN
Somatostatin receptor type 3 MC5R_HUMAN Melanocortin-5 receptor
5H7_HUMAN 5-hydroxytryptamine 7 receptor CB2R_HUMAN Cannabinoid
receptor 2 CRF1_HUMAN Corticotropin releasing factor receptor 1
precursor SSR5_HUMAN Somatostatin receptor type 5 OPRM_HUMAN
Mu-type opioid receptor OPRD_HUMAN Delta-type opioid receptor
MC3R_HUMAN Melanocortin-3 receptor PI2R_HUMAN Prostacyclin receptor
CXC1_HUMAN Chemokine XC receptor 1 ML1A_HUMAN Melatonin receptor
type 1A ML1B_HUMAN Melatonin receptor type 1B 5H6_HUMAN
5-hydroxytryptamine 6 receptor ACTR_HUMAN Adrenocorticotropic
hormone receptor MSHR_HUMAN Melanocyte stimulating hormone receptor
PTRR_HUMAN Parathyroid hormone/parathyroid hormone-related Peptide
receptor precursor 5H4_HUMAN 5-hydroxytryptamine 4 receptor
CGRR_HUMAN Calcitonin gene-related peptide type 1 receptor
precursor EDG7_HUMAN Lysophosphatidic acid receptor Edg-7
HH3R_HUMAN Histamine H3 receptor Htr7 RGD 5-hydroxytryptamine
(serotonin) receptor 7 G-protein signaling, coupled to cAMP
nucleotide second messenger PE23_MOUSE Prostaglandin E2 receptor,
EP3 subtype CYA4_MOUSE Adenylate cyclase, type IV P2YC_MOUSE P2Y
purinoceptor 12 GALS_HUMAN Galanin receptor type 2 GLP2_HUMAN
Glucagon-like peptide 2 receptor precursor CAL0_HUMAN Calcitonin
precursor [Contains: Calcitonin; Katacalcin SLIB_HUMAN
Somatoliberin precursor CAL1_HUMAN Calcitonin gene-related peptide
I precursor B2AR_HUMAN Beta-2 adrenergic receptor ACM2_HUMAN
Muscarinic acetylcholine receptor M2 B3AR_HUMAN Beta-3 adrenergic
receptor FMLR_HUMAN fMet-Leu-Phe receptor A1AD_HUMAN Alpha-1D
adrenergic receptor AA2A_HUMAN Adenosine A2a receptor V2R_HUMAN
Vasopressin V2 receptor PE23_MOUSE Prostaglandin E2 receptor, EP3
subtype MC4R_HUMAN Melanocortin-4 receptor GRK5_HUMAN G
protein-coupled receptor kinase GRK5 CRF1_HUMAN Corticotropin
releasing factor receptor 1 precursor A1AB_HUMAN Alpha-1B
adrenergic receptor PE24_HUMAN Prostaglandin E2 receptor, EP4
subtype GLR_HUMAN Glucagon receptor precursor CKR3_HUMAN C-C
chemokine receptor type 3 CRF2_HUMAN Corticotropin releasing factor
receptor 2 precursor Q8BZV8 P2Y12 platelet ADP receptor homolog
CYA4_MOUSE Adenylate cyclase, type IV Q99188 ORF OR107W from
chromosome XV P2YC_MOUSE P2Y purinoceptor 12 WAS2_HUMAN
Wiskott-Aldrich syndrome protein family member 2 Adcy2 MGI
adenylate cyclase 2 Adcy4 MGI adenylate cyclase 4 P2ry12 MGI With
purinergic receptor P2Y, G-protein coupled 12 Ptger3 MGI
prostaglandin E receptor 3 (subtype EP3) Crhr1 RGD I corticotropin
releasing hormone 1 CYR1 SGD adenylate cyclase RGS2 SGD GTPase
activating protein (GAP) acy-1 IMP - [cgc3038] acy-2 IMP -
[cgc3207] C44F1.5 [cgc3038] acy-4 [cgc3207] G-protein signaling,
adenylate cyclase activating pathway GBQ_MOUSE Guanine
nucleotide-binding protein G(q), alpha subunit Q9D1X2 Thyroid
stimulating hormone, receptor TSHR_MOUSE Thyrotropin receptor
precursor Q9WUC0 Extra large alpha stimulating guanine-nucleotide
Binding polypeptide Q9Z0H2 Neuroendocrine-specific golgi protein
P55 isoform 2 Q9Z0L1 G protein-coupled receptor precursor Q9Z1N8
G-protein XLalphas Q9Z1R7 Guanine nucleotide-binding protein
AA2A_MOUSE Adenosine A2a receptor LGR8_MOUSE Relaxin receptor 2
UCN3_MOUSE Urocortin III precursor RAS1_YEAST Ras-like protein 1
GBQ_MOUSE Guanine nucleotide-binding protein G(q), alpha subunit
CALR_HUMAN Calcitonin receptor precursor GPR3_HUMAN Probable G
protein-coupled receptor GPR3 TSHR_MOUSE Thyrotropin receptor
precursor Q14455 Alpha subunit of GsGTP binding protein (Fragment)
AA2A_MOUSE Adenosine A2a receptor GBAF_MOUSE Guanine
nucleotide-binding protein G(OLF), Alpha subunit (Fragment)
GB10_MOUSE Guanine nucleotide-binding protein, alpha-10 subunit
Fragment) Q80ZK6 Similar to GNAS (Fragment) Q8BIR3 XLALPHAS protein
homolog Q8BM77 Similar to G protein coupled receptor AFFECTING
testicular DESCENT Q8BUB2 GNAS Q8BXD1 Similar to G protein coupled
receptor AFFECTING testicular DESCENT Q8C6E2 Inferred: endothelial
differentiation Q8CAU3 Adenosine A2a receptor (Fragment) LGR8_MOUSE
Relaxin receptor 2 UCN3_MOUSE Urocortin III precursor Q9D1X2
Thyroid stimulating hormone, receptor Q9D697 Thyroid stimulating
hormone, receptor Q9JJX0 Xlalphas protein (Fragment) Q9QXW5 Nesp
Q9QYZ0 Extra large alpha stimulating guanine-nucleotide binding
protein (Fragment) Q9WUC0 Extra large alpha stimulating
guanine-nucleotide binding polypeptide Q9Z0H2
Neuroendocrine-specific golgi protein P55 isoform 2 Q9Z0L1 G
protein-coupled receptor precursor Q9Z1N8 G-protein XLalphas Q9Z1R7
Guanine nucleotide-binding protein Adora2a adenosine A2a receptor
Edg6 endothelial differentiation, G-protein-coupled receptor 6 Gnal
guanine nucleotide binding protein, alpha stimulating, olfactory
type Gnaq guanine nucleotide binding protein, alpha q polypeptide
Gnas GNAS (guanine nucleotide binding protein, alpha stimulating)
complex locus Gnas GNAS (guanine nucleotide binding protein, alpha
stimulating) complex locus Gpr106 G protein-coupled receptor 106
Ptger4 prostaglandin E receptor 4 (subtype EP4) Tshr thyroid
stimulating hormone receptor Ucn3 urocortin 3 RAS1 ras homolog
adenylate cyclase activation piaA cytosolic regulator of adenylyl
cyclase pianissimo G-salpha60A G-salpha60A Pacap38 F Pacap38
DADR_MOUSE D(1A) dopamine receptor O43190 Not Available CAL0_HUMAN
Calcitonin precursor [Contains: Calcitonin; Katacalcin GBAS_HUMAN
Guanine nucleotide-binding protein G(S), alpha subunit CAL1_HUMAN
Calcitonin gene-related peptide I precursor B2AR_HUMAN Beta-2
adrenergic receptor B1AR_HUMAN Beta-1 adrenergic receptor
PACA_HUMAN Pituitary adenylate cyclase activating polypeptide
precursor ET1R_HUMAN Endothelin-1 receptor precursor AA2A_HUMAN
Adenosine A2a receptor AA2B_HUMAN Adenosine A2b receptor V2R_HUMAN
Vasopressin V2 receptor CALR_HUMAN Calcitonin receptor precursor
AA3R_HUMAN Adenosine A3 receptor CRF1_HUMAN Corticotropin releasing
factor receptor 1 precursor CAP2_HUMAN Adenylyl cyclase-associated
protein 2 GLP1_HUMAN Glucagon-like peptide 1 receptor precursor
GIPR_HUMAN Gastric inhibitory polypeptide receptor precursor
CAP1_HUMAN Adenylyl cyclase-associated protein 1 GRFR_HUMAN Growth
hormone-releasing hormone receptor precursor GBAF_MOUSE Guanine
nucleotide-binding protein G(OLF), alpha sub-unit (Fragment)
GB10_MOUSE Guanine nucleotide-binding protein, alpha-10 subunit
(Fragment) DADR_MOUSE D(1A) dopamine receptor B2AR_ONCMY Beta-2
adrenergic receptor Adcy1 adenylate cyclase 1 Adcy2 adenylate
cyclase 2 Adcy3 adenylate cyclase 3 Adcy4 adenylate cyclase 4 Adcy5
adenylate cyclase 5 Adcy6 adenylate cyclase 6 Adcy7 adenylate
cyclase 7 Adcy9 adenylate cyclase 9 Adcyap1 adenylate cyclase
activating polypeptide 1 Drd1a dopamine receptor D1A Gnal guanine
nucleotide binding protein, alpha stimulating, olfactory type RAS2
small GTP-binding protein dopamine receptor, adenylate cyclase
activating pathway Q8CH75 Mu opioid receptor variant P Q8VI69 Mu
opioid receptor variant BII OPRM_MOUSE Mu-type opioid receptor
Q9JIY1 Mu opioid receptor variant F Q9R0D1 Mu opioid receptor
variant C Q9R1L9 Mu opioid receptor MOR1E DADR_MOUSE D(1A) dopamine
receptor DADR_HUMAN D(1A) dopamine receptor DBDR_HUMAN D(1B)
dopamine receptor OPRM_MOUSE Mu-type opioid receptor DADR_MOUSE
D(1A) dopamine receptor Q8CAN5 Opioid receptor Q8CGW2 Mu opioid
receptor variant MOR-1R Q8CH73 Mu opioid receptor variant R Q8CH74
Mu opioid receptor variant Q Q8CH75 Mu opioid receptor variant P
Q8VI69 Mu opioid receptor variant BII Q8VI70 Mu opioid receptor
variant BI Q8VI71 Mu opioid receptor variant A OPR2_MOUSE Mu-type
opioid receptor, isoforms 1G to 1M Q9JIY1 Mu opioid receptor
variant F Q9R0D1 Mu opioid receptor variant C Q9R0D2 Mu opioid
receptor variant 110222 (Fragment) Q9R1L9 Mu opioid receptor MOR1E
Q9R1M0 Mu opioid receptor MOR1D Drd1a dopamine receptor D1A NOT
Oprd1 opioid receptor, delta 1 Oprm opioid receptor, mu Tar1 trace
amine receptor 1 Serotonin receptor, adenylate cyclase activating
pathway 5-HT7 5-HT7 5-HT7 5-HT7 5-HT7 5-HT7 Htr7
5-hydroxytryptamine (serotonin) receptor 7 G-protein signaling,
adenylate cyclase inhibiting pathway GBI2_MOUSE Guanine
nucleotide-binding protein G(i),
alpha-2 subunit Q8CH75 Mu opioid receptor variant P Q8VI69 opioid
receptor variant BII SSR2_MOUSE Somatostatin receptor type 2
OPRD_MOUSE Delta-type opioid receptor Q9DC35 Endothelial
differentiation sphingolipid G-protein-coupled receptor 1
OPRM_MOUSE Mu-type opioid receptor Q9JIY1 Mu opioid receptor
variant F Q9R0D1 Mu opioid receptor variant C Q9R1L9 Mu opioid
receptor MOR1E Q9Z0U9 LYSOPHOSPHOLIPID receptor B3 EDG1_MOUSE
Probable G protein-coupled receptor Edg-1 CORT_HUMAN Cortistatin
precursor [Contains: Cortistatin-29; Cortistatin-17] EDG1_MOUSE
Probable G protein-coupled receptor Edg-1 GBI2_MOUSE Guanine
nucleotide-binding protein G(i), alpha-2 subunit NY1R_HUMAN
Neuropeptide Y receptor type 1 SSR2_MOUSE Somatostatin receptor
type 2 OPRD_MOUSE Delta-type opioid receptor OPRK_HUMAN Kappa-type
opioid receptor OPRX_HUMAN Nociceptin receptor OPRM_MOUSE Mu-type
opioid receptor NY2R_HUMAN Neuropeptide Y receptor type 2
RGS1_HUMAN Regulator of G-protein signaling 1 Q8BLP9 Delta-type
opioid receptor Q8BP20 Endothelial differentiation Q8C4A3
Endothelial differentiation sphingolipid G-protein-coupled receptor
1 Q8CAN5 Opioid receptor Q8CGW2 Mu opioid receptor variant MOR-1R
Q8CH73 Mu opioid receptor variant R Q8CH74 Mu opioid receptor
variant Q Q8CH75 Mu opioid receptor variant P Q8JZT4 Similar to
guanine nucleotide binding protein, alpha inhibiting 2 Q8VI69 Mu
opioid receptor variant BII Q8VI70 Mu opioid receptor variant BI
Q8VI71 Mu opioid receptor variant A OPR2_MOUSE Mu-type opioid
receptor, isoforms 1G to 1M Q922Y6 Hypothetical protein (Fragment)
MCR1_HUMAN Melanin-concentrating hormone receptor 1 Q9DC35
Endothelial differentiation sphingolipid G-protein-coupled receptor
1 Q9JIY1 Mu opioid receptor variant F Q9R0D1 Mu opioid receptor
variant C Q9R0D2 Mu opioid receptor variant 110222 (Fragment)
Q9R1L9 Mu opioid receptor MOR1E Q9R1M0 Mu opioid receptor MOR1D
Q9R235 Lysophospholipid receptor B1 Q9Z0U9 LYSOPHOSPHOLIPID
receptor B3 Edg1 endothelial differentiation sphingolipid
G-protein-coupled receptor 1 Edg3 endothelial differentiation,
sphingolipid G-protein-coupled receptor, Gnai2 guanine nucleotide
binding protein, alpha inhibiting 2 Npb neuropeptide B Oprd1 opioid
receptor, delta 1 Oprm opioid receptor, mu Sstr2 somatostatin
receptor 2 Oprm1 "Opioid receptor, mu 1" dopamine receptor,
adenylate cyclase inhibiting pathway D2DR_HUMAN D(2) dopamine
receptor muscarinic acetyl choline receptor, adenylate cyclase
inhibiting pathway ACM2_HUMAN Muscarinic acetylcholine receptor M2
ACM5_HUMAN Muscarinic acetylcholine receptor M5 Negative regulation
of adenylate cyclase activity MGR8_HUMAN Metabotropic glutamate
receptor 8 precursor GALT_HUMAN Galanin receptor type 3 GBR2_HUMAN
Gamma-aminobutyric acid type B receptor, subunit 2 precursor
GBI2_HUMAN Guanine nucleotide-binding protein G(i), alpha-2 subunit
GBAK_HUMAN Guanine nucleotide-binding protein G(k), alpha subunit
A2AA_HUMAN Alpha-2A adrenergic receptor ETBR_HUMAN Endothelin B
receptor precursor CKR2_HUMAN C-C chemokine receptor type 2
GALR_HUMAN Galanin receptor type 1 MGR2_HUMAN Metabotropic
glutamate receptor 2 precursor MGR7_HUMAN Metabotropic glutamate
receptor 7 precursor MGR3_HUMAN Metabotropic glutamate receptor 3
precursor MGR4_HUMAN Metabotropic glutamate receptor 4 precursor
Q9NPE5 Not Available GBR1_HUMAN Gamma-aminobutyric acid type B
receptor, subunit 1 precursor positive regulation of adenylate
cyclase activity dagA cytosolic regulator of adenylyl cyclase
serotonin receptor, adenylate cyclase inhibiting pathway 5-HT1A
5-HT1A 5-HT1B 5-HT1B 5HTA_DROME 5-hydroxytryptamine receptor 2A
5HTB_DROME 5-hydroxytryptamine receptor 2B Q9V8Q3 CG15113-PA Q9V8Q9
CG16720 protein G-protein signaling, coupled to cGMP nucleotide
second messenger TBL3_HUMAN WD-repeat protein SAZD 4933400B15Rik
RIKEN cDNA 4933400B15 gene Gnat1 guanine nucleotide binding
protein, alpha transducing 1 Gnat2 guanine nucleotide binding
protein, alpha transducing 2 Tbl3 transducin (beta)-like 3 Nos2
nitric oxide synthase 2 G-protein signaling, coupled to IP3 second
messenger (phospholipase C activating) GB15_MOUSE Guanine
nucleotide-binding protein, alpha-15 subunit Q9ERT2
Thyrotropin-releasing hormone receptor 2 GBGD_MOUSE Guanine
nucleotide-binding protein G(I)/G(S)/G(O) gamma-13 subunit O43190
Not Available O76067 Not Available ETBR_HUMAN Endothelin B receptor
precursor IL8B_HUMAN High affinity interleukin-8 receptor B
NK1R_HUMAN Substance-P receptor NMBR_HUMAN Neuromedin-B receptor
AG2R_HUMAN Type-1 angiotensin II receptor PE23_MOUSE Prostaglandin
E2 receptor, EP3 subtype OXYR_HUMAN Oxytocin receptor GB15_MOUSE
Guanine nucleotide-binding protein, alpha-15 subunit CCKR_HUMAN
Cholecystokinin type A receptor GASR_HUMAN Gastrin/cholecystokinin
type B receptor HH1R_HUMAN Histamine H1 receptor P2Y2_HUMAN P2Y
purinoceptor 2 5H2B_HUMAN 5-hydroxytryptamine 2B receptor
MC3R_HUMAN Melanocortin-3 receptor P2YR_HUMAN P2Y purinoceptor 1
P2Y4_HUMAN P2Y purinoceptor 4 P2Y6_HUMAN P2Y purinoceptor 6
L4R1_HUMAN Leukotriene B4 receptor 1 Q61621 G-protein beta subunit
(Fragment) Q9ERT1 Thyrotropin-releasing hormone receptor 2
(Fragment) Q9ERT2 Thyrotropin-releasing hormone receptor 2
GBGD_MOUSE Guanine nucleotide-binding protein G(I)/G(S)/G(O)
gamma-13 subunit Q9NYK7 CCK-B/gastrin receptor Gna15 guanine
nucleotide binding protein, alpha 15 Gnb1 guanine nucleotide
binding protein, beta 1 Gng13 guanine nucleotide binding protein
13, gamma Ptger3 prostaglandin E receptor 3 (subtype EP3) Trhr2
thyrotropin releasing hormone receptor 2 Agtr1a angiotensin
receptor 1a cytosolic calcium ion concentration elevation norpA
norpA Q99L49 Similar to transient receptor protein 2 PE23_MOUSE
Prostaglandin E2 receptor, EP3 subtype TRP6_MOUSE Short transient
receptor potential channel 6 JPH2_MOUSE Junctophilin 2 SY28_MOUSE
Small inducible cytokine A28 precursor CKRA_MOUSE C-C chemokine
receptor type 10 TRP2_MOUSE Short transient receptor potential
channel 2 O43431 Not Available GALS_HUMAN Galanin receptor type 2
SZ13_HUMAN Small inducible cytokine B13 precursor O95977 Putative
G-protein coupled receptor, EDG6 precursor CAL0_HUMAN Calcitonin
precursor [Contains: Calcitonin; Katacalcin CAL1_HUMAN Calcitonin
gene-related peptide I precursor DADR_HUMAN D(1A) dopamine receptor
C5AR_HUMAN C5a anaphylatoxin chemotactic receptor ET1R_HUMAN
Endothelin-1 receptor precursor BRB2_HUMAN B2 bradykinin receptor
AG2R_HUMAN Type-1 angiotensin II receptor PE23_MOUSE Prostaglandin
E2 receptor, EP3 subtype CCR4_HUMAN C--X--C chemokine receptor type
4 CCKR_HUMAN Cholecystokinin type A receptor GASR_HUMAN
Gastrin/cholecystokinin type B receptor CKR1_HUMAN C-C chemokine
receptor type 1 CKR7_HUMAN C-C chemokine receptor type 7 precursor
V1AR_HUMAN Vasopressin V1a receptor CKR2_HUMAN C-C chemokine
receptor type 2 CXC1_HUMAN Chemokine XC receptor 1 BRB1_HUMAN B1
bradykinin receptor V1BR_HUMAN Vasopressin V1b receptor CCR3_HUMAN
C--X--C chemokine receptor type 3 P2Y4_HUMAN P2Y purinoceptor 4
CKR3_HUMAN C-C chemokine receptor type 3 CKR4_HUMAN C-C chemokine
receptor type 4 CKR5_HUMAN C-C chemokine receptor type 5 (CCR5)
CKR6_HUMAN C-C chemokine receptor type 6 CKR8_HUMAN C-C chemokine
receptor type 8 CKR9_HUMAN C-C chemokine receptor type 9 PAR2_HUMAN
Proteinase activated receptor 2 precursor C3AR_HUMAN C3a
anaphylatoxin chemotactic receptor Q61057 Trp-related protein 2
(Fragment) TRP6_MOUSE Short transient receptor potential channel 6
Q8BRU2 Transient receptor protein 2 Q8CDC6 Transient receptor
protein 2 Q8CEM7 Transient receptor protein 2 (Fragment) EDG2_HUMAN
Lysophosphatidic acid receptor Edg-2 EDG3_HUMAN Sphingosine
1-phosphate receptor Edg-3 MCR1_HUMAN Melanin-concentrating hormone
receptor 1 Q99L49 Similar to transient receptor protein 2
JPH2_MOUSE Junctophilin 2 SY28_MOUSE Small inducible cytokine A28
precursor CKRA_MOUSE C-C chemokine receptor type 10 Q9NYK7
CCK-B/gastrin receptor TRP2_MOUSE Short transient receptor
potential channel 2 EDG7_HUMAN Lysophosphatidic acid receptor Edg-7
CLT1_HUMAN Cysteinyl leukotriene receptor 1 Ccl28 chemokine (C-C
motif) ligand 28 Edg3 endothelial differentiation, sphingolipid
G-protein-coupled receptor, 3 Gpr2 G protein-coupled receptor 2
Jph2 junctophilin 2 Ptger3 prostaglandin E receptor 3 (subtype EP3)
Trpc2 transient receptor potential cation channel, subfamily C,
member 2 Trpc6 transient receptor potential cation channel,
subfamily C, member 6 Itpr3 inositol 1, 4, 5-triphosphate receptor
3'' Trrp6 "transient receptor potential cation channel, subfamily
C, member 6" itr-1 Not Available Metabotropic glutamate receptor,
phospholipase C activating pathway O96003 SYN47 protein MGR5_HUMAN
Metabotropic glutamate receptor 5 precursor Grm5 "glutamate
receptor, metabotropic 5" Muscarinic acetyl choline receptor,
