U.S. patent application number 16/104733 was filed with the patent office on 2018-12-20 for method of synthesis and testing of cominatorial libraries using microcapsules.
The applicant listed for this patent is Medical Research Council. Invention is credited to Chris Abell, Andrew David Griffiths, Florian Hollfelder, Enrico Mastrobattista.
Application Number | 20180361346 16/104733 |
Document ID | / |
Family ID | 9955903 |
Filed Date | 2018-12-20 |
United States Patent
Application |
20180361346 |
Kind Code |
A1 |
Griffiths; Andrew David ; et
al. |
December 20, 2018 |
METHOD OF SYNTHESIS AND TESTING OF COMINATORIAL LIBRARIES USING
MICROCAPSULES
Abstract
Methods for use in the synthesis and identification of molecules
which bind to a target component of a biological system or modulate
the activity of a target are described.
Inventors: |
Griffiths; Andrew David;
(Strasbourg, FR) ; Abell; Chris; (Cambridge,
GB) ; Hollfelder; Florian; (Cambridge, GB) ;
Mastrobattista; Enrico; (Cambridge, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Medical Research Council |
Swindon |
|
GB |
|
|
Family ID: |
9955903 |
Appl. No.: |
16/104733 |
Filed: |
August 17, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12770006 |
Apr 29, 2010 |
10052605 |
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16104733 |
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11238257 |
Sep 29, 2005 |
7718578 |
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12770006 |
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PCT/GB2004/001352 |
Mar 31, 2004 |
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11238257 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01J 19/0093 20130101;
C40B 20/04 20130101; B01J 2219/00725 20130101; B01J 2219/005
20130101; B01J 2219/00722 20130101; B82Y 5/00 20130101; B01J
2219/00596 20130101; B82Y 30/00 20130101; B01J 2219/00894 20130101;
B01J 2219/00497 20130101; B01J 2219/00743 20130101; B01J 2219/00576
20130101; B01J 2219/00835 20130101; B01J 2219/00585 20130101; C12N
15/1075 20130101; C40B 30/04 20130101; C40B 50/08 20130101; B01J
2219/0086 20130101; G01N 33/5432 20130101; C40B 40/06 20130101;
B01J 19/0046 20130101; B01J 2219/00707 20130101; B01J 2219/00599
20130101; B01J 2219/00903 20130101; C40B 40/10 20130101; C07K 1/047
20130101; B82Y 10/00 20130101 |
International
Class: |
B01J 19/00 20060101
B01J019/00; B82Y 30/00 20110101 B82Y030/00; C07K 1/04 20060101
C07K001/04; C12N 15/10 20060101 C12N015/10; C40B 20/04 20060101
C40B020/04; C40B 30/04 20060101 C40B030/04; C40B 50/08 20060101
C40B050/08; G01N 33/543 20060101 G01N033/543 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 31, 2003 |
GB |
0307428.3 |
Claims
1. A method of forming a small molecule compound comprising:
providing at least a first aqueous fluid comprising at least a
first small molecule compound having a molecular weight of less
than 500 Daltons; providing at least a second aqueous fluid
comprising at least a second small molecule compound having a
molecular weight of less than 500 Daltons; encapsulating said at
least first small molecule compound into at least a first aqueous
microcapsule within an immiscible fluorocarbon oil; encapsulating
said at least second small molecule compound into at least a second
aqueous microcapsule within an immiscible fluorocarbon oil; and
fusing said at least first and said at least second aqueous
microcapsules, thereby causing a chemical reaction between said at
least first and at least second small molecule compounds to form at
least a third small molecule compound.
2. The method of claim 1, wherein said at least first small
molecule compound is attached to a microbead.
3. The method of claim 1, wherein said at least second small
molecule compound is attached to a microbead.
4. The method of claim 1, further comprising isolating said at
least third small molecule compound.
5. The method of claim 1, further comprising identifying said at
least third small molecule compound.
6. The method of claim 5, wherein said at least third small
molecule compound is identified by its optical properties.
7. The method of claim 1, further comprising identifying said at
least first and at least second small molecule compounds which
reacted to form said at least third small molecule compound.
8. The method of claim 1, wherein said at least first small
molecule compound comprises at least a first label and said at
least second small molecule compound comprises at least a second
label, wherein said at least first and said at least second labels
are different.
9. The method of claim 1, wherein said at least first aqueous
microcapsule comprises no more than one said at least first small
molecule compound.
10. The method of claim 1, wherein said at least first aqueous
microcapsule comprises a plurality of said at least first small
molecule compounds.
11. The method of claim 1, wherein said at least second aqueous
microcapsule comprises no more than one said at least second small
molecule compound.
12. The method of claim 1, wherein said at least second aqueous
microcapsule comprises a plurality of said at least second small
molecule compounds.
Description
[0001] This application is a continuation of U.S. Ser. No.
11/238,257, filed Sep. 29, 2005, allowed, which is a continuation
of PCT/GB2004/001352, filed Mar. 31, 2004, which claims priority to
United Kingdom application GB0307428.3, filed Mar. 31, 2003, the
entire contents of each of which are incorporated herein by
reference.
[0002] The present invention relates to methods for use in the
synthesis and identification of molecules which bind to a target
component of a biochemical system or modulate the activity of a
target.
[0003] Over the past decade, high-throughput screening (HTS) of
compound libraries has become a cornerstone technology of
pharmaceutical research. Investment into HTS is substantial. A
current estimate is that biological screening and preclinical
pharmacological testing alone account for .about.14% of the total
research and development (R&D) expenditures of the
pharmaceutical industry (Handen, Summer 2002). HTS has seen
significant improvements in recent years, driven by a need to
reduce operating costs and increase the number of compounds and
targets that can be screened. Conventional 96-well plates have now
largely been replaced by 384-well, 1536-well and even 3456-well
formats. This, combined with commercially available plate-handling
robotics allows the screening of 100,000 assays per day, or more,
and significantly cuts costs per assay due to the miniaturisation
of the assays.
[0004] HTS is complemented by several other developments.
Combinatorial chemistry is a potent technology for creating large
numbers of structurally related compounds for HTS. Currently,
combinatorial synthesis mostly involves spatially resolved parallel
synthesis. The number of compounds that can be synthesised is
limited to hundreds or thousands but the compounds can be
synthesised on a scale of milligrams or tens of milligrams,
enabling full characterisation and even purification. Larger
libraries can be synthesised using split synthesis on beads to
generate one-bead-one compound libraries. This method is much less
widely adopted due to a series of limitations including: the need
for solid phase synthesis; difficulties characterising the final
products (due to the shear numbers and small scale); the small
amounts of compound on a bead being only sufficient for one or a
few assays; the difficulty in identifying the structure of a hit
compound, which often relies on tagging or encoding methods and
complicates both synthesis and analysis. Despite this split
synthesis and single bead analysis still has promise. Recently
there have been significant developments in miniaturised screening
and single bead analysis. For example, printing techniques allow
protein-binding assays to be performed on a slide containing 10,800
compound spots, each of 1 nl volume (Hergenrother et al., 2000).
Combichem has so far, however, generated only a limited number of
lead compounds. As of April 2000, only 10 compounds with a
combinatorial chemistry history had entered clinical development
and all but three of these are (oligo)nucleotides or peptides
(Adang and Hermkens, 2001). Indeed, despite enormous investments in
both HTS and combinatorial chemistry during the past decade the
number of new drugs introduced per year has remained constant at
best.
[0005] Dynamic combinatorial chemistry (DCC) can also be used to
create dynamic combinatorial libraries (DCLs) from a set of
reversibly interchanging components, however the sizes of libraries
created and screened to date are still fairly limited
(.ltoreq.40,000) (Ramstrom and Lehn, 2002).
[0006] Virtual screening (VS) (Lyne, 2002), in which large compound
bases are searched using computational approaches to identify a
subset of candidate molecules for testing may also be very useful
when integrated with HTS. However, there are to date few studies
that directly compare the performance of VS and HTS, and further
validation is required.
[0007] Despite all these developments, current screening throughput
is still far from adequate. Recent estimates of the number of
individual genes in the human genome (.about.30,000) and the number
of unique chemical structures theoretically attainable using
existing chemistries suggests that an enormous number of assays
would be required to completely map the structure-activity space
for all potential therapeutic targets (Burbaum, 1998).
[0008] Hence, a method with the capability to both create and
screen vast numbers (.gtoreq.10.sup.10) of compounds quickly, using
reaction volumes of only a few femtolitres, and at very low cost
should be of enormous utility in the generation of novel drug
leads.
SUMMARY OF THE INVENTION
[0009] The invention, in a first aspect, provides a method for
preparing a repertoire of compounds comprising the steps of:
(a) compartmentalising two or more sets of primary compounds into
microcapsules; such that a proportion of the microcapsules contains
multiple copies of one or more compounds representative of each of
said sets, and wherein said one or more compounds represents a
subset of the set of primary compounds; and (b) forming secondary
compounds in the microcapsules by chemical reactions between
primary compounds from different sets.
[0010] A compound is "representative" of a set where is is member
of said set; advantageously, therefore, each microcapsule contains
compound(s) from each set. Although a microcapsule may contain more
than one different compound from each set, it contains only a
proportion of said set--i.e. a subset. The subset of a set
advantageously represents no more that 10% of the members of the
set; preferably, this figure is 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%
or less.
[0011] Most advantageously a microcapsule contains only a single
compound from each set of primary compounds.
[0012] The sets of primary compounds used in the method of the
invention can consist of any number of different compounds. At
least a first set comprises two or more compounds; but the other
set may be a single compound. Preferably, if a first set is a
single compound, at least one further set comprises a repertoire of
compounds. The larger this repertoire, the greater the number of
different secondary compounds that will be generated.
[0013] Preferably, at least one set of compounds comprises a
repertoire of different compounds. At least one set, however, may
consist of a single compound, such that secondary compounds are all
constructed based on or containing the single compound used in one
set. The greater the number of sets, and the greater the diversity
of each set, the greater the final diversity of the secondary
compounds generated.
[0014] Advantageously, in step (a) the number of different
compounds per compartment will be equivalent to the the number of
primary compounds forming the secondary compound in step (b).
[0015] In a second aspect, the invention provides a method for
identifying primary compounds which react together to form
secondary compounds capable of binding to or modulating the
activity of a target, comprising the steps of:
(a) compartmentalising two or more sets of primary compounds into
microcapsules; such that a proportion of the microcapsules contains
two or more compounds; (b) forming secondary compounds in the
microcapsules by chemical reactions between primary compounds from
different sets; and (c) identifying subsets of primary compounds
which react to form secondary compounds which bind to or modulate
the activity of the target.
[0016] In a third aspect, the invention provides a method for
synthesising compounds with enhanced ability to bind to or modulate
the activity of the target, comprising the steps of:
(a) compartmentalising into microcapsules subsets of primary
compounds identified in step (c) of the second aspect of the
invention and, optionally, compartmentalising additional sets of
primary compounds; (b) forming secondary compounds in the
microcapsules by chemical reactions between primary compounds from
different sets; and (c) identifying subsets of primary compounds
which react to form secondary compounds which bind to or modulate
the activity of the target.
[0017] Advantageously, steps (a) to (c) can be repeated, but after
the first cycle, step (a) comprises compartmentalising subsets of
primary compounds identified in step (c) into microcapsules and,
optionally, compartmentalising additional sets of compounds.
[0018] In a fourth aspect, the invention provides a method for
identifying individual compounds which bind to or modulate the
activity of the target, comprising the steps of:
(a) compartmentalising into microcapsules a primary compound
identified in step (c) of the second or third aspect of the
invention and additional sets of primary compounds; (b) forming
secondary compounds in the microcapsules by chemical reactions
between primary compounds from different sets; and (c) identifying
subsets of primary compounds which react to form secondary
compounds which bind to or modulate the activity of the target.
[0019] If the secondary compound is formed by the chemical reaction
between more than two primary compounds it can be identified by
iteratively repeating steps (a) to (c), but after the first cycle,
step (c) comprises compartmentalising the primary compound
identified in step (c) of the second or third aspect of the
invention, a primary compound identified in step (c) of each of the
previous cycles (of the fourth aspect of the invention) and
additional sets of primary molecules.
[0020] Preferably, the desired activity is selected from the group
consisting of a binding activity and the modulation of the activity
of a target. Advantageously, the target is compartmentalised into
microcapsules together with the compounds.
[0021] Sets of compounds may be compartmentalised in different ways
to achieve encapsulation of multiple copies of two or more
compounds into microcapsules.
[0022] For example, small aliquots of an aqueous solution of each
compound can be deposited into an oil phase (advantageously
containing surfactants and/or other stabilising molecules) whilst
applying mechanical energy, thereby dispersing each compound into
multiple aqueous microcapsules, each of which contains (for the
most part) a single sort of compound but multiple copies thereof.
Advantageously, the compounds can be deposited into the oil phase
in the form of droplets generated using inkjet printing technology
(Calvert, 2001; de Gans et al., 2004), and more advantageously by
piezoelectric drop-on-demand (DOD) inkjet printing technology.
Inkjet printing technology can be used to mix primary compounds
and, optionally, other reagents (e.g the target and reagents to
assay target activity) immediately prior to forming the emulsion.
Advantageously, multiple compounds can be mixed with multiple
targets in a combinatorial manner.
[0023] Thus, step (a) above can be modified such that it comprises
forming separate emulsion compartments containing two or more
compounds and mixing the emulsion compartments to form an
emulsified compound repertoire wherein a subset of the repertoire
is represented in multiple copies in any one microcapsule.
[0024] Moreover, compound libraries can be be compartmentalised in
highly monodisperse microcapsules produced using microfluidic
techniques. For example, aliquots of each compound can be
compartmentalised into one or more aqueous microcapsules (with less
than 3% polydispersity) in water-in-oil emulsions created by
droplet break off in a co-flowing steam of oil (Umbenhowar et al.,
2000). Advantageously, the aqueous microcapsules are then
transported by laminar-flow in a stream of oil in microfluidic
channels (Thorsen et al., 2001). The microcapsules containing
single compounds can, optionally, be split into two or more smaller
microcapsules using microfluidics (Link et al., 2004; Song et al.,
2003). Microcapsules containing primary compounds can be fused with
other microcapsules (Song et al., 2003) to form secondary
compounds. Microcapsules containing compounds can also, optionally,
be fused with microcapsules containing a target. A single
microcapsule containing a target can, optionally, be split into two
or more smaller microcapsules which can subsequently be fused with
microcapsules containing different compounds, or compounds at
different concentrations. Advantageously, a compound and a target
can be mixed by microcapsule fusion prior to a second microcapsule
fusion which delivers the reagents necessary to assay the activity
of the target (e.g. the substrate for the target if the target is
an enzyme). This allows time for the compound to bind to the
target. The microcapsules can be analysed and, optionally, sorted
using microfluidic devices (Fu et al., 2002).
[0025] In a further aspect, there is provided a method for
preparing a repertoire of compounds comprising the steps of:
(a) attaching two or more sets of primary compounds onto
microbeads; (b) compartmentalising the microbeads into
microcapsules such that a proportion of the microcapsules contains
two or more microbeads; (c) releasing at least one of the sets of
primary compounds from the microbeads; (d) forming secondary
compounds in the microcapsules by chemical reactions between
primary compounds from different sets.
[0026] Advantageously, the compounds are cleavable from the beads.
Where more than two sets of compounds are used, all the sets with
the exception of one are cleavable; preferably, they are all
cleavable. The compounds may be attached to the microbeads by
photochemically cleavable linkers.
[0027] In a still further aspect, the invention provides a method
for identifying primary compounds which react together to form
secondary compounds capable of binding to or modulating the
activity of a target, comprising the steps of:
(a) attaching two or more sets of primary compounds onto
microbeads; (b) compartmentalising the microbeads into
microcapsules together with the target such that many compartments
contain two or more microbeads; (c) releasing the primary compounds
from the microbeads; (d) forming secondary compounds in the
microcapsules by chemical reactions between primary compounds from
different sets; and (e) identifying subsets of primary compounds
which react to form secondary compounds which bind to or modulate
the activity of the target.
