U.S. patent application number 10/386198 was filed with the patent office on 2003-12-04 for method for producing and screening mass-coded combinatorial libraries for drug discovery and target validation.
This patent application is currently assigned to Neogenesis, Inc., a Delaware corporation. Invention is credited to Birnbaum, Seth, Jindal, Satish, Kalghatgi, Krishna, Nash, Huw M., Shipps, Gerald, Wintner, Edward A..
Application Number | 20030224409 10/386198 |
Document ID | / |
Family ID | 31996411 |
Filed Date | 2003-12-04 |
United States Patent
Application |
20030224409 |
Kind Code |
A1 |
Nash, Huw M. ; et
al. |
December 4, 2003 |
Method for producing and screening mass-coded combinatorial
libraries for drug discovery and target validation
Abstract
The present invention provides a method for producing a
mass-coded combinatorial library comprising a set of compounds
having the general formula X(Y).sub.n, where X is a scaffold, each
Y is, independently, a peripheral moiety, and n is an integer
greater than 1. The method comprises selecting a peripheral moiety
precursor subset from a peripheral moiety precursor set. The subset
includes a sufficient number of peripheral moiety precursors that
at least about 50 distinct combinations of n peripheral moieties
derived from the peripheral moiety precursors in the subset exist.
The subset of peripheral moiety precursors is selected so that at
least about 90% of all possible combinations of n peripheral
moieties derived from the subset have a molecular mass sum which is
distinct from the molecular mass sums of all of the other
combinations of n peripheral moieties. The method further comprises
contacting the peripheral moiety precursor subset with a scaffold
precursor which has n reactive groups. Methods of use of the
mass-coded combinatorial library produced by this method for
identifying a ligand to a particular biomolecule are also
disclosed.
Inventors: |
Nash, Huw M.; (Cambridge,
MA) ; Birnbaum, Seth; (Boston, MA) ; Wintner,
Edward A.; (Cambridge, MA) ; Kalghatgi, Krishna;
(Westborough, MA) ; Shipps, Gerald; (Stoneham,
MA) ; Jindal, Satish; (Milton, MA) |
Correspondence
Address: |
FISH & RICHARDSON PC
225 FRANKLIN ST
BOSTON
MA
02110
US
|
Assignee: |
Neogenesis, Inc., a Delaware
corporation
|
Family ID: |
31996411 |
Appl. No.: |
10/386198 |
Filed: |
March 11, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10386198 |
Mar 11, 2003 |
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09373018 |
Aug 11, 1999 |
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09373018 |
Aug 11, 1999 |
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09024592 |
Feb 17, 1998 |
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6207861 |
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60070456 |
Jan 5, 1998 |
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Current U.S.
Class: |
435/6.12 ;
435/6.13; 435/7.1; 530/324; 536/23.1; 560/1 |
Current CPC
Class: |
B01J 2219/00725
20130101; B01J 2219/00585 20130101; C40B 20/04 20130101; G01N
2458/15 20130101; C40B 70/00 20130101; B01J 2219/00722 20130101;
C40B 40/06 20130101; G01N 33/6845 20130101; B01J 2219/00599
20130101; B01J 2219/00695 20130101; B01J 2219/00596 20130101; G01N
33/6848 20130101; B01J 19/0046 20130101; C40B 30/10 20130101; B01J
2219/00581 20130101 |
Class at
Publication: |
435/6 ; 435/7.1;
560/1; 530/324; 536/23.1 |
International
Class: |
C12Q 001/68; G01N
033/53; C07H 021/04; C07C 069/74 |
Claims
We claim:
1. A method for producing a mass-coded set of compounds of the
general formula X(Y).sub.n, wherein X is a scaffold, n is from 2 to
about 6, and each Y is, independently, a peripheral moiety,
comprising the steps of: (a) selecting a peripheral moiety
precursor subset from a peripheral moiety precursor set, said
subset comprising a sufficient number of peripheral moiety
precursors that there exist at least about 250 distinct
combinations of n peripheral moieties derived from said subset,
wherein at least about 90% of said combinations of n peripheral
moieties derived from said subset have molecular mass sums which
are distinct from the molecular mass sums of all other combinations
of n peripheral moieties derived from said subset; and (b)
contacting said peripheral moiety precursor subset with a scaffold
precursor, said scaffold precursor having n reactive groups,
wherein each reactive group is capable of reacting with at least
one peripheral moiety precursor to form a covalent bond, under
conditions sufficient for the reaction of each reactive group with
a peripheral moiety precursor, thereby producing a mass-coded set
of compounds of the general formula X(Y).sub.n.
2. The method of claim 1 wherein the scaffold precursor comprises
one or more saturated, partially unsaturated or aromatic cyclic
groups.
3. The method of claim 2 wherein at least one cyclic group is
substituted by one or more reactive groups.
4. The method of claim 3 wherein the reactive groups are attached
to the cyclic group directly or via an intervening
C.sub.1-6-alkylene group.
5. The method of claim 4 wherein each reactive group is
independently selected from the group consisting of: reactive
carbonyl groups, reactive sulfonyl groups, reactive phosphonyl
groups, terminal epoxide group and the isocyanate group.
6. The method of claim 5 wherein the reactive group is selected
from the group consisting of: carbonyl chloride, carbonyl
pentafluorophenyl ester and sulfonyl chloride.
7. The method of claim 5 wherein at least one peripheral moiety
precursor comprises a primary amino group, a secondary amino group
or a hydroxyl group.
8. The method of claim 4 wherein each reactive group is
independently selected from the group consisting of: primary amino,
secondary amino and hydroxyl.
9. The method of claim 8 wherein at least one peripheral moiety
precursor comprises a reactive carbonyl group, reactive sulfonyl
group, reactive phosphonyl group, terminal epoxide group or an
isocyanate group.
10. The method of claim 9 wherein at least one peripheral moiety
precursor comprises a carbonyl chloride, a carbonyl
pentafluorophenyl ester or a sulfonyl chloride group.
11. A method as claimed in claim 1 wherein the step of selecting
includes the steps of: (a) choosing every set of two different
peripheral moiety precursors from the peripheral moiety precursor
set, said choosing performed in a manner such that for each set of
two, if the two peripheral moiety precursors have equal molecular
masses then one of the two is removed forming a remaining set; (b)
from the remaining set, choosing every set of four peripheral
moiety precursors, including for a given set of four, removing one
of the four peripheral moiety precursors if a sum of the molecular
masses of a first two precursors in the given set of four equals a
sum of the molecular masses of a second two precursors in the given
set of four peripheral moiety precursors, said choosing forming a
remainder set; (c) from the remainder set, choosing every set of
six different peripheral moiety precursors, including for a given
set of six, removing one of the six peripheral moiety precursors if
a sum of the molecular masses of a first three precursors in the
given set of six equals a sum of the molecular masses of a second
three precursors in the given set of six, said choosing forming a
working selection set of peripheral moiety precursors from which to
select a desired subset; and (d) from the working selection set,
choosing a desired subset so as to provide the selected subset by
(i) choosing a possible selected subset from the working selection
set, (ii) from the chosen possible subset, generating all possible
combinations of n peripheral moiety precursors, and (iii)
determining whether the generated combinations have an acceptable
percent mass redundancy, and if so, selecting the chosen possible
subset as the selected subset.
12. A method as claimed in claim 11 wherein the step of selecting
is performed by a digital processor assembly.
13. A method as claimed in claim 1 wherein the step of selecting
includes selecting a peripheral moiety precursor subset which
includes peripheral moiety precursors that simultaneously produce
mass-coded compounds when contacted with a scaffold precursor.
14. A method as claimed in claim 13 wherein the step of selecting
comprises the steps of: (a) choosing every set of two different
peripheral moiety precursors from the peripheral moiety precursor
set, said choosing performed in a manner such that for each set of
two, if the two peripheral moiety precursors have equal molecular
masses then one of the two is removed forming a remaining set; (b)
from the remaining set, choosing every set of four different
peripheral moiety precursors, including for a given set of four,
removing one of the four peripheral moiety precursors if a sum of
the molecular masses of a first two precursors in the given set of
four equals a sum of the molecular masses of a second two
precursors in the given set of four peripheral moiety precursors,
said choosing forming a remainder set; (c) from the remainder set,
choosing every set of six different peripheral moiety precursors,
including for a given set of six, removing one of the six
peripheral moiety precursors if a sum of the molecular masses of a
first three precursors in the given set of six equals a sum of the
molecular masses of a second three precursors in the given set of
six, said choosing forming a working selection set of peripheral
moiety precursors from which to select a desired subset; and (d)
from the working selection set, choosing a desired subset so as to
provide the selected subset by (i) choosing a possible selected
subset from the working selection set, (ii) from the chosen
possible subset, generating all possible combinations of n
peripheral moiety precursors, and (iii) determining whether the
generated combinations have an acceptable percent mass redundancy,
and if so, selecting the chosen possible subset as the selected
subset.
15. A method as claimed in claim 14 wherein the step of selecting
is performed by a digital processor assembly.
16. A method for identifying a member of a mass-coded combinatorial
library which is a ligand for a biomolecule, said mass-coded
molecular library comprising compounds of the general formula
XY.sub.n, wherein n is an integer from 2 to about 6, X is a
scaffold and each Y is, independently, a peripheral moiety, wherein
said mass-coded combinatorial library is produced by reacting a
scaffold precursor with a sufficient number of distinct peripheral
moiety precursors such that there exist at least about 250 distinct
combinations of n peripheral moieties derived from said peripheral
moiety precursors, said method comprising the steps of: (a)
contacting the biomolecule with the mass-coded molecular library,
whereby members of the mass-coded molecular library which are
ligands for the biomolecule bind to the biomolecule to form
biomolecule-ligand complexes and members of the mass-coded library
which are not ligands for the biomolecule remain unbound; (b)
separating the biomolecule-ligand complexes from the unbound
members of the mass-coded molecular library; (c) dissociating the
biomolecule-ligand complexes; and (d) determining the molecular
mass of each ligand to identify the set of n peripheral moieties
present in each ligand, wherein the molecular mass of each ligand
corresponds to a set of n peripheral moieties present in that
ligand, thereby identifying a member of the mass-coded
combinatorial library which is a ligand for the biomolecule.
17. The method of claim 16 wherein the biomolecule is immobilized
on a solid support.
18. The method of claim 17 wherein the solid support is a
water-insoluble matrix contained within a chromatographic
column.
19. The method of claim 16 wherein a solution comprising the
biomolecule is contacted with the mass-coded molecular library to
form, if one or members of the mass-coded molecular library are
ligands for the biomolecule, a solution comprising
biomolecule-ligand complexes and unbound members of the mass-coded
molecular library.
20. The method of claim 19 wherein the unbound members of the
mass-coded molecular library are separated from the
biomolecule-ligand complexes by directing the solution comprising
biomolecule-ligand complexes and the unbound members of the
mass-coded molecular library through a size exclusion
chromatography column, whereby the unbound members of the
mass-coded molecular library elute from said column after the
biomolecule-ligand complexes.
21. The method of claim 19 wherein the unbound members of the
mass-coded molecular library are separated from the
biomolecule-ligand complexes by contacting the solution comprising
biomolecule-ligand complexes and the unbound members of the
mass-coded molecular library with a size-exclusion membrane,
whereby the unbound combounds pass through said membrane and the
biomolecule-ligand complexes do not pass through said membrane.
22. The method of claim 16 wherein the biomolecule is a protein or
a nucleic acid molecule.
23. A method for identifying a member of a mass-coded combinatorial
library which is a ligand for a biomolecule and assessing the the
effect of the binding of the ligand to the biomolecule, said
mass-coded molecular library comprising compounds of the general
formula XY.sub.n, wherein n is an integer from 2 to about 6, X is a
scaffold and each Y is, independently, a peripheral moiety, wherein
said mass-coded combinatorial library is produced by reacting a
scaffold precursor with a sufficient number of distinct peripheral
moiety precursors such that there exist at least about 250 distinct
combinations of n peripheral moieties derived from said peripheral
moiety precursors, said method comprising the steps of: (a)
contacting the biomolecule with the mass-coded molecular library,
whereby members of the mass-coded molecular library which are
ligands for the biomolecule bind to the biomolecule to form
biomolecule-ligand complexes and members of the mass-coded library
which are not ligands for the biomolecule remain unbound; (b)
separating the biomolecule-ligand complexes from the unbound
members of the mass-coded molecular library; (c) dissociating the
biomolecule-ligand complexes; (d) determining the molecular mass of
each ligand to identify the set of n peripheral moieties present in
each ligand, wherein the molecular mass of each ligand corresponds
to a set of n peripheral moieties present in that ligand, thereby
identifying a member of the mass-coded combinatorial library which
is a ligand for the biomolecule; and (e) assessing in an in vitro
assay the effect of the binding of the ligand to the biomolecule on
the function of the biomolecule.
24. The method of claim 23 wherein the in vitro assay is a cell
proliferation assay, a cell death assay or a viral replication
assay.
25. The method of claim 23 wherein the biomolecule is a protein or
a nucleic acid molecule.