phospholipase C activating pathway ACM2_HUMAN Muscarinic
acetylcholine receptor M2 ACM1_HUMAN Muscarinic acetylcholine
receptor M1 (herein also designated Muscarinic M1) GB15_HUMAN
Guanine nucleotide-binding protein, alpha-15 subunit Chrm3
cholinergic receptor, muscarinic 3, cardiac phospholipase C
activation Galpha49B Galpha49B Gbeta76C Gbeta76C O43431 Not
Available O95977 Putative G-protein coupled receptor, EDG6
precursor CAL0_HUMAN Calcitonin precursor [Contains: Calcitonin;
Katacalcin CAL1_HUMAN Calcitonin gene-related peptide I precursor
C5AR_HUMAN C5a anaphylatoxin chemotactic receptor GBQ_DROME Guanine
nucleotide-binding protein G(q), alpha subunit ET1R_HUMAN
Endothelin-1 receptor precursor GBB2_DROME Guanine
nucleotide-binding protein beta subunit 2 GB15_HUMAN Guanine
nucleotide-binding protein, alpha-15 subunit CALR_HUMAN Calcitonin
receptor precursor GASR_HUMAN Gastrin/cholecystokinin type B
receptor V1AR_HUMAN Vasopressin V1a receptor V1BR_HUMAN Vasopressin
V1b receptor
GBQ_HUMAN Guanine nucleotide-binding protein G(q), alpha subunit
PIB2_HUMAN 1-phosphatidylinositol-4,5-bisphosphate phospho
diesterase beta 2 EDG2_HUMAN Lysophosphatidic acid receptor Edg-2
Q9I7C8 G protein alpha 49B Q9NYK7 CCK-B/gastrin receptor Q9TXA4
Signal-transducing G protein alpha Q subunit Q9VW29 GBETA76C
protein Adra1a "adrenergic receptor, alpha 1a" Adcyap1r1 adenylate
cyclase activating polypeptide 1 receptor 1 protein kinase C
activation PF14_0681 diacylglycerol kinase, putative Pfalciparum
GBLP_HUMAN Guanine nucleotide-binding protein beta subunit-like
protein 12.3 PIC1_MOUSE PRKCA-binding protein GBLP_HUMAN Guanine
nucleotide-binding protein beta subunit-like protein 12.3 PIC1_RAT
PRKCA-binding protein KPCN_HUMAN Protein kinase C, nu type
ACM1_HUMAN Muscarinic acetylcholine receptor M1 NEUM_HUMAN
Neuromodulin CAP7_HUMAN Azurocidin precursor ET2_HUMAN Endothelin-2
precursor GBLP_HUMAN Guanine nucleotide-binding protein beta
subunit-like protein 12.3 143F_HUMAN 14-3-3 protein eta PIC1_MOUSE
PRKCA-binding protein Q80VC8 Similar to protein that interacts with
C kinase 1 Q8C1W2 Protein that interacts with C kinase 1 PIC1_RAT
PRKCA-binding protein PIC1_HUMAN PRKCA-binding protein
C130010K08Rik RIKEN cDNA C130010K08 gene Cerk ceramide kinase
Gnb2-rs1 guanine nucleotide binding protein, beta 2, related
sequence 1 F13G24.120 diacylglycerol kinase 1 (DGK1) F17I23.320
diacylglycerol kinase family F18E5.160 diacylglycerol kinase family
F26K10.10 diacylglycerol (DAG) kinase accessory domain protein
F5H14.13 diacylglycerol kinase, putative K19M13.8 diacylglycerol
kinase family MBK5.25 diacylglycerol kinase, putative MRI1.5
diacylglycerol kinase, putative MSF3.11 diacylglycerol kinase,
putative F13G24.120 diacylglycerol kinase 1 (DGK1) F26K10.10
diacylglycerol (DAG) kinase accessory domain protein T3F17.26
diacylglycerol kinase family serotonin receptor, phospholipase C
activating pathway 5-HT1A 5-HT1A 5-HT1A 5-HT1A 5-HT1B 5-HT1B 5-HT1B
5-HT1B 5HTA_DROME 5-hydroxytryptamine receptor 2A 5HTB_DROME
5-hydroxytryptamine receptor 2B Q9V8Q3 CG15113-PA Q9V8Q9 CG16720
protein Htr2b 5-hydroxytryptamine (serotonin) receptor 2B Gene
symbol refers to the symbol used in the Gene Ontology Blast server
available 25 May 2005 at http://
godatabase.org/cgi-bin/go.cgi?view=blast&session_id=87201075891145.
[0173] In an even further embodiment of the invention the GPCR is
coupled to a G-protein, such as G.sub.Q, that activates
phospholipase C. Examples of such GPCRs are given in table 3.
[0174] Other receptors than GPCR may be used with the present
invention, for example the cell surface molecule may be a receptor
selected from the group consisting of receptors belonging to the
family of protein kinase coupled receptors and receptors belonging
to the family of receptor kinases.
[0175] The family of Protein kinase coupled receptors for example
includes receptors for cytokines, interferons and HGF. These
receptors do not have intrinsic kinase activity, but are associated
with a kinase.
[0176] Activation of preferred protein kinase coupled receptors
results in activation of AP-1, i.e. in increased transcription from
genes comprising one or more AP-1 sites in their regulatory
sequences. This is in particular true for receptors activated by a
cytokine.
[0177] Receptor kinases are receptors having an intrinsic kinase
activity. Frequently said activity may be modulated by association
of a ligand. The family for example includes receptors for Insulin,
NGF, PDGF, FGF, EGF and GH.
[0178] Activation of preferred receptor kinases results in
activation of SRE, i.e. in an increase in transcription from genes
comprising one or more SRE in their regulatory sequences. This is
in particular true for receptor kinases activated by growth
hormones.
[0179] The receptor may also be an orphan receptor, i.e. a receptor
for which no ligand has yet been identified. The methods of the
present invention may also be useful for identifying ligands of
orphan receptors.
[0180] The cell surface molecule may in one embodiment of the
invention be a channel which is accessible from the extracellular
surface, such as a transmembrane channel. Examples of channels are
ion-channels, such as Ca.sup.2+ channels.
Cellular Response
[0181] 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.
[0182] In a particularly preferred embodiment of the invention, the
cellular response is mediated through a cell surface molecule, for
example the cellular response may be activation of a receptor.
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 agonists or antagonist of a receptor.
[0183] Examples includes: [0184] Upregulation or downregulation of
the level of a member of the pathway [0185] Relocalisation of a
member of the pathway [0186] Complex formation between members of
the pathway or between members of the pathway with other cellular
compounds [0187] Enhanced or reduced transcription from genes
regulated by the pathway [0188] Modification by for example
phosphorylation of a member of the pathway [0189] Activation or
inhibition of an enzyme of the pathway [0190] Degradation of a
cellular compounds due to upregulation or downregulation of the
pathway [0191] Altered secretion of a compound [0192] Change in
ion-flux [0193] Morphological changes [0194] Change in
viability
[0195] In a preferred embodiment the signal transduction pathway is
a pathway modulated by any of the receptors described in the
section herein above. Hence, the cellular response may for example
be any of the following: [0196] Activation of adenylate cyclase;
i.e. increase in adenylate cyclase activity [0197] Increased levels
of cAMP [0198] Increased transcription of genes regulated by a CRE
[0199] Inhibition of adenylate cyclase; i.e. decrease in adenylate
cyclase activity [0200] Decreased levels of cAMP [0201] Decreased
transcription of genes regulated by a CRE [0202] Increased activity
of phospholipase C [0203] Increased level of inositol
1,4,5-trisphosphate [0204] Increased activity of Protein kinase
C(PKC) [0205] Phosphorylation of proteins, which are phosphorylated
by protein kinase C
[0206] 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.
[0207] Hence, the cellular response may also be an increased or
decreased level of a particular mRNA within a cell.
[0208] 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 and/or operably
associated therewith.
[0209] In another embodiment of the invention the cellular response
is: [0210] change in the intracellular level of a compound; or
[0211] 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
[0212] 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.
[0213] In yet another embodiment of the invention the cellular
response is relocalisation of a compound. Relocalisation may for
example be [0214] concentration of a compound otherwise dispersed
in one or more specific locations [0215] relocalisation from one
cellular compartment to another, for example relocalisation from
the cellular membrane to the cytoplasma. [0216] relocalisation from
one location within a compartment to another location within the
same compartment [0217] internalisation of an extracellular
compound
[0218] 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.
[0219] 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.
[0220] In another embodiment of the invention the cellular response
is change in phosphorylation of a compound.
[0221] In another embodiment of the invention the cellular response
is formation or disruption of a complex between compounds.
[0222] In another embodiment of the invention the cellular response
is change in the concentration of a compound.
[0223] 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.
[0224] In another embodiment of the invention the cellular response
is change in pH in an intracellular compartment, for example in the
cytoplasm.
[0225] 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.
[0226] 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) under specific conditions.
[0227] 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
[0228] 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.
[0229] 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.
[0230] Hence, the reporter system may be a system endogenous to
said cells. For example, the reporter system may comprise the
endogenous system regulating the intra-cellular 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.
[0231] In another example, the reporter system comprises the
intracellular localisation of an endogenous compound.
[0232] 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.
[0233] 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.
[0234] In embodiments of the invention, wherein the cellular
response is modulation of a signal transduction pathway, the
reporter system may comprise 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.
[0235] 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.
[0236] 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 transduction 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.
[0237] 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).
[0238] 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.
[0239] 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
[0240] The detectable polypeptide may be any detectable
polypeptide, however preferably the detectable polypeptide is
selected from the group consisting of fluorescent proteins and
enzymes.
[0241] 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.
[0242] 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 luminiscent 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.
[0243] In one embodiment of the invention the reporter system may
detect complex formation between two cellular proteins. This may
for example be achieved by linking a bioluminescent moiety, such as
luciferase, to the one protein and a fluorescent moiety, such as a
fluorescent protein, to the other protein. Direct interaction
between the proteins can after expression of the two chimeric
proteins be detected through occurrence of BRET (Bioluminescence
Resonance Energy Transfer). If the two proteins are linked to
fluorescent moieties it is possible to detect the complex formation
through the occurrence of FRET (Fluorescence Resonance Energy
Transfer). Complex formation may also be detecting using
scintillation proximity assays.
[0244] 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.
[0245] 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.
Detectable Output
[0246] 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, luminiscense, activity of an enzyme or the
like:
[0247] In preferred embodiments of the invention the detectable
output is luminiscense, such as fluorescence, bioluminescence, FRET
or BRET. Bioluminiscence may be detected by any conventional
methods, for example with the aid of a Plate reader. BRET may be
performed as described herein above. In one embodiment, BRET2
technology is 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).
A protein-protein interaction between Rluc and GFP2 fusion 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 (solid line). With the Rluc:GFP2 fusion construct, there is
efficient energy transfer between Rluc and GFP2 and the 510 nm
signal represents a major peak (dashed line). The BRET2 signal is
expressed as the 515 nm to 410 nm ratio, since filters centered at
those wavelengths are used for detection. FRET technology is based
on the distance-dependent energy transfer between two fluorescence
groups that are each coupled to a protein.
[0248] Alternatively, the detectable output may preferably be
linked (directly or indirectly) to a bioluminiscent signal.
[0249] 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.
[0250] 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.
[0251] 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,
[0252] 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 emmision of light upon oxidation of
the added substrate, luciferin.
[0253] 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.
[0254] 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. Non-limiting
examples of methods of determining cytosolic free Ca.sup.2+ are
given in examples 13 and 13a herein below. Other ion concentrations
can be monitored using suitable fluorescent compounds, which for
example are available from Molecular Probes Inc.
[0255] 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.
[0256] 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.
[0257] 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 chemiluminiscent product as discussed herein
above.
[0258] 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.
[0259] 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.
[0260] 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.
[0261] 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 a antibody that
specifically bind the phosphorylated protein said antibody can then
be quantified by specific fluorescence labelling.
[0262] 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
emmision 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.
[0263] 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,
[0264] 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
[0265] 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.
[0266] For example the predetermined selection criteria may be a
quantitative criterium, such as a quantitative level of
bioluminiscence above or below a specific threshold value.
[0267] 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.
[0268] 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. Then resin
beads may be selected using a method comprising the steps of:
[0269] 1. Determining the fluorescence intensity of positive
control resin beads and setting this fluorescence intensity to 100%
[0270] 2. Determining the fluorescence intensity of negative
control resin beads and setting this fluorescence intensity to 0%
[0271] 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%.
[0272] 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 a signal transduction pathway through a cell surface receptor,
then the positive control may be a resin bead comprising a known
ligand of said receptor, 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.
[0273] 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. Then resin beads may be selected using a method
comprising the steps of: [0274] 1. Determining the fluorescence
intensity of positive control resin beads and setting this
fluorescence intensity to 0% [0275] 2. Determining the fluorescence
intensity of negative control resin beads and setting this
fluorescence intensity to 100% [0276] 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%.
[0277] The positive control may for example be a resin bead (or
resin beads) comprising a compound known to influence the cellular
response. By way of example, if the cellular response is inhibition
of a signal transduction pathway through a cell surface receptor,
then the positive control may be a resin bead comprising a known
antagonist of said receptor. The negative control may be a resin
bead (or resin beads) optionally comprising a cell adhesion
compound, but otherwise comprising no library member or other test
compound.
[0278] One method of selecting resin beads using FABS is
illustrated in FIG. 1A.
[0279] 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.
[0280] 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.
[0281] 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.
[0282] 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 criterion
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.
[0283] 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.
[0284] 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 colorimetric method, such as a
spectrophotometer,
[0285] 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.