[0028] Advantageously, in step (b) the modal number of microbeads
per compartment will be equivalent to the number of primary
compounds forming the secondary compound in step (d).
[0029] In a further aspect, the invention provides a method for
synthesising compounds with enhanced ability to bind to or modulate
the activity of the target, comprising the steps of:
(a) attaching onto microbeads subsets of primary compounds
identified in step (e) of the second aspect of the invention and,
optionally, attaching additional sets of primary compounds; (b)
compartmentalising the microbeads into microcapsules together with
the target such that many compartments contain two or more
microbeads; (c) releasing the primary compounds from the
microbeads; (d) forming secondary compounds in the microcapsules by
chemical reactions between primary compounds from different sets;
and (e) identifying subsets of primary compounds which react to
form secondary compounds which bind to or modulate the activity of
the target.
[0030] Advantageously, steps (a) to (e) can be repeated, but after
the first cycle, step (a) comprises attaching onto microbeads
subsets of primary compounds identified in step (e) and,
optionally, attaching additional sets of compounds.
[0031] In a further aspect, the invention provides a method for
identifying individual compounds which bind to or modulate the
activity of the target, comprising the steps of:
(a) attaching onto microbeads a primary compound identified in step
(e) of the second or third aspect of the invention and additional
sets of primary compounds; (b) compartmentalising the microbeads
into microcapsules together with the target such that many
compartments contain two or more microbeads; (c) releasing the
primary compounds from the microbeads; (d) forming secondary
compounds in the microcapsules by chemical reactions between
primary compounds from different sets; and (e) identifying subsets
of primary compounds which react to form secondary compounds which
bind to or modulate the activity of the target;
[0032] If the secondary compound is formed by the chemical reaction
between more than two primary compounds it can be identified by
iteratively repeating steps (a) to (e), but after the first cycle,
step (a) comprises attaching onto microbeads the primary compound
identified in step (e) of the second or third aspect of the
invention, a primary compound identified in step (e) of each of the
previous cycles (of the fourth aspect of the invention) and
additional sets of primary molecules.
[0033] Preferably, the desired activity is selected from the group
consisting of a binding activity and the modulation of the activity
of a target. Advantageously, the target is compartmentalised into
microcapsules together with the microbeads.
[0034] According to a preferred implementation of the present
invention, the compounds may be screened according to an activity
of the compound or derivative thereof which makes the microcapsule
detectable as a whole. Accordingly, the invention provides a method
wherein a compound with the desired activity induces a change in
the microcapsule, or a modification of one or more molecules within
the microcapsule, which enables the microcapsule containing the
compound and, optionally, the microbead carrying it to be
identified. In this embodiment, therefore, the microcapsules are
either: (a) physically sorted from each other according to the
activity of the compound(s) contained therein, and the contents of
the sorted microcapsules analysed to determine the identity of the
compound(s) which they contain; or (b) analysed directly without
sorting to determine the identity of the compound(s) which the
microcapsules contain.
[0035] According to a preferred embodiment of the present
invention, the screening of compounds may be performed by, for
example:
(I) In a first embodiment, the microcapsules are screened according
to an activity of the compound or derivative thereof which makes
the microcapsule detectable as a whole. Accordingly, the invention
provides a method wherein a compound with the desired activity
induces a change in the microcapsule, or a modification of one or
more molecules within the microcapsule, which enables the
microcapsule containing the compound and the microbead carrying it
to be identified. In this embodiment, therefore, the microcapsules
are either: (a) physically sorted from each other according to the
activity of the compound(s) contained therein, the contents of the
sorted microcapsules optionally pooled into one or more common
compartments, and the microcapsule contents analysed to determine
the identity of the compound(s); or (b) analysed directly without
sorting to determine the identity of the compound(s) which the
microcapsules contained. Where the microcapsule contains
microbeads, the microbeads can be analysed to determine the
compounds with which they are coated. (II) In a second embodiment,
microbeads are analysed following pooling of the microcapsules into
one or more common compartments. In this embodiment, a compound
having the desired activity modifies the microbead which carried it
(and which resides in the same microcapsule) in such a way as to
make it identifiable in a subsequent step. The reactions are
stopped and the microcapsules are then broken so that all the
contents of the individual microcapsules are pooled. Modified
microbeads are identified and either: (a) physically sorted from
each other according to the activity of the compound(s) coated on
the microbeads, and the sorted microbeads analysed to determine the
identity of the compound(s) with which they are/were coated; or (b)
analysed directly without sorting to determine the identity of the
compound(s) with which the microbeads are/were coated. It is to be
understood, of course, that modification of the microbead may be
direct, in that it is caused by the direct action of the compound,
or indirect, in which a series of reactions, one or more of which
involve the compound having the desired activity, leads to
modification of the microbead. Advantageously, the target is bound
to the microbead and is a ligand and the compound within the
microcapsule binds, directly or indirectly, to said ligand to
enable the isolation of the microbead. In another configuration, a
substrate for the target is and is bound to the microbead, and the
activity of the compound within the microcapsule results, directly
or indirectly, in the conversion of said substrate into a product
which remains part of the microbead and enables its isolation.
Alternatively, the activity of the compound may prevent or inhibit
the conversion of said substrate into product. Moreover, the
product of the activity of the compound within the microcapsule can
result, directly or indirectly, in the generation of a product
which is subsequently complexed with the microbead and enables its
identification. (III) In a third embodiment, the microbeads are
analysed following pooling of the microcapsules into one or more
common compartments. In this embodiment, a compound with a desired
activity induces a change in the microcapsule containing the
compound and the microbead which carries it. This change, when
detected, triggers the modification of the microbead within the
compartment. The reactions are stopped and the microcapsules are
then broken so that all the contents of the individual
microcapsules are pooled. Modified microbeads are identified and
either: (a) physically sorted from each other according to the
activity of the compound(s) coated on the microbeads, and the
sorted microbeads analysed to determine the identity of the
compound(s) with which they are/were coated; or (b) analysed
directly without sorting to determine the identity of the
compound(s) with which the microbeads are/were coated.
[0036] The microcapsules or microbeads may be modified by the
action of the compound(s) such as to change their optical
properties. For example, the modification of the microbead can
enable it to be further modified outside the microcapsule so as to
induce a change in its optical properties.
[0037] In another embodiment, the change in optical properties of
the microcapsules or microbeads is due to binding of a compound
with distinctive optical properties to the target.
[0038] Moreover, the change in optical properties of the
microcapsules or microbeads can be due to binding of a target with
distinctive optical properties by the compound.
[0039] The change in the optical properties of the microcapsule may
be due to modulation of the activity of the target by the compound.
The compound may activate or inhibit the activity of the target.
For example, if the target is an enzyme, the substrate and the
product of the reaction catalysed by the target can have different
optical properties. Advantageously, the substrate and product have
different fluorescence properties. In the case where the
microcapsules contain microbeads, both the substrate and the
product can have similar optical properties, but only the product
of the reaction, and not the substrate, binds to, or reacts with,
the microbead, thereby changing the optical properties of the
microbead.
[0040] The change in optical properties of the microcapsules or
microbeads can also be due to the different optical properties of
the target and the product of the reaction being selected. Where
both target and product have similar optical properties, only the
product of the reaction being selected, and not the target, binds
to, or reacts with, the microbead, thereby changing the optical
properties of the microcapsules or microbeads.
[0041] In a further configuration, further reagents specifically
bind to, or specifically react with, the product (and not the
substrate) attached to or contained in the microcapsule or
microbead, thereby altering the optical properties of the
microcapsule or microbead.
[0042] Advantageously, microcapsules or microbeads are modified
directly or indirectly by the activity of the compound are further
modified by Tyramide Signal Amplification (TSA.TM.; NEN), resulting
directly or indirectly in a change in the optical properties of
said microcapsules or microbeads thereby enabling their
separation.
[0043] It is to be understood that the detected change in the
compartment may be caused by the direct action of the compound, or
indirect action, in which a series of reactions, one or more of
which involve the compound having the desired activity leads to the
detected change.
[0044] Where the compounds are attached to beads, the density with
which compounds are coated onto the microbeads, combined with the
size of the microcapsule will determine the concentration of the
compound in the microcapsule. High compound coating densities and
small microcapsules will both give higher compound concentrations
which may be advantageous for the selection of molecules with a low
affinity for the target. Conversely, low compound coating densities
and large microcapsules will both give lower compound
concentrations which may be advantageous for the selection of
molecules with a high affinity for the target.
[0045] Preferably, microencapsulation is achieved by forming an
emulsion.
[0046] The microbead can be nonmagnetic, magnetic or
paramagnetic.
[0047] Advantageously, the microcapsules or microbeads are analysed
by detection of a change in their fluorescence. For example,
microbeads can be analysed by flow cytometry and, optionally sorted
using a fluorescence activated cell sorter (FACS). The different
fluorescence properties of the target and the product can be due to
fluorescence resonance energy transfer (FRET).
[0048] In a further embodiment, the internal environment of the
microcapsules can be modified by the addition of one or more
reagents to the continuous phase of the emulsion. This allows
reagents to be diffused in to the microcapsules during the
reaction, if necessary.
[0049] The invention moreover relates to a method according to the
preceding aspects, further comprising the step of isolating the
secondary compound produced by reaction of the primary compounds
and optionally further comprising the step of manufacturing one or
more secondary compounds.
[0050] The invention also provides for a product when identified
according to the invention. As used in this context, a "product"
may refer to any compound, selectable according to the
invention.
[0051] Further embodiments of the invention are described in the
detailed description below and in the accompanying claims.
BRIEF DESCRIPTION OF THE FIGURES
[0052] FIG. 1. Examples of PTP1B inhibitors. Compounds with a
bis-difluoromethylene phosphonate moiety (e.g. 2) have
significantly more potency than those with a single moiety (e.g.
1).
[0053] FIG. 2. Screening PTP1B inhibitors using IVC. Polystyrene
beads with surface carboxylate groups, died with orange or red
fluorochromes (Fulton et al., 1997), are derivatised with a
phosphopeptide PTP1B substrate, and either PTP1B inhibitors or
non-inhibitory compounds, attached via a cleavable linker (1).
After mixing the beads, single beads and target enzyme (PTP1B) are
colocalised in a microcompartment by forming a water-in-oil
emulsion (2). The compound is released (photochemically) (3).
Inhibitors reduce the amount of substrate converted to product
(dephosphorylated peptide) (4). The enzyme reaction is stopped and
the emulsion is broken (5). After labelling with green fluorescent
anti-substrate antibodies, beads are analysed by 3-colour flow
cytometry to simultaneously determine extent of inhibition and the
compound on the beads (6). Compound libraries can be coupled to
optically tagged beads (see below) and rapidly decoded by flow
cytometry (at up to 100,000 beads s.sup.-1). Hit compounds are
re-synthesised for further characterisation (7) or elaborated and
rescreened in a process of synthetic evolution (8).
[0054] FIG. 3. Synthesizing PTP1B inhibitors in an emulsion. Two
types of beads are created, differentially labelled with orange and
red fluorochromes, and derivatised with two types of molecule, A or
B (neither, one, or both of which contain a difluoromethylene
phosphonate moiety), attached via a reversible connection (a Schiff
base). Beads are emulsified to give, on average, two beads per
compartment. The molecules, A & B, are released from the beads
in the compartment and react to form a new molecule, A-B, (in
solution). If A-B is a PTP1B inhibitor the PTP1B substrate also on
the beads is not dephosphorylated and these beads identified by
flow cytometry as FIG. 2.
[0055] FIG. 4. Small molecule evolution using four-component
reactions. Four sets of 25 beads are created, each derivatised with
one of 25 variants of molecules A, B, C or D, emulsified to give,
on average, 4 beads per compartment, the compounds released to
synthesise a large combinatorial repertoire (4.times.10.sup.5) in
situ and screened as FIG. 2. Low affinity inhibitors will be
`recombined` by re-screening mixtures of beads carrying moieties
identified in inhibitors. Beads carrying a moiety found in
inhibitors (e.g. A.sub.10) can also be mixed with complete sets of
beads coated with B, C and D and screened. If a moiety (say
B.sub.8) is then identified as a component of an inhibitor, beads
coated with A.sub.10 and B.sub.8 can be mixed with complete sets of
beads C and D and the process repeated. This process of `mutation`
also results in deconvolution. After fixing three of the four
moieties in active compounds, deconvolution can be completed using
multiplex bead analysis as above. Compounds can be re-diversified
or `mutated` using bead sets carrying variant, exploded sets of the
molecules used in the original libraries.
[0056] FIG. 5. Compartmentalisation of small molecules in
water-in-fluorocarbon emulsions. Water-in-perfluorooctyl bromide
emulsions were made containing texas red (1 mM) and calcein (1 mM)
in the aqueous phase by homogenisation as described in example 6
The two emulsions were mixed by vortexing and imaged by
epifluorescence microscopy after 24 hours. No exchange of texas-red
(red fluorescence) and calcein (green fluorescence) between
microdroplets could be observed.
[0057] FIG. 6. Primary compounds for the synthesis of PTP1B
inhibitors. An amine (A) and an aldehyde (B) with difluoromethylene
phosphonate moieties. Amine A reacts with aldehyde B in the aqueous
microcapsules of a water-in-oil emulsion to generate the imine C
which is a potent PTP1B inhibitor. C can be reduced in situ using
cyanoborohydride to generate the stable amine D.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0058] The term "microcapsule" is used herein in accordance with
the meaning normally assigned thereto in the art and further
described hereinbelow. In essence, however, a microcapsule is an
artificial compartment whose delimiting borders restrict the
exchange of the components of the molecular mechanisms described
herein which allow the identification of the molecule with the
desired activity. The delimiting borders preferably completely
enclose the contents of the microcapsule. Preferably, the
microcapsules used in the method of the present invention will be
capable of being produced in very large numbers, and thereby to
compartmentalise a library of compounds. Optionally, the compounds
can be attached to microbeads. The microcapsules used herein allow
mixing and sorting to be performed thereon, in order to facilitate
the high throughput potential of the methods of the invention.
Arrays of liquid droplets on solid surfaces, and multiwell plates,
are not microcapsules as defined herein.
[0059] A "proportion" of the microcapsules, which is defined as
comprising two or more compounds, or two or microbeads, is any
fraction of the microcapsules in question, including all of said
microcapsules. Advantageously, it is at least 25% thereof,
preferably 50%, and more preferably 60%, 70%, 80%, 90% or 95%.
[0060] The term "microbead" is used herein in accordance with the
meaning normally assigned thereto in the art and further described
hereinbelow. Microbeads, are also known by those skilled in the art
as microspheres, latex particles, beads, or minibeads, are
available in diameters from 20 nm to 1 mm and can be made from a
variety of materials including silica and a variety of polymers,
copolymers and terpolymers. Highly uniform derivatised and
non-derivatised nonmagnetic and paramagnetic microparticles (beads)
are commercially available from many sources (e.g. Sigma, Bangs
Laboratories, Luminex and Molecular Probes) (Fornusek and Vetvicka,
1986).
[0061] Microbeads can be "compartmentalised" in accordance with the
present invention by distribution into microcapsules. For example,
in a preferred aspect the microbeads can be placed in a water/oil
mixture and emulsified to form a water-in-oil emulsion comprising
microcapsules according to the invention. The concentration of the
microbeads can be adjusted to control the number of microbeads,
which on average, appear in each microcapsule.
[0062] The term "compound" is used herein in accordance with the
meaning normally assigned thereto in the art. The term compound is
used in its broadest sense i.e. a substance comprising two or more
elements in fixed proportions, including molecules and
supramolecular complexes. This definition includes small molecules
(typically <500 Daltons) which make up the majority of
pharmaceuticals. However, the definition also includes larger
molecules, including polymers, for example polypeptides, nucleic
acids and carbohydrates, and supramolecular complexes thereof.
[0063] The term "primary compound" is used herein to indicate a
compound which is compartmentalised in a microcapsule or coupled to
a bead.
[0064] The term "secondary compound" is used herein to indicate a
compound which is formed by the reaction between two or more
primary compounds in a microcapsule, optionally after the release
of at least one of the primary molecules from a microbead.