26. A method for identifying a member of a mass-coded combinatorial
library which is a ligand for a biomolecule and assessing the the
effect of the binding of the ligand to the biomolecule, said
mass-coded molecular library comprising compounds of the general
formula XY.sub.n, wherein n is an integer from 2 to about 6, X is a
scaffold and each Y is, independently, a peripheral moiety, wherein
said mass-coded combinatorial library is produced by reacting a
scaffold precursor with a sufficient number of distinct peripheral
moiety precursors such that there exist at least about 250 distinct
combinations of n peripheral moieties derived from said peripheral
moiety precursors, said method comprising the steps of: (a)
contacting the biomolecule with the mass-coded molecular library,
whereby members of the mass-coded molecular library which are
ligands for the biomolecule bind to the biomolecule to form
biomolecule-ligand complexes and members of the mass-coded library
which are not ligands for the biomolecule remain unbound; (b)
separating the biomolecule-ligand complexes from the unbound
members of the mass-coded molecular library; (c) dissociating the
biomolecule-ligand complexes; (d) determining the molecular mass of
each ligand to identify the set of n peripheral moieties present in
each ligand, wherein the molecular mass of each ligand corresponds
to a set of n peripheral moieties present in that ligand, thereby
identifying a member of the mass-coded combinatorial library which
is a ligand for the biomolecule; and (e) assessing in an in vivo
assay the effect of the binding of the ligand to the biomolecule on
the function of the biomolecule.
27. The method of claim 26 wherein the effect of the binding of the
ligand to the biomolecule on the function of the biomolecule is
assessed in an animal model, in an organism or in a human.
28. The method of claim 26 wherein the biomolecule is a protein or
a nucleic acid molecule.
29. A method for identifying a member of a mass-coded molecular
library which is a ligand for a biomolecule and bind to the
biomolecule at the binding site of a known second ligand for the
biomolecule, said mass-coded molecular library comprising compounds
of the general formula XY.sub.n, wherein n is an integer from 2 to
about 6, X is a scaffold and each Y is, independently, a peripheral
moiety, wherein said mass-coded molecular library is produced by
reacting a scaffold precursor with a sufficient number of distinct
peripheral moiety precursors such that there exist at least about
250 distinct combinations of n peripheral moieties derived from
said peripheral moiety precursors, said method comprising the steps
of: (a) contacting the biomolecule with the mass-coded molecular
library, whereby members of the mass-coded molecular library which
are ligands for the biomolecule bind to the biomolecule to form
biomolecule-ligand complexes and members of the mass-coded library
which are not ligands for the biomolecule remain unbound; (b)
separating the biomolecule-ligand complexes from the unbound
members of the mass-coded molecular library; (c) contacting the
biomolecule-ligand complexes with the second ligand to dissociate
biomolecule-ligand complexes in which the ligand binds to the
biomolecule at the binding site of the second ligand, thereby
forming biomolecule-second ligand complexes and dissociated
ligands; (d) separating the dissociated ligands and
biomolecule-ligand complexes; and (e) determining the molecular
mass of each dissociated ligand, wherein the molecular mass of each
dissociated ligand corresponds to a set of peripheral moieties
present in that ligand, thereby identifying a member of the
mass-coded molecular library which is a ligand for the biomolecule
and binds to the biomolecule at the binding site of the known
second ligand for the biomolecule.
30. The method of claim 29 wherein the second ligand is a
polypeptide, a nucleic acid molecule or a cofactor.
31. The method of claim 29 wherein wherein the biomolecule is
immobilized on a solid support.
32. The method of claim 31 wherein the solid support is a
water-insoluble matrix contained within a chromatographic
column.
33. The method of claim 29 wherein the biomolecule is a protein or
a nucleic acid molecule.
34. A method for identifying a member of a mass-coded combinatorial
library which is a ligand for a first biomolecule but is not a
ligand for a second biomolecule, said mass-coded molecular library
comprising compounds of the general formula XY.sub.n, wherein n is
an integer from 2 to about 6, X is a scaffold and each Y is,
independently, a peripheral moiety, wherein said mass-coded
combinatorial library is produced by reacting a scaffold precursor
with a sufficient number of distinct peripheral moiety precursors
such that there exist at least about 250 distinct combinations of n
peripheral moieties derived from said peripheral moiety precursors,
said method comprising the steps of: (a) contacting the first
biomolecule with the mass-coded molecular library, whereby members
of the mass-coded molecular library which are ligands for the first
biomolecule bind to the first biomolecule to form first
biomolecule-ligand complexes and members of the mass-coded library
which are not ligands for the first biomolecule remain unbound; (b)
separating the first biomolecule-ligand complexes from the unbound
members of the mass-coded molecular library; (c) dissociating the
first biomolecule-ligand complexes; (d) determining the molecular
mass of each ligand for the first biomolecule; (e) contacting the
second biomolecule with the mass-coded molecular library, whereby
members of the mass-coded molecular library which are ligands for
the second biomolecule bind to the second biomolecule to form
second biomolecule-ligand complexes and members of the mass-coded
library which are not ligands for the second biomolecule remain
unbound; (f) separating the second biomolecule-ligand complexes
from the unbound members of the mass-coded molecular library; (g)
dissociating the second biomolecule-ligand complexes; (h)
determining the molecular mass of each ligand for the second
biomolecule; and (i) determining which molecular mass or masses
determined in step (d) are not determined in step (h), thereby
providing the molecular masses of members of the mass-coded
combinatorial library which are ligands for the first biomolecule
but are not ligands for the second biomolecule, wherein the each
molecular mass determined in step (i) corresponds to a set of n
peripheral moieties present in a ligand for the first biomolecule
which is not a ligand for the second biomolecule, thereby
identifying a member of the mass-coded combinatorial library which
are ligands for the first biomolecule but are not ligands for the
second biomolecule.
35. The method of claim 34 wherein the first and second
biomolecules are each, independently, a protein or a nucleic acid
molecule.
36. The method of claim 35 wherein the first and second
biomolecules are each a protein and amino acid sequence of the
second biomolecule is derived from the amino acid sequence of the
first biomolecule by insertion, deletion or substitution of one or
more amino acid residues.
37. The method of claim 35 wherein the first biomolecule is a first
protein and the second biomolecule is a second protein, said first
and second proteins having the same amino acid sequence, wherein
said first and second proteins have different posttranslational
modifications.
38. The method of claim 37 wherein the first protein differs from
the second protein in extent of phosphorylation, glycosylation or
ubiquitination.
39. The method of claim 35 wherein the second biomolecule is a
complex of the first biomolecule with a ligand.
40. The method of claim 35 wherein the first and second
biomolecules are each immobilized on a solid support.
41. The method of claim 40 wherein the solid support is a
water-insoluble matrix contained within a chromatographic
column.
42. The method of claim 35 wherein a solution comprising the first
biomolecule is contacted with the mass-coded molecular library to
form a solution comprising first biomolecule-ligand complexes and
unbound members of the mass-coded molecular library and a solution
comprising the second biomolecule is contacted with the mass-coded
molecular library to form a solution comprising second
biomolecule-ligand complexes and unbound members of the mass-coded
molecular library.
43. The method of claim 42 wherein the unbound members of the
mass-coded molecular library are separated from the second
biomolecule-ligand complexes by directing the solution comprising
second biomolecule-ligand complexes and the unbound members of the
mass-coded molecular library through a size exclusion
chromatography column, whereby the unbound members of the
mass-coded molecular library elute from said column after the
second biomolecule-ligand complexes.
44. The method of claim 42 wherein the unbound members of the
mass-coded molecular library are separated from the second
biomolecule-ligand complexes by contacting the solution comprising
second biomolecule-ligand complexes and the unbound members of the
mass-coded molecular library with a size-exclusion membrane,
whereby the unbound combounds pass through said membrane and the
second biomolecule-ligand complexes do not pass through said
membrane.
45. A method for identifying a member of a mass-coded combinatorial
library which is a ligand for a first biomolecule but is not a
ligand for a second biomolecule, said mass-coded molecular library
comprising compounds of the general formula XY.sub.n, wherein n is
an integer from 2 to about 6, X is a scaffold and each Y is,
independently, a peripheral moiety, wherein said mass-coded
combinatorial library is produced by reacting a scaffold precursor
with a sufficient number of distinct peripheral moiety precursors
such that there exist at least about 250 distinct combinations of n
peripheral moieties derived from said peripheral moiety precursors,
said method comprising the steps of: (a) contacting the second
biomolecule with the mass-coded molecular library, whereby members
of the mass-coded molecular library which are ligands for the
second biomolecule bind to the second biomolecule to form second
biomolecule-ligand complexes and members of the mass-coded library
which are not ligands for the second biomolecule remain unbound;
(b) separating the second biomolecule-ligand complexes from the
unbound members of the mass-coded molecular library; (c) contacting
the first biomolecule with the unbound members of the mass-coded
molecular library of step (b), whereby members of the mass-coded
molecular library which are ligands for the first biomolecule bind
to the first biomolecule to form first biomolecule-ligand complexes
and members of the mass-coded library which are not ligands for the
first biomolecule remain unbound; (d) dissociating the first
biomolecule-ligand complexes; (e) determining the molecular mass of
each ligand for the first biomolecule; wherein each molecular mass
determined in step (e) corresponds to a set of n peripheral
moieties present in a ligand for the first biomolecule which is not
a ligand for the second biomolecule, thereby identifying a member
of the mass-coded combinatorial library which is a ligand for the
first biomolecule but are is not a ligand for the second
biomolecule.
46. The method of claim 45 wherein the first and second
biomolecules are each, independently, a protein or a nucleic acid
molecule.
47. The method of claim 45 wherein the second biomolecule is
immobilized on a solid support.
48. The method of claim 47 wherein the solid support is a
water-insoluble matrix contained within a chromatographic
column.
49. Apparatus for producing a mass-coded set of compounds of the
general formula X(Y).sub.n, wherein X is a scaffold, n is from 2 to
about 6, and each Y is, independently, a peripheral moiety,
comprising a digital processor assembly for selecting a peripheral
moiety precursor subset from a peripheral moiety precursor set,
said subset comprising a sufficient number of peripheral moiety
precursors that there exist at least about 250 distinct
combinations of n peripheral moieties derived from said subset,
wherein at least about 90% of said combinations of n peripheral
moieties derived from said subset have molecular mass sums which
are distinct from the molecular mass sums of all other combinations
of n peripheral moieties derived from said subset.
50. Apparatus as claimed in claim 49 wherein the digital processor
assembly employs a routine executed by a digital processor to: (a)
choose every set of two different peripheral moiety precursors from
the peripheral moiety precursor set, said choosing performed in a
manner such that for each set of two, if the two peripheral moiety
precursors have equal molecular masses then one of the two is
removed forming a remaining set; (b) from the remaining set, choose
every set of four peripheral moiety precursors, including for a
given set of four, removing one of the four peripheral moiety
precursors if a sum of the molecular masses of a first two
precursors in the given set of four equals a sum of the molecular
masses of a second two precursors in the given set of four
peripheral moiety precursors, said choosing forming a remainder
set; (c) from the remainder set, choose every set of six different
peripheral moiety precursors, including for a given set of six,
removing one of the six peripheral moiety precursors if a sum of
the molecular masses of a first three precursors in the given set
of six equals a sum of the molecular masses of a second three
precursors in the given set of six, said choosing forming a working
selection set of peripheral moiety precursors from which to select
a desired subset; and (d) from the working selection set, choose a
desired subset so as to provide the selected subset by (i) choosing
a possible selected subset from the working selection set, (ii)
from the chosen possible subset, generating all possible
combinations of n peripheral moiety precursors, and (iii)
determining whether the generated combinations have an acceptable
percent mass redundancy, and if so, selecting the chosen possible
subset as the selected subset.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/070,456, filed Jan. 5, 1998, the contents of
which are incorporated herein by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] Genomics is identifying the genes responsible for all human
functions and diseases. With 80,000 genes in the human genome, the
thousands of genes involved in development, stature, intelligence,
and other features of a human being are being defined. Humans
suffer from hundreds of inherited and infectious diseases, and the
genes involved in such are also being identified. Proteins encoded
by all these genes are targets for therapeutic drugs. However,
drugs that can be applied to human function and disease will not
simply emerge from genomic information. Conventional drug
development for a single disease is a lengthy, tedious and
extremely expensive process. Technologies that eliminate the major
hurdles facing drug development in the post-genomic era would be of
substantial value.
SUMMARY OF THE INVENTION
[0003] The present invention provides a method for producing a
mass-coded set of chemical compounds having the general formula
X(Y).sub.n, where X is a scaffold, each Y is, independently, a
peripheral moiety, and n is an integer greater than 1, typically
from 2 to about 6. The method comprises selecting a peripheral
moiety precursor subset from a peripheral moiety precursor set. The
subset includes a sufficient number of peripheral moiety precursors
that at least about 50, 100, 250 or 500 distinct combinations of n
peripheral moieties derived from the peripheral moiety precursors
in the subset exist. The subset of peripheral moiety precursors is
selected so that at least about 90% of all possible combinations of
n peripheral moieties derived from the subset of peripheral moiety
precursors have a molecular mass sum which is distinct from the
molecular mass sums of all of the other combinations of n
peripheral moieties. The method further comprises contacting the
peripheral moiety precursor subset with a scaffold precursor which
has n reactive groups, each of which is capable of reacting with at
least one peripheral moiety precursor to form a covalent bond. The
peripheral moiety precursor subset is contacted with the scaffold
precursor under conditions sufficient for the reaction of each
reactive group with a peripheral moiety precursor, resulting in a
mass-coded set of compounds of the general formula X(Y).sub.n.
[0004] In another embodiment, the invention provides a method of
identifying a member or members of a mass-coded combinatorial
library which are ligands for a biomolecule, for example, a protein
or a nucleic acid molecule, such as DNA or RNA. The method
comprises the steps of (1) contacting the biomolecule with the
mass-coded molecular library, whereby members of the mass-coded
molecular library which are ligands for the biomolecule bind to the
biomolecule to form biomolecule-ligand complexes and members of the
mass-coded library which are not ligands for the biomolecule remain
unbound; (2) separating the biomolecule-ligand complexes from the
unbound members of the mass-coded molecular library; (3)
dissociating the biomolecule-ligand complexes; and (4) determining
the molecular mass of each ligand to identify the set of n
peripheral moieties present in each ligand.