[0286] 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.
[0287] Resin beads meeting said at least one predetermined
selection criteria may be selected by manually sorting for example
with the aid of a microscope, 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 about
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.
[0288] 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 a cell surface receptor 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.
[0289] 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.
[0290] Non-limiting examples of methods of selecting resin beads
are illustrates in FIGS. 1 and 2.
Identification of Compound
[0291] Once a resin bead has been selected, the compound of 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.
[0292] 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.
[0293] 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.
[0294] 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, and then analyzed using
IR, MS, or NMR. If the library is attached to the resin bead by a
cleavable linker, then the compound can be cleaved by cleaving said
cleavable linker. 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.
[0295] 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 infra-red imaging. This method gives
limited information and while useful for pre-screening, is not
recommended for conclusive structural determination.
[0296] 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 sub-species to conclusively determine the structure. 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. MS-based methods include for example
QTOF MSMS, MSMS or QTOF LC/MSMS.
[0297] 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. For example, induction of a
signal transduction pathway by a G-protein coupled receptor
frequently involves internalization of the G-protein coupled
receptor as well as a transcriptional response. Reporter systems
for both internalization and transcription may thus be tested.
Multiplexing
[0298] The methods disclosed by the present invention may also be
used in multiplexing methods.
[0299] 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.
[0300] 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.
[0301] 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.
[0302] 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.
[0303] Examples of multiplexing methods are illustrated in FIGS. 2A
and 2B.
EXAMPLES
Example 1
Screening of Adhesion Peptide Library
[0304] 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 using the general method for coupling amino
acids outlined in example 5 below and involved [0305] Coupling HMBA
linker to PEGA-resin [0306] Coupling amino acid to HMBA linker
[0307] SPPS coupling
[0308] 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 non-attached cells were removed and
new growth medium added. Cells/beads were incubating for another 16
hrs. (37.degree. C., 5% CO.sub.2).
[0309] 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 1a
[0310] Identification of an Adhesion Peptide with Low Absorption of
Fluorescent Components from Growth Medium and High Adhesion
Properties:
[0311] An adhesion D-amino peptide library was synthesized (500,000
members) as described above in Example 1 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).
[0312] 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.
[0313] 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.
[0314] 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.
[0315] 22 peptides were sequenced and six of them were
re-synthesized. Based on Structure-Activity of the six peptides,
four additional ones (AP-7 and AP-10) were synthesized. The peptide
defined by SEQ IS 35 showed the best overall performance.
[0316] An example of a method of preparing a resin bead comprising
a useful adhesion peptide is described in example 5, section
"Synthesis of adhesion peptide".
Example 2
[0317] This example describes preparation of resin beads comprising
His-(D)phe-Arg-Trp. These beads are for example useful as positive
control for in methods for identification of compounds modulating a
cellular response mediated through the melanocortin 4 receptor
(MC4R). The synthesis is shown in FIG. 4.
Synthesis of Ac-His-(D)phe-Arg-Trp-NH.sub.2
[0318] An overview of the synthesis is given in FIG. 4A.
[0319] PEGA resin (35 mg, 0.056 mmol) was swollen in dry DMF (1 mL)
and treated with Fmoc-Rink amide linker (90.65 mg, 0.168 mmol, 3
equiv) in presence of TBTU (51.77 mg, 0.224 mmol, 2.88 equiv) and
NEM (28.3 .mu.L, 0.224 mmol, 4 equiv). After 3 h at room
temperature, the resin was washed with DMF (10.times.), MeOH
(10.times.), DCM (10.times.) and dried in vacuo. The resin was
negative to Kaiser amine test and a quantitative reaction was
observed by measuring the Fmoc group on the resin (5 mg) with 20%
Piperidine/DMF solution (8 mL) for 30 min at room temperature.
[0320] The resin was swollen in dry DMF (1 mL) and the Fmoc group
was removed by 20% Piperidine/DMF (1 mL) for 20 min at room
temperature. The resin was washed with DMF (10.times.) and the
amino acids Fmoc-Trp(Boc), Fmoc-Arg(Pmc), Fmoc-(D)Phe and
Fmoc-His(Trt) (3 equiv) were attached successively in presence of
TBTU (2.88 equiv) and NEM (4 equiv). After the incorporation of all
amino acids, the Fmoc protection was removed by 20% piperidine in
DMF (1 mL, 20 min) and the resin was washed with DMF (10.times.).
The peptide on the resin was then acetylated with
aceticanhydride/pyridine/DMF (2:4:4) (1 mL) and washed with DMF
(10.times.), MeOH (10.times.), DCM (10.times.) and dried in vacuo.
The peptide was cleaved from the resin by treating with a solution
of TFA (90%), water (5%), ethanedithiol (2%), triisopropyl silane
(2%) and thioanisole (1%) for 3 h at room temperature. The resin
was filtered off and washed with TFA (2.times.) and DCM (2.times.).
The combined filtrate was concentrated under vacuum and the peptide
was precipitated by ether. The peptide was washed with ether
(10.times.) and dried in vacuo to afford 36.93 mg (96%) of pure
peptide.
##STR00001##
[0321] HPLC: t.sub.R=9.61 min.
[0322] ESI-MS: calcd (M+H).sup.+=686.78 Da; found
(M+H).sup.+=686.4
[0323] MALDI TOF MS: calcd (M+H).sup.+=686.78 Da; found
(M+H).sup.+=686.98
[0324] .sup.1H NMR (600 MHz, MeOH-d.sub.4): .delta.=1.38-1.64 (m,
2H, Arg H.sup..beta.), 1.10-1.15 (m, 2H, Arg H.sup..gamma.), 2.00
(s, 3H, Acetyl CH.sub.3), 2.96 (m, 2H Arg H.sup..delta.), 3.00-3.09
(m, 2H Phe H.sup..beta.), 3.24-3.41 (m, 2H Trp H.sup..beta.),
3.04-3.23 (m, 2H His H.sup..beta.), 4.01 (m, 1H Arg H.sup..alpha.),
4.73 (m, 1H His H.sup..alpha.), 4.51 (m, 1H Phe H.sup..alpha.),
4.71 (m, 1H Trp H.sup..alpha.), 7.04-7.67 (br 5H Trp ring protons),
7.21, 8.76 (2H, His ring protons), 7.25-7.33 (br, 5H Phe ring
protons).
[0325] Another compound useful as positive control in methods for
identification of compounds modulating a cellular response mediated
through the melanocortin 4 receptor (MC4R) is alfa-MSH of the
sequence
Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-OH.
Synthesis of Ac-His-(D)phe-Arg-Trp-Gly-PEGA.sub.1900
[0326] These resin beads were used as positive controls in some of
the below mentioned examples.
[0327] An overview of the synthesis is given in FIG. 4B.
[0328] PEGA.sub.1900 library resin (100 mg, 0.02 mmol) was swollen
in dry DMF (3 mL) and treated with Fmoc-Gly (17.84 mg, 0.06 mmol, 3
equiv) in presence of TBTU (18.5 mg, 0.058 mmol, 2.88 equiv) and
NEM (10.2 .mu.L, 0.08 mmol, 4 equiv). After 3 h at room
temperature, the resin was washed with DMF (10.times.), MeOH
(10.times.), DCM (10.times.) and dried in vacuo. The resin was
negative to Kaiser amine test and a quantitative reaction was
observed by measuring the Fmoc group on the resin (5 mg) with 20%
Piperidine/DMF solution (8 mL) for 30 min at room temperature. The
PEGA1900 library resin had previously been coupled to an adhesion
peptide as described in Example 5a, in the section "Synthesis of
adhesion peptide".
[0329] The resin was swollen in dry DMF (1 mL) and the Fmoc group
was removed by 20% Piperidine/DMF (1 mL) for 20 min at room
temperature. The resin was washed with DMF (10.times.) and the
amino acids Fmoc-Trp(Boc), Fmoc-Arg(Pmc), Fmoc-(D)Phe and
Fmoc-His(Trt) (3 equiv) were attached successively in presence of
TBTU (2.88 equiv) and NEM (4 equiv). After the incorporation of all
amino acids, the Fmoc protection was removed by 20% piperidine in
DMF (1 mL, 20 min) and the resin was washed with DMF (10.times.).
The peptide on the resin was then acetylated with
aceticanhydride/pyridine/DMF (2:4:4) (1 mL) and washed with DMF
(10.times.), MeOH (10.times.), DCM (10.times.) and dried in vacuo.
The side chain protection of the peptide was removed by treating
with a solution of TFA (90%), water (5%), ethanedithiol (2%),
tri-isopropyl silane (2%) and thioanisole (1%) for 3 h at room
temperature and the resin was washed with DCM (10.times.), DMF
(10.times.) and water (10.times.).
Synthesis of Fmoc-Dap(N.sub.3)OH
[0330] An overview of the synthesis is given in FIG. 4C
[0331] Fmoc-Dap-OH (980 mg, 3 mmol) was dissolved in 80% aqueous
acetic acid (9 mL) and CuSO.sub.4.5H.sub.2O (15 mg, 0.06 mmol, 0.02
equiv) in water (1 mL) was added. The pH of the solution was
adjusted to 9-10 with K.sub.2CO.sub.3. Water (15 mL), MeOH (32 mL)
and trifluoromethanesulfonyl azide (6 mmol) in DCM (25 mL) was
added and the pH was readjusted to 9-10 with K.sub.2CO.sub.3. The
two-phase system was stirred vigorously for 20 h. The layers were
separated by addition of DCM and the organic phase was washed with
water (2.times.40 mL) and then the combined aqueous phases were
acidified with 3 M HCl (aqueous) to a pH 2. The aqueous phase was
extracted with DCM (4.times.50 mL) and the combined organic phases
were dried over sodium sulfate, filtered and concentrated under
vacuo (0.934 g, 88.2%).
[0332] HPLC: t.sub.R=10.08 min
[0333] ESI-MS: calcd (M+H).sup.+=353.34 Da; found
(M+H).sup.+=353.1
[0334] .sup.1H NMR (250 MHz, CDCl.sub.3): .delta.=3.75 (d, 2H),
4.14-4.9 (t, 1H), 4.36-4.39 (d, 2H), 4.50-4.54 (m, 1H), 5.50-5.54
(2H, NH and OH), 7.22-7.28 (4H, aromatic ring), 7.51-7.54 (d, 2H,
aromatic ring), 7.68-7.71 (d, 2H, aromatic ring).
Example 3
[0335] This example describes synthesis of resin beads comprising a
cyclic compound, which is capable of activating for example the
melanocortin 4 receptor. An overview of the synthesis is given in
FIG. 5A.
Synthesis of
Fmoc-Lys(Boc)-Dap(N.sub.3)-His(Trt)-(D)phe-Arg(Pmc)-Trp(Boc)-Pra-Met-HMBA-
-Gly-PEGA
[0336] PEGA-red-resin (150 mg, 0.24 mmol) was swollen in dry DMF (5
mL) and treated with Fmoc-Gly (215 mg, 0.72 mmol, 3 equiv) in
presence of TBTU (222 mg, 0.69 mmol, 2.88 equiv) and NEM (121.8
.mu.L, 0.96 mmol, 4 equiv). After 3 h at room temperature, the
resin was washed with DMF (10.times.), MeOH (10.times.), DCM
(10.times.) and dried in vacuo. The resin was negative to Kaiser
amine test and a quantitative reaction was observed by measuring
the Fmoc group on the resin (5 mg) with 20% piperidine/DMF solution
(8 mL) for 30 min at room temperature.
[0337] The resin was swollen in dry DMF (5 mL), Fmoc group was
removed by 20% Piperidine/DMF and treated with HMBA linker (109.5
mg, 0.72 mmol, 3 equiv) in presence of TBTU (222 mg, 0.69 mmol,
2.88 equiv) and NEM (121.8 .mu.L, 0.96 mmol, 4 equiv). After 3 h at
room temperature, the resin was washed with DMF (10.times.), MeOH
(10.times.), DCM (10.times.) and dried in vacuo. The resin was
negative to Kaiser amine test
[0338] The resin was swollen in dry DCM (2 mL), Fmoc-Met (267.5 mg,
0.72 mmol, 3 equiv), MSNT (213.4 mg, 0.72 mmol, 3 equiv) and MeIm
(43 .mu.L, 0.54 mmol, 2.25 equiv) were added. After 1 h, the resin
was filtered and washed with DCM (10.times.), MeOH (10.times.) and
DMF (10.times.). The Fmoc group was removed by 20% Piperidine/DMF
(1 mL) for 20 min at room temperature. The resin was washed with
DMF (10.times.) and the amino acids Fmoc-Pra, Fmoc-Trp(Boc),
Fmoc-Arg(Pmc), Fmoc-(D)Phe, Fmoc-His(Trt), Fmoc-Dap(N.sub.3) and
Fmoc-Lys(Boc) (3 equiv) were attached successively in presence of
TBTU (2.88 equiv) and NEM (4 equiv). After the incorporation of all
amino acids, the resin was washed with DMF (10.times.), MeOH
(10.times.), DCM (10.times.) and dried in vacuo.
Cyclisation of
Fmoc-Lys(Boc)-Dap(N.sub.3)-His(Trt)-(D)Phe-Arg(Pmc)-Trp(Boc)-Pra-Met-HMBA-
-Gly-PEGA
[0339] a. The peptidyl resin (20 mg) was treated with a solution of
TFA (90%), water (5%), ethanedithiol (2%), triisopropyl silane (2%)
and thioanisole (1%) for 3 h at room temperature for removing all
the side chain protection groups. The resin was washed with DCM
(10.times.), MeOH (10.times.) and DMF (10.times.). The Fmoc group
was removed by 20% Piperidine/DMF (2 mL) and the resin was washed
with DMF (10.times.), MeOH (10.times.), DCM (10.times.) and THF
(10.times.). DIPEA (61 .mu.L, 0.35 mmol, 50 equiv) and CuI (2.66
mg, 0.014 mmol, 2 equiv) in THF (300 .mu.L) were added to the
resin. The reaction was left for 16 h and then washed with THF,
water, DMF, MeOH, DCM and dried in vacuo. [0340] b. The peptidyl
resin (20 mg) was treated with DIPEA (61 .mu.L, 0.35 mmol, 50
equiv) and CuI (2.66 mg, 0.014 mmol, 2 equiv) in THF (300 .mu.L)
were added to the resin. The reaction was left for 16 h and then
washed with THF, water, DMF, MeOH, DCM and dried in vacuo. [0341]
c. Deprotection of the cyclic peptide. A solution of TFA (90%),
water (5%), ethanedithiol (2%), triisopropyl silane (2%) and
thioanisole (1%) were added to the resin for removing all the side
chain protection groups (3 h at room temperature). The resin was
washed with DCM (10.times.), MeOH (10.times.) and DMF (10.times.).
The Fmoc group was removed by 20% Piperidine/DMF (2 mL) and the
resin was washed with DMF (10.times.), MeOH (10.times.), DCM
(10.times.) and dried in vacuo. Cleavage of Peptide from the
Resin
[0342] The resin was treated with 0.1 M NaOH (100 .mu.L) for 2 h at
room temperature. The resin was filtered and the filtrate was
neutralised with 0.1 M HCl (100 .mu.L).
[0343] (a) Yield=8.1 mg (82.5%)
[0344] (b) Yield=7.8 mg (79%)
[0345] HPLC: t.sub.R=10.89 min
[0346] ES MS/MS: calcd (M+H).sup.+=1112.29 Da; found
(M+H).sup.+=1112.56 Da
[0347] .sup.1H NMR (600 MHz, DMSO-d.sub.6): 1.259-1.273 (m, 2H Arg
H.sup..gamma.), 1.311-1.332 (m, 2H Lys H.sup..gamma.), 1.508-1.514
(m, 2H Lys H.sup..delta.), 1.416-1.616 (m, 2H Arg H.sup..beta.),
1.650-1.669 (m, 2H Lys H.sup..beta.), 1.852-1.979 (m, 2H Met
H.sup..beta.), 2.022 (s, 3H Met --CH.sub.3), 2.461 (t, 2H Met
H.sup..gamma.), 2.484-2.577 (m, 2H Pra H.sup..beta.), 2.671-2.848
(m, 2H His H.sup..beta.), 2.721-2.944 (m, 2H Phe H.sup..beta.),
2.728-2.734 (t, 2H Lys H.sup..epsilon.), 2.961-3.157 (m, 2H Trp
H.sup..beta.), 2.998-3.004 (m, 2H Arg H.sup..delta.), 3.366-3.568
(m, 2H Dap H.sup..beta.), 3.794 (m, 1H Lys H.sup..alpha.), 4.282
(m, 1H Arg H.sup..alpha.), 4.309 (m, 1H Met H.sup..alpha.), 4.417
(m, 1H Pra H.sup..alpha.), 4.521 (m, 1H Dap H.sup..alpha.), 4.568
(m, 1H Trp H.sup..alpha.), 4.584 (m, 1H His H.sup..alpha.), 4.662
(m, 1H Phe H.sup..alpha.), 7.164-7.239 (br, 5H Phe ring protons),
7.201, 8.211 (2H, His ring protons), 7.447 (s, 1H Arg --NH),
6.955-7.312 (br, 5H Trp ring protons), 8.240 (s, 1H Triazole ring
proton), 8.094 (1H Trp amide H), 8.185 (1H Met amide H), 8.213 (1H
Phe amide H), 8.250 (1H Pra amide H), 8.329 (1H Arg amide H), 8.408
(1H His amide H), 8.805 (1H Dap amide H), 10.697 (1H Trp ring
NH).
Example 4
Synthesis of Cyclic Peptide Library
[0348] The cyclic peptide library of example 4 is for example
useful for identification of compounds capable of modulating a
cellular response mediated through the melacortin 4 receptor.
Fmoc-Lys(Boc)-Dap(N.sub.3)-Aa1-Aa2-Aa3-Aa4-Pra-Met-HMBA-Gly-PEGA-NH-Gly-Al-
loc
[0349] PEGA resin (1.5 g, 0.3 mmol) is swollen in dry DMF (15 mL)
and treated with a mixture of Fmoc-Gly (268 mg, 0.9 mmol, 3 equiv)
and Alloc-Gly (143 mg, 0.9 mmol, 3 equiv) by preactivation with
TBTU (277 mg, 0.86 mmol, 2.88 equiv) and NEM (152 .mu.L, 1.2 mmol,
4 equiv) and slow addition of the activated mixture to the resin.