Advantageously, all primary molecules are released from the
microbeads. The secondary compound may be the result of a covalent
or non-covalent reaction between the primary compounds.
[0065] The term "scaffold" is used herein in accordance with the
meaning normally assigned thereto in the art That is to say a core
portion of a molecule common to all members of a combinatorial
library (Maclean et al., 1999). Secondary compounds may optionally
comprise scaffolds.
[0066] A "repertoire" of compounds is a group of diverse compounds,
which may also be referred to as a library of compounds.
Repertoires of compounds may be generated by any means known in the
art, including combinatorial chemistry, compound evolution, or
purchased from commercial sources such as Sigma Aldrich, Discovery
Partners International, Maybridge and Tripos. A repertoire
advantageously comprises at least 10.sup.2, 10.sup.3, 10.sup.4,
10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10,
10.sup.11 or more different compounds, which may be related or
unrelated in structure or function.
[0067] A "set" of compounds may be a repertoire of compounds or any
part of a repertoire, including a single compound species. The
invention envisages the use of two or more sets of compounds, which
are reacted together. The sets may be derived from a single
repertoire, or a plurality of different repertoires.
[0068] Compounds can be "released" from a microbead by cleavage of
a linker which effects the attachment of the compound to the
microbead. Release of the compounds from the microbead allows the
compounds to interact more freely with other contents of the
microcapsule, and to be involved in reactions therein and
optionally to become combined with other reagents to form new
compounds, complexes, molecules or supramolecular complexes.
Cleavage of linkers can be performed by any means, with means such
as photochemical cleavage which can be effected from without the
microcapsule being preferred. Photochemically cleavable linkers are
known in the art (see for example (Gordon and Balasubramanian,
1999)) and further described below.
[0069] As used herein, the "target" is any compound, molecule, or
supramolecular complex. Typical targets include targets of medical
significance, including drug targets such as receptors, for example
G protein coupled receptors and hormone receptors; transcription
factors, protein kinases and phosphatases involved in signalling
pathways; gene products specific to microorganisms, such as
components of cell walls, replicases and other enzymes;
industrially relevant targets, such as enzymes used in the food
industry, reagents intended for research or production purposes,
and the like.
[0070] An "activity", as referred to herein in connection with the
modulation of an activity of a target, can be any activity of the
target or an activity of a molecule which is influenced by the
target, which is modulatable directly or indirectly by a compound
or compounds as assayed herein. The activity of the target may be
any measurable biological or chemical activity, including binding
activity, an enzymatic activity, an activating or inhibitory
activity on a third enzyme or other molecule, the ability to cause
disease or influence metabolism or other functions, and the like.
Activation and inhibition, as referred to herein, denote the
increase or decrease of a desired activity 1.5 fold, 2 fold, 3
fold, 4 fold, 5 fold, 10 fold, 100 fold or more. Where the
modulation is inactivation, the inactivation can be substantially
complete inactivation. The desired activity may moreover be purely
a binding activity, which may or may not involve the modulation of
the activity of the target bound to.
[0071] A compound defined herein as "low molecular weight" or a
"small molecule" is a molecule commonly referred to in the
pharmaceutical arts as a "small molecule". Such compounds are
smaller than polypeptides and other, large molecular complexes and
can be easily administered to and assimilated by patients and other
subjects. Small molecule drugs can advantageously be formulated for
oral administration or intramuscular injection. For example, a
small molecule may have a molecular weight of up to 2000 Dalton;
preferably up to 1000 Dalton; advantageously between 250 and 750
Dalton; and more preferably less than 500 Dalton.
[0072] A "selectable change" is any change which can be measured
and acted upon to identify or isolate the compound which causes it.
The selection may take place at the level of the microcapsule, the
microbead, or the compound itself optionally when complexed with
another reagent. A particularly advantageous embodiment is optical
detection, in which the selectable change is a change in optical
properties, which can be detected and acted upon for instance in a
FACS device to separate microcapsules or microbeads displaying the
desired change.
[0073] As used herein, a change in optical properties refers to any
change in absorption or emission of electromagnetic radiation,
including changes in absorbance, luminescence, phosphorescence or
fluorescence. All such properties are included in the term
"optical". Microcapsules or microbeads can be identified and,
optionally, sorted, for example, by luminescence, fluorescence or
phosphorescence activated sorting. In a preferred embodiment, flow
cytometry is employed to identify and, optionally, sort
microcapsules or microbeads. A variety of optical properties can be
used for analysis and to trigger sorting, including light
scattering (Kerker, 1983) and fluorescence polarisation (Rolland et
al., 1985). In a highly preferred embodiment microcapsules or
microbeads are analysed and, optionally, sorted using a
fluorescence activated cell sorter (FACS) (Norman, 1980; Mackenzie
and Pinder, 1986).
[0074] The compounds in microcapsules or on beads can be identified
using a variety of techniques familiar to those skilled in the art,
including mass spectroscopy, chemical tagging or optical
tagging.
General Techniques
[0075] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art (e.g., in cell culture, molecular
genetics, nucleic acid chemistry, hybridisation techniques and
biochemistry).
[0076] Standard techniques are used for molecular, genetic and
biochemical methods (see generally, Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al.,
Short Protocols in Molecular Biology (1999) 4.sup.th Ed, John Wiley
& Sons, Inc. which are incorporated herein by reference) and
chemical methods. In addition Harlow & Lane, A Laboratory
Manual Cold Spring Harbor, N.Y., is referred to for standard
Immunological Techniques.
(A) General Description
[0077] The microcapsules of the present invention require
appropriate physical properties to allow the working of the
invention.
[0078] First, to ensure that the compounds and the target may not
diffuse between microcapsules, the contents of each microcapsule
must be isolated from the contents of the surrounding
microcapsules, so that there is no or little exchange of compounds
and target between the microcapsules over the timescale of the
experiment.
[0079] Second, the method of the present invention requires that
there are only a limited number of beads per microcapsule. This
ensures that the compounds and the target will be isolated from
other beads.
[0080] Third, the formation and the composition of the
microcapsules must not abolish the activity of the target.
[0081] Consequently, any microencapsulation system used must fulfil
these three requirements. The appropriate system(s) may vary
depending on the precise nature of the requirements in each
application of the invention, as will be apparent to the skilled
person.
[0082] A wide variety of microencapsulation procedures are
available (see Benita, 1996) and may be used to create the
microcapsules used in accordance with the present invention.
Indeed, more than 200 microencapsulation methods have been
identified in the literature (Finch, 1993).
[0083] These include membrane enveloped aqueous vesicles such as
lipid vesicles (liposomes) (New, 1990) and non-ionic surfactant
vesicles (van Hal et al., 1996). These are closed-membranous
capsules of single or multiple bilayers of non-covalently assembled
molecules, with each bilayer separated from its neighbour by an
aqueous compartment. In the case of liposomes the membrane is
composed of lipid molecules; these are usually phospholipids but
sterols such as cholesterol may also be incorporated into the
membranes (New, 1990). A variety of enzyme-catalysed biochemical
reactions, including RNA and DNA polymerisation, can be performed
within liposomes (Chakrabarti et al., 1994; Oberholzer at al.,
1995a; Oberholzer et al., 1995b; Walde et al., 1994; Wick &
Luisi, 1996).
[0084] With a membrane-enveloped vesicle system much of the aqueous
phase is outside the vesicles and is therefore
non-compartmentalised. This continuous, aqueous phase should be
removed or the biological systems in it inhibited or destroyed in
order that the reactions are limited to the microcapsules (Luisi et
al., 1987).
[0085] Enzyme-catalysed biochemical reactions have also been
demonstrated in microcapsules generated by a variety of other
methods. Many enzymes are active in reverse micellar solutions (Bru
& Walde, 1991; Bru & Walde, 1993; Creagh et al., 1993;
Haber et al., 1993; Kumar et al., 1989; Luisi & B., 1987; Mao
& Walde, 1991; Mao et al, 1992; Perez et al., 1992; Walde et
al, 1994; Walde et al., 1993; Walde et al., 1988) such as the
AOT-isooctane-water system (Menger & Yamada, 1979).
[0086] Microcapsules can also be generated by interfacial
polymerisation and interfacial complexation (Whateley, 1996).
Microcapsules of this sort can have rigid, nonpermeable membranes,
or semipermeable membranes. Semipermeable microcapsules bordered by
cellulose nitrate membranes, polyamide membranes and
lipid-polyamide membranes can all support biochemical reactions,
including multienzyme systems (Chang, 1987; Chang, 1992; Lim,
1984). Alginate/polylysine microcapsules (Lim & Sun, 1980),
which can be formed under very mild conditions, have also proven to
be very biocompatible, providing, for example, an effective method
of encapsulating living cells and tissues (Chang, 1992; Sun et al.,
1992).
[0087] Non-membranous microencapsulation systems based on phase
partitioning of an aqueous environment in a colloidal system, such
as an emulsion, may also be used.
[0088] Preferably, the microcapsules of the present invention are
formed from emulsions; heterogeneous systems of two immiscible
liquid phases with one of the phases dispersed in the other as
droplets of microscopic or colloidal size (Becher, 1957; Sherman,
1968; Lissant, 1974; Lissant, 1984).
[0089] Emulsions may be produced from any suitable combination of
immiscible liquids. Preferably the emulsion of the present
invention has water (containing the biochemical components) as the
phase present in the form of finely divided droplets (the disperse,
internal or discontinuous phase) and a hydrophobic, immiscible
liquid (an oil`) as the matrix in which these droplets are
suspended (the nondisperse, continuous or external phase). Such
emulsions are termed water-in-oil` (W/O). This has the advantage
that the entire aqueous phase containing the biochemical components
is compartmentalised in discreet droplets (the internal phase). The
external phase, being a hydrophobic oil, generally contains none of
the biochemical components and hence is inert.
[0090] The emulsion may be stabilised by addition of one or more
surface-active agents (surfactants). These surfactants are termed
emulsifying agents and act at the water/oil interface to prevent
(or at least delay) separation of the phases. Many oils and many
emulsifiers can be used for the generation of water-in-oil
emulsions; a recent compilation listed over 16,000 surfactants,
many of which are used as emulsifying agents (Ash and Ash, 1993).
Suitable oils include light white mineral oil and decane. Suitable
surfactants include: non-ionic surfactants (Schick, 1966) such as
sorbitan monooleate (Span.TM.80; ICI), sorbitan monostearate
(Span.TM.60; ICI), polyoxyethylenesorbitan monooleate (Tween.TM.
80; ICI), and octylphenoxyethoxyethanol (Triton X-100); ionic
surfactants such as sodium cholate and sodium taurocholate and
sodium deoxycholate; chemically inert silicone-based surfactants
such as polysiloxane-polycetyl-polyethylene glycol copolymer (Cetyl
Dimethicone Copolyol) (e.g. Abil.TM.EM90; Goldschmidt); and
cholesterol.
[0091] Emulsions with a fluorocarbon (or perfluorocarbon)
continuous phase (Krafft et al., 2003; Riess, 2002) may be
particularly advantageous. For example, stable
water-in-perfluorooctyl bromide and water-in-perfluorooctylethane
emulsions can be formed using F-alkyl dimorpholinophosphates as
surfactants (Sadtler et al., 1996). Non-fluorinated compounds are
essentially insoluble in fluorocarbons and perfluorocarbons
(Curran, 1998; Hildebrand and Cochran, 1949; Hudlicky, 1992; Scott,
1948; Studer et al., 1997) and small drug-like molecules (typically
<500 Da and Log P<5) (Lipinski et al., 2001) are
compartmentalised very effectively in the aqueous microcapsules of
water-in-fluorocarbon and water-in-perfluorocarbon emulsions--with
little or no exchange between microcapsules.
[0092] Advantageously, compounds can be compartmentalised in
microcapsules comprising non-aqueous (organic) solvents.
Non-fluorinated organic solvents are essentially insoluble and
immiscible with fluorocarbons and perfluorocarbons (Curran, 1998;
Hildebrand and Cochran, 1949; Hudlicky, 1992; Scott, 1948; Studer
et al., 1997) allowing the formation of emulsions with a
fluorocarbon (or perfluorocarbon) continuous phase and a
discontinous phase formed from a non-aqueous solvent such as
dichloromethane, chloroform, carbon tetrachloride, toluene,
tetrahydrofuran, diethyl ether, and ethanol. The ability to form
secondary compounds in microcapsules comprising non-aqueous
solvents greatly expands the repertoire of chemical reactions that
can be performed and secondary molecules that can be synthesised
therein. Most of synthetic organic chemistry is carried out in
organic solvents including dichloromethane, chloroform, carbon
tetrachloride, toluene, tetrahydrofuran, diethyl ether, and
ethanol. Organic molecules dissolve better in organic solvents.
Electrostatic interactions are enhanced in organic solvents (due to
the low dielectric constant), whereas they can be solvated and made
less reactive in aqueous solvents. For example, much of
contemporary organic chemistry involves reactions relating to
carbonyl chemistry, including the use of metal enolates. Likewise
for a growing number of other organometallic interactions. These
reactions are often carried out under an inert atmosphere in
anhydrous solvents (otherwise the reagents would be quenched by
water). There are also a large number of reactions which use
palladium catalysis including the Suzuki reaction and the Heck
reaction.
[0093] Creation of an emulsion generally requires the application
of mechanical energy to force the phases together. There are a
variety of ways of doing this which utilise a variety of mechanical
devices, including stirrers (such as magnetic stir-bars, propeller
and turbine stirrers, paddle devices and whisks), homogenisers
(including rotor-stator homogenisers, high-pressure valve
homogenisers and jet homogenisers), colloid mills, ultrasound and
`membrane emulsification` devices (Becher, 1957; Dickinson,
1994).
[0094] Complicated biochemical processes, notably gene
transcription and translation are also active in aqueous
microcapsules formed in water-in-oil emulsions. This has enabled
compartmentalisation in water-in-oil emulsions to be used for the
selection of genes, which are transcribed and translated in
emulsion microcapsules and selected by the binding or catalytic
activities of the proteins they encode (Doi and Yanagawa, 1999;
Griffiths and Tawfik, 2003; Lee et al., 2002; Sepp et al., 2002;
Tawfik and Griffiths, 1998). This was possible because the aqueous
microcapsules formed in the emulsion were generally stable with
little if any exchange of nucleic acids, proteins, or the products
of enzyme catalysed reactions between microcapsules.
[0095] The technology exists to create emulsions with volumes all
the way up to industrial scales of thousands of litres (Becher,
1957; Sherman, 1968; Lissant, 1974; Lissant, 1984).
[0096] The preferred microcapsule size will vary depending upon the
precise requirements of any individual screening process that is to
be performed according to the present invention. In all cases,
there will be an optimal balance between the size of the compound
library and the sensitivities of the assays to determine the
identity of the compound and target activity.
[0097] The size of emulsion microcapsules may be varied simply by
tailoring the emulsion conditions used to form the emulsion
according to requirements of the screening system. The larger the
microcapsule size, the larger is the volume that will be required
to encapsulate a given compound library, since the ultimately
limiting factor will be the size of the microcapsule and thus the
number of microcapsules possible per unit volume.
[0098] Water-in-oil emulsions can be re-emulsified to create
water-in-oil-in water double emulsions with an external
(continuous) aqueous phase. These double emulsions can be analysed
and, optionally, sorted using a flow cytometer (Bernath et al.,
2004).
[0099] Highly monodisperse microcapsules can be produced using
microfluidic techniques. For example, water-in-oil emulsions with
less than 3% polydispersity can be generated by droplet break off
in a co-flowing steam of oil (Umbanhowar et al., 2000).
Microfluidic systems can also be used for laminar-flow of aqueous
microdroplets dispersed in a stream of oil in microfluidic channels
(Thorsen et al., 2001). This allows the construction of
microfluidic devices for flow analysis and, optionally, flow
sorting of microdroplets (Fu et al., 2002).
[0100] Microcapsules can, advantageously, be fused or split. For
example, aqueous microdroplets can be merged and split using
microfluidics systems (Link et al., 2004; Song et al., 2003).