[0005] In a further embodiment, the invention provides a method for
identifying a member or members of a mass-coded molecular library
which are ligands for a biomolecule and bind to the biomolecule at
the binding site of a ligand known to bind the biomolecule (a known
ligand). The method comprises the steps of: (1) contacting the
biomolecule with the mass-coded molecular library, so that members
of the mass-coded molecular library which are ligands for the
biomolecule bind to the biomolecule to form biomolecule-ligand
complexes and members of the mass-coded library which are not
ligands for the biomolecule remain unbound; (2) separating the
biomolecule-ligand complexes from the unbound members of the
mass-coded molecular library; (3) contacting the biomolecule-ligand
complexes with a ligand known to bind the biomolecule, to
dissociate biomolecule-ligand complexes in which the ligand binds
to the biomolecule at the binding site of the known ligand, thereby
forming biomolecule-known ligand complexes and dissociated ligands;
(4) separating the dissociated ligands and biomolecule-ligand
complexes; and (5) determining the molecular mass of each
dissociated ligand to identify the set of n peripheral moieties
present in each dissociated ligand.
[0006] In a yet further embodiment, the invention provides a method
for identifying a member or members of a mass-coded combinatorial
library which are ligands for a first biomolecule but are not
ligands for a second biomolecule. The method comprises the steps
of: (1) contacting the first biomolecule with the mass-coded
molecular library, whereby members of the mass-coded molecular
library which are ligands for the first biomolecule bind to the
first biomolecule to form first biomolecule-ligand complexes and
members of the mass-coded library which are not ligands for the
first biomolecule remain unbound; (2) separating the first
biomolecule-ligand complexes from the unbound members of the
mass-coded molecular library; (3) dissociating the first
biomolecule-ligand complexes; (4) determining the molecular mass of
each ligand for the first biomolecule; (5) contacting the second
biomolecule with the mass-coded molecular library, whereby members
of the mass-coded molecular library which are ligands for the
second biomolecule bind to the second biomolecule to form second
biomolecule-ligand complexes and members of the mass-coded library
which are not ligands for the second biomolecule remain unbound;
(6) separating the second biomolecule-ligand complexes from the
unbound members of the mass-coded molecular library; (7)
dissociating the second biomolecule-ligand complexes; (8)
determining the molecular mass of each ligand for the second
biomolecule; and (9) determining which molecular masses determined
in step (4) are not determined in step (8). This provides the
molecular masses of members of the mass-coded combinatorial library
which are ligands for the first biomolecule, but are not ligands
for the second biomolecule.
[0007] In another embodiment, the method for identifying a member
or members of a mass-coded combinatorial library which are ligands
for a first biomolecule but are not ligands for a second
biomolecule comprises the steps of: (1) contacting the second
biomolecule with the mass-coded molecular library, so that members
of the mass-coded molecular library which are ligands for the
second biomolecule bind to the second biomolecule to form second
biomolecule-ligand complexes and members of the mass-coded library
which are not ligands for the second biomolecule remain unbound;
(2) separating the second biomolecule-ligand complexes from the
unbound members of the mass-coded molecular library; (3) contacting
the first biomolecule with the unbound members of the mass-coded
molecular library of step (2), whereby members of the mass-coded
molecular library which are ligands for the first biomolecule bind
to the first biomolecule to form first biomolecule-ligand complexes
and members of the mass-coded library which are not ligands for the
first biomolecule remain unbound; (4) dissociating the first
biomolecule-ligand complexes; and (5) determining the molecular
mass of each ligand for the first biomolecule. Each molecular mass
determined corresponds to a set of n peripheral moieties present in
a ligand for the first biomolecule which is not a ligand for the
second biomolecule.
[0008] In yet another embodiment, the present invention relates to
a method for identifying a member of a mass-coded combinatorial
library which is a ligand for a biomolecule and assessing the the
effect of the binding of the ligand to the biomolecule. The method
comprises the steps of: contacting the biomolecule with the
mass-coded molecular library, whereby members of the mass-coded
molecular library which are ligands for the biomolecule bind to the
biomolecule to form biomolecule-ligand complexes and members of the
mass-coded library which are not ligands for the biomolecule remain
unbound; separating the biomolecule-ligand complexes from the
unbound members of the mass-coded molecular library; dissociating
the biomolecule-ligand complexes; determining the molecular mass of
each ligand to identify the set of n peripheral moieties present in
each ligand. The molecular mass of each ligand corresponds to a set
of n peripheral moieties present in that ligand, thereby
identifying a member of the mass-coded combinatorial library which
is a ligand for the biomolecule. The method further comprisies
assessing in an in vivo or in vitro assay the effect of the binding
of the ligand to the biomolecule on the function of the
biomolecule.
[0009] The method of the invention allows rapid production of
mass-coded combinatorial libraries comprising large numbers of
compounds. The mass-coding enables the identification of individual
combinations of scaffold and peripheral moieties by molecular mass.
The libraries prepared by the method of the invention also allow
the rapid identification of compounds which are ligands for a given
biomolecule.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIGS. 1A and 1B are flow charts illustrating a procedure and
alternative procedure, respectively, for selecting a subset of
peripheral moiety precursors from among a larger set of peripheral
moiety precursors for the production of a mass-coded combinatorial
library.
[0011] FIG. 2A is a graph illustrating the mass redundancy of the
combinatorial libraries resulting from a computer selected set of
peripheral moiety precursors selected using a mass-coding
algorithm.
[0012] FIG. 2B is a graph illustrating the mass redundancy of the
combinatorial libraries resulting from a set of peripheral moiety
precursors selected randomly.
[0013] FIG. 2C presents graphs illustrating the mass redundancy of
the combinatorial libraries resulting from (1) a computer optimized
set of peripheral moiety precursors selected using a mass-coding
algorithm ( . . . ) and (2) a set of peripheral moiety precursors
selected randomly (-).
[0014] FIG. 3 is a schematic diagram of a computer system employing
a digital processor assembly embodying the invention method of
selecting a subset of peripheral moiety precursors which minimize
or eliminate mass redundancy in a library.
DETAILED DESCRIPTION OF THE INVENTION
[0015] The major hurdles in drug development include a need for: 1)
combinatorial chemistry technology that enables rapid production of
nearly unlimited numbers of compounds while incorporating the
ability to identify efficiently single chemical compounds that bind
tightly to a specific biomolecule target, such as a protein or
nucleic acid molecule; 2) extremely efficient target-based
screening technologies that permit rapid identification of chemical
compounds within a large library mixture that become tightly
associated with a target biomolecule, even when the function of
that biomolecule is not well understood and 3) an information data
set that describes how chemical components interact with
biomolecules of medical importance.
[0016] The present invention provides a method of producing a
mass-coded set of compounds, such as a mass-coded combinatorial
library. The compounds are of the general formula X(Y), wherein X
is a scaffold, each Y is a peripheral moiety and n is an integer
greater than 1, typically from 2 to about 6. The term "scaffold",
as used herein, refers to a molecular fragment to which two or more
peripheral moieties are attached via a covalent bond. The scaffold
is a molecular fragment which is common to each member of the
mass-coded set of compounds. The term "peripheral moiety", as used
herein, refers to a molecular fragment which is bonded to a
scaffold. Each member of the set of mass-coded compounds will
include a combination of n peripheral moieties bonded to the
scaffold and this set of compounds forms a mass coded combinatorial
library.
[0017] The term "combination", as used herein, refers to all
permutations of m moieties having n members where m is an integer
greater than 2, n is an integer greater than 1 and m is greater
than or equal to n, such that:
[0018] (1) Permutations having n members in which a given moiety is
present from 0 to n times are included.
[0019] (2) Permutations having the same n moieties but ordered
differently are included once and only once.
[0020] The number of combinations of all permutations of m moieties
having n members may be calculated from the formula:
Combinations=k!/((k-n)!*n!) where k=m+(n-1)
[0021] For example, the combinations of the four moieties labeled
A, B, C, D which have 3 members are: A A A; A A B; A A C; A A D; A
B B; A B C; A B D; A C C; A C D; A D D; B B B; B B C; B B D; B C C;
B C D; B D D; C C C; C C D; C D D and D D D. B A A and A B A, for
example, are not counted as separate combinations; only A A B is
counted. In this example, m=4, n=3 and the number of combinations
is given by
6!/((6-3)!*3!)=20.
[0022] The terms "mass-coded set of compounds" and "mass-coded
combinatorial library", as used herein, refer to a set of compounds
of the formula XY.sub.n, where X is a scaffold, each Y is,
independently, a peripheral moiety and n is an integer greater than
1, typically from 2 to about 6. Such a set of compounds is
synthesized as a mixture by the combination of a set of peripheral
moiety precursors with a scaffold precursor, and is designed to
possess minimum mass redundancy, given the requirement that a fixed
number (subset) of peripheral moiety precursors must be chosen from
a set of available peripheral moiety precursors.
[0023] The term "mass" or "molecular mass", as used herein, refers
to the exact mass of a molecule or collection of chemical moieties
in which each atom is the most abundant naturally occurring isotope
for the particular element. Exact masses and their determination by
mass spectrometry are discussed by Pretsch et al., Tables of
Spectral Data for Structure Determination of Organic Compounds,
second edition, Springer-Verlag (1989), and Holden et al., Pure
Appl. Chem. 55: 1119-1136 (1983), the contents of each of which are
incorporated herein by reference in their entirety.
[0024] "Minimum mass redundancy", as the term is used herein, is
exhibited by a set of compounds of the formula X(Y).sub.n formed by
reaction of a scaffold precursor having n reactive groups, where n
is an integer greater than 1, typically from 2 to about 6, with a
subset of peripheral moiety precursors in which at least about 90%
of the possible combinations of n peripheral moieties derived from
the subset of peripheral moiety precursors have a molecular mass
sum which is distinct from the molecular mass sum of any other
combination of n peripheral moieties derived from the subset. The
molecular mass sum of a combination of peripheral moieties is the
sum of the masses of each peripheral moiety within the combination.
For the present purposes, two molecular masses are distinct if they
can be distinguished by mass spectrometry or high resolution mass
spectrometry. For example, molecular masses which differ by at
least 0.001 atomic mass units can be distinguished by high
resolution mass spectrometry.
[0025] It is to be understood that the molecular mass sum of the
combination of the n peripheral moieties in a particular compound
of the formula X(Y).sub.n is the collective contribution of the n
peripheral moieties to the molecular mass of the compound. As each
compound within the set includes a constant scaffold, the
difference in the molecular masses of two compounds within the
mass-coded set of compounds is the difference in the molecular mass
sums of the set of peripheral moieties in each compound.
[0026] The method of the invention comprises selecting a peripheral
moiety precursor subset from a larger peripheral moiety precursor
set. Details of the preferred selection process are discussed later
with reference to FIGS. 1A, 1B and 3. The subset includes a
sufficient number of peripheral moiety precursors so that, in one
embodiment, at least about 50 distinct combinations of n peripheral
moieties derived from the peripheral moiety precursors in the
subset can be formed. In another embodiment, at least about 100
distinct combinations of n peripheral moieties can be formed. In a
further embodiment, at least about 250 distinct combinations of n
peripheral moiety precursors can be formed, and, in yet another
embodiment, at least about 500 distinct combinations of n
peripheral moieties can be formed.
[0027] The subset of peripheral moiety precursors is selected so
that at least about 90% of all possible combinations of n
peripheral moieties derived from the subset have a molecular mass
sum which is distinct from the molecular mass sums of all of the
other combinations of n peripheral moieties. The method further
comprises contacting the peripheral moiety precursor subset with a
scaffold precursor which has n reactive groups, each of which is
capable of reacting with at least one peripheral moiety precursor
to form a covalent bond. The peripheral moiety precursor subset is
contacted with the scaffold precursor under conditions sufficient
for the reaction of each reactive group with a peripheral moiety
precursor, resulting in a mass-coded set of compounds.
[0028] In one embodiment, at least about 95% of all possible
combinations of n peripheral moieties derived from the peripheral
moiety precursor subset have a molecular mass sum which is distinct
from the molecular mass sums of all of the other combinations of n
peripheral moieties. In another embodiment, each of the possible
combinations of n peripheral moieties derived from the subset has a
molecular mass sum which is distinct from the molecular mass sums
of all of the other combinations of n peripheral moieties.
[0029] The scaffold precursor can be any molecule comprising two or
more reactive groups which are capable of reacting with a
peripheral moiety precursor reactive group to form a covalent bond.
For example, suitable scaffold precursors can have a wide range of
sizes, shapes, degrees of flexibility and charges. The reactive
groups should be incapable of intramolecular reaction under the
conditions employed. Further, a scaffold precursor molecule should
not react with another scaffold precursor molecule under the
conditions employed. The scaffold precursor can also include any
additional functional groups which are masked or protected or which
do not interfere with the reaction of the reactive groups with the
peripheral moiety precursors.
[0030] Preferably, the scaffold precursor comprises one or more
saturated, partially unsaturated or aromatic cyclic groups, such as
a cyclic hydrocarbon or heterocyclic group. In scaffold precursors
comprising two or more cyclic groups, the cyclic groups can be
fused, connected via a direct bond or connected via an intervening
group, such as an oxygen atom, an NH group or a C.sub.1-6-alkylene
group. At least one cyclic group is substituted by one or more
reactive groups. The reactive groups can be attached to the cyclic
group directly or via an intervening group, such as a
C.sub.1-6-alkylene group, preferably a methylene group.
[0031] Examples of suitable scaffold precursors include reactive
group-substituted benzene, biphenyl, cyclohexane, bipyridyl,
N-phenylpyrrole, diphenyl ether, naphthalene and benzophenone.