After 3 h at room temperature, the resin is washed with DMF
(10.times.), MeOH (10.times.), DCM (10.times.) and dried in vacuo.
The resin is negative to Kaiser amine test and a 1:2 ratio of
Alloc:Fmoc is observed by measuring the Fmoc group on the resin (5
mg) with 20% Piperidine/DMF solution (15 mL) for 30 min at room
temperature and determination of the absorption of the eluate at
305 nm.
[0350] The resin is swollen in dry DMF (15 mL), Fmoc group is
removed by 20% Piperidine/DMF and treated with HMBA linker (92 mg,
0.6 mmol, 3 equiv) in presence of TBTU (185 mg, 0.58 mmol, 2.88
equiv) and NEM (102 .mu.L, 0.8 mmol, 4 equiv). After 3 h at room
temperature, the resin is washed with DMF (10.times.), MeOH
(10.times.), DCM (10.times.) and dried in vacuo. The resin is
negative to Kaiser amine test.
[0351] The resin is swollen in dry DCM (20 mL), Fmoc-Met-OH (223
mg, 0.6 mmol, 3 equiv), MSNT (178 mg, 0.6 mmol, 3 equiv) and MeIm
(36 .mu.L, 0.45 mmol, 2.25 equiv) were added. After 1 h, the resin
is filtered and washed with DCM (10.times.), MeOH (10.times.) and
DMF (10.times.). The Fmoc group is removed by 20% Piperidine/DMF
(15 mL) for 20 min at room temperature. The resin is washed with
DMF (10.times.) and Fmoc-Pra is attached to the resin in presence
of TBTU and NEM. The resin is transferred to a 20 well multiple
column peptide synthesiser and distributed equally in to each
wells. Amino acids Fmoc-Aa4-OH, Fmoc-Aa3-OH, Fmoc-Aa2-OH,
Fmoc-Aa1-OH, Fmoc-Dap(N.sub.3) and Fmoc-Lys(Boc) (3 equiv) are
attached successively in presence of TBTU (2.88 equiv) and NEM (4
equiv). After the incorporation of all amino acids, the resin is
washed with DMF (10.times.), MeOH (10.times.), DCM (10.times.) and
dried in vacuo.
[0352] The synthesis is illustrated in FIG. 5B.
[0353] Fmoc-NH--CH(R.sub.1)--CO may be any natural amino acids
coupled to Fmoc
[0354] Fmoc-NH--CH(R.sub.2)--CO; Fmoc-NH--CH(R.sub.3)--CO;
Fmoc-NH--CH(R.sub.4)--CO may be any of the following amino acids
coupled to Fmoc: Cys, Phe, His, Lys, Met, Pro, Arg, Ser, Thr, Val,
Trp, Tyr, Homophenyl alanine, Tic, 4-Phenyl pyrrolidone
2-carboxylic acid, 1-Aminocyclohexane carboxylic acid, 4-Pyridyl
alanine, (D)-Orn Hyp, 4-Phenyl peperidine carboxylic acid.
[0355] Cyclisation of
Fmoc-Lys(Boc)-Dap(N.sub.3)-Aa1-Aa2-Aa3-Aa4-Pra-Met-HMBA-Gly-PEGA-NH-Alloc
[0356] The peptidyl resin is treated with a solution of TFA (90%),
water (5%), ethanedithiol (2%), triisopropyl silane (2%) and
thioanisole (1%) for 3 h at room temperature for removing all the
side chain protection groups. The resin is washed with DCM
(10.times.), MeOH (10.times.) and DMF (10.times.). The Fmoc group
is removed by 20% piperidine/DMF (15 mL) and the resin is washed
with DMF (10.times.), MeOH (10.times.), DCM (10.times.) and THF
(10.times.). DIPEA (1.75 mL, 10 mmol, 50 equiv) and CuI (76.2 mg,
0.4 mmol, 2 equiv) in THF (10 mL) are added to the resin. The
reaction is left for 16 h and then washed with THF, water, DMF,
MeOH, DCM and dried in vacuo.
[0357] The cyclisation process is illustrated in FIG. 5B
Synthesis of Adhesion Peptide ((D)Arg-(D)Arg-(D)Ile-(D)Arg-Gly) on
Cyclic Peptide Library Beads
[0358] a. Alloc Deprotection [0359] Pd(PPh.sub.3).sub.4 (346.5 mg,
0.3 mmol, 3 equiv) is dissolved in acetic acid (5%) and NEM (2.5%)
in chloroform (15 mL) and degassed by purging with Ar for 10 min.
The reaction mixture is added to the lyophilised resin under Ar
atmosphere and kept for 20 min at room temperature. b. Synthesis of
Adhesion Peptide [0360] The resin is washed with DMF (10.times.)
and Fmoc-Lys(Boc) (141 mg, 0.3 mmol, 3 equiv) is attached using
TBTU (92 mg, 0.288 mmol, 2.88 equiv) and NEM (51 .mu.L, 0.4 mmol, 4
equiv). The resin is washed with DMF (10.times.) and the
.alpha.-Fmoc and side chain Boc protections are removed by 20%
piperidine (15 mL) and 30% TFA in DCM (20 mL) respectively. The
resin is again treated with Fmoc-Lys(Boc) (282 mg, 0.6 mmol, 3
equiv) and TBTU (184 mg, 0.576 mmol, 2.88 equiv) and the Fmoc and
Boc protections are removed by 20% piperidine in DMF and 30% TFA in
DCM. The resin is washed with DMF (10.times.) and two residues of
Ahx are attached by adding TBTU activated Fmoc-Ahx (425 mg, 1.2
mmol, 3 equiv). The amino acids Fmoc-Gly, Fmoc-(D) Arg(Pmc) and
Fmoc-(D) Ile (3 equiv) are attached according to the sequence in
presence of TBTU (2.88 equiv) and NEM (4 equiv). After the
incorporation of all amino acids, the N-terminal Fmoc group is
removed by 20% piperidine in DMF (15 mL) and the resin is washed
with DMF (10.times.), MeOH (10.times.), DCM (10.times.) and dried
in vacuo. [0361] The peptidyl resin is treated with a solution of
TFA (90%), water (5%), ethanedithiol (2%), triisopropyl silane (2%)
and thioanisole (1%) for 3 h at room temperature for removing all
the side chain protection groups. The resin is washed with DCM
(10.times.), MeOH (10.times.) and DCM (10.times.) and dried in
vacuo.
Example 5
Library of Oligocyclic Ureas as Peptidomimetics.
[0362] This library is for example useful for identification of
compounds modulating a cellular response mediated through a
G-protein coupled receptor.
Synthesising Combinatorial Library of Potential Urea GPCR Agonist
Via SPPS and the Pictet-Spengler Reaction:
Experimental:
General:
[0363] All chemicals described, apart from the building block O-Pfp
carbamates are commercially available and used without further
purification. The building block O-Pfp carbamates are prepared as
described in: Diness, F.; Beyer, J.; Meldal, M.; J. Combi. Chem.
and QSAR. 2004, 23, 1-15. All solvents are HPLC-grade.
PEGA.sub.900-resin is purchased from VersaMatrix A/S. Each washing
step lasts 2 min unless otherwise stated.
Coupling of HMBA Linker to PEGA.sub.900-Resin:
[0364] Dry PEGA.sub.900-resin is swelled in DCM and washed with DMF
(3.times.). 3.0 eq. HMBA, 2.9 eq. TBTU and 3.0 eq. NEM are mixed in
appropriate DMF and allowed to react for 10 min. The mixture is
added to resin and after 2 h the resin is washed with DMF
(6.times.), DCM (6.times.) and lyophilised.
General Procedure for Coupling of Amino Acid to HMBA-Linker
[0365] Dry PEGA.sub.900-resin with HMBA-linker is swelled in DCM.
3.0 eq. Fmoc-protected amino acid, 2.25 eq. MeIm and 3.0 eq. MSNT
are mixed in appropriate amount of DCM and added to resin. After 1
h the resin is washed with DCM (3.times.) and the coupling is
repeated as above once. After coupling for 1 h the resin is washed
with DCM (6.times.), DMF (6.times.), DCM (6.times.) and
lyophilised.
General SPPS Coupling Procedure
[0366] The terminal amino acid on the resin is 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 are mixed in appropriate
amount of DMF and allowed to react for 10 min. The mixture is added
to the resin and after 2 h the resin is washed with DMF
(6.times.).
General Building Block Coupling Procedure
[0367] The terminal amino acid on the PEGA.sub.900-resin with HMBA
linker and tetrapeptide is Fmoc-deprotected by treatment with 20%
piperidine in DMF (1.times.2 min+1.times.17 min) followed by
washing with DMF (6.times.). 3.0 eq. building block --O-Pfp
carbamate is dissolved in appropriate amount of DMF and the
solution is added to resin. After ended coupling the resin is
washed with DMF (6.times.), DCM (6.times.) and lyophilised.
General Pictet-Spengler Reaction Procedure
[0368] Dry PEGA.sub.900-resin with HMBA linker, peptide and
building block is swelled in 10% TFA (aq) (1.times.1 h and
1.times.11 h). The resin is washed with H.sub.2O until washing
water has pH=6-7 and washed with DMF (6.times.), DCM (6.times.) and
lyophilised.
General Side Chain Deprotection Procedure
[0369] Dry PEGA.sub.900-resin with HMBA linker, peptide and
Pictet-Spengler product is swelled in H.sub.2O and the side chains
are deprotected with 95% TFA (aq) (2.times.15 min). The resin is
washed with H.sub.2O until washing water had pH=5-7. The resin is
then washed with DMF (6.times.), DCM (6.times.) and
lyophilised.
General HMBA Cleavage Procedure
[0370] Dry PEGA.sub.900-resin with HMBA linker and attached
compound is swelled in water and NaOH (aq.) 0.1 M is added. After 2
h HCl (aq.) 0.1 M is used for neutralisation and then AcN was added
until the H.sub.2O/AcN ratio is 1:1 by volume. The resin is
filtered off and the liquid is used direct for RP-HPLC or/and Q-TOF
MS analysis.
Synthesising the Combinatorial Library:
[0371] Dry PEGA.sub.900-resin (1.0 g, 0.2 mmol) is coupled with
HMBA linker as described in "Coupling of HMBA linker to
PEGA.sub.900-resin" an equimolar mixture of Fmoc-glycine and Alloc
glycine is the coupled to the HMBA functionalised
PEGA.sub.900-resin as described in "General Procedure for Coupling
of amino acid to HMBA linker". An analytical sample is cleaved by
"General HMBA Cleavage Procedure" and tested by RP-HPLC. The
Fmoc-Gly-HMBA-PEGA.sub.900-resin is swelled in DCM, washed with DMF
(6.times.) and divided into the wells of a 20-welled peptide
synthesiser. The 10 different amino acids are coupled to the
glycine using "General SPPS Coupling Procedure". The resin from all
the wells is mixed again and divided into the wells of a 20-welled
peptide synthesiser and the 10 different amino acids are coupled to
the terminal amino acid using "General SPPS Coupling Procedure".
The resins from all the wells are mixed again and divided into the
wells of a 20-welled peptide synthesiser and the 10 different
trypthophan derivatives acids are coupled to the terminal amino
acid using "General SPPS Coupling Procedure". The resins from all
the wells is mixed again and divided into the wells of a 20-welled
peptide synthesiser and the 10 different building blocks are
coupled to the terminal amino acid using "General Building Block
Coupling Procedure". The resin from all the wells is mixed again
and the Pictet-Spengler reaction is performed as described in
"General Pictet-Spengler Reaction Procedure". The Alloc group is
removed from amino groups with 5 mol % Pd(P(Ph.sub.3)).sub.4 in DMF
containing 1% morpholinium acetate. Boc/tBu/Pcm protected adhesion
peptide 4 (2 eqv) is coupled using TBTU/NEM preactivation (5 min,
0.degree. C.) for 14 h, until Kaiser test showed complete reaction.
This is followed by Boc-, Bu.sup.t and Pmc-deprotection as
described in "General Side Chain Deprotection Procedure". Finally
analytical samples are cleaved from single beads by "General HMBA
Cleavage Procedure" and tested by Q-TOF MS and MSMS analysis.
[0372] The structure of the resulting library members is given
below.
##STR00002##
R.sub.1=dipeptide. Any combination of Gly, L-Trp, L-Arg, D-Arg,
L-His, L-Phe, D-Phe, L-Lys, L-Asn, 4-amino-L-Phe
R.sub.2=H, 5-OH, 5-Br, 6-F, 7-N.sub.3, 5-OMe
##STR00003##
[0373] R.sub.1=dipeptide. Any combination of Gly, L-Trp, L-Arg,
D-Arg, L-His, L-Phe, D-Phe, L-Lys, L-Asn, 4-amino-L-Phe
R.sub.2=H, 5-OH, 5-Br, 6-F, 7-N.sub.3, 5-OMe
R.sub.3=R.sub.4=Me
[0374] or R.sub.3=H and R.sub.4=H, iPr, H.sub.2N--CH.sub.2,
Ph-CH.sub.2, (4-HO--)Ph-CH.sub.2 or indo-2-ly-CH.sub.2 or
R.sub.3=Phe and R.sub.4=H
[0375] iPr=
##STR00004##
H.sub.2N--CH.sub.2=
##STR00005##
[0376] Ph-CH.sub.2=
##STR00006##
[0377] (4-HO--)Ph-CH.sub.2=
##STR00007##
[0378] indo-2-ly-CH.sub.2=
##STR00008##
Example 5a
Library of Oligocyclic Ureas as Peptidomimetics 2.
[0379] This library is for example useful for identification of
compounds modulating a cellular response mediated through a
G-protein coupled receptor.
Synthesising Combinatorial Library of Potential Urea GPCR Agonist
Via SPPS and the Pictet-Spengler Reaction:
Experimental:
General:
[0380] All chemicals described, apart from the building block O-Pfp
carbamates are commercially available and used without further
purification. The building block O-Pfp carbamates are prepared as
described in: Diness, F.; Beyer, J.; Meldal, M.; J. Combi. Chem.
and QSAR. 2004, 23, 1-15. All solvents are HPLC-grade.
PEGA.sub.900-resin is purchased from VersaMatrix A/S, Denmark. Each
washing step lasts 2 min unless otherwise stated.
General SPPS Coupling Procedure
[0381] The terminal amino acid on the resin is 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 or HMBA, 2.9 eq. TBTU and 4.0 eq. NEM are mixed in
appropriate amount of DMF and allowed to react for 10 min. The
mixture is added to the resin and after 2 h the resin is washed
with DMF (6.times.).
General Procedure for Coupling of Amino Acid to HMBA-Linker
[0382] Dry PEGA.sub.1900-resin with HMBA-linker is swelled in DCM.
3.0 eq. Fmoc-protected amino acid, 2.25 eq. MeIm and 3.0 eq. MSNT
are mixed in appropriate amount of DCM and added to resin. After 1
h the resin is washed with DCM (3.times.) and the coupling is
repeated as above once. After coupling for 1 h the resin is washed
with DCM (6.times.), DMF (6.times.), DCM (6.times.) and
lyophilised.
General Building Block Coupling Procedure
[0383] The terminal amino acid on the PEGA.sub.1900-resin with HMBA
linker and tetrapeptide is Fmoc-deprotected by treatment with 20%
piperidine in DMF (1.times.2 min+1.times.17 min) followed by
washing with DMF (6.times.). 3.0 eq. building block --O-Pfp
carbamate is dissolved in appropriate amount of DMF and the
solution is added to resin. After ended coupling the resin is
washed with DMF (6.times.), DCM (6.times.) and lyophilised.
General Pictet-Spengler Reaction Procedure
[0384] Dry PEGA.sub.900-resin with HMBA linker, peptide and
building block is swelled in 10% TFA (aq) (1.times.1 h and
1.times.11 h). The resin is washed with H.sub.2O until washing
water has pH=6-7 and washed with DMF (6.times.), DCM (6.times.) and
lyophilised.
General Side Chain Deprotection Procedure
[0385] Dry PEGA.sub.1900-resin with HMBA linker and attached
compounds is swelled in H.sub.2O and the side chains are
deprotected with 95% TFA (aq) (2.times.15 min). The resin is washed
with H.sub.2O until washing water had pH=5-7. The resin is then
washed with DMF (6.times.), DCM (6.times.) and lyophilised.
General HMBA Cleavage Procedure
[0386] Dry PEGA.sub.1900-resin with HMBA linker and attached
compounds is swelled in water and NaOH (aq.) 0.1 M is added. After
2 h HCl (aq.) 0.1 M is used for neutralisation and then AcN was
added until the H.sub.2O/AcN ratio is 1:1 by volume. The resin is
filtered off and the liquid is used direct for RP-HPLC or/and Q-TOF
MS analysis.
Synthesising the Combinatorial Library:
[0387] Dry PEGA.sub.1900-resin (1.0 g, 0.2 mmol) is coupled with an
equimolar mixture of Fmoc-glycine and Alloc glycine as described in
"General SPPS Coupling Procedure". HMBA is coupled as described in
"General SPPS Coupling Procedure". Fmoc-glycine is the coupled to
the HMBA functionalised PEGA.sub.1900-resin as described in
"General Procedure for Coupling of amino acid to HMBA linker". The
Fmoc-Gly-HMBA-Gly-PEGA.sub.1900-Gly-Alloc resin is swelled in DCM,
washed with DMF (6.times.) and divided into the wells of a
20-welled peptide synthesiser. The 20 different natural L-amino
acids are coupled to the glycine using "General SPPS Coupling
Procedure". The resin from all the wells is mixed again and divided
into the wells of a 20-welled peptide synthesiser and the 20
different natural L-amino acids are coupled to the terminal amino
acid using "General SPPS Coupling Procedure". The resins from all
the wells are mixed again and divided into 10 wells of a 20-welled
peptide synthesiser and the 10 different Fmoc-protected tryptophane
derivatives (shown in Table 6) are coupled to the terminal amino
acid using "General SPPS Coupling Procedure". The resins from all
the wells is mixed again and divided into 8 wells of a 20-welled
peptide synthesiser and the 8 different building blocks (shown in
Table 6) are coupled to the terminal amino acid using "General
Building Block Coupling Procedure". The resin from each well is
transferred into a syringe with a filter in the bottom. The Alloc
group is removed from the resin bound glycine by using 5 mol %
Pd(P(Ph.sub.3)).sub.4 in chloroform containing 5% AcOH and 2.5% NEM
under argon for 12 h. The resin is then washed with chloroform
(6.times.), 0.5% Et.sub.2NCS.sub.2Na-3H.sub.2O and 0.5% DIPEA in
DMF (6.times.) and DMF (10.times.). 1.5 eq. protected adhesion
peptide (AP4), 1.4 eq. TBTU and 2.0 eq. NEM are mixed in
appropriate amount of DMF and allowed to react for 10 min. The
mixture is added to the resin and added to resin. When the Kaiser
test shows complete reaction the resin is washed with DMF
(6.times.) and DCM (6.times.). The Pictet-Spengler reaction is
performed as described in "General Pictet-Spengler Reaction
Procedure". This is followed by side chain deprotection as
described in "General Side Chain Deprotection Procedure". Finally
analytical samples are cleaved from single beads by "General HMBA
Cleavage Procedure" and tested by Q-TOF MS and MSMS analysis.