Microcapsule fusion allows the mixing of reagents. Fusion, for
example, of a microcapsule containing the target with a
microcapsule containing the compound could initiate the reaction
between target and compound. Microcapsule splitting allows single
microcapsules to be split into two or more smaller microcapsules.
For example a single microcapsule containing a compound can be
split into multiple microcapsules which can then each be fused with
a different microcapsule containing a different target. A single
microcapsule containing a target can also be split into multiple
microcapsules which can then each be fused with a different
microcapsule containing a different compound, or compounds at
different concentrations.
[0101] Microcapsules can be optically tagged by, for example,
incorporating fluorochromes. In a preferred configuration, the
microcapsules are optically tagged by incorporating quantum dots:
quantum dots of 6 colours at 10 concentrations would allow the
encoding of 10.sup.6 microcapsules (Han et al., 2001).
Microcapsules flowing in an ordered sequence in a microfluidic
channel can be encoded (wholly or partially) by their sequence in
the stream of microcapsules (positional encoding).
[0102] Microbeads, also known by those skilled in the art as
microspheres, latex particles, beads, or minibeads, are available
in diameters from 20 nm to 1 mm and can be made from a variety of
materials including silica and a variety of polymers, copolymers
and terpolymers including polystyrene (PS), polymethylmethacrylate
(PMMA), polyvinyltoluene (PVT), styrene/butadiene (S/B) copolymer,
and styrene/vinyltoluene (S/VT) copolymer (www.bangslabs.com). They
are available with a variety of surface chemistries from
hydrophobic surfaces (e.g. plain polystyrene), to very hydrophilic
surfaces imparted by a wide variety of functional surface groups:
aldehyde, aliphatic amine, amide, aromatic amine, carboxylic acid,
chloromethyl, epoxy, hydrazide, hydroxyl, sulfonate and tosyl. The
functional groups permit a wide range of covalent coupling
reactions for stable or reversible attachment of compounds to the
microbead surface.
[0103] Microbeads can be optically tagged by, for example,
incorporating fluorochromes. For example, one hundred different
bead sets have been created, each with a unique spectral address
due to labelling with precise ratios of red (>650 nm) and orange
(585 nm) fluorochromes (Fulton et al., 1997) (www.luminex.com) and
sets of up to 10.sup.6 beads can be encoded by incorporating
quantum dots of 10 intensities and 6 colours (Han et al.,
2001).
[0104] The compounds can be connected to the microbeads either
covalently or non-covalently by a variety of means that will be
familiar to those skilled in the art (see, for example, (Hermanson,
1996)). Advantageously, the compounds are attached via a cleavable
linker. A variety of such linkers are familiar to those skilled in
the art (see for example (Gordon and Balasubramanian, 1999)),
including for example, linkers which can be cleaved photochemically
and reversible covalent bonds which can be controlled by changing
the pH (e.g. imines and acylhydrazones), by adjusting the
oxido-reductive properties (e.g. disulphides), or using an external
catalyst (e.g. cross-metathesis and transamidation).
[0105] The method of the present invention permits the
identification of compounds which modulate the activity of the
target in a desired way in pools (libraries or repertoires) of
compounds.
[0106] In a highly preferred application, the method of the present
invention is useful for screening libraries of compounds. The
invention accordingly provides a method according to preceding
aspects of the invention, wherein the compounds are identified from
a library of compounds.
[0107] The compounds identified according to the invention are
advantageously of pharmacological or industrial interest, including
activators or inhibitors of biological systems, such as cellular
signal transduction mechanisms suitable for diagnostic and
therapeutic applications. In a preferred aspect, therefore, the
invention permits the identification of clinically or industrially
useful products. In a further aspect of the invention, there is
provided a product when isolated by the method of the
invention.
[0108] The selection of suitable encapsulation conditions is
desirable. Depending on the complexity and size of the compound
library to be screened, it may be beneficial to set up the
encapsulation procedure such that one or less than one secondary
compound is formed per microcapsule. This will provide the greatest
power of resolution. Where the library is larger and/or more
complex, however, this may be impracticable; it may be preferable
to form several secondary compounds together and rely on repeated
application of the method of the invention to identify the desired
compound. A combination of encapsulation procedures may be used to
identify the desired compound.
[0109] Theoretical studies indicate that the larger the number of
compounds created the more likely it is that a compound will be
created with the properties desired (see (Perelson and Oster, 1979)
for a description of how this applies to repertoires of
antibodies). It has also been confirmed practically that larger
phage-antibody repertoires do indeed give rise to more antibodies
with better binding affinities than smaller repertoires (Griffiths
et al., 1994). To ensure that rare variants are generated and thus
are capable of being identified, a large library size is desirable.
Thus, the use of optimally small microcapsules is beneficial.
[0110] The largest repertoires of compounds that can be screened in
a single experiment to date, using two dimensional microarrays of 1
nl volume spots, is .about.10.sup.3 (Hergenmother et al., 2000).
Using the present invention, at a microcapsule diameter of 2.6 mm
(Tawfik and Griffiths, 1998), by forming a three-dimensional
dispersion, a repertoire size of at least 10.sup.11 can be screened
using 1 ml aqueous phase in a 20 ml emulsion.
[0111] In addition to the compounds, or microbeads coated with
compounds, described above, the microcapsules according to the
invention will comprise further components required for the
screening process to take place. They will comprise the target and
a suitable buffer. A suitable buffer will be one in which all of
the desired components of the biological system are active and will
therefore depend upon the requirements of each specific reaction
system. Buffers suitable for biological and/or chemical reactions
are known in the art and recipes provided in various laboratory
texts, such as (Sambrook and Russell, 2001).
[0112] Other components of the system will comprise those necessary
for assaying the activity of the target. These may for example
comprise substrate(s) and cofactor(s) for a reaction catalysed by
the target, and ligand(s) bound by the target. They may also
comprise other catalysts (including enzymes), substrates and
cofactors for reactions coupled to the activity of the target which
allow for the activity of the target to be detected.
(B) Screening Procedures
[0113] To screen compounds which bind to or modulate the activity
of a target, the target is compartmentalised in microcapsules
together with one or more compounds or compound-coated microbeads.
Advantageously each microcapsule contains only a single sort of
secondary compound, but many copies thereof. Advantageously each
microbead is coated with only a single sort of compound, but many
copies thereof. Advantageously the compounds are connected to the
microbeads via a cleavable linker, allowing them to be released
from the microbeads in the compartments. Advantageously, each
microcapsule or microbead is optically tagged to allow
identification of the compounds contained within the microcapsule
of attached to the microbead.
(i) Screening for Binding
[0114] Compounds can be screened directly for binding to a target.
In this embodiment, if the compound is attached to a microbead and
has affinity for the target it will be bound by the target. At the
end of the reaction, all of the microcapsules are combined, and all
microbeads pooled together in one environment. Microbeads carrying
compounds exhibiting the desired binding can be selected by
affinity purification using a molecule that specifically binds to,
or reacts specifically with, the target.
[0115] In an alternative embodiment, the target can be attached to
microbeads by a variety of means familiar to those skilled in the
art (see for example (Hermanson, 1996)). The compounds to be
screened contain a common feature--a tag. The compounds are
released from the microbeads and if the compound has affinity for
the target, it will bind to it. At the end of the reaction, all of
the microcapsules are combined, and all microbeads pooled together
in one environment. Microbeads carrying compounds exhibiting the
desired binding can be selected by affinity purification using a
molecule that specifically binds to, or reacts specifically with,
the "tag".
[0116] In an alternative embodiment, microbeads may be screened on
the basis that the compound, which binds to the target, merely
hides the ligand from, for example, further binding partners. In
this eventuality, the microbead, rather than being retained during
an affinity purification step, may be selectively eluted whilst
other microbeads are bound.
[0117] Sorting by affinity is dependent on the presence of two
members of a binding pair in such conditions that binding may
occur. Any binding pair may be used for this purpose. As used
herein, the term binding pair refers to any pair of molecules
capable of binding to one another. Examples of binding pairs that
may be used in the present invention include an antigen and an
antibody or fragment thereof capable of binding the antigen, the
biotin-avidin/streptavidin pair (Savage et al., 1994), a
calcium-dependent binding polypeptide and ligand thereof (e.g.
calmodulin and a calmodulin-binding peptide (Montigiani et al.,
1996; Stofko et al., 1992), pairs of polypeptides which assemble to
form a leucine zipper (Tripet et al., 1996), histidines (typically
hexahistidine peptides) and chelated Cu.sup.2+' Zn.sup.2+ and
Ni.sup.2+, (e.g. Ni-NTA; (Hochuli et al., 1987)), RNA-binding and
DNA-binding proteins (Klug, 1995) including those containing
zinc-finger motifs (Klug and Schwabe, 1995) and DNA
methyltransferases (Anderson, 1993), and their nucleic acid binding
sites.
[0118] In an alternative embodiment, compounds can be screened for
binding to a target using a change in the optical properties of the
microcapsule or the microbead.
[0119] The change in optical properties of the microcapsule or the
microbead after binding of the compound to the target may be
induced in a variety of ways, including: [0120] (1) the compound
itself may have distinctive optical properties, for example, it is
fluorescent [0121] (2) the optical properties of the compound may
be modified on binding to the target, for example, the fluorescence
of the compound is quenched or enhanced on binding (Voss, 1993;
Masui and Kuramitsu, 1998). [0122] (3) the optical properties of
the target may be modified on binding of the compound, for example,
the fluorescence of the target is quenched or enhanced on binding
(Guixe et al., 1998; Qi and Grabowski, 1998) [0123] (4) the optical
properties of both target and compound are modified on binding, for
example, there can be a fluorescence resonance energy transfer
(FRET) from target to compound (or vice versa) resulting in
emission at the "acceptor" emission wavelength when excitation is
at the "donor" absorption wavelength (Heim & Tsien, 1996;
Mahajan et al., 1998; Miyawaki et al., 1997).
[0124] The invention provides a method wherein a compound with the
desired activity induces a change in the optical properties of the
microcapsule, which enables the microcapsule containing the
compound and the microbeads contained therein to be identified, and
optionally, sorted.
[0125] In an alternative embodiment, the invention provides a
method wherein microbeads are analysed following pooling of the
microcapsules into one or more common compartments. In this
embodiment, a compound having the desired activity modifies the
optical properties of the microbead which carried it (and which
resides in the same microcapsule) to allow it to be identified, and
optionally, sorted.
[0126] In this embodiment, it is not necessary for binding of the
compound to the target to directly induce a change in optical
properties.
[0127] In this embodiment, if the compound attached to the
microbead has affinity for the target it will be bound by the
target. At the end of the reaction, all of the microcapsules are
combined, and all microbeads pooled together in one environment.
Microbeads carrying compounds exhibiting the desired binding can be
identified by adding reagents that specifically bind to, or react
specifically with, the target and thereby induce a change in the
optical properties of the microbeads allowing their identification.
For example, a fluorescently-labelled anti-target antibody can be
used, or an anti-target antibody followed by a second fluorescently
labelled antibody which binds the first.
[0128] In an alternative embodiment, the target can be attached to
the microbeads by a variety of means familiar to those skilled in
the art (see for example (Hermanson, 1996)). The compounds to be
screened contain a common feature--a tag. The compounds are
released from the microbeads and if the compound has affinity for
the target, it will bind to it. At the end of the reaction, all of
the microcapsules are combined, and all microbeads pooled together
in one environment. Microbeads carrying compounds exhibiting the
desired binding can be identified by adding reagents that
specifically bind to, or react specifically with, the "tag" and
thereby induce a change in the optical properties of the microbeads
allowing their identification. For example, a
fluorescently-labelled anti-"tag" antibody can be used, or an
anti-"tag" antibody followed by a second fluorescently labelled
antibody which binds the first.
[0129] In an alternative embodiment, microbeads may be identified
on the basis that the gene product, which binds to the ligand,
merely hides the ligand from, for example, further binding partners
which would otherwise modify the optical properties of the
microbeads. In this case microbeads with unmodified optical
properties would be selected.
[0130] Fluorescence may be enhanced by the use of Tyramide Signal
Amplification (TSA.TM.) amplification to make the microbeads
fluorescent (Sepp et al., 2002). This involves peroxidase (linked
to another compound) binding to the microbeads and catalysing the
conversion of fluorescein-tyramine in to a free radical form which
then reacts (locally) with the microbeads. Methods for performing
TSA are known in the art, and kits are available commercially from
NEN.
[0131] TSA may be configured such that it results in a direct
increase in the fluorescence of the microbeads, or such that a
ligand is attached to the microbeads which is bound by a second
fluorescent molecule, or a sequence of molecules, one or more of
which is fluorescent.
(ii) Screening for Regulation of Binding
[0132] In an alternative embodiment, the invention can be used to
screen compounds which act to regulate a biochemical process. If
the compound activates a binding activity of a target, a ligand for
the target which is activated can be attached to microbeads by a
variety of means familiar to those skilled in the art (see for
example (Hermanson, 1996)). At the end of the reaction, all of the
microcapsules are combined, and all microbeads pooled together in
one environment. Microbeads carrying compounds exhibiting the
desired binding can be selected by affinity purification using a
molecule that specifically binds to, or reacts specifically with,
the target.
[0133] In an alternative embodiment, microbeads may be screened on
the basis that the compound inhibits the binding activity of a
target. In this eventuality, the microbead, rather than being
retained during an affinity purification step, may be selectively
eluted whilst other microbeads are bound.
[0134] In an alternative embodiment, compounds can be screened for
the ability to modulates a binding activity of a target using a
change in the optical properties of the microcapsule or the
microbead.
[0135] The change in optical properties of the microcapsule or the
microbead after binding of the target to its ligand may be induced
in a variety of ways, including: [0136] (1) the ligand itself may
have distinctive optical properties, for example, it is fluorescent
[0137] (2) the optical properties of the ligand may be modified on
binding to the target, for example, the fluorescence of the ligand
is quenched or enhanced on binding (Voss, 1993; Masui and
Kuramitsu, 1998). [0138] (3) the optical properties of the target
may be modified on binding of the ligand, for example, the
fluorescence of the target is quenched or enhanced on binding
(Guixe et al., 1998; Qi and Grabowski, 1998) [0139] (4) the optical
properties of both target and ligand are modified on binding, for
example, there can be a fluorescence resonance energy transfer
(FRET) from target to ligand (or vice versa) resulting in emission
at the "acceptor" emission wavelength when excitation is at the
"donor" absorption wavelength (Heim & Tsien, 1996; Mahajan et
al., 1998; Miyawaki et al., 1997).
[0140] The invention provides a method wherein a compound with the
desired activity induces a change in the optical properties of the
microcapsule, which enables the microcapsule containing the
compound and the microbeads contained therein to be identified, and
optionally, sorted.
[0141] In an alternative embodiment, the invention provides a
method wherein microbeads are analysed following pooling of the
microcapsules into one or more common compartments. In this
embodiment, a compound having the desired activity modifies the
optical properties of the microbead which carried it (and which
resides in the same microcapsule) to allow it to be identified, and
optionally, sorted.
[0142] In this embodiment, it is not necessary for binding of the
target to the ligand to directly induce a change in optical
properties.
[0143] In this embodiment, if a ligand attached to the microbead
has affinity for the target it will be bound by the target. At the
end of the reaction, all of the microcapsules are combined, and all
microbeads pooled together in one environment. Microbeads carrying
compounds which modulate the binding activity can be identified by
adding reagents that specifically bind to, or react specifically
with, the target and thereby induce a change in the optical
properties of the microbeads allowing their identification. For
example, a fluorescently-labelled anti-target antibody can be used,
or an anti-target antibody followed by a second fluorescently
labelled antibody which binds the first.
[0144] In an alternative embodiment, the target can be attached to
the microbeads by a variety of means familiar to those skilled in
the art (see for example (Hermanson, 1996)). The ligand to be
screened contains a feature--a tag. At the end of the reaction, all
of the microcapsules are combined, and all microbeads pooled
together in one environment. Microbeads carrying compounds which
modulate binding can be identified by adding reagents that
specifically bind to, or react specifically with, the "tag" and
thereby induce a change in the optical properties of the microbeads
allowing their identification. For example, a
fluorescently-labelled anti-"tag" antibody can be used, or an
anti-"tag" antibody followed by a second fluorescently labelled
antibody which binds the first.