Other suitable classes of scaffold precursors are shown below.
1
[0032] In these examples, each of the indicated substituents R is,
independently, a reactive group, and the scaffold precursor can
include one or more additional functional groups which are either
(1) masked or protected to prevent their reaction with a peripheral
moiety precursor (e.g., scaffold precursors f and g, above) or (2)
do not react either with R or with a peripheral moiety precursor
under the given reaction conditions (e.g., scaffold precursor h,
above, in which R.dbd.C(O)O(C.sub.6F.sub.5) and the peripheral
moiety precursors include primary amino groups).
[0033] A peripheral moiety precursor is a compound which includes a
reactive group which is complementary to one or more of the
reactive groups of the scaffold precursor. In addition to the
reactive group, a peripheral moiety precursor can include a wide
variety of structural features. For example, the peripheral moiety
precursor can include one or more functional groups in addition to
the reactive group. Any additional functional group should be
appropriately masked or not interfere with the reaction between the
scaffold precursor and the peripheral moiety precursor. In
addition, two peripheral moiety precursors should not react
together under the conditions employed. For example, a subset of
peripheral moiety precursors can include, in addition to the
reactive groups, functionalities selected from groups spanning a
range of charge, hydrophobicity/hydrophilicity, and sizes. For
example, the peripheral moiety precursor can include a negative
charge, a positive charge, a hydrophilic group or a hydrophobic
group.
[0034] In addition to the reactive groups, peripheral moiety
precursors can include, for example, functionalities selected from
among amino acid side chains, a nucleotide base or nucleotide base
analogue, sugar moieties, sulfonamides, peptidomimetic groups,
charged or polar functional groups, alkyl groups and aryl
groups.
[0035] For the present purposes, two reactive groups are
complementary if they are capable of reacting together to form a
covalent bond. In a preferred embodiment, the bond forming
reactions occur rapidly under ambient conditions without
substantial formation of side products.
[0036] Preferably, a given reactive group will react with a given
complementary reactive group exactly once.
[0037] In one embodiment, the reactive group of the scaffold
precursor and the reactive group of the peripheral moiety precursor
react, for example, via nucleophilic substitution, to form a
covalent bond. In one embodiment, the reactive group of the
scaffold precursor is an electrophilic group and the reactive group
of the peripheral moiety precursor is a nucleophilic group. In
another embodiment, the reactive group of the scaffold precursor is
a nucleophilic group, while the reactive group of the peripheral
moiety precursor is an electrophilic group.
[0038] Complementary electrophilic and nucleophilic groups include
any two groups which react via nucleophilic substitution under
suitable conditions to form a covalent bond. A variety of suitable
bond-forming reactions are known in the art. See, for example,
March, Advanced Organic Chemistry, fourth edition, New York: John
Wiley and Sons (1992), Chapters 10 to 16; Carey and Sundberg,
Advanced Organic Chemistry, Part B, Plenum (1990), Chapters 1-11;
and Collman et al., Principles and Applications of Organotransition
Metal Chemistry, University Science Books, Mill Valley, Calif.
(1987), Chapters 13 to 20; each of which is incorporated herein by
reference in its entirety. Examples of suitable electrophilic
groups include reactive carbonyl groups, such as carbonyl chloride
(acyl chloride) and carbonyl pentafluorophenyl ester groups,
reactive sulfonyl groups, such as the sulfonyl chloride group, and
reactive phosphonyl groups. Other electrophilic groups which can be
used include terminal epoxide groups and the isocyanate group.
Suitable nucleophilic groups include primary and secondary amino
groups and alcohol (hydroxyl) groups.
[0039] Examples of suitable scaffold precursors with specified
reactive groups are shown below. 2
[0040] In these examples, each R is, independently, an additional
reactive group which can be the same as the specified reactive
group or a different group.
[0041] Illustrated below are examples of suitable peripheral moiety
precursors having amino groups. 3
[0042] R in this case is an amino acid side chain, .sup.tBoc is
.sup.tbutoxycarbonyl, Ac is acetyl and .sup.tBu is tertiary
butyl.
[0043] Examples of scaffold precursors and peripheral moiety
precursors which have complementary reactive groups include the
following, which are provided for the purposes of illustration and
are not to be construed as limiting in any way:
[0044] 1. The scaffold precursor includes from two to about six
reactive carbonyl groups, reactive sulfonyl groups or reactive
phosphonyl groups, or a combination thereof. Each peripheral moiety
precursor includes a primary or secondary amino group which reacts
with the scaffold precursor to form an amide, sulfonamide or
phosphonamidate bond.
[0045] 2. The scaffold precursor includes from two to about six
primary or secondary amino groups or a combination thereof. Each
peripheral moiety precursor includes a reactive carbonyl group, a
reactive sulfonyl group or a reactive phosphonyl group.
[0046] 3. The scaffold precursor includes from two to about six
terminal epoxide groups. Each peripheral moiety precursor includes
a primary or secondary amino group. In the presence of a suitable
Lewis acid, the scaffold precursor and the peripheral moiety
precursors react to form C-amino alcohols.
[0047] 4. The scaffold precursor includes from two to about six
primary or secondary amino groups. Each peripheral moiety precursor
contains a terminal epoxide group.
[0048] 5. The scaffold precursor includes from two to about six
isocyanate groups. Each peripheral moiety precursor contains a
primary or secondary amino group which reacts with the scaffold
precursor to form a urea.
[0049] 6. The scaffold precursor includes from two to about six
primary or secondary amino groups, or a combination thereof. Each
peripheral moiety precursor contains an isocyanate group.
[0050] 7. The scaffold precursor includes from two to about six
isocyanate groups. Each peripheral moiety precursor contains an
alcohol group which reacts with the scaffold precursor to form a
carbamate.
[0051] 8. The scaffold precursor includes from 2 to about 6
aromatic bromides. Each peripheral moiety precursor is an
organo-tributyl-tin compound. The scaffold precursor and the
peripheral moiety precursors are reacted in the presence of a
suitable palladium catalyst to form one or more carbon-carbon
bonds.
[0052] 9. The scaffold precursor includes from 2 to about 6
aromatic halides or triflates. Each peripheral moiety precursor
includes a primary or secondary amino groups. The scaffold
precursor and the peripheral moiety precursors are reacted in the
presence of a suitable palladium catalyst to form one or more
carbon-nitrogen bonds.
[0053] 10. The scaffold precursor includes from two to about six
amino groups. Each peripheral moiety precursor contains an aldehyde
or ketone group which reacts with the scaffold precursor under
reducing conditions (reductive amination) to form an amine.
[0054] 11. The scaffold precursor includes from two to about six
aldehyde or ketone groups. Each peripheral moiety precursor
contains an amino group which reacts with the scaffold precursor
under reducing conditions (reductive amination) to form an
amine.
[0055] 12. The scaffold precursor includes from two to about six
phosphorous ylide groups. Each peripheral moiety precursor contains
an aldehyde or ketone group which reacts with the scaffold
precursor (Wittig type reaction) to form an alkene.
[0056] 13. The scaffold precursor includes from two to about six
aldehyde or ketone groups. Each peripheral moiety precursor
contains a phosphorous ylide group which reacts with the scaffold
precursor (Wittig type reaction) to form an alkene.
[0057] The scaffold is that portion of the scaffold precursor which
remains after each reactive group of the scaffold precursor has
reacted with a peripheral moiety precursor. A peripheral moiety is
that portion of the peripheral moiety precursor which is bonded to
the scaffold following the bond-forming reaction. A peripheral
moiety which results from the reaction of a particular peripheral
moiety precursor with a reactive functional group of a scaffold
precursor is said to be "derived" from that peripheral moiety
precursor.
[0058] A peripheral moiety precursor can include one or more
functional groups in addition to the reactive group. One or more of
these additional functional groups can be protected to prevent
undesired reactions of these functional groups. Suitable protecting
groups are known in the art for a variety of functional groups
(Greene and Wuts, Protective Groups in Organic Synthesis, second
edition, New York: John Wiley and Sons (1991), incorporated herein
by reference). Particularly useful protecting groups include
t-butyl esters and ethers, acetals, trityl ethers and amines,
acetyl esters, trimethylsilyl ethers and trichloroethyl ethers and
esters.
[0059] The compounds within the set are mass-coded as a result of
the selection of a subset of suitable peripheral moiety precursors.
The subset of peripheral moiety precursors is selected such that
for a scaffold precursor having n reactive groups, where n is an
integer from 2 to about 6, there exist at least about 50, 100, 250
or 500 different combinations of n peripheral moieties derived from
the peripheral moiety precursor subset. At least about 90% of the
possible combinations of n peripheral moieties derived from the
peripheral moiety precursors within the subset will have a distinct
mass sum. In one embodiment, the selection of suitable peripheral
moiety precursors for the production of a mass-coded set of
compounds includes one or more automated steps utilizing hardware
apparatus, software apparatus or any combination thereof. In the
preferred embodiment, a digital processor assembly employs a
suitable software routine which selects a subset of peripheral
moiety precursors which minimize or eliminate mass redundancy in
the library. FIG. 3 is illustrative of such apparatus employing a
digital processor assembly for carrying out the present invention
method.
[0060] Referring to FIG. 3, there is shown a computer system 25
formed of (a) a digital processor 11 having working memory 17 for
executing programs, routines, procedures and the like, (b) input
means 21 coupled to the digital processor 11 for providing data,
parameters and the like to support execution of the programs,
routines and/or procedures in the digital processor working memory
17, and (c) output means 23 coupled to the digital processor 11 for
displaying results, prompts, messages and the like from operation
of the digital processor 11. The input means 21 include a keyboard,
mouse and the like common in the art. The output means 23 include a
viewing monitor, printer and the like common in the art. The
invention software routine 27 is executed in the working memory 17
by the digital processor 11 as follows.
[0061] First, a user interface prompts the end-user to input
indications of an initial set 13 of peripheral moiety precursors
and the exact masses of the peripheral moieties which are derived
therefrom. This initial set 13 may be copied, transferred or
otherwise obtained from a database or other source such as is known
in the art. The user interface also obtains from the end-user a set
of user determined/desired criteria 19. In the preferred
embodiment, the user selected criteria 19 includes (i) the total
count j of peripheral moiety precursors in the initial set, (ii)
the value of n indicating the number of reactive groups of a
subject scaffold precursor for which the invention software routine
27 is to select a subset of peripheral moiety precursors from the
input initial set 13 and (iii) the number of members of the subset,
k. Preferably, the user interface enables the end-user to
interactively provide the user selected criteria 19 through the
input means 21 as indicated at 15 in FIG. 3.
[0062] The digital processor 11 is responsive to the foregoing
input and stores the indications of the initial set 13 of
peripheral moiety precursors in a memory area 29 or data storage
system associated locally or off disk with the software routine 27.
That is, the memory area 29 or data storage system supports the
invention software routine 27. For each peripheral moiety precursor
in the initial set 13 as indicated in memory area 29, an identifier
and indication of respective exact mass of the the peripheral
moiety derived from the peripheral moiety precursor is provided to
the software routine 27. Upon receipt of the peripheral moiety
precursor identifiers, indications of exact mass, and user selected
criteria (n, j and k), the software routine 27 determines and
generates a subset of k peripheral moiety precursors which minimize
or eliminate mass redundancy in a resulting library of compounds of
the formula XY.sub.n, wherein X is a scaffold, each Y is,
independently, a peripheral moiety, and n is an integer greater
than 1, typically from 2 to about 6. Preferably, the software
routine 27 determines a subset of peripheral moiety precursors in
which at least about 90% of the possible combinations of n
peripheral moieties derived from the subset have a distinct mass
sum. The details of the software routine 27 employed in the
preferred embodiment are discussed next for purposes of
illustration and not limitation. It is understood that other
software or firmware routines for accomplishing the present
invention method of selecting a subset of the initial set 13 of
peripheral moiety precursors are suitable and within the purview of
one skilled in the art given this disclosure.
[0063] A typical situation involves a scaffold precursor with n
reactive groups, where n is an integer, a set of j peripheral
moiety precursors, where j is an integer 6 or greater, where the
peripheral moieties derived from the peripheral moiety precursors
have molecular masses y.sub.1, y.sub.2, . . . y.sub.j. An example
of a software routine which can be employed to select a suitable
subset of k peripheral moiety precursors (k.ltoreq.j) from the set
of j peripheral moiety precursors includes the following steps:
[0064] 1. From an initial set of j peripheral moiety precursors,
choose every set of two peripheral moiety precursors. If
y.sub.a=y.sub.b, randomly remove either y.sub.a or y.sub.b.
[0065] 2. From the remaining set of peripheral moiety precursors,
choose every set of four peripheral moiety precursors. If
y.sub.a+y.sub.b=y.sub.c+y.sub.d randomly remove either y.sub.a,
y.sub.b, y.sub.c or y.sub.d.
[0066] 3. From the remaining set of peripheral moiety precursors,
choose every set of six peripheral moiety precursors. If
y.sub.a+y.sub.b+y.sub.c- =y.sub.d+y.sub.e+y.sub.f, randomly remove
either y.sub.a, y.sub.b, y.sub.c, y.sub.d, y.sub.e or y.sub.f.
[0067] If at any step 1 through 3 the remaining number of
peripheral moiety precursors becomes <k, then there is no mass
coded subset k which can be made from set j, and a new set j must
be employed.
[0068] 4. From the remaining computer selected set of peripheral
moiety precursors, choose any or all subsets of k peripheral moiety
precursors.
[0069] 5. Generate all possible combinations of n peripheral moiety
precursors from this subset.