TABLE-US-00004 TABLE 6 Tryptophane derivatives Building Blocks
##STR00009## ##STR00010## ##STR00011## ##STR00012## ##STR00013##
##STR00014## ##STR00015## ##STR00016## ##STR00017## ##STR00018##
##STR00019## ##STR00020## ##STR00021## ##STR00022## ##STR00023##
##STR00024## ##STR00025## ##STR00026##
[0388] The structure of the resulting library members is given
below.
##STR00027##
R.sub.1=dipeptide. Any combination of the 20 natural occurring L
amino acids
R.sub.2=H, 5-OH, 5-OMe, 5-OBn, 5-Br, 5-F, 6-F, 5-Me, 6-Me, 7-Me
[0389] R.sub.3=H, iPr, H.sub.2N--CH.sub.2, Ph-CH.sub.2,
(4-HO--)Ph-CH.sub.2 or indol-2-ly-CH.sub.2 iPr=
##STR00028##
H.sub.2N--CH.sub.2=
##STR00029##
[0390] Ph-CH.sub.2=
##STR00030##
[0391] (4-HO--)Ph-CH.sub.2=
##STR00031##
[0392] indo-2-ly-CH.sub.2=
##STR00032##
Synthesis of Protected Adhesion Peptide
[0393] PEGA.sub.900-resin is swelled in DMF. 3.0 eq. HMBA, 2.9 eq.
TBTU and 4.0 eq. NEM are mixed in appropriate amount of DMF and
allowed to react for 5 min. The mixture is added to resin and after
2 h the resin is washed with DMF (6.times.), DCM (6.times.) and
lyophilised. The resin is swelled in DCM and 3.0 eq. Fmoc-Gly-OH,
2.25 eq. MeIm and 3.0 eq. MSNT are mixed in appropriate amount of
DCM and added to resin. After 1 h the resin is washed with DCM
(3.times.) and the coupling is repeated as above once. After
coupling for 1 h the resin is washed with DCM (6.times.), DMF
(6.times.), DCM (6.times.) and lyophilised. The resin is swelled in
DMF and a sequence of Fmoc-gln(trt)-OH, Fmoc-arg(Pmc)-OH,
Fmoc-ile-OH, Fmoc-arg(Pmc)-OH, Fmoc-lys(Boc)-OH and Boc-ala-OH is
coupled as described in "General SPPS Coupling Procedure". The
resin is then washed with DMF (6.times.), DCM (6.times.) and
lyophilised. The final peptide
(Boc-ala-arg(Pmc)-lys(Boc)-arg(Pmc)-ile-arg(Pmc)-gln(trt)-GlyOH) is
cleaved from the resin as described in "General HMBA Cleavage
Procedure".
Example 6a
Library of Multi-Heterocyclic Peptidomimetics for GPCR Receptors
(Library 6a).
[0394] This library is for example useful for identification of
compounds modulating a cellular response mediated through a
G-protein coupled receptor.
Library Design and Synthesis
[0395] All Pictet-Spengler reaction methodology has been developed
and tested on the synthesis resin PEGA.sub.800,.sup.1 wherefore the
analogous library resin PEGA.sub.1900 is chosen for the library
synthesis. In order to screen for active compounds, the library is
prepared following a "one-bead-two-compounds" strategy. This is
accomplished by treating the amino-functionalized resin with a
mixture of Fmoc-Gly-OH:Alloc-Gly-OH (10:1) activated by the TBTU
procedure.sup.2 to provide orthogonal reaction sites for (a)
split-and-mix library synthesis (via the Fmoc handle); and (b)
attachment of an adhesion molecule (AM) (via the Alloc handle). The
library synthesis of Pictet-Spengler reaction precursor 1 is
carried out according to standard Fmoc amino acid coupling
protocols for solid-phase peptide synthesis (FIG. 6a). Due to the
requirement of acidic reaction conditions for the Pictet-Spengler
reaction step (q), the base labile HMBA (hydroxymethylbenzoic acid)
linker is employed. Prior to attachment of HMBA to
H.sub.2N-Gly-PEGA.sub.1900 via the TBTU activation procedure, the
Fmoc protecting group is removed by standard piperidine treatment.
The HMBA linker provides a convenient cleavage site for
quantitative release from the solid support via basic hydrolysis.
Cleavage of product from a single bead is routinely achieved by
treating the bead with 0.1 M NaOH (aq) overnight, thus providing
amounts of material sufficient for structure elucidation via QTOF
ES-MSMS analysis. After splitting the resin portion into 10
different wells, the hydroxy handle of the linker is esterified by
treatment with 10 MSNT-activated Fmoc amino acids
(Fmoc-AA.sub.1-OH),.sup.3 thus attaching the first amino acid
residue of the peptidomimetic sequence. Subsequent analogous
split-and-mix synthesis and 3 cycles of Fmoc
deprotection/TBTU-mediated couplings of 10 Fmoc amino acids as the
second amino acid residue (Fmoc-AA.sub.2-OH), 15 Fmoc amino acids
incorporating the reactive aromatic side-chain (Fmoc-AA.sub.3-OH),
and 7 masked aldehyde building blocks (R.sup.4-MABB-OH) (Table 5a),
prepared as previously reported,.sup.4,5 afford the Pictet-Spengler
reaction precursor 1. In this coupling sequence, one fifth of the
resin is withdrawn prior to the coupling of Fmoc-AA.sub.2-OH (steps
e and f), and remixed with the remaining resin from step g and
forth. Ultimately, this affords a library composed of tripeptoidal
(n=0) and tetrapeptoidal (n=1) substructures. The Alloc protecting
group of 1 is removed with Pd(PPh.sub.3).sub.4, and subsequent TBTU
coupling of Fmoc-Lys(Fmoc)-OH/Fmoc deprotection (.times.2) provided
the amino handles for attachment of the adhesion molecule AM, which
is accomplished via the TBTU activation procedure. The adhesion
molecule is synthesized via standard solid-phase peptide synthesis,
and purified by preparative HPLC prior to attachment to resin. To
finalize the library synthesis, the resin 2 is treated with 10% TFA
(aq), which simultaneously facilitates the intramolecular
N-acyliminium Pictet-Spengler reaction and removal of the
Boc-protecting groups in the side-chains of AA.sub.1 (R.sup.1) and
AA.sub.2 (R.sup.2). As a consequence of the structurally diverse
aromatic heterocycles undergoing the intramolecular N-acyliminium
Pictet-Spengler reaction, the library is graphically represented by
the six sublibraries (Ia-VIa) below (FIG. 6a). Theoretically, the
library is composed by 11270 different cornpounds (32890 when all
stereoisomers are counted).
[0396] An overview of the synthesis of a combinatorial library via
the intramolecular N-acyliminium Pictet-Spengler reaction.sup.a,b
is given in FIG. 6a. The amino acids and building blocks used for
the library synthesis are indicated in table 5a.
[0397] Reagents and conditions: (a) Fmoc-Gly-OH:Alloc-Gly-OH (9:1),
TBTU, NEM, DMF; (b) 20% piperidine (DMF); (c) HMBA, TBTU, NEM, DMF;
(d) Fmoc-AA.sub.1-OH, MSNT, MeIm, CH.sub.2Cl.sub.2; (e) 20%
piperidine (DMF); (f) Fmoc-AA.sub.2-OH, TBTU, NEM, DMF; (g) 20%
piperidine (DMF); (h) Fmoc-AA.sub.3-OH, TBTU, NEM, DMF; (i) 20%
piperidine (DMF); (j) R.sup.4-MABB-OH, TBTU, NEM, DMF; (k)
Pd(PPh.sub.3).sub.4 (CHCl.sub.3:AcOH:NEM (925:50:25); (l)
Fmoc-Lys(Fmoc)-OH, TBTU, NEM, DMF; (m) 20% piperidine (DMF); (n)
Fmoc-Lys(Fmoc)-OH, TBTU, NEM, DMF; (o) 20% piperidine (DMF); (p)
AM-OH, TBTU, NEM, DMF; (q) 10% TFA (aq);.sup.a Sublibraries Ia,
IIIa, IVa, Va and VIa each consists of 700 different compounds
(1300 when all stereoisomers are counted) with n=1, and 70
different compounds (130 when all stereoisomers are counted) with
n=0;.sup.b Sublibrary IIa consists of 7000 different compounds
(23400 when all stereoisomers are counted) with n=1, and 700
different compounds (2340 when all stereoisomers are counted) with
n=0.
TABLE-US-00005 TABLE 5a Amino acids and building blocks for
combinatorial library synthesis ##STR00033## ##STR00034##
##STR00035## ##STR00036## AA.sub.1 AA.sub.2 AA.sub.3 (Sublibrary
structure) R.sup.4 D-Phe Phe L-3,4-Dimethoxyphe (Ia) H D-Tyr(t-Bu)
Tyr(t-Bu) Trp (IIa) Me D-Arg(Boc).sub.2 Arg(Boc).sub.2
D/L-(5-Br)Trp (IIa) i-Bu D-Lys(Boc) Lys(Boc) L-(5-OH)Trp (IIa) Bn
D-His(Boc) His(Boc) D/L-(5-MeO)Trp (IIa) Ph D-Trp Trp D/L-(4-Me)Trp
(IIa) 4-Br--Ph L-(1-Np)Ala L-(1-Np)Ala D/L-(5-Me)Trp (IIa)
3-CF.sub.3--Ph L-Homophe L-Homophe D/L-(6-Me)Trp (IIa) L-(3-CN)Phe
L-(3-CN)Phe D/L-(5-BnO)Trp (IIa) L-(4-CF.sub.3)Phe
L-(4-CF.sub.3)Phe D/L-(5-F)Trp (IIa) D/L-(6-F)Trp (IIa)
L-(2-Thi)Ala (IIIa) L-(3-Thi)Ala (IVa) L-(2-Fur)Ala (Va)
L-(3-BzThi)Ala (VIa)
[0398] General Methods. All solvents are of HPLC quality and stored
over molecular sieves. Solid-phase organic combinatorial chemistry
is routinely carried out using a 20-well peptide synthesizer
equipped with sintered teflon filters (50 .mu.m pores), teflon
tubing, and valves, which allow suction to be applied below the
wells. For all reactions on solid support, PEGA.sub.1900 resin (0.2
mmol/g, VersaMatrix A/S) is used. Prior to use, the resin is washed
with methanol (.times.6), DMF (.times.6), and CH.sub.2Cl.sub.2
(.times.6). All commercially available reagents are used as
received without further purification. Analysis of all solid-phase
reactions is performed after cleaving the products as their free
acids from the resin. A single bead is treated with 0.1 M aqueous
NaOH (10 .mu.L) in a 0.5 mL Eppendorf tube overnight, then diluted
with CH.sub.3CN (20 .mu.L), before filtering the solution, thereby
providing a sample for ES MSMS 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.
Spectra (FIG. 7) are analyzed by generating the exact mass
differences between fragment ions and tabulated (FIG. 8) to provide
the fragmentation pathway (FIG. 9) and therefore structure of the
compound released from the single bead.
Solid-Phase Synthesis of Combinatorial Library (6a).
[0399] Attachment of Fmoc-Gly-OH/Alloc-Gly-OH to the
amino-functionalized PEGA.sub.1900 resin (1.00 g) is carried out by
premixing Fmoc-Gly-OH (0.62 mmol, 185 mg):Alloc-Gly-OH (0.07 mmol,
9.9 mg) (9:1, 3.0 equiv in total), N-ethyl morpholine (NEM, 0.92
mmol, 106 mg, 4.0 equiv), and
N-[1H-benzotriazol-1-yl)-(dimethylamino)methylene]-N-methylmethanaminium
tetrafluoroborate N-oxide (TBTU, 0.66 mmol, 213 mg, 0.88 equiv) for
5 min in DMF. The resulting solution is added to the DMF preswollen
resin and allowed to react for 5 h, followed by washing with DMF
(.times.6), and CH.sub.2Cl.sub.2 (.times.6). Completion of the
reaction is monitored using the Kaiser test. Prior to attachment of
the HMBA linker via the procedure above, Fmoc-deprotection was
accomplished with 20% piperidine in DMF, first for 2 min, and then
for 18 min, followed by washing with DMF (.times.6). Coupling of
the first amino acid (Fmoc-AA.sub.1-OH) to the HMBA derivatized
resin is accomplished by treating the freshly lyophilized resin,
split in 20 (2.times.10) wells via dry CH.sub.2Cl.sub.2, with a
mixture of the Fmoc-AA.sub.1-OH (4.5 equiv), MeIm (3.4 equiv), and
MSNT (4.5 equiv) in CH.sub.2Cl.sub.2:THF (5:1)..sup.3 The coupling
is carried out for 1 h. When split in 20 wells, each well is
assumed to hold ca. 50 mg resin, and accordingly added reagents
relative to 0.01 mmol of material on the solid phase. Excess
reagents are removed with suction below each well, followed by
washing with dry DMF (.times.1), and dry CH.sub.2Cl.sub.2
(.times.1), before repeating the MSNT coupling of Fmoc-AA.sub.1-OH
once. Subsequent split-and-mix peptide syntheses with
Fmoc-AA.sub.2-OH, Fmoc-AA.sub.3-OH, and R.sup.4-MABB-OH,
respectively, are accomplished following the coupling procedure
described above for the attachment of Fmoc-Gly-OH (via TBTU and NEM
in DMF)..sup.2 The usual washing protocol followed each coupling
and deprotection step, and all couplings are checked via the Kaiser
test. The Alloc group of 1 is removed by treating the resin with
Pd(PPh.sub.3).sub.4 (0.06 mmol, 69 mg, 3.0 equiv) in
CHCl.sub.3:AcOH:NEM (925:50:25) for 2 h. Washing is carried out
with CHCl.sub.3 (.times.6), a mixture of 5% sodium
diethyldithiocarbamate trihydrate and 5% DIPEA in DMF (.times.2),
and DMF (.times.10). The free amino group of the resin (ca. 0.02
mmol) is coupled with Fmoc-Lys(Fmoc)-OH (0.06 mmol, 35 mg, 3.0
equiv.) via the TBTU activation procedure, using TBTU (0.058 mmol,
19 mg, 2.88 equiv), and NEM (0.08 mmol, 9 mg, 4.0 equiv). Following
Fmoc-deprotection with 20% piperidine in DMF, first for 2 min, and
then for 18 min, followed by washing with DMF (.times.6), the two
newly liberated amino handles are coupled with Fmoc-Lys(Fmoc)-OH
(0.12 mmol, 71 mg, 3.0 equiv pr amino handle) via the TBTU
activation procedure, using TBTU (0.115 mmol, 37 mg, 2.88 equiv.)
and NEM (0.16 mmol, 18 mg, 4.0 equiv). Another round of
Fmoc-deprotection with 20% piperidine in DMF, first for 2 min, and
then for 18 min, followed by washing with DMF (.times.6), provided
four amino handles, which are coupled to the adhesion molecule
AM-OH (0.24 mmol, 534 mg, 3.0 equiv) via the TBTU activation
procedure, using TBTU (0.23 mmol, 73 mg, 2.88 equiv.) and NEM (0.32
mmol, 37 mg, 4.0 equiv). The resin is washed with DMF (.times.6),
and CH.sub.2Cl.sub.2 (.times.6), and lyophilized overnight.
Finally, the library synthesis is finished by treating the resin
with 10% TFA (aq) for 24 h, followed by washing with water
(.times.6), DMF (.times.6), and CH.sub.2Cl.sub.2 (.times.6). The
resin is lyophilized overnight, and kept in the freezer
(-18.degree. C.).
Example 6b
Library of Multi-Heterocyclic Peptidomimetics for GPCR Receptors
(Library 6b).
[0400] This library is for example useful for identification of
compounds modulating a cellular response mediated through a
G-protein coupled receptor.
Library Design and Synthesis
[0401] All Pictet-Spengler reaction methodology has been developed
and tested on the synthesis resin PEGA.sub.800,.sup.1 wherefore the
analogous library resin PEGA.sub.1900 is chosen for the library
synthesis. In order to screen for active compounds, the library is
prepared following a "one-bead-two-compounds" strategy. This is
accomplished by treating the amino-functionalized resin with a
mixture of Fmoc-Gly-OH:Alloc-Gly-OH (1:1) activated by the TBTU
procedure.sup.2 to provide orthogonal reaction sites for (a)
split-and-mix library synthesis (via the Fmoc handle); and (b)
attachment of an adhesion molecule (AM) (via the Alloc handle). The
library synthesis of Pictet-Spengler reaction precursors 3 is
carried out according to standard Fmoc amino acid coupling
protocols for solid-phase peptide synthesis (FIG. 6b). Due to the
requirement of acidic reaction conditions for the Pictet-Spengler
reaction step (q), the base labile HMBA (hydroxymethylbenzoic acid)
linker is employed. Prior to attachment of HMBA to
H.sub.2N-Gly-PEGA.sub.1900 via the TBTU activation procedure, the
Fmoc protecting group is removed by standard piperidine treatment.
The HMBA linker provides a convenient cleavage site for
quantitative release from the solid support via basic hydrolysis.