[0145] Fluorescence may be enhanced by the use of Tyramide Signal
Amplification (TSA.TM.) amplification to make the microbeads
fluorescent (Sepp et al., 2002), as above.
(III) Screening for Regulation of Catalysis
[0146] In an alternative embodiment, the invention provides a
method wherein a compound with the desired activity induces a
change in the optical properties of the microcapsule, which enables
the microcapsule containing the compound and, optionally, the
microbeads contained therein to be identified, and optionally,
sorted. The optical properties of microcapsules can be modified by
either: [0147] (a) the substrate and product of the regulated
reaction having different optical properties (many fluorogenic
enzyme substrates are available commercially, see for example
(Haugland, 1996 and www.probes.com) including substrates for
glycosidases, phosphatases, peptidases and proteases, or [0148] (b)
the presence of reagents which specifically bind to, or react with,
the product (or substrate) of the regulated reaction in the
microcapsule and which thereby induce a change in the optical
properties of the microcapsules allowing their identification.
[0149] A wide range of assays for screening libraries of compounds
for those which modulate the activity of a target are based on
detecting changes in optical properties and can be used to screen
compounds according to this invention. Such assays are well known
to those skilled in the art (see for example Haugland, 1996 and
www.probes.com).
[0150] Alternatively, selection may be performed indirectly by
coupling a first reaction to subsequent reactions that takes place
in the same microcapsule. There are two general ways in which this
may be performed. First, the product of the first reaction could be
reacted with, or bound by, a molecule which does not react with the
substrate(s) of the first reaction. A second, coupled reaction will
only proceed in the presence of the product of the first reaction.
A regulatory compound can then be identified by the properties of
the product or substrate of the second reaction.
[0151] Alternatively, the product of the reaction being selected
may be the substrate or cofactor for a second enzyme-catalysed
reaction. The enzyme to catalyse the second reaction can be
incorporated in the reaction mixture prior to microencapsulation.
Only when the first reaction proceeds will the coupled enzyme
generate an identifiable product.
[0152] This concept of coupling can be elaborated to incorporate
multiple enzymes, each using as a substrate the product of the
previous reaction. This allows for selection of regulators of
enzymes that will not react with an immobilised substrate. It can
also be designed to give increased sensitivity by signal
amplification if a product of one reaction is a catalyst or a
cofactor for a second reaction or series of reactions leading to a
selectable product (for example, see (Johannsson, 1991; Johannsson
and Bates, 1988). Furthermore an enzyme cascade system can be based
on the production of an activator for an enzyme or the destruction
of an enzyme inhibitor (see (Mize et al., 1989)). Coupling also has
the advantage that a common screening system can be used for a
whole group of enzymes which generate the same product and allows
for the selection of regulation of complicated multi-step chemical
transformations and pathways.
[0153] In an alternative embodiment, if the target is itself an
enzyme, or regulates a biochemical process which is enzymatic, the
microbead in each microcapsule may be coated with the substrate for
the enzymatic reaction. The regulatory compound will determine the
extent to which the substrate is converted into the product. At the
end of the reaction the microbead is physically linked to the
product of the catalysed reaction. When the microcapsules are
combined and the reactants pooled, microbeads which were coated
with activator compounds can be identified by any property specific
to the product. If an inhibitor is desired, selection can be for a
chemical property specific to the substrate of the regulated
reaction.
[0154] It may also be desirable, in some cases, for the substrate
not to be attached to the microbead. In this case the substrate
would contain an inactive "tag" that requires a further step to
activate it such as photoactivation (e.g. of a "caged" biotin
analogue, (Pirrung and Huang, 1996; Sundberg et al., 1995)). After
convertion of the substrate to product the "tag" is activated and
the "tagged" substrate and/or product bound by a tag-binding
molecule (e.g. avidin or streptavidin) attached to the microbead.
The ratio of substrate to product attached to the nucleic acid via
the "tag" will therefore reflect the ratio of the substrate and
product in solution. A substrate tagged with caged biotin has been
used to select for genes encoding enzymes with phosphotriesterase
activity using a procedure based on compartmentalisation in
microcapsules (Griffiths and Tawfik, 2003). The phosphotriesterase
substrate was hydrolysed in solution in microcapsules containing
active enzyme molecules, and after the reaction was completed, the
caging group was released by irradiation to allow the product to
bind, via the biotin moiety, to microbeads to which the gene
encoding the enzyme was attached.
[0155] After the microbeads and the contents of the microcapsules
are combined, those microbeads coated with regulators can be
selected by affinity purification using a molecule (e.g. an
antibody) that binds specifically to the product or substrate as
appropriate.
[0156] In an alternative embodiment, the invention provides a
method wherein the microbeads are analysed following pooling of the
microcapsules into one or more common compartments. Microbeads
coated with regulator compounds can be identified using changes in
optical properties of the microbeads. The optical properties of
microbeads with product (or substrate) attached can be modified by
either. [0157] (1) the product-microbead complex having
characteristic optical properties not found in the
substrate-microbead complex, due to, for example; [0158] (a) the
substrate and product having different optical properties (many
fluorogenic enzyme substrates are available commercially (see for
example Haugland, 1996 and www.probes.com) including substrates for
glycosidases, phosphatases, peptidases and proteases, or [0159] (b)
the substrate and product having similar optical properties, but
only the product, and not the substrate binds to, or reacts with,
the microbead; [0160] (2) adding reagents which specifically bind
to, or react with, the product (or substrate) and which thereby
induce a change in the optical properties of the microbeads
allowing their identification (these reagents can be added before
or after breaking the microcapsules and pooling the microbeads).
The reagents; [0161] (a) bind specifically to, or react
specifically with, the product, and not the substrate, (or vice
versa) if both substrate and product are attached to the
microbeads, or [0162] (b) optionally bind both substrate and
product if only the product, and not the substrate binds to, or
reacts with, the microbeads (or vice versa).
[0163] In this scenario, the substrate (or one of the substrates)
can be present in each microcapsule unlinked to the microbead, but
has a molecular "tag" (for example biotin, DIG or DNP or a
fluorescent group). When the regulated enzyme converts the
substrate to product, the product retains the "tag" and is then
captured in the microcapsule by the product-specific antibody. When
all reactions are stopped and the microcapsules are combined, these
microbeads will be "tagged" and may already have changed optical
properties, for example, if the "tag" was a fluorescent group.
Alternatively, a change in optical properties of "tagged"
microbeads can be induced by adding a fluorescently labelled ligand
which binds the "tag" (for example fluorescently-labelled
avidin/streptavidin, an anti-"tag" antibody which is fluorescent,
or a non-fluorescent anti-"tag" antibody which can be detected by a
second fluorescently-labelled antibody).
(IV) Screening for Compound Specificity/Selectivity
[0164] Compounds with specificity or selectivity for certain
targets and not others can be specifically identified by carrying
out a positive screen for regulation of a reaction using one
substrate and a negative screen for regulation of a reaction with
another substrate. For example, two substrates, specific for two
different target enzymes, are each labelled with different
fluorogenic moieties. Each target enzymes catalyse the generation
of a product with with a different fluorescence spectrum resulting
in different optical properties of the microcapsules depending on
the specificity of the compound for two targets.
(v) Screening Using Cells
[0165] In the current drug discovery paradigm, validated
recombinant targets form the basis of in vitro high-throughput
screening (HTS) assays. Isolated proteins cannot, however, be
regarded as representative of complex biological systems; hence,
cell-based systems can provide greater confidence in compound
activity in an intact biological system. A wide range of cell-based
assays for drug leads are known to those skilled in the art. Cells
can be compartmentalised in microcapsules, such as the aqeous
microdroplets of a water-in-oil emulsion (Ghadessy, 2001). The
effect of a compound(s) on a target can be determined by
compartmentalising a cell (or cells) in a microcapsule together
with a compound(s) and using an appropriate cell-based assay to
identify those compartments containing compounds with the desired
effect on the cell(s). The use of water-in-fluorocarbon emulsions
may be particularly advantageous: the high gas dissolving capacity
of fluorocarbons can support the exchange of respiratory gases and
has been reported to be beneficial to cell culture systems (Lowe,
2002).
(vi) Flow Cytometry
[0166] In a preferred embodiment of the invention the microcapsules
or microbeads will be analysed and, optionally, sorted by flow
cytometry. Many formats of microcapsule can be analysed and,
optionally, sorted directly using flow cytometry. Some formats of
microcapsule may require that the microcapsules be further
processed before analysis or sorting. For example, water-in-oil
emulsions can be converted into water-in-oil-in-water double
emulsions to facilitate analysis by flow cytometry (Bernath et al.,
2004). Multiple emulsions are prepared by the re-emulsification of
a simple primary water-in-oil (or oil-in-water) emulsion to provide
water-in-oil-in-water (or oil-in-water-in-oil) emulsions (Davis and
Walker, 1987).
[0167] Highly monodisperse microcapsules can be produced using
microfluidic techniques. For example, water-in-oil emulsions with
less than 3% polydispersity can be generated by droplet break off
in a co-flowing steam of oil (Umbanhowar, 2000). Microfluidic
systems can also be used for laminar-flow of aqueous microdroplets
dispersed in a stream of oil in microfluidic channels (Thorsen,
2001). This allows the construction of microfluidic devices for
flow analysis and, optionally, flow sorting of microdroplets (Fu,
2002).
[0168] A variety of optical properties can be used for analysis and
to trigger sorting, including light scattering (Kerker, 1983) and
fluorescence polarisation (Rolland et al., 1985). In a highly
preferred embodiment the difference in optical properties of the
microcapsules or microbeads will be a difference in fluorescence
and, if required, the microcapsules or microbeads will be sorted
using a fluorescence activated cell sorter (Norman, 1980; Mackenzie
and Pinder, 1986), or similar device. Flow cytometry has a series
of advantages: [0169] (1) commercially available fluorescence
activated cell sorting equipment from established manufacturers
(e.g. Becton-Dickinson, Coulter, Cytomation) allows the analysis
and sorting at up to 100,000 microcapsules or microbeads s.sup.-1.
[0170] (2) the fluorescence signal from each microcapsule or
microbead corresponds tightly to the number of fluorescent
molecules present. As little as few hundred fluorescent molecules
per microcapsules or microbeads can be quantitatively detected;
[0171] (3) the wide dynamic range of the fluorescence detectors
(typically 4 log units) allows easy setting of the stringency of
the sorting procedure, thus allowing the recovery of the optimal
number microcapsules or microbeads from the starting pool (the
gates can be set to separate microcapsules or microbeads with small
differences in fluorescence or to only separate out microcapsules
or microbeads with large differences in fluorescence, dependant on
the selection being performed); [0172] (4) commercially available
fluorescence-activated cell sorting equipment can perform
simultaneous excitation and detection at multiple wavelengths
(Shapiro, 1995). allowing positive and negative selections to be
performed simultaneously by monitoring the labelling of the
microcapsules or microbeads with two to thirteen (or more)
fluorescent markers, for example, if substrates for two alternative
targets are labelled with different fluorescent tags the
microcapsules or microbeads can labelled with different
fluorophores dependent on the target regulated.
[0173] If the microcapsules or microbeads are optically tagged,
flow cytometry can also be used to identify the compound or
compounds in the microcapsule or coated on the microbeads (see
below). Optical tagging can also be used to identify the
concentration of the compound in the microcapsule (if more than one
concentration is used in a single experiment) or the number of
compound molecules coated on a microbead (if more than one coating
density is used in a single experiment). Furthermore, optical
tagging can be used to identify the target in a microcapsule (if
more than one target is used in a single experiment). This analysis
can be performed simultaneously with measuring activity, after
sorting of microcapsules containing microbeads, or after sorting of
the microbeads.
(VII) Microcapsule Identification and Sorting
[0174] The invention provides for the identification and,
optionally, the sorting of intact microcapsules where this is
enabled by the sorting techniques being employed. Microcapsules may
be identified and, optionally, sorted as such when the change
induced by the desired compound either occurs or manifests itself
at the surface of the microcapsule or is detectable from outside
the microcapsule. The change may be caused by the direct action of
the compound, or indirect, in which a series of reactions, one or
more of which involve the compound having the desired activity
leads to the change. For example, where the microcapsule is a
membranous microcapsule, the microcapsule may be so configured that
a component or components of the biochemical system comprising the
target are displayed at its surface and thus accessible to reagents
which can detect changes in the biochemical system regulated by the
compound on the microbead within the microcapsule.
[0175] In a preferred aspect of the invention, however,
microcapsule identification and, optionally, sorting relies on a
change in the optical properties of the microcapsule, for example
absorption or emission characteristics thereof, for example
alteration in the optical properties of the microcapsule resulting
from a reaction leading to changes in absorbance, luminescence,
phosphorescence or fluorescence associated with the microcapsule.
All such properties are included in the term "optical". In such a
case, microcapsules can be identified and, optionally, sorted by
luminescence, fluorescence or phosphorescence activated sorting. In
a highly preferred embodiment, flow cytometry is employed to
analyse and, optionally, sort microcapsules containing compounds
having a desired activity which result in the production of a
fluorescent molecule in the microcapsule.
[0176] In an alternative embodiment, a change in microcapsule
fluorescence, when identified, is used to trigger the modification
of the microbead within the compartment. In a preferred aspect of
the invention, microcapsule identification relies on a change in
the optical properties of the microcapsule resulting from a
reaction leading to luminescence, phosphorescence or fluorescence
within the microcapsule. Modification of the microbead within the
microcapsules would be triggered by identification of luminescence,
phosphorescence or fluorescence. For example, identification of
luminescence, phosphorescence or fluorescence can trigger
bombardment of the compartment with photons (or other particles or
waves) which leads to modification of the microbead or molecules
attached to it. A similar procedure has been described previously
for the rapid sorting of cells (Keij et al., 1994). Modification of
the microbead may result, for example, from coupling a molecular
"tag", caged by a photolabile protecting group to the microbeads:
bombardment with photons of an appropriate wavelength leads to the
removal of the cage. Afterwards, all microcapsules are combined and
the microbeads pooled together in one environment. Microbeads
coated with compounds exhibiting the desired activity can be
selected by affinity purification using a molecule that
specifically binds to, or reacts specifically with, the "tag".
(C) Compound Libraries
(i) Primary Compound Libraries
[0177] Libraries of primary compounds can be obtained from a
variety of commercial sources. The compounds in the library can be
made by a variety of means well known to those skilled in the art.
Optionally, compound libraries can be made by combinatorial
synthesis using spatially resolved parallel synthesis or using
split synthesis, optionally to generate one-bead-one-compound
libraries. The compounds can, optionally, be synthesised on beads.
These beads can be compartmentalised in microcapsules directly or
the compounds released before compartmentalisation.
[0178] Advantageously, only a single type of compound, but multiple
copies thereof is present in each microcapsule.
[0179] The compounds can, optionally, be connected to microbeads
either covalently or non-covalently by a variety of means that will
be familiar to those skilled in the art (see, for example,
(Hermanson, 1996)).
[0180] Microbeads are available with a variety of surface
chemistries from hydrophobic surfaces (e.g. plain polystyrene), to
very hydrophilic surfaces imparted by a wide variety of functional
surface groups: aldehyde, aliphatic amine, amide, aromatic amine,
carboxylic acid, chloromethyl, epoxy, hydrazide, hydroxyl,
sulfonate and tosyl. The functional groups permit a wide range of
covalent coupling reactions, well known to those skilled in the
art, for stable or reversible attachment of compounds to the
microbead surface.
[0181] Advantageously, the compounds are attached to the microbeads
via a cleavable linker. A variety of such linkers are familiar to
those skilled in the art (see for example (Gordon and
Balasubramanian, 1999)), including for example, linkers which can
be cleaved photochemically and reversible covalent bonds which can
be controlled by changing the pH (e.g. imines and acylhydrazones),
by adjusting the oxido-reductive properties (e.g. disulphides), or
using an external catalyst (e.g. cross-metathesis and
transamidation).