[0070] 6. If the % mass redundancy of the resulting set of
combinations is found to be unacceptable, repeat step 5 until a
desired mass coded library has been obtained or no further possible
combinations of peripheral moiety precursors remain. In the latter
case, begin again with step 1.
[0071] Once an above subset of mass-coded peripheral moiety
precursors is determined, the scaffold precursor is contacted with
the subset of complementary peripheral moiety precursors under
conditions suitable for bond-forming reactions to occur between the
peripheral moiety precursors and the scaffold precursor. The
mass-coded set of compounds is, preferably, synthesized in solution
as a combinatorial library.
[0072] The foregoing selection of a subset from a larger peripheral
moiety precursor set and generation of a mass-coded set of
compounds using the selected subset is more generally illustrated
in FIGS. 1A and 1B. Referring to FIG. 1A, the larger set of
peripheral moiety precursors is provided at 31 from known sources.
The end-user (e.g., chemist) selects an initial set of j peripheral
moiety precursors from the larger set 31 at step 33. Typically the
chemist chooses all of the larger set to form the initial set at
33. The invention mass coding selection procedure 35 is applied to
the initial set. The result of the mass-coding procedure 35 is a
subset 37 of peripheral moiety precursors that satisfies the
mass-coding criteria outlined above. In step 39, this subset of
peripheral moiety precursors is used to generate all theoretical
subsets of of k peripheral moiety precursors. Also in step 39, the
mass redundancies of the libraries obtained from all theoretical
subsets of k peripheral moieties are calculated, and only those
subsets which yield mass-coded libraries, as defined above, are
passed to 41. The net result is one or more subsets 41 of k
peripheral moiety precursors in which there are 50, 100, 250, or
500 distinct combinations of n peripheral moiety precursors in a
given subset and at least 90% of all possible combinations of n
peripheral moieties derived from a given subset have a molecular
mass sum which is distinct from the molecular mass sums of all of
the other combinations of n peripheral moieties, as discussed
above. The subset(s) 41 of peripheral moiety precursors would
subsequently yield mass-coded sets of compounds when contacted with
an appropriate scaffold precursor in the manner discussed
above.
[0073] As an alternative to the single-step application of the
invention mass-coding selection procedure 35 in FIG. 1A, multiple
or stepped application of procedure 35 is suitable and in certain
cases may be advantageous. For instance, using mass-coding
procedures at each level allows for rapid sorting into distinct
sets, each of which may yield optimal mass-coding. During the
mass-coding process, certain criteria reduce the set size as it is
passed into the next layer through mass-coding. This multi-layer
approach yields advantages in speed and the elimination of mass
redundancy.
[0074] Multiple application of mass-coding selection procedure 35
on initial set 33 is illustrated in FIG. 11. Here initial set 33 is
divided into plural parts (the starting larger set of peripheral
moiety precursors 31 and chemist selection 33 being similar to that
in FIG. 1A) The mass-coding selection procedure 35 is applied to
each plural part and results in intermediate resultant sets 43A,
43B, 43C. The mass-coded selection procedure 35 is applied in a
second round/level, but this time with intermediate resultant sets
43A, 43B, 43C. This produces final sets 45A, 45B, 45C. Step 39 is
as in FIG. 1A and generates the subsets 47A, 47B, 47C of k
peripheral moiety precursors that.would subsequently yield
mass-coded stes of compounds when contacted with an appropriate
scaffold precursor in a manner discussed above.
[0075] It is understood that other variations between the approach
illustrated in FIG. 1A and that in FIG. 1B are within the purview
of one skilled in the art. The foregoing discussion and Figures are
for purposes of illustrating and not limiting the present invention
method.
[0076] In one embodiment, the scaffold precursor is contacted with
all members of the peripheral moiety precursor subset
simultaneously. In general, a scaffold precursor having n reactive
groups, where n is an integer from 2 to about 6, will be contacted
with at least about n molar equivalents relative to the scaffold
precursor of peripheral moiety precursors from the selected subset.
For example, the scaffold precursor can be contacted with a
solution comprising each member of the subset in approximately
equal concentrations. For example, if the scaffold precursor
includes n reactive groups, where n is an integer greater than 1,
and the number of peripheral moiety precursors in the subset is
denoted by p, the scaffold precursor can be contacted with about
n/p to about (1.1)n/p molar equivalents of each peripheral moiety
precursor.
[0077] In another embodiment, the scaffold precursor is contacted
with the members of the peripheral moiety precursor subset
sequentially. This results in the formation of intermediate
partially reacted scaffold precursor molecules which include at
least one peripheral moiety and at least one reactive group. For
example, the scaffold precursor can be contacted with one or more
peripheral moiety precursors under conditions suitable for bond
formation to occur. The resulting intermediates can then be
contacted with one or more additional peripheral moiety precursors
under suitable conditions for bond formation to occur. These steps
can be repeated until each scaffold precursor reactive group has
reacted with a peripheral moiety precursor.
[0078] In one embodiment, the reactive groups of the scaffold
precursor can react sequentially with the subset of peripheral
moiety precursors using a suitable reactive group
protection/deprotection scheme. For example, the scaffold precursor
can include two or more sets of reactive groups, where one set is
unprotected and another set is protected, or where two sets are
masked by different protecting groups. An example is the use of the
scaffold precursor 4
[0079] which contains one unprotected, reactive group and two
protected reactive groups. In this case, the unprotected
pentafluorophenyl ester can react with a peripheral moiety
precursor first (e.g., a primary amine). Either the
Cl.sub.3CCH.sub.2O-protected group or the benzyloxy-protected group
can then be deprotected using standard methods and reacted with a
set of peripheral moiety precursors. Finally, the remaining
protected group or groups can be deprotected and reacted with a set
of peripheral moiety precursors.
[0080] Following the reaction of each scaffold precursor reactive
group with a peripheral moiety precursor, any peripheral moiety
having a protected functional group can be deprotected using
methods known in the art.
[0081] The ability to identify individual scaffold plus peripheral
moiety combinations derived from such a mixture is a consequence of
the mass-coding of the library and the ability of mass spectrometry
to identify a molecular mass. This allows the identification of
individual scaffold plus peripheral moiety combinations within the
set which have a particular activity, such as binding to a
particular biomolecule.
[0082] In one embodiment, the present invention provides a method
for identifying a compound or compounds within a mass-coded
combinatorial library which bind to, or are ligands for, a
biomolecule, such as a protein or nucleic acid molecule. The
mass-coded combinatorial library can be produced, for example, by
the method of the invention disclosed above. The target
biomolecule, such as a protein, is contacted with the mass-coded
combinatorial library, and, if any members of the library are
ligands for the biomolecule, biomolecule-ligand complexes form.
Compounds which do not bind the biomolecule are separated from the
biomolecule-ligand complexes. The biomolecule-ligand complexes are
dissociated and the ligands are separated and their molecular
masses are determined. Due to the mass-coding of the combinatorial
library, a given molecular mass is characteristic of a unique
combination of peripheral moieties or only a small number of such
combinations. Thus, a ligand's molecular mass allows the
determination of its composition.
[0083] In one embodiment, the target is immobilized on a solid
support by any known immobilization technique. The solid support
can be, for example, a water-insoluble matrix contained within a
chromatography column or a membrane. The mass-coded set of
compounds can be applied to a water-insoluble matrix contained
within a chromatography column. The column is then washed to remove
non-specific binders. Target-bound compounds (ligands) can then be
dissociated by changing the pH, salt concentration, organic solvent
concentration, or other methods, such as competition with a known
ligand to the target. The dissociated ligands are injected directly
onto a reverse phase column. The reverse phase column acts as a
concentrator/collector and can be interfaced directly to a mass
spectrometer, such as an electrospray mass spectrometer
(ES-MS).Mass information provided by the mass spectrometer is
sufficient for identifying the combination of scaffold and
peripheral moieties within the ligand.
[0084] In another embodiment, the target is free in solution and is
incubated with the mass-coded set of compounds. Compounds which
bind to the target (ligands) are selectively isolated by a size
separation step such as gel filtration or ultrafiltration. In one
embodiment, the mixture of mass-coded compounds and the target
biomolecule are passed through a size exclusion chromatography
column (gel filtration), which separates any ligand-target
complexes from the unbound compounds. The ligand-target complexes
are transferred to a reverse-phase chromatography column, which
dissociates the ligands from the target. The dissociated ligands
are then analyzed by mass spectrometry. Mass information provided
by the mass spectrometer is sufficient for identifying the scaffold
and peripheral moiety composition of the ligand. This approach is
particularly advantageous in situations where immobilization of the
target may result in a loss of activity.
[0085] Once single ligands are identified by the above-described
process, various levels of analysis can be applied to yield SAR
information and to guide further optimization of the affinity,
specificity and bioactivity of the ligand. For ligands derived from
the same scaffold, three-dimensional molecular modeling can be
employed to identify significant structural features common to the
ligands, thereby generating families of small-molecule ligands that
presumably bind at a common site on the target biomolecule.
[0086] In order to identify a consensus, highest affinity, ligand
for a particular binding site, this analysis should include a
ranking of the members of a given ligand family with respect to
their affinities for the target. This process can provide this
information by identifying both low and high affinity ligands for a
target biomolecule in one experiment. For example, when the screen
utilizes an immobilized target, the dissociation rate of the ligand
is inversely correlated with the number of column volumes employed
during of the ligand from its target. When the screen utilizes the
target free in solution, weak affinity ligands can be selected by
using a higher concentration of the target.
[0087] Given that each mass-coded set of compounds is synthesized
with a limited number of peripheral moiety precursors, the
disclosed approach can, in certain cases, identify a superior
ligand which combines structural features of molecules synthesized
in separate libraries.
[0088] When possible, the analysis of ligand structural features is
based on information regarding the target biomolecule's structure,
wherein the hypothetical consensus ligand is computationally docked
with the putative binding site. Further computational analysis can
involve a dynamic search of multiple lowest energy conformations,
which allows comparison of high affinity ligands that are derived
from different scaffolds. The end goal is the identification of
both the optimal functionality and the optimal vectorial
presentation of the peripheral moieties that yields the highest
binding affinity/specificity. This may provide the basis for the
synthesis of an improved, second-generation scaffold.
[0089] Due to the modular design of the mass-coded compounds,
computational analysis may identify the point of attachment on the
scaffold that has the least functional importance with respect to
affinity for the target. In many cases, the ligand will not be
completely engulfed by the target biomolecule, and one peripheral
moiety will be pointed away from the biomolecule towards the bulk
solvent. Three-dimensional alignment of a family of ligands will
reveal a high degree of functional variability at the site that is
presented to the solvent. Modification at this site can then be
used to optimize the affinity. For example, the noncritical
reactive site can be removed and replaced with a small unreactive
group, such as a hydrogen atom or a methyl group. A set of
compounds structurally identical except for the peripheral moiety
at this position can be examined to identify compounds that most
effectively inhibit or promote the binding of another
protein/DNA/RNA molecule. Also, the peripheral moiety at this
position can be modified to link two ligands together. The joining
of two ligands could in certain cases yield a ligand with improved
affinity and specificity, if one joins molecules that bind to
adjacent sites, or yield a designed biomolecule dimerizer.
[0090] A variety of screening approaches can be used to obtain
ligands that possess high affinity for one target but significantly
weaker affinity for another closely related target. One screening
strategy is to identify ligands for both biomolecules in parallel
experiments and to subsequently eliminate common ligands by a
cross-referencing comparison. In this method, ligands for each
biomolecule can be separately identified as disclosed above. This
method is compatible with both immobilized target biomolecules and
target biomolecules free in solution.
[0091] For immobilized target biomolecules, another strategy is to
add a preselection step that eliminates all ligands that bind to
the non-target biomolecule from the library. For example, a first
biomolecule can be contacted with a mass-coded combinatorial
library as described above. Compounds which do not bond to the
first biomolecule are then separated from any first
biomolecule-ligand complexes which form. The second biomolecule is
then contacted with the compounds which did not bind to the first
biomolecule. Compounds which bind to the second biomolecule can be
identified as described above and have significantly greater
affinity for the second biomolecule than to the first
biomolecule.
[0092] The screening approach detailed above can also be applied to
identify ligands that selectively interact with an altered version
of the same biomolecule, wherein the first biomolecule is the
unaltered biomolecule and the second biomolecule is an altered or
variant version of the biomolecule. The second biomolecule can, for
example, have an amino acid sequence which differs from the amino
acid sequence of the first biomolecule by the insertion, deletion
or substitution of one or more amino acid residues. For example,
the second biomolecule can include a specific amino acid mutation
that is linked to the progression of a particular disease.
Alternatively, the second biomolecule can also differ from the
first biomolecule in having a different post-translational
modification, such as an extra site of phosphorylation or
glycosylation, or it may be truncated or aberrantly fused with
another biomolecule.
[0093] The screening approach detailed above can also serve as a
method for identifying small molecule ligands that bind at the same
site on a biomolecule as another known, biologically relevant
ligand. This known ligand can be another biomolecule, such as a
protein or peptide, or it can be a DNA or RNA molecule, or a
substrate or cofactor involved in an enzymatic reaction. In one
embodiment, the first and second biomolecules are both proteins.
The first protein is a complex of the protein and the known ligand,
while the second protein is the protein alone. Compounds which bind
to the protein alone, but not to the complex of the protein with
the known ligand, bind to the protein at the binding site of the
known ligand. This approach is especially well suited to the
development of small molecule replacements of known therapeutic
ligands, such as peptides or proteins.
[0094] An advantage of the present method is that it can be used to
identify chemical compounds that bind tightly to any biomolecule of
interest, even when the function of that biomolecule is not well
understood, as is often the case with gene products defined through
genomics, or when a functional assay is not available. The
screening technologies described can be miniaturized to provide
massive parallel screening capabilities.