Cleavage of product from a single bead is routinely achieved by
treating the bead with 0.1 M NaOH (aq) overnight, thus providing
amounts of material sufficient for structure elucidation via QTOF
ES-MSMS analysis. The hydroxy handle of the linker is esterified by
treatment with MSNT-activated Fmoc-Gly-OH.sup.3 thus placing
glycine as the first amino acid residue of the peptidomimetic
sequence. Subsequent analogous split-and-mix synthesis and 4 cycles
of Fmoc deprotection/TBTU-mediated couplings of 20 Fmoc amino acids
as the first amino acid residue (Fmoc-AA.sub.1-OH), 20 Fmoc amino
acids as the second amino acid residue (Fmoc-AA.sub.2-OH), 15 Fmoc
amino acids incorporating the reactive aromatic side-chain
(Fmoc-AA.sub.3-OH), and 6 masked aldehyde building blocks
(R.sup.4-MABB-OH) (table 5b), prepared as previously
reported,.sup.4,5 afford the Pictet-Spengler reaction precursor 3.
The Alloc protecting group of 3 is removed with
Pd(PPh.sub.3).sub.4, and subsequent TBTU coupling of
Fmoc-Lys(Fmoc)-OH/Fmoc deprotection provided the amino handles for
attachment of the adhesion molecule AM, which is accomplished via
the TBTU activation procedure. The adhesion molecule is synthesized
via standard solid-phase peptide synthesis, and purified by
preparative HPLC prior to attachment to resin. To finalize the
library synthesis, the resin 4 is treated with 10% TFA (aq) to
facilitate the intramolecular N-acyliminium Pictet-Spengler
reaction and
TFA:CH.sub.2Cl.sub.2:H.sub.2O:MeSPh:(CH.sub.2SH).sub.2:TIPS
(66.5:20:5:5:2.5:1) to remove residual protecting groups in the
side-chains of AA.sub.1 (R.sup.1) and AA.sub.2 (R.sup.2). As a
consequence of the structurally diverse aromatic heterocycles
undergoing the intramolecular N-acyliminium Pictet-Spengler
reaction, the library is graphically represented by the six
sublibraries (Ib-VIb) below (FIG. 6b). Theoretically, the library
is composed by 38400 different compounds (118800 different
compounds when all stereoisomers are counted).
[0402] An overview of the synthesis of a combinatorial library via
the intramolecular N-acyliminium Pictet-Spengler reaction.sup.a,b,c
is given in FIG. 6b. The amino acids and building blocks used for
the library synthesis are indicated in table 5b.
[0403] Reagents and conditions: (a) Fmoc-Gly-OH:Alloc-Gly-OH (1:1),
TBTU, NEM, DMF; (b) 20% piperidine (DMF); (c) HMBA, TBTU, NEM, DMF;
(d) Fmoc-Gly-OH, MSNT, MeIm, CH.sub.2Cl.sub.2; (e) 20% piperidine
(DMF); (f) Fmoc-AA.sub.1OH, TBTU, NEM, DMF; (g) 20% piperidine
(DMF); (h) Fmoc-AA.sub.2-OH, TBTU, NEM, DMF; (i) 20% piperidine
(DMF); (j) Fmoc-AA.sub.3-OH, TBTU, NEM, DMF; (k) 20% piperidine
(DMF); (l) R.sup.4-MABB-OH, TBTU, NEM, DMF; (m) Pd(PPh.sub.3).sub.4
(CHCl.sub.3:AcOH:NEM (925:50:25); (n) Fmoc-Lys(Fmoc)-OH, TBTU, NEM,
DMF; (o) 20% piperidine (DMF); (p) AM-OH, TBTU, NEM, DMF, 20 h; (q)
10% TFA (aq); (r)
TFA:CH.sub.2Cl.sub.2:H.sub.2O:MeSPh:(CH.sub.2SH).sub.2:TIPS
(66.5:20:5:5:2.5:1)..sup.a Sublibrary Ib consists of 26400
different compounds (92400 when all stereoisomers are
counted)..sup.b Sublibraries IIb, IIIb, IVb, and Vb each consists
of 2400 different compounds (4400 when all stereoisomers are
counted). Sublibrary VIb consists of 2400 different compounds (8800
when all stereoisomers are counted).
TABLE-US-00006 TABLE 5b Amino acids and building blocks for
combinatorial library synthesis ##STR00037## ##STR00038##
##STR00039## ##STR00040## AA.sub.1 AA.sub.2 AA.sub.3 (Sublibrary
structure) R.sup.4 His(Boc) His(Boc) Trp (Ib) H Asp(t-Bu) Asp(t-Bu)
D/L-(5-Br)Trp (Ib) Me Arg(Pmc) Arg(Pmc) L-(5-OH)Trp (Ib) i-Bu Phe
Phe D/L-(5-MeO)Trp (Ib) Bn Ala Ala D/L-(4-Me)Trp (Ib) Ph Cys(Trt)
Cys(Trt) D/L-(5-Me)Trp (Ib) CH.sub.2OH Gly Gly D/L-(6-Me)Trp (Ib)
Gln(Trt) Gln(Trt) D/L-(5-BnO)Trp (Ib) Glu(t-Bu) Glu(t-Bu)
D/L-(5-F)Trp (Ib) Lys(Boc) Lys(Boc) D/L-(6-F)Trp (Ib) Leu Leu
L-(2-Thi)Ala (IIb) Met Met L-(3-Thi)Ala (IIIb) Asn(Trt) Asn(Trt)
L-(2-Fur)Ala (IVb) Ser(t-Bu) Ser(t-Bu) L-(3-BzThi)Ala (Vb)
Tyr(t-Bu) Tyr(t-Bu) D/L-(7-Aza)Trp (VIb) Thr(t-Bu) Thr(t-Bu) Ile
Ile Trp(Boc) Trp(Boc) Pro Pro Val Val
[0404] General Methods. All solvents are of HPLC quality and stored
over molecular sieves. Solid-phase organic combinatorial chemistry
is routinely carried out using a 20-well peptide synthesizer
equipped with sintered teflon filters (50 .mu.m pores), teflon
tubing, and valves, which allow suction to be applied below the
wells. For all reactions on solid support, PEGA.sub.1900 resin
(0.24 mmol/g, VersaMatrix A/S) is used. Prior to use, the resin is
washed with methanol (.times.6), DMF (.times.6), and
CH.sub.2Cl.sub.2 (.times.6). All commercially available reagents
are used as received without further purification.
[0405] Analysis of all solid-phase reactions is performed after
cleaving the products as their free acids from the resin. A single
bead is treated with 0.1 M aqueous NaOH (10 .mu.L) in a 0.5 mL
Eppendorf tube overnight, then diluted with CH.sub.3CN (20 .mu.L),
before filtering the solution, thereby providing a sample for ES
MSMS 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. Spectra (FIG. 7) are
analyzed by generating the exact mass differences between fragment
ions and tabulated (FIG. 8) to provide the fragmentation pathway
(FIG. 9) and therefore structure of the compound released from the
single bead.
[0406] Solid-phase synthesis of combinatorial library (6b).
Attachment of Fmoc-Gly-OH/Alloc-Gly-OH to the amino-functionalized
PEGA.sub.1900 resin (0.24 mmol/g, 1.68 mmol, 7.00 g). The resin
swelled in DMF is added solutions (i)+(ii) of TBTU-activated
N-protected glycines; (i) Fmoc-Gly-OH (1.5 equiv., 2.52 mmol, 749
mg)+NEM (2.0 equiv., 3.36 mmol, 426 .mu.L)+TBTU (1.44 equiv., 2.42
mmol, 809 mg) in 5 mL DMF (activation in the usual way); and (ii)
Alloc-Gly-OH (1.5 eq, 2.52 mmol, 401 mg)+NEM (2.0 eq, 3.36 mmol,
426 .mu.L)+TBTU (1.44 eq, 2.42 mmol, 809 mg) in 5 mL DMF
(activation in the usual way). Both solutions are simultaneously
added to the resin in 100.times.50 .mu.L portions with vigorous
shaking, maintaining the rate at 1 addition from each solution pr.
minute. After addition of solutions (i) and (ii), the reaction
mixture is further shaken for 30 min, followed by washing with DMF
(.times.6), and CH.sub.2Cl.sub.2 (.times.6) in a syringe fitted
with a Teflon filter. Completion of the reaction is monitored using
the Kaiser test. Prior to attachment of the HMBA linker via the
procedure above, Fmoc-deprotection is accomplished with 20%
piperidine in DMF, first for 2 min, and then for 18 min, followed
by washing with DMF (.times.6). Coupling of the first amino acid
(Fmoc-Gly-OH) to the HMBA derivatized resin is accomplished by
treating the freshly lyophilized resin (0.84 mmol) with a mixture
of the Fmoc-Gly-OH (4 eq, 3.4 mmol, 999 mg), MeIm (8 eq, 6.8 mmol,
533 .mu.L), and MSNT (4 eq, 3.4 mmol, 996 mg) in dry
CH.sub.2Cl.sub.2 (30 mL)..sup.3 The coupling is carried out for 2
h, then the resin is washed with dry DMF (.times.1), and dry
CH.sub.2Cl.sub.2 (.times.1), before repeating the MSNT coupling of
Fmoc-Gly-OH once. The resin is washed with DMF (.times.6) and
CH.sub.2Cl.sub.2 (.times.6) prior to lyophilization for removal of
all solvent traces. A batch of resin (1.00 g) is subjected to
split-and-mix peptide syntheses with Fmoc-AA.sub.1-OH,
Fmoc-AA.sub.2-OH, Fmoc-AA.sub.3-OH, and R.sup.4-MABB-OH,
respectively, following the coupling procedure described above for
the attachment of Fmoc-Gly-OH (via TBTU and NEM in DMF)..sup.2 The
usual washing protocol follows each coupling and deprotection step,
and all couplings are checked via the Kaiser test. The Alloc group
of 3 is removed by treating the resin batch twice with
Pd(PPh.sub.3).sub.4 (3.0 equiv., 0.36 mmol, 416 mg) in
CHCl.sub.3:AcOH:NEM (925:50:25) for 3 h. Washing was carried out
with CHCl.sub.3 (.times.6) and DMF (.times.10). The free amino
group of the resin (0.12 mmol) was coupled with Fmoc-Lys(Fmoc)-OH
(3.0 equiv., 0.36 mmol, 210 mg) via the TBTU activation procedure,
using TBTU (2.88 equiv., 0.348 mmol, 114 mg) and NEM (4.0 equiv.,
0.48 mmol, 54 mg). Following Fmoc-deprotection with 20% piperidine
in DMF, first for 2 min, and then for 18 min, followed by washing
with DMF (.times.6), newly liberated amino handle is coupled to the
adhesion molecule AM-OH (1.5 equiv., 0.36 mmol, 801 mg) via the
TBTU activation procedure, using TBTU (2.88 equiv., 0.691 mmol, 222
mg) and NEM (4.0 equiv., 0.96 mmol, 122 .mu.L). The resin was
washed with DMF (.times.6), and CH.sub.2Cl.sub.2 (.times.6), and
lyophilized overnight. The library synthesis was finished by first
treating the resin with 10% TFA (aq) for 24 h, followed by washing
with water (.times.6), DMF (.times.6), and CH.sub.2Cl.sub.2
(.times.6), and finally with
TFA:CH.sub.2Cl.sub.2:H.sub.2O:MeSPh:(CH.sub.2SH).sub.2:TIPS
(66.5:20:5:5:2.5:1) for 5 h, before washing with CH.sub.2Cl.sub.2
(.times.6), DMF (.times.6), water (.times.6), DMF (.times.6), and
CH.sub.2Cl.sub.2 (.times.6). The resin was lyophilized overnight,
and stored in the freezer (-18.degree. C.).
REFERENCES
[0407] (1) Meldal, M. Tetrahedron Lett. 1993, 33, 3077-3080. [0408]
(2) Knorr, R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D.
Tetrahedron Lett. 1989, 30, 1927-1930. [0409] (3)
Blankemeyer-Menge, B.; Nimtz, M.; Frank, R. Tetrahedron Lett. 1990,
31, 1701-1704. [0410] (4) Groth, T.; Meldal, M. J. Comb. Chem.
2001, 3, 34-44. [0411] (5) Nielsen, T. E.; Meldal, M. J. Org. Chem.
2004, 69, 3765-3773.
Example 7
[0412] Gs Coupled Receptor (MC4R): Agonist Assay (Cre-GFPreporter
Assay Detected with a Fluorescence Activated Bead Sorter)
Cre-GFP:
[0413] 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:
[0414] 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:
[0415] U2OS 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.
[0416] 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:
[0417] 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
1) Positive control: 50 ml Growth medium+5000 positive control
beads+2.times.10E7 cells. 2) Negative control: 50 ml Growth
medium+5000 negative control beads+2.times.10E7 cells. 3) Screening
library (eg. 100,000 compounds): 1000 ml Growth medium+100,000
library beads+4.times.10E8 cells.
[0418] 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. 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:
[0419] 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.
[0420] 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 in
Example 2 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.
[0421] 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.
[0422] This assay may also be performed using HEK cells essentially
as described herein below in Example 7a, except that the HEK cells
should be transfected with the Cre-GFP and pDEST1.2.2MC4R
constructs. Positive resin beads may also preferably be selected
using a fluorescence microscope, as described in Example 7a.
Example 7a
Gs Coupled Receptor (MC4R): Agonist Assay (MC4R-GFP
Internalization: Microscopy)
Construction of MC4R-GFP:
[0423] 996 bp of MC4R ORF sequence without stop codon is inserted
into pGFP2-N1 vector (Biosignal Packard Cat. # 6310013-001) with
cloning sites EcoRI/BamHI.
Cell Line Establishment:
[0424] Hek293 cells are transfected with MC4R-GFP using standard
procedure for Fugene6 transfection. Cells are put under zeocin
selection for 4 weeks to obtain a cell line stably expressing the
MC4R-GFP.
Cell/Bead Preparation:
[0425] Cells were cultured on respectively 1) Negative control
beads (prepared as described in example 1), 2) Positive control
beads (prepared as described in example 2;
Ac-His-(D)phe-Arg-Trp-Gly-PEGA.sub.1900) and Library beads
(prepared as described in example 6b). Each batch of beads was
handled separately.
[0426] Cells were trypsinized and mixed with Negative control
beads/Positive control beads/Library beads in growth medium (Hams
F12 containing 5% FCS) [0427] Add 500 beads in 500 ul Hams to a 14
ml Nunc tube [0428] Add 2500 ul cell suspension 1.times.10E6/ml
Hams w. 5% FCS [0429] Leave tube vertically in incubator (37
degrees, 5% CO2) for 16-24 hrs--rock tube gently every 15 min for
the first hour [0430] Remove medium. Wash loose cells away by
gently adding and removing 4 ml Hams .times.2 (Turn the tube upside
down and back again--as soon as beads have sedimented suck away
medium) [0431] Add 2 ml Hams w. FCS 5% [0432] Incubate o/n at 37
degrees, 5% CO2 [0433] Decant beads to a 1 well Lab-Tek Chambered
Coverglass (#155361)
Hit Identification and Isolation
[0434] The LabTek 1 well chambered coverglass was placed on a Zeiss
Axiovert 200 fluorescence microscope equipped with filters optimal
for GFP fluorescence. The microscope was further more equipped with
a micromanipulator (Eppendorf Transferman NK2)) capable of picking
out single beads. Using 40.times. objective chambers were scanned
for positive hit beads, which appeared as cells with green dots
located in the cytoplasma in contrast to negative beads where GFP
is located in the plasma membrane of the cells. Positive and
negative control beads were used to set cut off for positive hit
beads. Such hit beads were picked out using the micromanipulator.
MC4R-GFP internalization was quantified and the results are shown
in FIG. 11.
Example 8
Gs Coupled Receptor (MC4R): Agonist Assay (Multiplexed Cre-YFP
Reporter and MC4R-GFPinternalization: FABS and Microscopy)
[0435] Construction of pCRE-d2EYFP:
[0436] A 732 bp of EYFP fragment from pd2EYFP-1 (Clontech Cat.
#6912-1) is ligated to a 3.5 kb fragment from pCRE-d2EGFP (Clontech
Cat #6034-1). Both fragments are excised from the two vectors by a
common restriction enzyme digestion.
Construction of MC4R-GFP:
[0437] 996 bp of MC4R ORF sequence without stop codon is inserted
into pGFP2-N1 vector (Biosignal Packard Cat. # 6310013-001) with
cloning sites EcoRI/BamHI.
Cell Line Establishment:
[0438] U2OS cells are transfected with MC4R-GFP using standard
procedure for Fugene6 transfection. Cells are put under zeocin
selection for 4 weeks to obtain a cell line stably expressing the
MC4R-GFP.
[0439] The U2OS cell line stably expressing the human MC4R-GFP
(melanocortin4 receptor-GFP) is further transfected with Cre-YFP
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:
[0440] Cells are trypsinized and mixed with beads in growth medium
(DMEM containing 10% FCS, in the proportion 4000 cells/bead and
app. 50 ml growth medium/5000 beads.
1) Positive control: 50 ml Growth medium+5000 positive control
beads+2.times.10E7 cells. 2) Negative control: 50 ml Growth
medium+5000 negative control beads+2.times.10E7 cells. 3) Screening
library (eg. 100,000 compounds): 1000 ml Growth medium+100,000
library beads+4.times.10E8 cells
[0441] 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.
[0442] 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 for Cre-YFP Response:
[0443] A Fluorescence Activated Bead Sorter (FABS) equipped with
514 nm excitation laser line and 528-572 nm emission filter is used
to identify and isolate positive hit beads.
[0444] The FABS is calibrated to identify and isolate positive hit
beads (increased YFP fluorescence) by determining the dynamic range
of the assay using positive control beads as Smax (maximum
response) and negative control beads 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.
[0445] Positive hit beads are isolated into a 1 well Nunc chamber
and are hereafter ready to test for receptor internalisation.
MC4R-GFPinternalisation: Microscope Analysis
[0446] The Nunc chamber with positive Cre-YFP hits is placed on an
imaging microscope (Zeiss Axiovert 200M) equipped with filters
allowing separation of YFP and GFP. Further more the microscope is
equipped with a micromanipulator (Eppendorf Transferman NK2))
capable of picking out single beads. Using 20.times. objective the
chamber is scanned for positive MC4R-GFPinternalisation (appear as
intracellular spots as compared to membrane distribution in non
positive MC4R-GFP internalisation) and such hit beads are picked
out using the micromanipulator for compound structure
elucidation.