[0182] Advantageously, only a single type of compound, but multiple
copies thereof is attached to each bead.
(ii) Secondary Compound Libraries
[0183] Secondary compound libraries are created by reactions
between primary compounds in microcapsules. Secondary compounds can
be created by a variety of two component, and multi-component
reactions well known to those skilled in the art (Armstrong et al.,
1996; Domling, 2002; Domling and Ugi, 2000; Ramstrom and Lehn,
2002).
[0184] To form secondary compound libraries by a two-component
reaction, two sets of compounds are compartmentalised in
microcapsules such that many compartments contain two or more
compounds. Advantageously, the modal number of compounds per
microcapsule is two. Advantageously, the microcapsules contain at
least one type of compound from each set of compounds.
Advantageously, the microcapsules contain one type of compound from
each set of compounds. The secondary compounds are formed by
chemical reactions between primary compounds from different sets.
The secondary compound may be the result of a covalent or
non-covalent reaction between the primary compounds.
[0185] A variety of chemistries, familiar to those skilled in the
art, are suitable to form secondary compounds in two-component
reactions. For example, reversible covalent bonds which can be
controlled by changing the pH (e.g. imines and acylhydrazones), by
adjusting the oxido-reductive properties (e.g. disulphides), or
using an external catalyst (e.g. cross-metathesis and
transamidation), can be used (Ramstrom and Lehn, 2002).
[0186] In a further embodiment, the method can also be used to
create secondary compound libraries using three-component,
four-component and higher order multi-component reactions. Three,
four or more sets of compounds (as appropriate) are
compartmentalised in microcapsules. The compounds are
compartmentalised in microcapsules such that many compartments
contain multiple compounds. Advantageously, the modal number of
compounds per microcapsule is equal to the number of components in
the reaction.
[0187] Advantageously, the microcapsules contain at least one type
of compound from each set of compounds. Advantageously, the
microcapsules contain one type of compound from each set of
compounds. The secondary compounds are formed by chemical reactions
between primary compounds from different sets. The secondary
compound may be the result of covalent or non-covalent reactions
between the primary compounds.
[0188] Examples of suitable multi-component reactions are the
Strecker, Hantzsch, Biginelli, Mannich, Passerini, Bucherer-Bergs
and Pauson-Khand three-component reactions and the Ugi
four-component reaction (Armstrong et al., 1996; Domling, 2002;
Domling and Ugi, 2000).
[0189] Secondary compound libraries may also be built using a
scaffold molecule which is common to all the secondary compounds
(Ramstrom and Lehn, 2002). This scaffold molecule may be
compartmentalised into microcapsules together with the other
primary compounds.
[0190] In a further embodiment, to form secondary compound
libraries by a two-component reaction, two sets of compounds are
attached to microbeads, advantageously to give only a single type
of molecule per microbead. The microbeads are compartmentalised in
microcapsules such that many compartments contain two or more
microbeads. Advantageously, the modal number of beads per
microcapsule is two. The compounds comprising at least one of the
two sets are released from the microbeads. The secondary compounds
are formed by chemical reactions between primary compounds from
different sets. The secondary compound may be the result of a
covalent or non-covalent reaction between the primary
compounds.
[0191] In a further embodiment, the method can also be used to
create secondary compound libraries using three-component,
four-component and higher order multi-component reactions. Three,
four or more sets of compounds (as appropriate) are attached to
microbeads, advantageously to give only a single type of molecule
per microbead. The microbeads are compartmentalised in
microcapsules such that many compartments contain multiple
microbeads. Advantageously, the modal number of beads per
microcapsule is equal to the number of components in the reaction.
The compounds comprising either all, or all bar one, of the sets
are released from the microbeads. The secondary compounds are
formed by chemical reactions between primary compounds from
different sets. The secondary compound may be the result of
covalent or non-covalent reactions between the primary
compounds.
[0192] Advantageously, the same reversible covalent bond can used
to couple the primary compound to the microbead as is used to form
the secondary compound.
[0193] Secondary compound libraries may also be built using a
scaffold molecule which is common to all the secondary compounds
(Ramstrom and Lehn, 2002). This scaffold molecule may be
compartmentalised into microcapsules together with the
microbeads.
(D) Identification of Compounds
[0194] The compounds in microcapsules or on microbeads can be
identified in a variety of ways.
[0195] If the identified microcapsules are sorted (e.g. by using a
fluorescence activated cell sorter--FACS) the compounds can be
identified by by direct analysis, for example by mass-spectroscopy.
If the compounds remain attached to beads isolated as a result of
selection (for example by affinity purification) or sorting (for
example using a FACS) they can also be identified by direct
analysis, for example by mass-spectroscopy. The microcapsules or
beads can also be tagged by a variety of means well known to those
skilled in the art and the tag used to identify the compound
attached to the beads (Czarnik, 1997). Chemical, spectrometric,
electronic, and physical methods to encode the compounds may all be
used. In a preferred embodiment microcapsules or beads have
different optical properties and are thereby optically encoded. In
a preferred embodiment encoding is based on microcapsules or beads
having different fluorescence properties. In a highly preferred
embodiment the microcapsules or beads are encoded using fluorescent
quantum dots present at different concentrations in the
microcapsule or bead (Han, 2001). Microcapsules flowing in an
ordered sequence in a microfluidic channel can also be encoded
(wholly or partially) by their sequence in the stream of
microcapsules (positional encoding).
[0196] Advantageously, each compounds is present in different
microcapsules at different concentrations (typically at
concentrations varying from mM to nM) allowing the generation of a
dose-response curve. This would, for example, allow the
determination of the inhibition constant (K.sub.i) of an inhibitory
compound. The concentration of the compounds in the microcapsules
can be determined by, for example, optical encoding or positional
encoding of the microcapsules or microbeads as above.
(E) Identification of Targets
[0197] Advantageously, multiple different targets can be
compartmentalised in microcapsules such that each microcapsule
contains multiple copies of the same target. For example, multiple
protein kinases, or multiple polymorphic variants of a single
target, can be compartmentalised to allow the specificity of
compounds to be determined. The identity of the target in a
microcapsule can be determined by, for example, optical encoding or
positional encoding of the microcapsules or microbeads as
above.
[0198] Expressed in an alternative manner, there is provided a
method for the synthesis and identification of compounds which bind
to a target component of a biochemical system or modulate the
activity of the target, comprising the steps of: [0199] (a)
compartmentalising two or more sets of primary compounds into
microcapsules together with the target such that many compartments
contain two or more primary compounds; [0200] (b) forming secondary
compounds in the microcapsules by chemical reactions between
primary compounds from different sets; and [0201] (c) identifying
subsets of primary compounds which react to form secondary
compounds which bind to or modulate the activity of the target.
[0202] There is also provided a method for the synthesis and
identification of compounds which bind to a target component of a
biochemical system or modulate the activity of the target,
comprising the steps of [0203] (1) attaching two or more sets of
primary compounds onto microbeads; [0204] (2) compartmentalising
the microbeads into microcapsules together with the target such
that many compartments contain two or more microbeads; [0205] (3)
releasing the primary compounds from the microbeads; [0206] (4)
forming secondary compounds in the microcapsules by chemical
reactions between primary compounds from different sets; and [0207]
(5) identifying subsets of primary compounds which react to form
secondary compounds which bind to or modulate the activity of the
target.
[0208] If the primary compounds react, not only with other primary
compounds in the same compartment, but also with other microbeads
in the compartment, the primary compounds which react together to
form a secondary compound can be identified by direct analysis of
the compounds present on a microbeads isolated as a result of
selection or sorting. For example, if the primary compounds are
linked to the beads via a disulphide bond when they are released in
the compartment the primary compounds will react both with each
other to form a secondary compound and with the sulphydryl groups
on the beads. Hence, if two beads are co-compartmentalised, each
bead will end up carrying both primary compounds. After isolation
of these beads both primary compounds which reacted to form the
secondary compound can be identified.
[0209] Various aspects and embodiments of the present invention are
illustrated in the following examples. It will be appreciated that
modification of detail may be made without departing from the scope
of the invention.
EXAMPLES
Example 1
Screening for Inhibitors of the Enzyme Protein Tyrosine Phosphatase
1B (PTP1B)
[0210] PTP1B is a negative regulator of insulin and leptin signal
transduction. Resistance to insulin and leptin are hallmarks of
type 2 diabetes mellitus and obesity and hence PTP1B is an
attractive drug target for diabetes and obesity therapy (Johnson et
al., 2002). Two water-in-oil emulsions are made as follows.
[0211] A solution of 1% (w/v) Span 60 and 1% (w/v) cholesterol in
decane (all from Sigma Aldrich) is prepared by dissolving 80 mg of
Span 60 and 80 mg of cholesterol into 7.84 ml of decane. The decane
is heated to 45.degree. C. to allow complete solubilization of the
surfactant and cholesterol. The surfactant/decane solution is
divided over batches of 200 .mu.l and placed in a block-heater at
37.degree. C.
[0212] A hand-extruding device (Mini extruder, Avanti Polar Lipids
Inc, Alabaster, Ala., USA) is assembled according to the
manufacturer's instructions. For extrusion, a single 19 mm
Track-Etch polycarbonate filter with average pore size of 14 .mu.m
(Whatman Nuclepore, Whatman, Maidstone, UK) is fitted inside the
mini extruder. Two gas-tight 1 ml Hamilton syringes (Gastight
#1001, Hamilton Co, Reno, Nev., USA) are used for extrusion. The
extruder was pre-rinsed with 3.times.1 ml of decane by loading one
of the Hamilton syringes with 1 ml of decane, placing the syringe
at one and of the mini extruder and extruding it through the
filters into the empty Hamilton syringe on the other side of the
extruder.
[0213] The first emulsion is made by loading 50 .mu.l of 100 .mu.M
compound 2 (FIG. 1), which has a bis-difluoromethylene phosphonate
and is a known PTP1B inhibitor (Johnson et al., 2002), the target
enzyme (human recombinant PTP1B, residues 1-322; Biomol Research
Laboratories, Inc.) at 5 mU/ml, the fluorogenic PTP1B substrate
6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) (Molecular
Probes), and 100 .mu.M Texas Red (Sigma; excitation/emmission
maxima 595/615 nm; red fluorescence) in a buffer compatible with
PTP1B activity (25 mM HEPES, pH 7.4, 125 mM NaCl, 10% glycerol, 1
mM EDTA) (Doman et al., 2002) into one of the Hamilton syringes,
and 200 .mu.l of the pre-heated decane/surfactant mix into the
other Hamilton syringe. The syringes are fitted into the openings
on both sides of the filter holder of the extruder. The compound
mix is forced through the filter holder into the alternate syringe
containing the decane/surfactant mix and directly forced back into
the original syringe to complete one round of extrusion. In total,
7.5 rounds of extrusion are completed. The filled syringe is
removed from the extruder and emptied into a 1.7 ml Axygen tube (#
MCT-175-C, Axygen Scientific, Inc., Union City, Calif., USA).
[0214] A second water-in-oil emulsion is made identical to the
emulsion above but containing 100 .mu.M hydrocinnamic acid
(Aldrich), a compound that is not a PTP1B inhibitor, in place of
compound 2, and 100 .mu.M calcein (Sigma; excitation/emmission
maxima 470/509 nm; green fluorescence) in place of Texas Red.
[0215] The two emulsions are mixed by vortexing in ratios varying
from 1:1000 to 1:1 (compound 2 emulsion:hydrocinnamic acid
emulsion) and incubated at 37.degree. C. for 30 min. Inhibitors
reduce the amount of non-fluorescent substrate (DiFMUP) converted
to the dephosphorylated product (DiFMU; excitation/emmission maxima
358/452 nm; blue fluorescence).
[0216] The water-in-oil emulsions are then converted into
water-in-oil-in water double emulsions as follows. The extruder
(see above) is disassembled, cleaned extensively with soap and
reversed-osmosis water, and re-assembled. A single 19 mm Track-Etch
polycarbonate filter with an average pore size of 8 .mu.m is
fitted. The extruder is pre-rinsed with 3.times.1 ml
phosphate-buffered saline solution (PBS). 750 .mu.l of PBS
containing 0.5% (w/v) Tween 80 (Sigma Aldrich) is loaded into a 1
ml gas-tight Hamilton syringe and fitted into the extruder. 250
.mu.l of the water-in-oil emulsion is loaded into the alternate 1
ml Hamilton syringe and fitted into the extruder. The emulsion is
forced through the filter into the alternate syringe containing the
PBS/0.5% Tween 80 and immediately forced back into the original
syringe to complete one cycle of extrusion. In total, 4.5 cycles of
extrusion are performed. The filled syringe is removed from the
extruder and emptied into a 1.7 ml Axygen tube. The
water-in-oil-in-water double emulsions formed are placed on
ice.
[0217] The double emulsions are then analysed by multi-colour flow
cytometery using a MoFlo (Cytomation) flow cytometer.
Predominantly, microcapsules exhibiting green fluorescence (and
containing hydrocinnamic acid) also show blue fluorescence due to
dephosphorylation of DiFMUP by PTP1B. Predominantly, microcapsules
exhibiting red fluorescence (and containing containing compound 2)
also show little or no blue fluorescence due to inhibition of
PTP1B.
Example 2
[0218] Two aqueous mixtures are made on ice (to prevent reaction).
The first mixture contains 100 .mu.M compound 2 (FIG. 1), which has
a bis-difluoromethylene phosphonate and is a known PTP1B inhibitor
(Johnson et al., 2002), the target enzyme (human recombinant PTP1B,
residues 1-322; Biomol Research Laboratories, Inc.) at 5 mU/ml, the
fluorogenic PTP1B substrate 6,8-difluoro-4-methylumbelliferyl
phosphate (DiFMUP) (Molecular Probes), and 100 .mu.M Texas Red
(Sigma; excitation/emmission maxima 595/615 nm; red fluorescence)
in a buffer compatible with PTP1B activity (25 mM HEPES, pH 7.4,
125 mM NaCl, 10% glycerol, 1 mM EDTA) (Doman et al., 2002). A
second mixture is created identical to the above but containing 100
.mu.M hydrocinnamic acid (Aldrich), a compound that is not a PTP1B
inhibitor, in place of compound 2, and 100 .mu.M calcein (Sigma;
excitation/emmission maxima 470/509 nm; green fluorescence) in
place of Texas Red.
[0219] 50 .mu.l of each of the compound mixtures is added
sequentially to a solution of 1% (w/v) Span 60 and 1% (w/v)
cholesterol in decane, made and held at 37.degree. C. as example 1,
whilst homogenising at 25,000 r.p.m. using an Ultra-Turrax T8
Homogenizer (IKA) with a 5 mm dispersing tool. Homogenisation is
continued for 3 minutes after the addition of the second aliquot.
The coarse emulsion produced is then extruded as in example 1 to
create a fine water-in-oil emulsion and incubated at 37.degree. C.
for 30 min. Inhibitors reduce the amount of non-fluorescent
substrate (DiFMUP) converted to the dephosphorylated product
(DiFMU; excitation/emmission maxima 358/452 nm; blue fluorescence).
The water-in-oil emulsion is then converted into a water-in-oil-in
water double emulsion and analysed by multi-colour flow cytometery
as in example 1. Predominantly, microcapsules exhibiting green
fluorescence (and containing hydrocinnamic acid) also show blue
fluorescence due to dephosphorylation of DiFMUP by PTP1B.
Predominantly, microcapsules exhibiting red fluorescence (and
containing containing compound 2) also show little or no blue
fluorescence due to inhibition of PTP1B.