[0095] A ligand for a biomolecule of unknown function which is
identified by the method disclosed above can also be used to
determine the biological function of the biomolecule. This is
advantageous because although new gene sequences continue to be
identified, the functions of the proteins encoded by these
sequences and the validity of these proteins as targets for new
drug discovery and development are difficult to determine and
represent perhaps the most significant obstacle to applying genomic
information to the treatment of disease. Target-specific ligands
obtained through the process described in this invention can be
effectively employed in whole cell biological assays or in
appropriate animal models to understand both the function of the
target protein and the validity of the target protein for
therapeutic intervention. This approach can also confirm that the
target is specifically amenable to small molecule drug discovery.
The ligands obtained through the process described in this
invention are small molecules and are, thus, similar to actual
human therapeutics (small molecule drugs).
[0096] In one embodiment, a member of a combinatorial library is
identified as a ligand for a particular biomolecule using the
method described above. The ligand can then be, assessed in an in
vitro assay for the effect of the binding of the ligand to the
biomolecule on the function of the biomolecule. For a biomolecule
having a known function, the assay can include a comparison of the
activity of the biomolecule in the presence and absence of the
ligand. If the biomolecule is of unknown function, a cell which
expresses the biomolecule can be contacted with the ligand and the
effect of the ligand on the viability or function of the cell is
assessed. The in vitro assay can be, for example, a cell death
assay, a cell proliferation assay or a viral replication assay. For
example, if the biomolecule is a protein expressed by a virus, the
a cell infected with the virus can be contacted with a ligand for
the protein. The affect of the binding of binding of the ligand to
the protein on viral viability can then be assessed.
[0097] A ligand identified by the method of the invention can also
be assessed in an in vivo model or in a human. For example, the
ligand can be evaluated in an animal or organism which produces the
biomolecule. Any resulting change in the health status (e.g.,
disease progression) of the animal or organism can be
determined.
[0098] For a biomolecule, such as a protein or a nucleic acid
molecule, of unknown function, the effect of a ligand which binds
to the biomolecule on a cell or organism which produces the
biomolecule can provide information regarding the biological
function of the biomolecule. For example, the observation that a
particular cellular process is inhibited in the presence of the
ligand indicates that the process depends, at least in part, on the
function of the biomolecule.
[0099] The mass-coded libraries provided by the present method
enable the development of an information set that describes how the
universe of small molecules interacts with any biomolecule encoded
within the human and other genomes. This information set would
include data regarding: 1) those libraries and components therein
which bind to the target biomolecule, 2) quantitative
structure-activity relationships (SAR) on chemical functionalities
which contribute to the binding affinity of a compound for a
biomolecule target, and 3) the domains of the biomolecule that are
bound by chemical compounds. The database can be used to expedite
drug development in a number of ways, for example, by identifying
chemical pharmacophores that interact with high affinity with a
specific drug binding site.
[0100] The invention will now be further and more specifically
described in the following examples.
EXAMPLES
Example 1
Application of Mass-Coding by Computer Algorithms: Comparison of
Mass-Coded and Non-Mass-Coded Combinatorial Libraries
[0101] The following is an analysis of the application of
mass-coding algorithms towards the design of combinatorial
libraries. The sequence of steps involved in identifying subsets of
peripheral moiety precursors that can be allowed to react with a
predetermined scaffold precursor to yield a mass-coded
combinatorial library of compounds with the molecular formula
X(Y).sub.n is shown in FIG. 1A; FIG. 1B is an alternate sequence of
steps. It is to be understood that the molecular mass sum of the
combination of the n peripheral moieties in a particular compound
of the formula X(Y).sub.n is the collective contribution of the n
peripheral moieties to the molecular mass of the compound. As each
compound within the library includes a constant scaffold, the mass
redundancy of the mass-coded library is equivalent to the molecular
mass sum redundancy of all combinations of n peripheral moieties
derived from the identified subset of peripheral moiety
precursors.
[0102] The mass-coding analysis was performed on the initial set of
22 peripheral moieties shown below. This initial set was selected
arbitrarily. Included were peripheral moiety precursors having the
same exact mass. The master set consisted of the peripheral moiety
precursors shown below, along with the exact masses of the
resulting peripheral moieties. The molecular masses given are the
exact molecular masses and not the isotope averages. The exact
molecular masses are also adjusted for any atoms which are lost as
a result of the reaction with the scaffold precursor (in this case
the loss of a hydrogen atom). From the initial set of 22 peripheral
moiety precursors, two sets of 16 peripheral moiety precursors were
generated. One set was chosen by the computer using the mass coding
algorithm described herein (computer selected set). The other set
was randomly chosen.
[0103] From each set of 16 peripheral moiety precursors the
computer generated every possible subset of 12 peripheral moiety
precursors. These subsets were used to generate all combinations of
peripheral moiety precursors taken 4 at a time (representing
libraries synthesized with a scaffold precursor having four
reactive groups, such as four pentafluorophenyl esters). This
process yielded two sets of 16 peripheral moiety precursors
containing 1820 subsets of 12 each. Theoretically, these subsets of
12 peripheral moiety precursors would each yield a library of 1365
compounds containing different peripheral moiety combinations when
allowed to react simultaneously with an appropriate scaffold
precursor containing four reactive groups (15!/[(15-4)!*4!]=1365).
The computer sorted every precursor subset and checked for mass
redundancy in the resultant libraries (in this example mass
redundancies were checked to the second significant digit after the
decimal point).
[0104] It is noteworthy that the mass coding algorithms and the
mass redundancy check are both flexible in that it is possible to
adjust the computational filter to check mass redundancy to any
significant figure. This architecture for mass-coding allows for
rapid automated mass-coding, insures that a significant portion of
the libraries generated with the computer selected set have less
than 10% redundancy, and includes parameters for peripheral moiety
precursor selection outside of exact mass. The computational
requirements for this selection are fairly significant. The
mass-coding algorithms are essential because it is computationally
intractable to brute force calculate and check every possible set
of peripheral moiety precursors from a master set of 60 or more
peripheral moiety precursors. 56
[0105] Results
[0106] The computer selected set of 16 peripheral moiety precursors
contained 86a, 79a, 13a, 108a, 76a, 20a, 69a, la, 70a, 26a, 24a,
36a, 97a, 94a, 104a, and 21a. The set of 16 randomly chosen
peripheral moiety precursors contained 79a, 13a, 20a, 69a, 1a, 26a,
24a, 104a, 52a, 54a, 19a, 77a, 53a, 21a, 55a, 36a. The libraries
generated from the computer selected set of peripheral moiety
precursors had an average mass redundancy of 11.5% per library with
234 libraries having mass redundancies of less than 5% and 972
libraries having mass redundancies of less than 10% (FIG. 2A). The
libraries generated from the randomly chosen set of peripheral
moiety precursors had an average mass redundancy of 60.7% with no
libraries having a mass redundancy of less than 10% (FIG. 2B). A
direct graphical comparison of the mass redundancies of the two
sets of libraries is shown in FIG. 2C. The libraries derived from
the computer-selected set of peripheral moiety precursors and the
corresponding mass redundancies are listed in the Table below.
Example 2
Development of Ligands for a Monofunctional Protein
[0107] A mass-coded combinatorial library can be used to identify
ligands that have a high affinity for a monofunctional protein. One
such monofunctional protein is the serine protease trypsin. Ligands
that exhibit a high affinity for trypsin would be candidates to
screen further for their ability to inhibit the proteolytic
activity of trypsin. The identification of ligands to trypsin
involves the following steps: trypsin is covalently biotinylated by
incubation of the protein with a chemically activated biotin
precursor. The biotin-trypsin conjugate is immobilized by binding
to a streptavidin-derivatized water-insoluble column matrix. The
mass-coded combinatorial library is solubilized in an appropriate
binding buffer and injected onto a column containing the
trypsin+streptavidin complex. Compounds that do not bind to the
column are washed off with binding buffer. Compounds that bind to
the column are dissociated by a change in the buffer conditions,
such as a change in the pH or an increase in the percentage of
organic solvent. These compounds are then loaded onto a
reversed-phase column that is placed downstream of the
trypsin+streptavidin column. The compounds are eluted from the
reversed-phase column and analyzed by mass spectrometry. Molecular
masses that correspond to ligands for trypsin are identified by
eliminating those masses which are also observed when the library
is similarly screened with a streptavidin column. The molecular
mass of each trypsin ligand identifies one combination of
peripheral moieties plus scaffold. The individual compound or
compounds that result from the identified combination of peripheral
moieties plus scaffold are synthesized and tested for their in
vitro activity as inhibitors of trypsin.
Example 3
Development of Ligands for a Multifunctional Protein
[0108] Many proteins, especially human proteins, are
multifunctional, and these functions are often mediated through
interactions with multiple proteins. Ligands that bind to different
sites on the protein might therefore yield different therapeutic
results. The human protein HSP70 is one such example of a
multifunctional protein. HSP70 has been shown to interact with
multiple polypeptides, which are largely unfolded, to facilitate
their translocation and folding. This role of HSP70 has been
implicated in a variety of physiological processes, including
antigen processing/presentation, development of certain cancers,
and replication of a variety of human viruses. A mass-coded
combinatorial library can be used to identify ligands that have a
high affinity for HSP70 and bind at different sites. These ligands
for HSP70 can be further evaluated in secondary assays to establish
their effects on the immune response, cancer progression, and viral
infection.
[0109] The identification of ligands to HSP70 involves the
following steps: HSP70 is covalently biotinylated by incubation of
the protein with a chemically activated biotin precursor. The
biotin-HSP70 conjugate is immobilized by binding to a
streptavidin-derivatized water-insoluble column matrix. The
mass-coded library is solubilized in an appropriate binding buffer
and injected onto a column containing the HSP70-streptavidin
complex. Compounds that do not bind to the column are washed off
with binding buffer. Compounds that bind to the column are
dissociated by a change in the buffer conditions, such as a change
in the pH or an increase in the percentage of organic solvent.
Compounds that are dissociated from the column are loaded onto a
reversed-phase column that is placed downstream of the
HSP70-streptavidin column. Compounds are eluted from the
reversed-phase column and analyzed by mass spectrometry. Masses
that correspond to ligands for HSP70 are identified by eliminating
those masses which are also observed when the library is similarly
screened with a streptavidin column. The mass of each HSP70 ligand
identifies one combination of peripheral moieties plus scaffold.
The individual compound(s) that result from the identified
combination of peripheral moieties plus scaffold are synthesized
and tested for their in vivo ability to affect the immune response,
cancer progression, and viral infection.
Example 3
Development of Ligands that Affect the Binding of a Known Ligand to
a Protein
[0110] It is often the situation that a biologically important
ligand is known for a target protein, but development of a
high-throughput screen for molecules that modulate the binding of
that ligand is not practical. For instance, it is known that HSP70
binds unfolded polypeptides in the presence of ADP, and that the
binding of ATP to HSP70 leads to the dissociation of the
polypeptide. Mass-coded combinatorial libraries can be used in the
discovery of small molecule ligands that affect the binding of ATP,
ADP, or unfolded peptides to HSP70, and one configuration is listed
below: HSP70 is covalently biotinylated by incubation of the
protein with a chemically activated biotin precursor. The
biotin-HSP70 conjugate is immobilized by binding to a
streptavidin-derivatized water-insoluble column matrix. The
mass-coded library is solubilized in an appropriate binding buffer
and injected onto a column containing the HSP70-streptavidin
complex. Compounds that do not bind to the column are washed off
with binding buffer. Compounds that bind to the column are
dissociated upon addition of ATP, ADP, or ADP plus an unfolded
peptide only compounds that bind to the same sites on HSP70 as
these known ligands will be eluted under these conditions.
Compounds that are dissociated from the column are loaded onto a
reversed-phase column that is placed downstream of the
HSP70-streptavidin column. Compounds are eluted from the
reversed-phase column and analyzed by mass spectrometry. Masses
that correspond to ligands for HSP70 are identified by eliminating
those masses which are also observed when the library is similarly
screened with a streptavidin column. The mass of each HSP70 ligand
identifies one combination of peripheral moieties plus scaffold.
The individual compound(s) that result from the identified
combination of peripheral moieties plus scaffold are synthesized
and tested in vitro for the ability to compete with these known
ligands to HSP70 and for their in vivo ability to affect the immune
response, cancer progression, and viral infection.
Example 4
Discovery of Small Molecule Replacements for Protein
Therapeutics
[0111] In some instances, the known ligand to a target protein is
in fact another protein, and the binding of these two proteins
confers a therapeutic benefit. An example of such an interaction is
the binding of granulocyte colony stimulating factor (G-CSF) to the
G-CSF receptor (G-CSF-R). Replacement of G-CSF with a non-peptide
small molecule can be undertaken using a mass-coded combinatorial
library, and one approach is detailed below: in two separate and
parallel experiments, the mass-coded library is solubilized in an
appropriate binding buffer and incubated with either the G-CSF-R
alone or the G-CSF-R plus G-CSF. Compounds that bind to the
protein(s) are separated from the unbound compounds by rapid size
exclusion chromatography. The binding compounds are loaded with the
protein(s) onto a reversed-phase column that is placed downstream
of the size exclusion column. The binding compounds are dissociated
from the protein(s) and are eluted from the reversed-phase column
and analyzed by mass spectrometry. Masses that correspond to
compounds that bind to the G-CSF/G-CSF-R interface are identified
as those masses which are only observed when the library is
screened with G-CSF-R alone; masses which are also observed in the
screen with the G-CSF/G-CSF-R complex are ignored. The mass of each
interface-specific compound identifies one combination of
peripheral moieties plus scaffold. The individual compound(s) that
result from the identified combination of peripheral moieties plus
scaffold are then synthesized and tested for their in vitro or in
vivo ability to mimic G-CSF.