[0447] A multiplexed screening like this is expected to give very
low rate of false positive hits since hits picked out for structure
elucidation are giving rise to both specific receptor activation
(internalisation of receptor) as well as a functional response
(activation of transcription of Cre-YFP).
Example 9
Gs Coupled Receptor (MC4R): Agonist Assay (Cre-YFP Reporter and
HA-MC4R Internalization: FABS and Microscopy)
[0448] Construction of pCRE-d2EYFP:
[0449] A 732 bp of EYFP fragment from pd2EYFP-1 (Clontech Cat.
#6912-1) is ligated to a 3.5 kb fragment from pCRE-d2EGFP (Clontech
Cat #6034-1). Both fragments are excised from the two vectors by a
common restriction enzyme digestion.
Construction of HA-MC4R:
[0450] 999 bp of MC4R ORF sequence is first inserted into pCMV-HA
vector (Clontech Cat#6003-1) with cloning sites EcoRI/XhoI, then
the fusion fragment of HA-MC4R is further cloned into pcDNA3.1/Zeo
(Invitrogen Cat.#V86520) with the cloning sites HindIII/XhoI.
Cell Line Establishment:
[0451] U2OS cells are transfected with HA-MC4R using standard
procedure for Fugene6 transfection. Cells are put under zeocin
selection for 4 weeks to obtain a cell line stably expressing the
HA-MC4R.
[0452] The U2OS cell line stably expressing the human HA-MC4R
(melanocortin4 receptor-GFP) is further transfected with Cre-YFP
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:
[0453] Cells are trypsinized and mixed with beads in growth medium
(DMEM containing 10% FCS, in the proportion 4000 cells/bead and
app. 50 ml growth medium/5000 beads.
1) Positive control: 50 ml Growth medium+5000 positive control
beads+2.times.10E7 cells. 2) Negative control: 50 ml Growth
medium+5000 negative control beads+2.times.10E7 cells. 3) Screening
library (eg. 100,000 compounds): 1000 ml Growth medium+100,000
library beads+4.times.10E8 cells
[0454] 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.
[0455] 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 for Cre-YFP Response:
[0456] A Fluorescence Activated Bead Sorter (FABS) equipped with a
multiline Argon laser adjusted to the 514 nm excitation line and
528-572 nm emission filter is used to identify and isolate positive
hit beads.
[0457] The FABS is calibrated to identify and isolate positive hit
beads (increased YFP fluorescence) by determining the dynamic range
of the assay using positive control beads as Smax (maximum
response) and negative control beads 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.
[0458] Positive hits are isolated into a 1 well Nunc chamber and
are hereafter ready to test for receptor internalisation.
HA-MC4R Internalisation: Microscope Analysis
[0459] Beads isolated as positive hits in Cre-YFP transcription
reporter assay by FABS are treated with Triton-x to permeabilize
cells followed by incubation with HA-tag poly-clonal antibody
followed by staining with appropriate TRITC conjugated secondary
antibody. A Nunc 1 well chamber holding the labelled beads are
placed on an imaging microscope (Zeiss Axiovert 200M) equipped with
filters allowing separation of YFP and TRITC. Further more the
microscope is equipped with a micromanipulator (Eppendorf
Transferman NK2)) capable of picking out single beads. Using
20.times. objective the chamber is scanned for positive
HA-internalisation (appear as intracellular spots as compared to
membrane distribution in non positive HA-MC4Rinternalisation and
such hit beads are picked out for compound structure
elucidation.
[0460] A multiplexed screening like this is expected to give very
low rate of false positive hits since hits picked out for structure
elucidation is giving rise to both specific receptor activation
(observed as internalisation of receptor) as well as a functional
response (observed as transcription of Cre-YFP construct).
Example 10
Gs Coupled Receptor (MC4R): Agonist Assay (Cre-YFP Reporter and
HA-MC4R Internalization: FABS+FABS)
[0461] Construction of pCRE-d2EYFP:
[0462] A 732 bp of EYFP fragment from pd2EYFP-1 (Clontech
Cat.#6912-1) is ligated to a 3.5 kb fragment from pCRE-d2EGFP
(Clontech Cat #6034-1). Both fragments are excised from the two
vectors by a common restriction enzyme digestion.
Construction of HA-MC4R:
[0463] 999 bp of MC4R ORF sequence is first inserted into pCMV-HA
vector (Clontech Cat#6003-1) with cloning sites EcoRI/XhoI, then
the fusion fragment of HA-MC4R is further cloned into pcDNA3.1/Zeo
(Invitrigen Cat.#V86520) with the cloning sites HindIII/XhoI.
Cell Line Establishment:
[0464] U2OS cells are transfected with HA-MC4R using standard
procedure for Fugene6 transfection. Cells are put under zeocin
selection for 4 weeks to obtain a cell line stably expressing the
HA-MC4R.
[0465] The U2OS cell line stably expressing the human HA-MC4R
(HA-melanocortin4 receptor) is further transfected with Cre-YFP 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:
[0466] Cells are trypsinized and mixed with beads in growth medium
(DMEM containing 10% FCS, in the proportion 4000 cells/bead and
app. 50 ml growth medium/5000 beads.
1) Positive control: 50 ml Growth medium+5000 positive control
beads+2.times.10E7 cells. 2) Negative control: 50 ml Growth
medium+5000 negative control beads+2.times.10E7 cells. 3) Screening
library (eg. 100,000 compounds): 1000 ml Growth medium+100,000
library beads+4.times.10E8 cells
[0467] 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. 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 for Cre-YFP Response:
[0468] A Fluorescence Activated Bead Sorter (FABS) equipped with
514 nm laser excitation line and 528-572 nm emission filter is used
to identify and isolate positive hit beads.
[0469] The FABS is calibrated to identify and isolate positive hit
beads (increased YFP fluorescence) by determining the dynamic range
of the assay using positive control beads as Smax (maximum
response) and negative control beads 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.
[0470] Positive hits are isolated into a 10 ml tube and are
hereafter ready to test for receptor internalisation.
HA-MC4R Internalisation: FABS Analysis
[0471] A Fluorescence Activated Bead Sorter (FABS) equipped with
568 nm laser line excitation and 584-640 nm emission filter is used
to identify and isolate positive hit beads.
[0472] Beads isolated as positive hits in Cre-YFP transcription
reporter assay by FABS as well as positive and negative control
beads are incubated with HA-tag polyclonal antibody followed by
staining with appropriate TRITC conjugated secondary antibody.
[0473] 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 response) and negative control beads
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.
Positive hits are giving less TRITC fluorescence than negative hits
caused by receptor internalization resulting in inability of the
TRITC conjugated sec. antibody to reach the HA-tag (no
permeabilization of the plasma membrane).
[0474] Positive hits are separated into each 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.
[0475] A multiplexed screening like this is expected to give very
low rate of false positive hits since hits picked out for structure
elucidation is giving rise to both specific receptor activation as
well as a functional response
Example 11
[0476] Gs coupled Receptor (.beta.2AR): Antagonist Assay
(Cre-Reporter)
Cre-GFP:
[0477] Cre-GFP (c-AMP Response Element-Green Fluorescent Protein)
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. .beta.2 adrenergic receptor
(.beta.2AR):
[0478] A 1776 bp cDNA fragment containing .beta.2AR ORF sequence is
PCR-amplified from human kidney and fetal brain cDNA libraries
(Clontech Cat#639305, 6393029) using primers designed from
.beta.2AR mRNA sequence (accession # NM.sub.--000024), and cloned
into pCR-XL-TOPO vector (invitrogen). A 1274 bp of .beta.2AR gene
containing a kozak sequence and a stop codon is further cloned into
pcDNA3.1/zeo(+) vector (invitrogen) with the restriction sites
HindIII/XhoI. The .beta.2AR gene is sequencing confirmed.
Cell Line Establishment:
[0479] U2OS cells are transfected with .beta.2AR using standard
procedure for Fugene6 transfection. Cells are put under zeocin
selection for 4 weeks to obtain a cell line stably expressing the
.beta.2AR.
[0480] The U2OS cell line stably expressing the human .beta.2AR 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 displaying adhesion
peptide and isoproterenol) and 3) Library compounds. The three
cultures are handled separately in each their culture flask.
Bead/Cell Preparation:
[0481] Cells are trypsinized and mixed with beads in growth medium
(DMEM containing 10% FCS), added Isoproterenol 10 uM, in the
proportion 4000 cells/bead and app. 50 ml growth medium w.
isoproterenol 10 uM/5000 beads.
1) Positive control: 50 ml Growth medium w. proterenol 10 uM+5000
positive control beads+2.times.10E7 cells. 2) Negative control: 50
ml Growth medium w. proterenol 10 uM+5000 Negative control
beads+2.times.10E7 cells. 3) Screening library (eg. 100,000
compounds): 1000 ml Growth medium w. proterenol 10 uM+100,000
library beads+4.times.10E8 cells
[0482] 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.
[0483] 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:
[0484] A Fluorescence Activated Bead Sorter (FABS) equipped with
488 nm laser excitation line and 500-550 nm emission filter is used
to identify and isolate positive hit beads (=inhibition of
isoproterenol induced Cre-GFP transcription (=decreased
fluorescence compared to negative control).
[0485] 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). A cut off at 30% inhibition compared to negative
control beads is set as threshold for a positive hit bead.
[0486] 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
Gi Coupled Receptor (CCR5): Agonist Assay (Cre-GFP Reporter)
Cre-GFP:
[0487] 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.
C--C Chemokine Receptor5 (CCR5):
Accession no. AAB57793
[0488] U2OS cells are transfected with CCR5 using standard
procedure for Fugene6 transfection. Cells are put under zeocin
selection for 4 weeks to obtain a cell line stably expressing the
CCR5.
[0489] The U2OS cell line stably expressing the human CCR5 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
and RANTES) and 3) Library compounds. The three cultures are
handled separately in each their culture flask.
Bead/Cell Preparation:
[0490] Cells are trypsinized and mixed with beads in DMEM
containing 10% FCS, 10 uM forskolin and 500 uM IBMX, in the
proportion 4000 cells/bead and app. 50 ml DMEM/5000 beads.
1) Positive control: 50 ml DMEM+5000 positive control
beads+2.times.10E7 cells. 2) Negative control: 50 ml DMEM+5000
negative control beads+2.times.10E7 cells. 3) Screening library
(eg. 100,000 compounds): 1000 ml DMEM+100,000 library
beads+4.times.10E8 cells
[0491] 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.
[0492] 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:
[0493] A Fluorescence Activated Bead Sorter (FABS) equipped with
488 nm laser excitation line and 500-550 nm emission filter and
sorting capability into 96 well plate is used to identify and
isolate positive hit beads.
[0494] The FABS is calibrated to identify and isolate positive hit
beads (decreased GFP fluorescence compared to negative control) by
determining the dynamic range of the assay using positive control
beads (RANTES) as Smax (maximum response=minimal fluorescence) and
negative control beads as 5 min (minimum response=maximal
fluorescence). A cut off at 30% response compared to negative
control beads is set as threshold for a positive hit bead.
[0495] 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 13
Gq Coupled Receptor (Muscarinic M1): Antagonist Assay (Ca++
Mobilization Using Fluo-4)
Ca++ Antagonist Assay:
[0496] 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++. 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.
[0497] 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:
[0498] 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.
1) Positive control: 50 ml Growth medium+5000 positive control
beads+2.times.10E7 cells. 2) Negative control: 50 ml Growth
medium+5000 negative control beads+2.times.10E7 cells. 3) Screening
library (eg. 100,000 compounds): 1000 ml Growth medium+100,000
library beads+4.times.10E8 cells
[0499] 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.
[0500] 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.
[0501] 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 are 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 is 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 are
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 is challenged with 1 uM ionomycin immediately
before the fluorescence detection. Similarly F.sub.max is 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.21.5 mM, HEPES 10 mM) and challenged with 1 uM ionomycin
immediately before detection.
[0502] 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:
[0503] 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).
[0504] 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 steam
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
hit beads may preferably be identified using a plate reader
essentially as described in Example 13a herein below.
[0505] 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 13a
[0506] Gq Coupled Receptor (Muscarinic M1): Antagonist Assay (Ca++
Mobilization using Fluo-4)
Ca++Antagonist Assay:
[0507] This assay is designed to identify muscarinic M1
antagonistic compounds. The readout is changes in intracellular
Ca++ conc. detected using the Fluo-4 probe from Molecular probes.
Positive hits are compounds that inhibit Carbacol (muscarinic M1
agonist) induced increase in intracellular Ca++.
[0508] For calculation of the exact [Ca.sup.2+].sub.i the
fluorescence intensity is 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 Fluo4
is saturated with Ca.sup.2+. To obtain F.sub.min the cells are
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, probenecid 2 mM) and is challenged with 1 uM ionomycin
immediately before the fluorescence detection. Similarly F.sub.max
is 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.21.5 mM, HEPES 10 mM, probenecid 2 mM) and
challenged with 1 uM ionomycin immediately before detection.
[0509] In general it is not required to use exact ion
[Ca.sup.2+].sub.i. Rather, the response of screening compounds may
be expressed as relative to control compounds (see below).
Cell Line Establishment:
[0510] BHK cells are transfected with the muscarinic M1 receptor
using standard procedure for Fugene6 transfection. Cells are put
under G418 selection for 4 weeks to obtain a cell line stably
expressing the muscarinic M1 receptor.
Cell/Bead Preparation:
[0511] BHK cells expressing the Muscarinic M1 receptor are cultured
on Negative control beads (prepared as described in example 1)
using the following procedure: [0512] Trypsinize cells and adjust
cell conc. to 1.times.10E6/ml Hams F12 growth medium [0513] Add 500
beads in 500 ul Hams to a 14 ml Nunc tube [0514] Add 2500 ul cell
suspension 1.times.10E6/ml Hams w. 5% FCS [0515] Leave tube
vertically in incubator (37 degrees, 5% CO2) for 16-24 hrs--rock
tube gently every 15 min for the first hour [0516] Remove medium.
Wash loose cells away by gently adding and removing 4 ml Hams F12
twice (Turn the tube upside down and back again--as soon as beads
have sedimented suck away medium) [0517] Add 2 ml Hams F12 w. FCS
10% [0518] Incubate o/n at 37 degrees, 10% CO2
[0519] Measurement of changes in the cytoplasmic free calcium
concentration [Ca.sup.2].sub.i. [0520] Beads, now covered with
cells, are allowed to sediment for 10 min (no centrifugation
needed) and the growth medium is removed using a pipette. [0521] 2
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.2 1.5 mM, HEPES 10 mM, probenecid 2 mM) added 1 uM Fluo-4
(Molecular Probes F-14201)+0.02% Pluronic (Molecular Probes F-127)
per 500 beads is added, mixed gently and cells/beads are incubated
at 37.degree. c. for 30 min. [0522] Beads are hereafter washed w. 5
ml KRW/500 beads .times.2 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.
[0523] The fluorescence is monitored in a fluorescence plate-reader
(Polarstar Optima from BMG Labtech, Germany) equipped with a flash
Xenon blitz lamp and 490.+-.5 nm excitation filter and 510.+-.5 nm
emission filter. The plate reader is furthermore equipped with a
dispenser allowing injection of agonist.
[0524] The measurement can equally well be performed on a
microscope equipped with a fluorescence illuminator (E.g. HBO 100 W
lamp) and 480/30 nm excitation filter, 505 nm LP dicroic mirror and
535/40 nm emission filter.
[0525] Approx. 50 beads covered with BHK cells expressing the M1
receptor now loaded with the fluorescent Ca++ indicator Fluo-4 are
pipetted into each well of a 96 well plate. The plate is placed in
the fluorescence plate reader and Carbacol 1 uM is injected
resulting in an increase in fluorescence for negative control
beads.
[0526] This assay can be used to screen for Carbacol inhibitors.
Positive hits are compounds that inhibit the Carbacol (muscarinic
M1 agonist)
[0527] FIG. 12 shows the intracellular Ca++ mobilization in BHK-M1
cells on beads treated with Carbamylcholin 100 uM versus control
(buffer).
Example 14
[0528] Gs Coupled Receptor (MC4R): Agonist Assay (Cre-GFP Reporter
Assay Detected with Fluorescence Plate Reader or Fluorescence
Imaging Equipment)
Cre-GFP:
[0529] 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:
[0530] 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:
[0531] U2OS 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
the MC4R.
[0532] 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 ex-ample 2) and 3) Library compounds. The three cultures are
handled separately in each their culture flask.
Bead/Cell Preparation:
[0533] 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.
1) Positive control: 50 ml Growth medium+5000 positive control
beads+2.times.10E7 cells. 2) Negative control: 50 ml Growth
medium+5000 negative control beads+2.times.10E7 cells. 3) Screening
library (eg. 100,000 compounds): 1000 ml Growth medium+100.000
library beads+4.times.10E8 cells
[0534] 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.
[0535] 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:
[0536] 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 reseeding in a new 384 well plate, this
time having one bead per well. Smin=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 Analysis:
[0537] Control beads as well as library beads are seeded in 384
well black plates (eg.
[0538] 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. Smin=one negative control bead
and Smax=one positive control bead. Image acquisition and analysis
described above is repeated and final hit beads are identified.
[0539] 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). This method is
preferred.
Example 14a
Gs Coupled Receptor (MC4R) Agonist Screening (Cre-YFP Reporter
Assay Detected Using a Fluorescence Microscope)
[0540] pCre-d.sub.2YFP:
[0541] A 732 bp of EYFP fragment from pd2EYFP-1 (Clontech
Cat.#6912-1) is ligated to a 3.5 kb fragment from pCRE-d2EGFP
(Clontech Cat #6034-1). Both fragments are excised from the two
vectors by a common restriction enzyme digestion.
MC4R:
[0542] 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:
[0543] Hek293 cells are co-transfected with pDEST1.2.2MC4R and
Cre-YFP (using standard procedure for Fugene6 transfection) and
cells are cultured on PEGA beads displaying adhesion peptide and
respectively 1) Negative control (PEGA beads with adhesion peptide)
and 2) Positive control (PEGA beads of example 2) by mixing appr.
400 beads with 400,000 cells in 1 ml Hams F12 medium containing 10%
FCS in a 1.8 ml Eppendorf tube. Tubes are shaked gently every 15
min for 2 hrs. Cells/beads are incubated in a CO2 incubator (5%
CO2, m 37 degrees) for 20 hrs. The level of CRE-YFP expression was
detected using a Zeiss Axiovert 200M microscope equipped with
appropriate filters for YFP detection (Excitation 500/20 nm,
Dicroic 515 LP EM 535/30).