Example 3
[0220] Screening of PTP1B Inhibitors from a Compound Library
[0221] 100 water-in-oil emulsions are made on ice (to prevent
reaction) as in example 1. The first emulsion is made by dispersing
a mixture of 100 .mu.M compound 2 (FIG. 1), which has a
bis-difluoromethylene phosphonate and is a known PTP1B inhibitor
(Johnson et al., 2002), the target enzyme (human recombinant PTP1B,
residues 1-322; Biomol Research Laboratories, Inc.) at 5 mU/ml, the
fluorogenic PTP1B substrate 6,8-difluoro-4-methylumbelliferyl
phosphate (DiFMUP) (Molecular Probes), and a pre-defined ratio of
Qdot.TM. Streptavidin Conjugates with emmission maxima at 585 nm,
655 nm and 705 nm (Quantum Dot Corporation, Hayward Calif.) in a
buffer compatible with PTP1B activity (25 mM HEPES, pH 7.4, 125 mM
NaCl, 10% glycerol, 1 mM EDTA) (Doman et al., 2002). The 99 other
water-in-oil emulsions are identical to the above but each contain
one of 99 carboxylic acids from the Carboxylic Acid Organic
Building Block Library (Aldrich) in place of compound 2, and
different ratios of Qdot.TM. Streptavidin Conjugates with emmission
maxima at 585 nm, 655 nm and 705 nm. In all emulsions the
concentration of the 705 nm Qdot.TM. Streptavidin Conjugates is 100
nM, and the concentrations of the 585 nm and 655 nm Qdot.TM.
Streptavidin Conjugates is either 0, 11, 22, 33, 44, 55, 66, 77, 88
or 100 nM. Hence, there are 100 (10.times.10) permutations of
Qdot.TM. Streptavidin Conjugate concentrations which allows the
microcapsules containing each compound to have a unique
fluorescence signature which is read by determining the
fluorescence ratios of fluorescence at at 705 nm, 585 nm and 655
nm.
[0222] The 100 emulsions are mixed in equal ratios by vortexing and
the temperature raised to 25.degree. C. for 30 min. Inhibitors
reduce the amount of non-fluorescent substrate (DiFMUP) converted
to the dephosphorylated product (DiFMU; excitation/emmission maxima
358/452 nm; blue fluorescence). The water-in-oil emulsion is then
converted into a water-in-oil-in water double emulsion and analysed
by multi-colour flow cytometery as in example 1n. Predominantly,
all microcapsules exhibited blue fluorescence due to
dephosphorylation of DiFMUP by PTP1B except those with the Qdot
fluorescence signature of the microcapsules containing compound
2.
Example 4
[0223] Screening of PTP1B Inhibitors from a Compound Library
[0224] 100 aqueous mixtures are made on ice (to prevent reaction).
The first mixture contains 100 .mu.M compound 2 (FIG. 1), which has
a bis-difluoromethylene phosphonate and is a known PTP1B inhibitor
(Johnson et al., 2002), the target enzyme (human recombinant PTP1B,
residues 1-322; Biomol Research Laboratories, Inc.) at 5 mU/ml, the
fluorogenic PTP1B substrate 6,8-difluoro-4-methylumbelliferyl
phosphate (DiFMUP) (Molecular Probes), and a pre-defined ratio of
Qdot.TM. Streptavidin Conjugates with emmission maxima at 585 nm,
655 nm and 705 nm (Quantum Dot Corporation, Hayward Calif.) in a
buffer compatible with PTP1B activity (25 mM HEPES, pH 7.4, 125 mM
NaCl, 10% glycerol, 1 mM EDTA) (Doman et al., 2002). The 99 other
aqueous mixtures are identical to the above but each contain one of
99 carboxylic acids from the Carboxylic Acid Organic Building Block
Library (Aldrich) in place of compound 2, and different ratios of
Qdot.TM. Streptavidin Conjugates with emission maxima at 585 nm,
655 nm and 705 nm. In all mixtures the concentration of the 705 nm
Qdot.TM. Streptavidin Conjugates is 100 nM, and the concentrations
of the 585 nm and 655 nm Qdot.TM. Streptavidin Conjugates is either
0, 11, 22, 33, 44, 55, 66, 77, 88 or 100 nM. Hence, there are 100
(10.times.10) permutations of Qdot.TM. Streptavidin Conjugate
concentrations which allows the microcapsules containing each
compound to have a unique fluorescence signature which is read by
determining the fluorescence ratio of fluorescence at at 705 nm,
585 nm and 655 nm.
[0225] 0.5 .mu.l of each of the compound mixtures is added
sequentially to a solution of 1% (w/v) Span 60 and 1% (w/v)
cholesterol in decane, made and held at 37.degree. C. as example 1,
whilst homogenising at 25,000 r.p.m. using an Ultra-Turrax T8
Homogenizer (IKA) with a 5 mm dispersing tool. Homogenisation is
continued for 3 minutes after the addition of the second aliquot.
The coarse emulsion produced is then extruded as in example 1 to
create a fine water-in-oil emulsion and incubated at 37.degree. C.
for 30 min. Inhibitors reduce the amount of non-fluorescent
substrate (DiFMUP) converted to the dephosphorylated product
(DiFMU; excitation/emmission maxima 358/452 nm; blue fluorescence).
The water-in-oil emulsion is then converted into a water-in-oil-in
water double emulsion and analysed by multi-colour flow cytometery
as in example 1. Predominantly, all microcapsules exhibited blue
fluorescence due to dephosphorylation of DiFMUP by PTP1B except
those with the Qdot fluorescence signature of the microcapsules
containing compound 2.
Example 5
Screening for PTP1B Inhibitors Using Microcapsules in Microfluidic
Systems
[0226] Microchannels are fabricated with rectangular cross-sections
using rapid prototyping in poly(dimethylsiloxane) (PDMS) (McDonald
and Whitesides, 2002) and rendered hydrophobic as (Song and
Ismagilov, 2003). Syringe pumps were used to drive flows (Harvard
Apparatus PHD 2000 Infusion pumps). For aqueous solutions, 50 .mu.l
Hamilton Gastight syringes (1700 series, TLL) with removeable
needles of 27-gauge are used with 30-gauge Teflon tubing (Weico
Wire and Cable). For the carrier fluid, 1 ml Hamilton Gastight
syringes (1700 series, TLL) are used with 30-gauge Teflon needles
with one hub from Hamilton (Song and Ismagilov, 2003). The carrier
fluid is 9% (v/v) C.sub.6F.sub.11C.sub.2H.sub.4OH in
perfluorodecaline (PFD) (Song et al., 2003). All water-soluble
reagents were dissolved in (25 mM HEPES, pH 7.4, 125 mM NaCl, 1 mM
EDTA), a buffer compatible with PTP1B activity.
[0227] A solution of the target enzyme (human recombinant PTP1B,
residues 1-322; Biomol Research Laboratories, Inc.) at 50 mU/ml and
a solution of either a) 100 .mu.M compound 2 (FIG. 1), which has a
bis-difluoromethylene phosphonate and is a known PTP1B inhibitor
(Johnson et al., 2002), or b) 100 .mu.M hydrocinnamic acid
(Aldrich), a compound that is not a PTP1B inhibitor are flowed in a
microchannel as two laminar streams, with an inert centre stream
(of 25 mM HEPES, pH 7.4, 125 mM NaCl, 1 mM EDTA) to separate them
and prevent the enzyme and compound coming into contact prior to
droplet microcapsule formation (Song et al., 2003). These three
steams are continuously injected into a flow of water immiscible
fluorocarbon carrier fluid (9% (v/v)
C.sub.6F.sub.11C.sub.2H.sub.4OH in PFD). Inlet channels for the
aqeous solutions are 50 .mu.m.sup.2 wide and channel for PFD is 28
.mu.m wide. A variety of PFD/water volumetric flow rates (in .mu.l
min.sup.-1) can be used including 0.6:0.3, 1.0:0.6, 12.3:3.7, 10:6
and 20:6, resulting in flow rates of 10, 19, 190, 190 and 300 mm
s.sup.-1 respectively. Aqueous microcapsules which occupy the
entire width of the channel are formed by drop-breakoff in the PFD
stream (Song et al., 2003). Microcapsules containing either
compound 2 or hydrocinnamic acid can be formed by switching between
injection with syringes containing compound 2 and hydrocinnamic
acid.
[0228] The channel immediately downstream of the point of droplet
formation is winding with a peak to peak distance of 50 .mu.m for a
distance of 1 mm. This results in rapid mixing of the contents of
the microcapsule by chaotic advection (Song et al., 2003). After
this point the microcapsules are run for up to 1 min through a 60
cm long microchannel (to allow inhibitor binding). This
microchannel is then merged with a 60.times.50 .mu.m.sup.2
microchannel containing aqueous microcapsules in (9% (v/v)
C.sub.6F.sub.11C.sub.2H.sub.4OH in PFD) formed as above. These
larger microcapsules contain the fluorogenic PTP1B substrate
6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) (Molecular
Probes) in 25 mM HEPES, pH 7.4, 125 mM NaCl, 1 mM EDTA. After the
junction between the microchannels the expanded main channel is
100.times.50 .mu.m.sup.2 and the microcapsules do not block the
channel and can move at different speeds until a large microcapsule
(containing DiFMUP) coalesces with a small microcapsule (containing
PTP1B and the compound) (Song et al., 2003). The frequency of
production of large and small microcapsules is equal such that each
large microcapsule has a small microcapsule with which to fuse. The
fused microcapsules are then run for up to 2 min through a 60 cm
long microchannel. Fluorescence of the microcapsules due to
production of DiFMU (excitation/emmission maxima 358/452 nm; blue
fluorescence) is measured using an epifluorescence microscope.
Predominantly, microcapsules exhibiting blue fluorescence are those
containing hydrocinnamic acid whereas microcapsules containing
compound 2 exhibit low fluorescence due to inhibition of PTP1B.
Example 6
Attachment of a Compound Library to Microbeads
[0229] 5.5 .mu.m diameter polystyrene microbeads that bear
carboxylate functional groups on the surface are commercially
available (www.luminexcorp.com) in an optically tagged form, as a
result of incorporation of precise ratios of orange (585 nm), and
red (>650 nm) fluorochromes (Fulton et al., 1997). A set of 100
such beads, each with a unique optical signature
(www.luminexcorp.com) are modified with an excess of
ethylenediamine and EDC
(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(Pierce) as (Hermanson, 1996) to create primary amino groups on the
surface. The photocleavable linker
4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy)butanoic acid
(NovaBiochem) (Holmes and Jones, 1995) is then attached to the
beads by forming an amide bond using EDC as above. 100 different
carboxylic acids from the Carboxylic Acid Organic Building Block
Library (Aldrich) are then coupled to the beads, by reacting with
the linker alcohol to form a carboxylate ester, each of the 100
different optically tagged beads being coupled to a different
carboxylic acid, and each bead being derivatised with
.about.10.sup.6 molecules of carboxylic acid. Irradiation for 4 min
on ice using a B100 AP 354 nm UV lamp (UVP) from a distance of
.about.5 cm results in release of the compounds from the beads as
carboxylic acids.
Example 7
Screening for Inhibitors of the Enzyme Protein Tyrosine Phosphatase
1B (PTP1B) Using Compounds Attached to Microbeads
[0230] PTP1B is a negative regulator of insulin and leptin signal
transduction. Resistance to insulin and leptin are hallmarks of
type 2 diabetes mellitus and obesity and hence PTP1B is an
attractive drug target for diabetes and obesity therapy (Johnson et
al., 2002). 5.5 .mu.m diameter polystyrene microbeads that bear
carboxylate functional groups on the surface are commercially
available (www.luminexcorp.com) in an optically tagged form, as a
result of incorporation of precise ratios of orange (585 nm), and
red (>650 nm) fluorochromes (Fulton et al., 1997). First, the
carboxylate functional groups on the microbeads are converted to
primary amines using ethylenediamine and EDC as in example 6. A
phosphopeptide substrate for PTP1B, the undecapaptide
EGFR.sub.988-998 (DADEpYLIPQQG) (Zhang et al., 1993), is then
coupled to both sets of microbeads via the surface amino groups
using EDC. This peptide is made by solid phase synthesis on Sieber
Amide resin (9-Fmoc-amino-xanthen-3-yloxy-Merrifield resin)
(Novabiochem) with orthogonal protection on the side chain
carboxylate groups using carboxylate-O-allyl esters. A linker
comprised of tetradecanedioic acid is coupled to the N-terminus and
the peptide cleaved from the beads using 1% TFA to yield a peptide
with a C-terminal amide The peptide is coupled to the beads (using
EDC) via the linker to give .about.10.sup.5 peptides per bead. The
remaining surface amino groups are then modified by attaching the
photochemically cleavable linker
4-(4-hydroxymethyl-2-methoxy-5-nitrophenoxy)butanoic acid as in
example 6. The protecting groups on the side chain carboxylates of
the peptide are then removed using
Pd(Ph.sub.3).sub.4/CHCl.sub.3/HOAc/N-methyl morpholine. A first set
of microbeads is derivatised with
3-(4-difluorophosphonomethylphenyl)propanoic acid (compound 1, FIG.
1), a compound that is a known PTP1B inhibitor (Johnson et al.,
2002). A second set of beads, with a distinct optical tag from the
first set of beads, is derivatised with hydrocinnamic acid
(Aldrich), a compound that is not a PTP1B inhibitor. In each case
the compound is coupled by reacting with the linker alcohol to form
a carboxylate ester as in example 6. Each microbead is derivatised
with .about.10.sup.6 molecules (Fulton et al., 1997).
[0231] The microbeads are then screened using the method outlined
in FIG. 2. The two sets of microbeads are mixed in ratios varying
from 1:1000 to 1:1 (compound 1 beads:hydrocinnamic acid beads) and
10.sup.8 total microbeads are mixed with the target enzyme (human
recombinant PTP1B, residues 1-322; Biomol Research Laboratories,
Inc.) at a concentration of 10 nM, on ice (to prevent reaction) in
a buffer compatible with PTP1B activity (25 mM HEPES, pH 7.4, 125
mM NaCl, 10% glycerol, 1 mM EDTA) (Doman et al., 2002). Single
beads and target enzyme (PTP1B) are then colocalised in
microcapsules by forming a water-in-oil emulsion (also on ice). The
concentration of beads is such that most microcapsules contain one
or no beads. The compound is released photochemically (as in
example 6) and the temperature raised to 25.degree. C. Inhibitors
reduce the amount of substrate converted to product
(dephosphorylated peptide). The emulsion is cooled to 4.degree. C.
and broken as (Griffiths and Tawfik, 2003) into 100 .mu.M vanadate
to stop the reaction (Harder et al., 1994). After labelling with an
anti-substrate (anti-phosphotyrosine) antibody labelled with the
green (530 nm) fluorochrome fluorescein isothiocyanate (mouse
monoclonal IgG.sub.2b PY20 (Santa Cruz) according to the
manufacturer's instructions, beads are analysed by 3-colour flow
cytometry using a FACScan (Becton-Dickinson), FACScalibur
(Becton-Dickinson) or MoFlo (Cytomation) flow cytometers to
simultaneously determine the extent of inhibition and the compound
on the beads. Predominantly, dephosphorylation of the peptide is
only observed on those microbeads which were coated with PTP1B
inhibitors, and not on other microbeads.
Example 8
[0232] Screening of PTP1B Inhibitors from a Compound Library
Attached to Microbeads
[0233] A set of 100 5.5 .mu.m diameter polystyrene microbeads,
bearing carboxylate functional groups on the surface and each with
a unique optical signature (www.luminexcorp.com) as a result of
incorporation of precise ratios of orange (585 nm), and red
(>650 nm) fluorochromes (Fulton et al., 1997) are derivatised
with a phosphopeptide substrate for PTP1B, the undecapaptide E
GFR.sub.988-998 (DADEpYLIPQQO) (Zhang et al., 1993), and 100
different carboxylic acids, each attached via a photochemically
cleavable linker, as in example 7. One of these carboxylic acids is
3-(4-difluorophosphonomethylphenyl) propanoic acid (compound 1,
FIG. 1), a compound that is a known PTP1B inhibitor (Johnson et
al., 2002). The other 99 carboxylic acids are from the Carboxylic
Acid Organic Building Block Library (Aldrich) as example 6. Equal
numbers of each of the 100 bead sets are then mixed and screened as
for example 7. Predominantly, dephosphorylation of the peptide is
only observed on those microbeads which were coated with the PTP1B
inhibitor 3-(4-difluorophosphonomethylphenyl) propanoic acid
(compound 1, FIG. 1), and not on microbeads coated with other
compounds.