Example 5
Development of Small Molecules that Dimerize Two Proteins
[0112] Certain therapeutic proteins, such as erythropoietin (EPO),
are multivalent and act by binding two molar equivalents of the
target protein, thereby dimerizing the target protein, which, in
the case of EPO is the EPO receptor (EPO-R). The protein
replacement strategy outlined in Example 3 can be extended to yield
non-peptide compounds that act therapeutically by inducing the
dimerization of two EPO-R molecules. In two separate and parallel
experiments, the mass-coded library is solubilized in an
appropriate binding buffer and incubated with either EPO-R alone or
EPO-R plus EPO. Compounds that bind to the protein(s) are separated
from the unbound compounds by rapid size exclusion chromatography.
The bound compounds are loaded with the protein(s) onto a
reversed-phase column that is placed downstream of the size
exclusion column. The bound compounds are dissociated from the
protein(s) and are eluted from the reversed-phase column and
analyzed by mass spectrometry. Masses that correspond to compounds
that bind to the EPO/EPO-R interface are identified as those masses
which are observed only when the library is screened with EPO-R
alone; masses which are also observed in the screen with the
EPO/EPO-R complex are ignored. The mass of each interface-specific
compound identifies one combination of peripheral moieties plus
scaffold. The individual compound(s) that result from the
identified combination of peripheral moieties plus scaffold are
synthesized and tested for their in vitro ability to bind to the
target protein, EPO-R. Those compounds exhibiting the highest
affinity for the target protein are compared to identify
similarities among them. Ideally, it is observed that one site of
derivatization on the scaffold is relatively unimportant for high
affinity binding. The peripheral moiety at this site is
subsequently replaced with a covalent tether that joins two
molecules of the highest affinity compound to yield a non-peptide
compound that dimerizes the target protein, EPO-R.
Example 6
Simultaneous Target Validation and Small-Molecule Drug
Discovery
[0113] An example of a class of target proteins whose roles in a
disease process can be validated by application of target-specific
ligands to a bioassay are the proteins encoded by the open reading
frames (ORF) of the Herpes Simplex Virus. The identification of
ligands to an ORF-encoded protein and the use of the resulting
ligands to determine the function of the ORF-encoded protein and
its validity as a target for anti-viral drug discovery involves the
following steps: the ORF-encoded protein is covalently biotinylated
by incubation of the ORF-encoded protein with a chemically
activated biotin precursor. The ORF-encoded protein-biotin
conjugate is immobilized by binding to a streptavidin-derivatized
water-insoluble column matrix. The mass-coded library is
solubilized in an appropriate binding buffer and injected onto a
column containing the ORF-encoded protein+streptavidin complex.
Compounds that do not bind to the column are washed off with
binding buffer. Compounds that bind to the column are dissociated
by a change in the buffer conditions, such as a change in the pH or
an increase in the percentage of organic solvent. These compounds
are loaded onto a reversed-phase column placed downstream of the
ORF-encoded protein+streptavidin column. The binding compounds are
eluted from the reversed-phase column and analyzed by mass
spectrometry. Molecular masses that correspond to ligands for the
ORF-encoded protein are identified by eliminating those masses that
are also observed when the library is similarly screened with a
streptavidin column. The molecular mass of each ligand for the
ORF-encoded protein identifies one combination of peripheral
moieties plus scaffold. The individual compound(s) that result from
the identified combination of peripheral moieties plus scaffold are
synthesized and tested for their ability to inhibit the replication
or transmission of the virus in a mammalian cell bioassay or animal
model.
[0114] The observation of a virus-specific inhibitory activity
implicates the ORF-encoded protein as a critical component of the
viral disease process and confirms that the ORF-encoded protein is
specifically amenable to small molecule anti-viral drug discovery.
Observation of a direct correlation between the relative binding
affinities of the ORF-encoded protein-specific ligands and the
relative inhibitory concentrations of the ORF-encoded
protein-specific ligands further strengthens the identification of
the ORF-encoded protein as a target for small molecule anti-viral
drug discovery.
Example 7
Development of Small Molecules that can be Applied to the Affinity
Purification of a Target Protein
[0115] A mass-coded combinatorial library can be used to identify
ligands that have a high affinity for a target protein. One such
target protein is human erythropoietin (EPO), which is expressed
and purified industrially for use as a therapeutic drug. Ligands
that exhibit a high affinity for EPO can be immobilized on a solid
support to generate an EPO-specific affinity matrix.
[0116] The identification of ligands to EPO and the construction of
an EPO-specific affinity matrix involves the following steps: the
mass coded library is solubilized in an appropriate binding buffer
and incubated with the EPO protein. Compounds that bind to the EPO
protein are separated from the unbound compounds by rapid size
exclusion chromatography. These compounds are loaded with the EPO
protein onto a reversed-phase column that is placed downstream of
the size exclusion column. The compounds are dissociated from the
EPO protein and are eluted from the reversed-phase column and
analyzed by mass spectrometry. The molecular mass of each EPO
protein-specific ligand identifies one combination of peripheral
moieties plus scaffold. The individual ligand(s) that result from
the identified combination of peripheral moieties plus scaffold are
synthesized and tested for their in vitro ability to bind to the
EPO protein. Compounds exhibiting the highest affinity for the EPO
protein are compared to identify similarities between the
compounds. If it is observed that one reactive site on the scaffold
is relatively unimportant for high affinity binding, the peripheral
moiety at this site is subsequently replaced with a covalent tether
that joins the EPO-specific ligand to a water insoluble matrix,
thereby generating an EPO-specific affinity matrix.
[0117] Alternatively, the covalent tether is used to join the
EPO-specific ligand to a another molecule, such as biotin, which
possesses a high affinity for a commercially available affinity
matrix (streptavidin-derivatized agarose). The biotin-streptavidin
interaction is used as a strong, non-covalent immobilization
technique.
Example 8
Development of Small Molecules that can be Applied to the
Visualization of a Target Protein
[0118] A mass-coded combinatorial library can be used to identify
ligands that have a high affinity for a target protein. One such
target protein is the human protein telomerase, the expression of
which is linked to cancer progression and aging. Ligands that
exhibit a high affinity for telomerase can be functionalized with a
radioactive or non-radioactive tag to thereby generate a
telomerase-specific affinity probe for visualization of the enzyme
in vitro or in vivo. The identification of ligands to telomerase
and the construction of a telomerase-specific affinity probe would
involve the following steps: a mass-coded library is solubilized in
an appropriate binding buffer and incubated with telomerase protein
alone. Compounds that bind to the telomerase protein are separated
from the unbound compounds by rapid size exclusion chromatography.
The binding compounds are loaded with the telomerase protein onto a
reversed-phase column that is placed downstream of the size
exclusion column. The compounds are dissociated from the telomerase
protein and are eluted from the reversed-phase column and analyzed
by mass spectrometry.mass of each telomerase protein-specific
ligand identifies one combination of peripheral moieties plus
scaffold. The individual ligand(s) that result from the identified
combination of peripheral moieties plus scaffold are synthesized
and tested for their in vitro ability to bind to the telomerase
protein. Compounds exhibiting the highest affinity for the
telomerase protein are compared to identify similarities between
the compounds. Ideally, it is observed that one reactive site on
the scaffold is relatively unimportant for high affinity binding.
The peripheral moiety at this site is subsequently replaced with a
covalent tether that joins the telomerase-specific ligand to a
radioactive moiety or a non-radioactive moiety such as a
fluorophore, thereby generating a telomerase-specific affinity
probe.
EQUIVALENTS
[0119] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
spirit and scope of the invention as defined by the appended
claims. Those skilled in the art will recognize or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
specifically herein. Such equivalents are intended to be
encompassed in the scope of the claims.
1TABLE Field1 1 2 3 4 5 6 7 8 9 10 11 12 2.197802 86a 79a 13a 108a
76a 20a 1a 70a 26a 94a 21a 97a 2.490843 86a 108a 76a 69a 1a 70a 26a
24a 36a 94a 21a 97a 2.637363 86a 13a 108a 76a 69a 1a 70a 26a 24a
36a 94a 97a 2.783883 86a 79a 13a 108a 76a 20a 69a 1a 70a 26a 94a
21a 2.783883 86a 79a 13a 108a 76a 20a 69a 1a 70a 36a 94a 21a
2.783883 86a 13a 108a 76a 20a 69a 1a 70a 24a 36a 94a 97a 2.783883
86a 79a 13a 108a 76a 70a 26a 24a 36a 94a 21a 97a 2.930403 86a 13a
108a 76a 20a 69a 1a 70a 36a 94a 21a 97a 2.930403 86a 13a 108a 76a
1a 70a 26a 24a 36a 94a 21a 97a 2.930403 86a 13a 108a 76a 69a 1a 70a
26a 36a 94a 21a 97a 2.930403 86a 79a 13a 108a 76a 20a 69a 1a 70a
26a 24a 104a 3.003663 86a 79a 13a 108a 76a 20a 69a 70a 26a 24a 94a
104a 3.003663 86a 79a 13a 108a 20a 69a 1a 70a 36a 94a 21a 97a
3.076923 86a 13a 108a 76a 20a 69a 1a 70a 26a 24a 94a 97a 3.076923
86a 13a 108a 76a 69a 1a 70a 26a 24a 36a 94a 104a 3.076923 86a 13a
108a 76a 20a 69a 1a 70a 26a 94a 21a 97a 3.076923 86a 108a 76a 20a
69a 1a 70a 24a 36a 94a 104a 97a 3.076923 79a 13a 108a 76a 20a 69a
1a 70a 36a 94a 21a 97a 3.076923 86a 79a 13a 108a 76a 20a 69a 70a
26a 94a 104a 21a 3.076923 86a 79a 13a 108a 76a 20a 1a 70a 36a 94a
21a 97a 3.223443 86a 13a 108a 76a 69a 1a 70a 26a 36a 94a 104a 21a
3.223443 86a 79a 13a 108a 76a 69a 1a 70a 26a 94a 104a 21a 3.223443
86a 79a 13a 108a 20a 69a 1a 70a 24a 36a 94a 97a 3.369963 86a 79a