[0544] Higher signal was observed for the Hek293 cells compared to
the U2OS, why Hek293 were used for further experiments (see FIG.
13).
Library Screening:
Synthesis of Library- and Control Beads:
[0545] Two libraries were synthesized according to examples 6a and
6b. Control beads were synthesized as described in example 5a
section "Synthesis of adhesion peptide".
Cell Line Establishment:
[0546] Hek293 cells were co-transfected with pDEST1.2.2MC4R and
pCRE-d2EYFP and put under G418 selection for 3 weeks. Hereafter
cells were FACSorted (Fluorescence Activated Bead sorted) for high
YFP expression after stimulation with aMSH 100 nM and 0.4 uM TSA
(Tricostatin A) for 20 hrs. Cells were propagated and subcultured
for 2 month and FACSorted again for high aMSH/TSA induced YFP
expression.
Cell/Bead Preparation:
[0547] Cells were cultured on respectively 1) Negative control
beads (prepared as described in example 1), 2) Positive control
beads (prepared as described in example 2) and Library beads
(prepared as described in example 6b). Each batch of beads was
handled separately.
[0548] Cells were trypsinized and mixed with Negative control
beads/Positive control beads/Library beads in growth medium (Hams
F12 containing 5% FCS):
Control Beads
[0549] Add 500 beads in 500 ul Hams to a 14 ml Nunc tube [0550] Add
2500 ul cell suspension 1.times.10E6/ml Hams w. 5% FCS [0551] Leave
tube vertically in incubator (37 degrees, 5% CO2) for 16-24
hrs--rock tube gently every 15 min for the first hour [0552] Remove
medium. Wash loose cells away by gently adding and removing 4 ml
Hams .times.2 (Turn the tube upside down and back again--as soon as
beads have sedimented suck away medium) [0553] Add 2 ml Hams w. FCS
5% and TSA (Tricostatin A) 0.4 uM [0554] Incubate o/n at 37
degrees, 5% CO2 [0555] Decant beads to a 1 well Lab-Tek Chambered
Coverglass (#155361)
Library Beads
[0555] [0556] Add 10,000 beads in 5 ml Hams to a 50 ml Nunc tube
[0557] Add 25 ml cell suspension 2.times.10E6/ml Hams w. 5% FCS
[0558] Leave tube vertically in incubator (37 degrees, 5% CO2) for
16-24 hrs--rock tube gently every 15 min for the first hour [0559]
Remove medium. Wash loose cells away by gently adding and removing
25 ml Hams .times.2 (Turn the tube upside down and back again--as
soon as beads have sedimented suck away medium) [0560] Add 25 ml
Hams w. FCS 5% and TSA (Tricostatin A) 0.4 uM [0561] Incubate o/n
at 37 degrees, 5% CO2 [0562] Decant beads to 2.times.1 well Lab-Tek
Chambered Coverglass (#155361)
Hit Identification and Isolation:
[0563] The LabTek one well chambered coverglass was placed on a
Zeiss Axiovert 200 fluorescence microscope equipped with filters
optimal for YFP fluorescence. The microscope was further more
equipped with a micromanipulator (Eppendorf Transferman NK2))
capable of picking out single beads. Using 10.times. objective
chambers were scanned for positive hit beads, which appeared as
green dotted beads caused by cells expressing CRE-YFP. Positive and
negative control beads were used to set cut off for positive hit
beads. Such hit beads were picked out using the micromanipulator
for further test in specificity assay (Receptor internalization)
before final structure elucidation.
[0564] Microscope detection was preferred for this screening
campaign. The throughput of this microscope-based method was app.
40,000 beads per day. 90,000 beads were screened in totally. 35
hits were identified and isolated, 15 were structure elucidated and
resynthesized.
[0565] Signal obtained from a sub-fraction of identified hits is
shown in the graph of FIG. 14.
Structure Elucidation and Resynthesis:
[0566] Hit beads are structure elucidated using the method
described in example 15. As an illustrative example identification
of hit designated TEN-636-33-26 is described om example 15b. Other
hits may be identified using a similar method.
Specificity Screening:
[0567] Hits are tested for MC4 receptor specificity using a Hek 293
cell line stably expressing the MC4R-GFP as described in example 7a
under cell line establishment. Cells are seeded in Hams F12 w. 10%
FCS in an 8 well Lab-tek Chambered Cover-glass to give 75%
confluency 24 hrs after seeding. Cells are challenged with hit
compounds for 30 min at 37 degrees. The chamber is placed on a
Zeiss Axiovert 200M equipped with filters suited for GFP
fluorescence and cells are inspected for MC4R-GFP internalization
using image acquisition (20.times.) followed by image analysis.
Negative and positive controls are Hams F12 respectively .alpha.MSH
100 nM.
Selectivity screening .beta.2-adrenergic receptor (.beta.2-AR-GFP
Internalization:
[0568] Hits are further tested for receptor selectivity using a
Hek293 cell line stably expressing the .beta.2-adrenergic receptor
fused to GFP (.beta.2-AR-GFP). Cells are tested as described above
for "Specificity determination".
[0569] MC4R specific hits are those showing a positive response in
CRE-YFP reporter assay, a positive response in the MC4R-GFP
internalization assay (specificity) and a negative response in
.beta.2-AR-GFP internalization assay (selectivity).
Example 15
Identification of Compound
[0570] Once a resin bead is selected, the library compound
comprised within the bead is identified. The selected, single resin
bead is treated with 0.1 M aqueous NaOH (10 .mu.L) in a 0.5 mL
Eppendorf tube overnight, then diluted with CH.sub.3CN (20 .mu.L),
before filtering the solution, thereby providing a sample for ES
MSMS analysis on a Micro-Mass 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.
Example 15b
Identification of Hit TEN-636-33-26 (From Library Prepared as
Described in Example 6b) from GPCR Assay
[0571] Hits selected in the GPCR assay call for unambiguous
structure assignment. High purity of compounds generated on the
solid support during library synthesis are preferred for single
bead analysis. For example for the chemistry utilized in Example
6a-b (indsaet evt. ref til scaffold-patent), it has demonstrated
that quantities of material cleaved from single synthesis and
library beads can be analyzed and identified by QTOF MSMS (ES). The
signal arising from the molecular ion (M+H) is first detected, and
MSMS is subsequently carried out to obtain a specific fragmentation
pattern.
[0572] A hit (bead) selected in the assay (FIG. 15a) is carefully
washed with 10% TFA (aq) and MiliQ water by successive rounds of
decantation. The bead is placed in a 1.5 mL Eppendorf tube and
treated with 0.1 M NaOH (3 mL) prepared in the usual way from solid
NaOH pellets and MiliQ water. Hydrolysis is effected during 2 h to
24 h in a sealed tube. After hydrolysis, 0.1 M HCl (3 mL) is added
to neutralize the alkaline cleavage mixture, followed by addition
of MiliQ water (40 .mu.L). Prior to loading, the wells of the OASIS
elution plate was carefully washed with 3.times.CH.sub.3CN:H.sub.2O
(4:1, 0.1% HCOOH), then 3.times.H.sub.2O (0.1% HCOOH), and
3.times.H.sub.2O. The selected well is loaded with the sample
solution by applying gentle suction. Salts are then washed out with
water (70 .mu.L) and 0.1% HCOOH (70 mL), before eluting the
compound with CH.sub.3CN:H.sub.2O (4:1, 0.1% HCOOH) (200 .mu.L).
The eluent is removed on the speed vac, and the resulting residue
is taken up in CH.sub.3CN:H.sub.2O (19:1, 0.05% TFA) (50 .mu.L)
before analysis by QTOF LC/MSMS (FIG. 15b).
Identification of Compound (from Libraries Described in Examples
6a-b)
[0573] Libraries containing heterocyclic scaffolds attached to
peptide sequences (see Example 6a-b) are very well applicable to
single-bead MSMS analysis (material cleaved from single library
beads). This compound class generally displays a high propensity to
afford unique detectable fragments corresponding to the
heterocyclic scaffold core structures. Recognizing this peak in
MSMS analysis of the anticipated molecular ion, and relying on the
general tendency of peptides to fragment at amide bonds, a
predictable fragmentation pattern is emerging (See FIG. 16), since
each randomized position of amino acids is given by their unique
masses (with the pairs of leucine/isoleucine and glutamine/lysine
as the only exceptions).
Example 16
Multiple GPCR Receptors
.beta.2-Adrenergic Receptor (.beta.2AR)-GFP (for Internalization
Studies):
[0574] Hek293 cells are transfected with .beta.2-adrenergic
receptor (.beta.2AR)-GFP using standard procedure for Fugene6
transfection. Cells are put under zeocin selection for 4 weeks to
obtain a cell line stably expressing 12-adrenergic receptor
(.beta.2AR)-GFP.
Bead/Cell Preparation
[0575] Hek293 cells stably expressing .beta.2AR-GFP are seeded in a
Nunc 8 well chambered coverglass in Hams F12 w. 10% FCS and
incubated at 37 degrees, 5% CO.sub.2 for 20 hrs.
[0576] Cells are stimulated with isoproterenol 100 uM (positive
control) and medium (negative control) for 30 min. Cells are imaged
on a Zeiss Axiovert 200M fluorescence microscope equipped with
optimal filters for GFP.
[0577] For negative control the .beta.2-adrenergic receptor
(.beta.2AR)-GFP is localized in the membrane whereas for positive
control .beta.2-adrenergic receptor (.beta.2AR)-GFP is localized in
intracellular spots as an indication of receptor activation and
consequently internalization.
[0578] Compounds modulating the .beta.2AR can be identified in a
similar manner by growing above-mentioned cells on resin beads
comprising library compounds, such as the resin beads used in
example 14a.
Abbreviations:
HGF: Hepatocyte Growth Factor
NGF: Nerve Growth Factor
PDGF: Platelet Derived Growth Factor
FGF: Fibroblast Growth Factor
[0579] EGF: epidermal Growth Factor GH: Growth hormone
TRE: TPA Response Element
[0580] SRE: serum response element CRE: cAMP response element AcN:
acetonitril; Boc: tert-butoxycarbonyl; Bu.sup.t: tert-butyl; DCM:
dichlormethane; DMF: dimethylformamide; Fmoc:
9-fluorenylmethoxycarbonyl; HMBA: 4-hydroxymethylbenzoic acid;
Q-TOF MS: quadrupole time-of-flight mass spectrometry; MeIm:
N-methyl imidazole; MSNT:
1-(mesitylene-2-sulphonyl)-3-nitro-1H-1,2,4-triazole; NEM: 4-ethyl
morpholine; PEGA: polyethylene glycol-polydimethyl acrylamide
resin; Pfp: pentafluorophenyl; Pmc:
2,2,5,7,8-pentamethylchroman-6-sulfonyl; RP-HPLC: reversed phase
high pressure liquid chromatography; SPPS: solid phase peptide
synthesis; TBTU: O-(benzotriazol-1-yl-N,N,N',N'-tetramethyluronium
tetrafluoroborate; TFA: trifluoro acetic acid; Thi: thienyl Fur:
furanyl BzThi: benzothienyl
Sequence CWU 1
1
7017PRTArtificialCell adhesion peptide 1Ala Arg Ile Arg Ile Gln
His1 527PRTArtificialCell adhesion peptide 2Ala Lys Cys Arg Trp Cys
Met1 537PRTArtificialCell adhesion peptide 3Ala Lys Ala Arg Cys Lys
Ser1 547PRTArtificialCell adhesion peptide 4Ala Lys Tyr Trp Ser Tyr
Lys1 557PRTArtificialCell adhesion peptide 5Ala His Leu Tyr Arg Asn
Lys1 567PRTArtificialCell adhesion peptide 6Ala Arg Arg Cys Phe Arg
Asp1 577PRTArtificialCell adhesion peptide 7Ala Ala Arg His Cys Tyr
Tyr1 587PRTArtificialCell adhesion peptide 8Ala Tyr Tyr Cys Gln Gln
Arg1 597PRTArtificialCell adhesion peptide 9Ala Asp Leu Lys Arg Pro
Met1 5107PRTArtificialCell adhesion peptide 10Ala Gly Gly Lys Arg
Lys Phe1 5117PRTArtificialCell adhesion protein 11Ala Pro Arg Lys
Arg Cys Gly1 5127PRTArtificialCell adhesion peptide 12Ala Thr Arg
Arg Val Ala Arg1 5137PRTArtificialCell adhesion peptide 13Ala Gly
Lys Lys Asn Lys Asn1 5147PRTArtificialCell adhesion peptide 14Ala
Ala Lys Arg Trp Lys Phe1 5157PRTArtificialCell adhesion peptide
15Ala Arg Trp Pro Tyr Arg Gly1 5167PRTArtificialCell adhesion
peptide 16Ala Leu Tyr Trp Thr Trp Arg1 5177PRTArtificialCell
adhesion peptide 17Ala Ala Tyr Arg Trp Tyr Arg1
5187PRTArtificialCell adhesion peptide 18Ala Arg Cys Ile Arg Gly
Asp1 5197PRTArtificialCell adhesion peptide 19Ala Thr Lys Cys Lys
Gly Arg1 5207PRTArtificialCell adhesion peptide 20Ala Val Tyr Met
Arg Asn Ile1 5217PRTArtificialCell adhesion peptide 21Ala Arg Lys
Arg Ile Arg Gln1 5227PRTArtificialCell adhesion peptide 22Ala Lys
Ile Arg Glu Lys Arg1 5237PRTArtificialCell adhesion peptide 23Ala
Arg Arg Phe Lys Met Tyr1 5244PRTArtificialCell adhesion peptide
24Arg Arg Phe Lys1254PRTArtificialCell adhesion peptide 25Arg Arg
Ile Arg1266PRTArtificialCell adhesion peptide 26Leu Arg His Arg Leu
Lys1 5275PRTArtificialCell adhesion peptide 27Lys Phe Gly Gln Lys1
5286PRTArtificialCell adhesion peptide 28Lys Val Tyr Met His Lys1
5296PRTArtificialCell adhesion peptide 29Ile Arg Tyr Arg Leu Arg1
5306PRTArtificialCell adhesion peptide 30Ala Gln Arg Pro Arg Trp1
5316PRTArtificialCell adhesion peptide 31Trp Tyr Ala Lys Arg Arg1
5328PRTArtificialCell adhesion peptide 32Lys Arg Ile Arg Gln Arg
Leu Arg1 5337PRTArtificialCell adhesion peptide 33Lys Arg Ile Arg
Gln Arg Leu1 5345PRTArtificialCell adhesion peptide 34Arg Ile Arg
Gln Arg1 5355PRTArtificialCell adhesion peptide 35Arg Gln Arg Ile
Arg1 5366PRTArtificialCell adhesion peptide 36Lys Phe Gly Gln Lys
Cys1 5376PRTArtificialCell adhesion peptide 37Arg Arg Leu Leu Pro
Ile1 5386PRTArtificialCell adhesion peptide 38Pro Phe Arg Lys Lys
Cys1 5396PRTArtificialCell adhesion peptide 39Tyr Arg Trp Arg Ile
Ala1 5406PRTArtificialCell adhesion peptide 40Arg Ser Lys Arg Ile
Asn1 5416PRTArtificialCell adhesion peptide 41Arg Ser Ala Lys Arg
Cys1 5426PRTArtificialCell adhesion peptide 42Lys Lys Gln Phe Trp
Phe1 5436PRTArtificialCell adhesion peptide 43Arg Met Lys Leu His
Lys1 5446PRTArtificialCell adhesion peptide 44Arg His Trp Gly Arg
Ile1 5456PRTArtificialCell adhesion peptide 45Thr Lys Arg Leu Lys
Thr1 5466PRTArtificialCell adhesion peptide 46Thr Lys Gly Lys Ala
Lys1 5476PRTArtificialCell adhesion peptide 47Ala Lys Thr Arg His
Arg1 5486PRTArtificialCell adhesion peptide 48Asn Arg Pro Arg Val
Arg1 5496PRTArtificialCell adhesion peptide 49Val Pro Arg Lys Val
Gln1 5506PRTArtificialCell adhesion peptide 50Lys Met Arg Tyr Cys
Gln1 5516PRTArtificialCell adhesion peptide 51Ile Arg Lys His Leu
Ile1 5526PRTArtificialCell adhesion peptide 52Pro Arg Arg Val Val
Ile1 5536PRTArtificialCell adhesion peptide 53Lys Arg Glu Ser Lys
Arg1 5546PRTArtificialCell adhesion peptide 54Ser Arg Lys Asp Arg
Lys1 5556PRTArtificialCell adhesion peptide 55Arg Cys Lys Lys Leu
Ile1 5566PRTArtificialCell adhesion peptide 56Arg Lys Leu Arg Val
Asn1 5576PRTArtificialCell adhesion peptide 57Val Arg Thr Val Arg
Val1 5586PRTArtificialCell adhesion peptide 58Arg Ala Phe Lys Tyr
Tyr1 5596PRTArtificialCell adhesion peptide 59Ile Thr Arg Arg Thr
Gln1 5606PRTArtificialCell adhesion peptide 60Lys Met Pro Lys Lys
Asn1 5616PRTArtificialCell adhesion peptide 61Lys Pro Leu Met Met
Cys1 5626PRTArtificialCell adhesion peptide 62Lys Lys Met Arg Phe
Trp1 5636PRTArtificialCell adhesion peptide 63Lys Lys Lys Phe Tyr
Tyr1 5646PRTArtificialCell adhesion peptide 64Lys Ser Asn Lys Val
Arg1 5656PRTArtificialCell adhesion peptide 65Lys Trp Pro His His
Arg1 5666PRTArtificialCell adhesion peptide 66Arg His Ile Gln Trp
Tyr1 5676PRTArtificialCell adhesion peptide 67Leu Arg Leu Lys Pro
Lys1 5686PRTArtificialCell adhesion peptide 68Glu Arg Lys Arg Cys
Thr1 5696PRTArtificialCell adhesion peptide 69Arg Arg Ala Arg Gln
Asp1 5706PRTArtificialCell adhesion peptide 70Arg Glu Lys Gly Ala
Arg1 5
* * * * *
References