Example 9
Synthesis of Secondary Compounds in Emulsion Microcapsules and
Screening for PTP1B Inhibition in Microfluidic Systems
[0234] Microchannels are fabricated with rectangular cross-sections
using rapid prototyping in poly(dimethylsiloxane) (PDMS) (McDonald
and Whitesides, 2002) and rendered hydrophobic as (Song and
Ismagilov, 2003). Syringe pumps were used to drive flows (Harvard
Apparatus PHD 2000 Infusion pumps). For aqueous solutions, 50 .mu.l
Hamilton Gastight syringes (1700 series, TLL) with removeable
needles of 27-gauge are used with 30-gauge Teflon tubing (Weico
Wire and Cable). For the carrier fluid, 1 ml Hamilton Gastight
syringes (1700 series, TLL) are used with 30-gauge Teflon needles
with one hub from Hamilton (Song and Ismagilov, 2003). The carrier
fluid is 9% (v/v) C.sub.6F.sub.11C.sub.2H.sub.4OH in
perfluorodecaline (PFD) (Song et al., 2003). All water-soluble
reagents were dissolved in (25 mM HEPES, pH 7.4, 125 mM NaCl, 1 mM
EDTA), a buffer compatible with PTP1B activity.
[0235] A solution of the target enzyme (human recombinant PTP1B,
residues 1-322; Biomol Research Laboratories, Inc.) at 50 mU/ml, a
solution of a compound which is a primary amine, and a solution of
a compound which is an aldehyde are flowed in a microchannel as
three laminar streams, with two inert separating streams (of 25 mM
HEPES, pH 7.4, 125 mM NaCl, 1 mM EDTA) to prevent the enzyme and
the compounds coming into contact prior to droplet microcapsule
formation (Song et al., 2003). These five streams are continuously
injected into a flow of water immiscible fluorocarbon carrier fluid
(9% (v/v) C.sub.6F.sub.11C.sub.2H.sub.4OH in PFD). The amines and
aldehydes can either a) contain a difluoromethylene phosphonate
moiety (FIG. 6, compounds A and B), or b) contain no
difluoromethylene phosphonate moiety.
[0236] Inlet channels for the aqeous solutions are 50 .mu.m.sup.2
wide and the channel for PFD is 28 .mu.m wide. A variety of
PFD/water volumetric flow rates (in .mu.l min.sup.-1) can be used
including 0.6:0.3, 1.0:0.6, 12.3:3.7, 10:6 and 20:6, resulting in
flow rates of 10, 19, 190, 190 and 300 mm s.sup.-1 respectively.
Aqueous microcapsules which occupy the entire width of the channel
are formed by drop-breakoff in the PFD stream (Song et al., 2003).
Microcapsules containing compounds with and without
difluoromethylene phosphonate moieties can be formed by switching
between injection with syringes containing amines or aldehydes with
or without a difluoromethylene phosphonate moiety.
[0237] The channel immediately downstream of the point of droplet
formation is winding with a peak to peak distance of 50 .mu.m for a
distance of 1 mm. This results in rapid mixing of the contents of
the microcapsule by chaotic advection (Song et al., 2003). After
this point the microcapsules are run for up to 1 min through a 60
cm long microchannel. This allows the amine and the aldehyde to
react together by formation of a Schiff base to create a secondary
compound and allows inhibitors to bind to PTP1B. This microchannel
is then merged with a 60.times.50 .mu.m.sup.2 microchannel
containing aqueous microcapsules in (9% (v/v)
C.sub.6F.sub.11C.sub.2H.sub.4OH in PFD) formed as above. These
larger microcapsules contain the fluorogenic PTP1B substrate
6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) (Molecular
Probes) in 25 mM HEPES, pH 7.4, 125 mM NaCl, 1 mM EDTA. After the
junction between the microchannels the expanded main channel is
100.times.50 .mu.m.sup.2 and the microcapsules do not block the
channel and can move at different speeds until a large microcapsule
(containing DiFMUP) coalesces with a small microcapsule (containing
PTP1B and the compound) (Song et al., 2003). The frequency of
production of large and small microcapsules is equal such that each
large microcapsule has a small microcapsule with which to fuse. The
fused microcapsules are then run for up to 2 min through a 60 cm
long microchannel. Fluorescence of the microcapsules due to
production of DiFMU (excitation/emmission maxima 358/452 nm; blue
fluorescence) is measured using an epifluorescence microscope.
[0238] Predominantly, when the amine and aldehyde concentrations
are low (<100 .mu.M) inhibition of PTP1B activity is only
observed in microcapsules containing both an amine with a
difluoromethylene phosphonate moiety (compound A, FIG. 6) and an
aldehyde with a difluoromethylene phosphonate moiety (compound B,
FIG. 6). This is because the Schiff base formed in these
microcapsules (compound C, FIG. 6) contains bis-difluoromethylene
phosphonate and is a much more potent PTP1B inhibitor than a
molecule with a single difluoromethylene phosphonate moiety (see
FIG. 1).
[0239] Predominantly, when the amine and aldehyde concentrations
are high (>100 .mu.M) inhibition of PTP1B activity is observed
in microcapsules containing either an amine with a
difluoromethylene phosphonate moiety (compound A, FIG. 6) or an
aldehyde with a difluoromethylene phosphonate moiety (compound B,
FIG. 6), or both, but not in other microcapsules. This is because
at higher concentrations molecules with either a single
difluoromethylene phosphonate moiety or a bis-difluoromethylene
phosphonate (compound C, FIG. 6) can inhibit PTP1B (see FIG.
1).
Example 10
Synthesis of Secondary Compounds in Emulsion Microcapsules and
Screening for PTP1B Inhibition Using Compounds Attached to
Microbeads
[0240] 5.5 .mu.m diameter polystyrene microbeads that bear
carboxylate functional groups on the surface are commercially
available (www.luminexcorp.com) in an optically tagged form, as a
result of incorporation of precise ratios of orange (585 nm), and
red (>650 nm) fluorochromes (Fulton et al., 1997). First, the
carboxylate functional groups on the microbeads are converted to
primary amines using ethylenediamine and EDC as in example 6. A
phosphopeptide substrate for PTP1B, the undecapaptide
EGFR.sub.988-998 (DADEpYLIPQQG) (Zhang et al., 1993), is then
coupled to both sets of microbeads via the surface amino groups
using EDC and the protecting groups on the side chain carboxylates
of the peptide removed as in example 7. A first set of microbeads
(set 1) is reacted with succinimidyl p-formylbenzoate to convert
the surface amino groups to aldehydes. A second set of microbeads
(set 2), with a distinct optical tag from the first set of
microbeads, is left unreacted (i.e. with primary amines on the
surface).
[0241] The first set of microbeads (set 1), are then reacted with a
compound containing a difluoromethylene phosphonate moiety and a
primary amine (compound A, FIG. 6) via reaction with the surface
aldehyde groups to form a Schiff base. The second set of microbeads
(set 2), with a distinct optical tag from the first set of beads,
are reacted with a compound containing a difluoromethylene
phosphonate moiety and an aldehyde (compound B, FIG. 6) via
reaction of the aldehyde with the surface amine groups to form a
Schiff base. The formation of Schiff bases is enhanced by reaction
at alkaline pH (i.e. pH9-10). Microbeads coated with compounds at
various densities are created.
[0242] The two sets of microbeads are mixed with the target enzyme
(human recombinant PTP1B, residues 1-322; Biomol Research
Laboratories, Inc.) at a concentration of 10 nM, on ice (to prevent
reaction) in a buffer compatible with PTP1B activity (25 mM HEPES,
pH 7.4, 125 mM NaCl, 10% glycerol, 1 mM EDTA) (Doman et al., 2002).
The beads and target enzyme (PTP1B) are immediately
compartmentalised in microcapsules by forming a water-in-oil
emulsion (also on ice).
[0243] The number of microbeads is varied such that, at one
extreme, most microcapsules contain one or no beads, and at the
other, the majority of microcapsules contain two or more
microbeads. The temperature raised to 25.degree. C. The Schiff base
is a relatively labile, reversible interaction, readily hydrolysed
at neutral pH, resulting in release of compounds from the beads. In
microcapsules containing a microbead from both set 1 and set 2, the
compounds released from the microbeads can react with each other,
forming a Schiff base and creating a new molecule in solution. This
new molecule (FIG. 6, compound C) contains a bis-difluoromethylene
phosphonate moiety and has significantly more potency as a PTP1B
inhibitor than compounds with a single difluoromethylene
phosphonate moiety (see FIG. 1). Inhibitors reduce the amount of
substrate converted to product (dephosphorylated peptide). The
emulsion is cooled to 4.degree. C. and broken as (Griffiths and
Tawfik, 2003) into 100 .mu.M vanadate to stop the reaction (Harder
et al., 1994). After labelling with an anti-substrate
(anti-phosphotyrosine) antibody labelled with the green (530 nm)
fluorochrome fluorescein isothiocyanate (mouse monoclonal
IgG.sub.2b PY20 (Santa Cruz) according to the manufacturer's
instructions, beads are analysed by 3-colour flow cytometry using a
FACScan (Becton-Dickinson), FACScalibur (Becton-Dickinson) or MoFlo
(Cytomation) flow cytometers to simultaneously determine the extent
of inhibition and the compound on the beads. With low microbead
numbers, most microcapsules contain only a single or no microbeads
and PTP1B inhibition is only detected on beads coated with a high
density of inhibitor, when the concentration of inhibitor released
into solution in each microcapsule is sufficiently high for
effective inhibition. In contrast, when the bead numbers are
higher, many microbeads are detected where little substrate has
been converted to product, even when the microbeads are coated with
inhibitor at low density. This is due to the formation of a highly
potent PTP1B inhibitor (FIG. 6, compound C) containing a
bis-difluoromethylene phosphonate moiety in microcapsules
containing a microbead each from set 1 and set 2.
Example 11
Synthesis of a Library 2500 Secondary Compounds in Emulsion
Microcapsules and Screening for PTP1B Inhibition Using Compounds
Attached to Microbeads
[0244] A set of 100 5.5 .mu.m diameter polystyrene microbeads,
bearing carboxylate functional groups on the surface and each with
a unique optical signature (www.luminexcorp.com) as a result of
incorporation of precise ratios of orange (585 nm), and red
(>650 nm) fluorochromes (Fulton et al., 1997) are modified to
convert the carboxylate functional groups to primary amines as in
example 6, then derivatised with a phosphopeptide substrate for
PTP1B, the undecapaptide EGFR.sub.988-998 (DADEpYLIPQQG) (Zhang et
al., 1993), as in example 10. The first 50 sets of microbeads are
reacted to convert a proportion of the surface carboxyl groups to
aldehydes as in example 10. The second 50 sets of microbeads are
left unreacted (i.e. with primary amines on the surface).
[0245] The first 50 sets of microbeads are each reacted with a
unique compound containing a primary amine via reaction with the
surface aldehyde groups to form a Schiff base which links the
compounds to the beads. One of these compounds (compound A, FIG. 6)
contains a difluoromethylene phosphonate moiety. The second 50 sets
of microbeads are each reacted with a unique compound containing an
aldehyde via reaction with the surface amine groups to form a
Schiff base which links the compounds to the beads. One of these
compounds (compound B, FIG. 6) contains a difluoromethylene
phosphonate moiety. The formation of Schiff bases is enhanced by
reaction at alkaline pH (i.e. pH9-10).
[0246] The two sets of microbeads are mixed with the target enzyme
(human recombinant PTP1B, residues 1-322; Biomol Research
Laboratories, Inc.) at a concentration of 10 nM, on ice (to prevent
reaction) in a buffer compatible with PTP1B activity (25 mM HEPES,
pH 7.4, 125 mM NaCl, 10% glycerol, 1 mM EDTA) (Doman et al., 2002).
The beads and target enzyme (PTP1B) are immediately
compartmentalised in microcapsules by forming a water-in-oil
emulsion (also on ice).
[0247] The number of microbeads is such that the modal number of
microcapsules per microcapsule is two. The temperature raised to
25.degree. C. The Schiff base is a relatively labile, reversible
interaction, readily hydrolysed at neutral pH, resulting in release
of compounds from the beads. In microcapsules containing a
microbead from one of the first 50 sets and a microbead from one of
the second 50 sets, the compounds released from the microbeads can
react with each other, forming a Schiff base and creating a new
molecule in solution. Inhibitors reduce the amount of substrate
converted to product (dephosphorylated peptide). The emulsion is
cooled to 4.degree. C. and broken as (Griffiths and Tawfik, 2003)
into 100 .mu.M vanadate to stop the reaction (Harder et al., 1994).
After labelling with an anti-substrate (anti-phosphotyrosine)
antibody labelled with the green (530 nm) fluorochrome fluorescein
isothiocyanate (mouse monoclonal IgG.sub.2b PY20 (Santa Cruz)
according to the manufacturer's instructions, beads are analysed by
3-colour flow cytometry using a FACScan (Becton-Dickinson),
FACScalibur (Becton-Dickinson) or MoFlo (Cytomation) flow
cytometers to simultaneously determine the extent of inhibition and
the compound on the beads. Beads which were coated with a primary
compound which is itself a PTP1B inhibitor, or which reacts with
another primary compound released from another co-compartmentalised
bead to form a second inhibitor are identified as little substrate
has been converted to product. The identified beads where as little
substrate has been converted to product include those carrying
compounds containing a difluoromethylene phosphonate moiety. When
the microbeads are coated with compounds at low density the
concentration of the released compounds containing a
difluoromethylene phosphonate moieties in the microcapsules is
insufficient to efficiently inhibit PTP1B (see example 7). However
in microcapsules containing two microbeads, one from the first set
of 50 beads and one from the second set of 50 beads, and where each
microbead carries a molecule with a difluoromethylene phosphonate
moiety, the released molecules can form a highly potent PTP1B
inhibitor (compound C, FIG. 6) containing a bis-difluoromethylene
phosphonate moiety in the microcapsules which inhibits conversion
of the PTP1B substrate to product.
Example 12
Compartmentalisation of Small Molecules in a Water-in-Fluorocarbon
Emulsions.
[0248] Water-in-fluorocarbon emulsions containing 95% (v/v)
perfluorooctyl bromide, 5% (v/v) phosphate buffered saline
containing the molecule of interest in solution, and 2% (w/v)
C.sub.8F.sub.17C.sub.11H.sub.22OP(O)[N(CH.sub.2CH.sub.2).sub.2O].sub.2
(F8H11DMP) as surfactant were formed essentially as (Sadder et al.,
1996) by extrusion (15 times) through 14 .mu.m filters (Osmonics)
or by homogenising for 5 min at 25,000 r.p.m. using an Ultra-Turrax
T8 Homogenizer (IKA) with a 5 mm dispersing tool. Emulsions were
made containing a series of small fluorescent molecules dissolved
in the aqueous phase at concentrations from 100 .mu.m to 2 mM.
These molecules, including calcein, texas red, fluorescein,
coumarin 102, 7-hydroxycoumarin-3-carboxylic acid and
7-diethylamino-4-methyl coumarin (coumarin 1), had molecular
weights from 203 to 625 Da and Log P values--calculated using SRC's
LogKow/KowWin Program (Meylan and Howard, 1995)--ranging from -0.49
to 4.09. Emulsions containing different coloured fluorochromes were
mixed by vortexing. Compartmentalisation was observed by
epifluorescence microscopy of the mixed emulsions. No exchange
between compartments was observed 24 hours after mixing (see FIG.
5).
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[0358] All publications mentioned in the above specification, and
references cited in said publications, are herein incorporated by
reference. Various modifications and variations of the described
methods and system of the invention will be apparent to those
skilled in the art without departing from the scope and spirit of
the invention. Although the invention has been described in
connection with specific preferred embodiments, it should be
understood that the invention as claimed should not be unduly
limited to such specific embodiments. Indeed, various modifications
of the described modes for carrying out the invention which are
obvious to those skilled in molecular biology or related fields are
intended to be within the scope of the following claims.
* * * * *
References