13a 108a 20a 69a 1a 70a 26a 94a 21a 97a 3.369963 86a 13a 108a 76a
20a 69a 1a 70a 26a 94a 104a 21a 3.369963 86a 79a 13a 108a 76a 20a
1a 70a 26a 94a 104a 21a 3.516484 86a 13a 108a 76a 20a 69a 1a 70a
26a 24a 36a 97a 3.516484 86a 79a 13a 108a 20a 69a 1a 70a 26a 24a
94a 97a 3.516484 86a 13a 108a 76a 20a 69a 1a 70a 26a 24a 94a 104a
3.516484 86a 79a 13a 108a 76a 20a 69a 1a 70a 26a 104a 21a 3.516484
86a 13a 108a 76a 20a 69a 1a 70a 26a 36a 21a 97a 3.663004 86a 79a
13a 108a 76a 20a 69a 1a 70a 26a 94a 104a 3.663004 79a 13a 108a 76a
69a 1a 70a 36a 94a 104a 21a 97a 3.663004 79a 13a 108a 76a 20a 1a
70a 26a 94a 104a 21a 97a 3.663004 86a 79a 108a 76a 69a 70a 26a 24a
36a 94a 21a 97a 3.663004 86a 79a 13a 108a 76a 20a 69a 1a 70a 94a
104a 21a 3.663004 86a 13a 108a 76a 20a 69a 1a 70a 26a 36a 94a 97a
3.809524 86a 13a 108a 76a 69a 1a 70a 24a 36a 94a 104a 97a 3.809524
79a 13a 108a 76a 69a 1a 70a 26a 94a 104a 21a 97a 3.809524 86a 79a
13a 108a 76a 1a 70a 26a 24a 94a 21a 97a 3.809524 79a 13a 108a 76a
20a 69a 1a 26a 94a 104a 21a 97a 3.809524 79a 13a 108a 76a 69a 70a
26a 24a 36a 94a 104a 97a 3.809524 79a 13a 108a 76a 20a 69a 1a 70a
26a 94a 21a 97a 3.882784 79a 13a 108a 76a 69a 70a 26a 36a 94a 104a
21a 97a 3.956044 86a 79a 108a 76a 20a 69a 70a 26a 24a 94a 104a 97a
3.956044 86a 79a 13a 108a 76a 20a 69a 1a 70a 24a 36a 94a 3.956044
86a 79a 13a 108a 76a 20a 69a 1a 36a 94a 21a 97a 3.956044 86a 13a
108a 76a 69a 1a 70a 26a 24a 36a 94a 21a 3.956044 86a 79a 13a 108a
76a 69a 1a 70a 26a 94a 21a 97a 3.956044 86a 108a 76a 69a 1a 70a 24a
36a 94a 104a 21a 97a 3.956044 79a 13a 108a 76a 20a 69a 1a 70a 26a
24a 104a 97a 3.956044 86a 79a 13a 108a 20a 69a 1a 70a 24a 94a 104a
97a 3.956044 86a 79a 108a 76a 20a 69a 1a 70a 26a 94a 104a 21a
3.956044 86a 79a 13a 108a 76a 20a 69a 1a 26a 94a 104a 21a 4.029304
86a 79a 13a 76a 20a 69a 1a 70a 26a 24a 94a 104a 4.102564 86a 79a
13a 108a 76a 20a 69a 1a 70a 26a 24a 94a 4.102564 86a 13a 108a 76a
69a 1a 70a 24a 36a 94a 21a 97a 4.102564 86a 108a 76a 69a 1a 70a 26a
24a 36a 94a 104a 21a 4.102564 79a 13a 108a 76a 20a 69a 1a 70a 94a
104a 21a 97a 4.102564 86a 79a 13a 108a 76a 20a 69a 1a 70a 26a 24a
36a 4.102564 86a 13a 108a 76a 20a 69a 1a 26a 24a 36a 94a 97a
4.175824 86a 79a 13a 108a 20a 69a 1a 70a 26a 24a 94a 104a 4.175824
86a 79a 13a 108a 76a 69a 70a 26a 36a 94a 21a 97a 4.175824 86a 79a
108a 76a 20a 69a 1a 70a 36a 94a 21a 97a 4.249084 86a 79a 13a 108a
76a 1a 70a 26a 36a 94a 21a 97a 4.249084 86a 13a 108a 76a 69a 1a 26a
24a 36a 94a 21a 97a 4.249084 86a 79a 13a 108a 76a 69a 70a 26a 36a
94a 104a 21a 4.249084 86a 13a 108a 76a 20a 69a 1a 70a 26a 24a 36a
94a 4.249084 86a 79a 13a 108a 76a 69a 1a 70a 36a 94a 21a 97a
4.249084 86a 79a 13a 108a 76a 20a 1a 70a 26a 24a 94a 97a 4.249084
79a 13a 108a 76a 20a 69a 70a 26a 24a 94a 104a 97a 4.249084 86a 79a
108a 76a 20a 69a 1a 70a 26a 94a 21a 97a 4.249084 86a 79a 13a 108a
76a 20a 69a 1a 70a 24a 36a 97a 4.322344 79a 13a 108a 76a 20a 69a
70a 26a 36a 94a 21a 97a 4.395605 86a 79a 13a 108a 69a 1a 70a 26a
24a 36a 94a 97a 4.395605 79a 13a 108a 76a 20a 69a 70a 26a 94a 104a
21a 97a 4.395605 86a 79a 13a 108a 76a 20a 69a 1a 70a 36a 94a 97a
4.395605 79a 13a 108a 76a 20a 69a 70a 26a 24a 36a 94a 97a 4.395605
86a 79a 13a 108a 76a 20a 69a 1a 70a 26a 24a 97a 4.395605 86a 79a
13a 108a 76a 20a 69a 1a 70a 26a 36a 94a 4.395605 79a 13a 108a 76a
20a 69a 1a 70a 26a 104a 21a 97a 4.395605 86a 79a 13a 108a 20a 69a
1a 70a 26a 94a 104a 21a 4.395605 79a 13a 108a 76a 20a 69a 1a 70a
26a 94a 104a 21a 4.395605 86a 79a 13a 108a 76a 20a 70a 26a 36a 94a
21a 97a 4.395605 86a 79a 13a 108a 76a 20a 70a 26a 24a 94a 104a 97a
4.395605 86a 79a 13a 108a 76a 69a 70a 26a 24a 36a 94a 21a 4.395605
13a 108a 76a 69a 1a 70a 26a 36a 94a 104a 21a 97a 4.395605 86a 79a
13a 108a 76a 20a 69a 1a 70a 94a 21a 97a 4.395605 86a 108a 76a 20a
69a 1a 70a 26a 24a 36a 94a 97a 4.395605 86a 79a 13a 108a 76a 20a 1a
70a 26a 36a 94a 97a 4.395605 86a 79a 13a 108a 76a 69a 70a 26a 24a
36a 94a 97a 4.468864 86a 79a 13a 108a 20a 69a 70a 26a 24a 94a 104a
97a 4.468864 86a 79a 13a 108a 76a 20a 1a 70a 26a 24a 36a 97a
4.468864 86a 108a 76a 20a 69a 1a 70a 36a 94a 104a 21a 97a 4.468864
86a 79a 13a 108a 76a 20a 69a 70a 26a 24a 36a 94a 4.468864 86a 79a
13a 76a 1a 70a 26a 24a 36a 94a 21a 97a 4.468864 86a 79a 13a 108a
76a 20a 70a 26a 24a 36a 94a 97a 4.542125 86a 79a 13a 108a 69a 1a
70a 24a 36a 94a 104a 97a 4.542125 86a 79a 13a 108a 76a 20a 69a 70a
26a 36a 94a 21a 4.542125 86a 79a 13a 108a 76a 69a 70a 26a 24a 36a
94a 104a 4.542125 86a 79a 13a 108a 76a 20a 69a 1a 26a 94a 21a 97a
4.542125 86a 79a 13a 76a 20a 69a 1a 70a 24a 36a 94a 97a 4.542125
86a 108a 76a 20a 69a 1a 70a 26a 36a 94a 21a 97a 4.542125 86a 13a
108a 76a 69a 70a 26a 24a 36a 94a 21a 97a 4.542125 86a 79a 13a 108a
76a 1a 70a 24a 36a 94a 21a 97a 4.542125 13a 108a 76a 20a 69a 1a 70a
26a 94a 104a 21a 97a 4.542125 86a 13a 108a 76a 20a 69a 1a 26a 36a
94a 21a 97a 4.542125 86a 79a 13a 108a 76a 69a 1a 70a 26a 24a 94a
104a 4.542125 86a 79a 13a 108a 76a 20a 69a 70a 36a 94a 21a 97a
4.542125 86a 79a 13a 108a 76a 20a 69a 1a 70a 26a 94a 97a 4.615385
79a 108a 76a 20a 69a 1a 70a 36a 94a 104a 21a 97a 4.688645 86a 79a
13a 108a 76a 20a 69a 1a 70a 26a 21a 97a 4.688645 86a 79a 13a 108a
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21a 97a 4.688645 13a 108a 76a 69a 1a 70a 26a 24a 36a 94a 104a 97a
4.688645 86a 79a 13a 108a 76a 20a 69a 70a 24a 36a 94a 97a 4.688645
86a 79a 13a 76a 20a 69a 1a 70a 26a 94a 104a 21a 4.688645 86a 108a
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69a 1a 36a 94a 104a 21a 4.688645 86a 79a 13a 108a 76a 69a 1a 70a
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86a 79a 13a 76a 20a 69a 1a 70a 36a 94a 21a 97a 4.835165 79a 13a
108a 76a 20a 69a 1a 26a 24a 94a 104a 97a 4.835165 86a 79a 13a 108a
76a 20a 69a 1a 26a 24a 94a 104a 4.835165 79a 13a 108a 76a 69a 1a
70a 24a 36a 94a 104a 97a 4.835165 86a 13a 108a 76a 20a 1a 70a 26a
36a 94a 21a 97a 4.835165 86a 79a 13a 108a 69a 1a 70a 26a 24a 94a
21a 97a 4.835165 79a 108a 76a 20a 69a 70a 26a 24a 36a 94a 104a 97a
4.835165 86a 108a 76a 69a 1a 70a 26a 24a 36a 104a 21a 97a 4.835165
86a 13a 108a 76a 20a 69a 1a 70a 24a 36a 94a 104a 4.835165 79a 13a
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94a 97a 4.835165 86a 79a 13a 108a 76a 20a 69a 70a 26a 24a 94a 97a
4.835165 86a 79a 13a 76a 69a 1a 70a 26a 24a 36a 94a 104a 4.835165
79a 13a 108a 76a 20a 1a 70a 26a 36a 94a 21a 97a 4.835165 86a 13a
76a 20a 69a 1a 70a 26a 24a 36a 94a 97a 4.835165 86a 13a 108a 76a
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69a 1a 70a 36a 21a 97a 4.908425 86a 79a 13a 108a 76a 1a 70a 26a 24a
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108a 76a 20a 69a 70a 26a 24a 36a 94a 97a 4.981685 86a 108a 76a 20a
69a 1a 70a 26a 24a 36a 94a 104a 4.981685 79a 13a 108a 76a 20a 69a
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104a 21a 97a 4.981685 86a 79a 13a 108a 69a 1a 70a 24a 36a 94a 21a
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4.981685 86a 13a 76a 69a 1a 70a 26a 24a 36a 94a 21a 97a 4.981685
86a 79a 13a 108a 76a 20a 69a 70a 26a 94a 21a 97a 4.981685 86a 79a
108a 20a 69a 1a 70a 24a 36a 94a 104a 97a 4.981685 86a 13a 108a 76a
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104a 21a 5.128205 13a 108a 76a 20a 69a 1a 70a 26a 24a 94a 104a 97a
5.128205 86a 79a 13a 76a 20a 69a 1a 70a 26a 24a 94a 97a 5.128205
86a 108a 76a 20a 69a 1a 70a 26a 36a 94a 104a 21a 5.128205 79a 13a
108a 20a 69a 1a 70a 26a 94a 104a 21a 97a 5.128205 86a 79a 13a 76a
69a 1a 70a 26a 24a 36a 94a 97a 5.128205 86a 79a 13a 108a 20a 69a 1a
70a 26a 24a 104a 97a 5.128205 79a 13a 76a 20a 69a 1a 70a 26a 24a
94a 104a 97a 5.128205 86a 13a 108a 76a 69a 1a 70a 26a 24a 94a 21a
97a 5.128205 86a 79a 76a 20a 69a 1a 70a 24a 36a 94a 104a 97a
5.128205 79a 13a 76a 20a 69a 1a 70a 26a 94a 104a 21a 97a 5.128205
86a 13a 76a 20a 69a 1a 70a 26a 36a 94a 21a 97a 5.128205 86a 13a
108a 69a 1a 70a 26a 24a 36a 94a 21a 97a 5.201465 86a 13a 108a 76a
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1a 26a 24a 94a 104a 97a 5.274725 79a 13a 108a 76a 20a 69a 70a 24a
36a 94a 104a 97a 5.274725 86a 79a 13a 108a 20a 69a 1a 70a 94a 104a
21a 97a 5.274725 86a 13a 108a 20a 69a 1a 70a 26a 36a 94a 21a 97a
5.274725 86a 79a 13a 108a 76a 69a 1a 70a 26a 24a 94a 21a 5.274725
86a 79a 13a 108a 76a 20a 69a 1a 70a 24a 94a 104a 5.274725 86a 79a
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76a 20a 1a 70a 26a 24a 94a 104a 5.274725 86a 79a 108a 76a 20a 69a
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94a 104a 21a 5.347985 86a 79a 108a 76a 20a 69a 70a 24a 36a 94a 104a
97a 5.347985 79a 13a 108a 76a 69a 70a 26a 24a 36a 94a 21a 97a
5.347985 86a 79a 13a 108a 20a 69a 1a 70a 26a 36a 94a 97a 5.347985
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108a 76a 20a 69a 1a 70a 26a 36a 94a 104a 5.421246 86a 79a 13a 76a
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70a 26a 24a 94a 104a 97a 5.421246 86a 79a 13a 108a 76a 69a 1a 70a
26a 36a 94a 97a 5.421246 79a 13a 108a 76a 20a 69a 1a 70a 26a 24a
94a 97a 5.421246 79a 13a 108a 76a 20a 69a 1a 70a 24a 36a 104a 97a
5.421246 86a 79a 108a 76a 69a 70a 26a 24a 36a 94a 104a 21a 5.421246
108 76a 69a 1a 70a 26a 24a 36a 94a 104a 21a 97a 5.421246 79a 13a
76a 20a 69a 1a 70a 26a 24a 36a 94a 97a 5.421246 86a 79a 108a 76a
20a 69a 70a 26a 36a 94a 21a 97a 5.421246 86a 79a 108a 76a 20a 69a
70a 26a 24a 36a 94a 97a 5.421246 86a 79a 108a 20a 69a 1a 70a 26a
24a 94a 104a 97a 5.421246 86a 79a 108a 76a 69a 70a 26a 24a 36a 94a
104a 97a 5.421246 79a 13a 108a 76a 20a 69a 70a 36a 94a 104a 21a 97a
5.421246 86a 79a 13a 108a 76a 69a 1a 70a 26a 24a 94a 97a 5.494505
86a 79a 13a 108a 20a 69a 70a 26a 24a 36a 94a 97a 5.494505 86a 79a
108a 76a 20a 69a 1a 70a 26a 24a 36a 97a 5.494505 86a 79a 108a 76a
20a 69a 70a 26a 36a 94a 104a 21a 5.494505 86a 79a 13a 76a 69a 1a
70a 24a 36a 94a 104a 97a 5.494505 86a 79a 13a 108a 76a 20a 70a 26a
94a 104a 21a 97a 5.494505 86a 79a 108a 76a 20a 69a 70a 26a 24a 36a
94a 104a 5.567766 79a 13a 108a 76a 20a 1a 70a 36a 94a 104a 21a 97a
5.567766 86a 79a 13a 108a 76a 69a 1a 70a 24a 36a 94a 97a 5.567766
86a 79a 13a 76a 20a 69a 1a 70a 26a 24a 36a 97a 5.567766 86a 79a
108a 76a 20a 69a 1a 70a 26a 24a 94a 97a 5.567766 86a 79a 108a 76a
69a 1a 70a 26a 24a 94a 21a 97a 5.567766 79a 108a 76a 20a 69a 70a
26a 36a 94a 104a 21a 97a 5.567766 86a 79a 13a 108a 20a 69a 70a 26a
36a 94a 21a 97a 5.567766 86a 79a 13a 108a 76a 20a 69a 70a 26a 24a
36a 97a 5.567766 86a 79a 13a 108a 76a 20a 69a 1a 24a 36a 94a 97a
5.567766 86a 79a 13a 108a 76a 20a 69a 1a 26a 24a 94a 97a 5.567766
86a 79a 13a 108a 76a 69a 26a 24a 36a 94a 104a 97a 5.567766 86a 13a
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21.90476 86a 13a 108a 20a 69a 1a 26a 24a 36a 94a 104a 21a
* * * * *