U.S. patent application number 09/836145 was filed with the patent office on 2002-05-30 for proteomic analysis.
This patent application is currently assigned to The Scripps Research Institute of an Assignment. Invention is credited to Adam, Gregory, Cravatt, Benjamin F., Lovato, Martha, Patricelli, Matthew P., Sorensen, Erik.
Application Number | 20020064799 09/836145 |
Document ID | / |
Family ID | 27393542 |
Filed Date | 2002-05-30 |
United States Patent
Application |
20020064799 |
Kind Code |
A1 |
Cravatt, Benjamin F. ; et
al. |
May 30, 2002 |
Proteomic analysis
Abstract
The present invention provides methods for analyzing proteomes,
as cells or lysates. The analysis is based on the use of probes
that have specificity to the active form of proteins, particularly
enzymes and receptors. The probes can be identified in different
ways. In accordance with the present invention, a method is
provided for generating and screening compound libraries that are
used for the identification of lead molecules, and for the parallel
identification of their biological targets. By appending specific
functionalities and/or groups to one or more binding moieties, the
reactive functionalities gain binding affinity and specificity for
particular proteins and classes of proteins. Such libraries of
candidate compounds, referred to herein as activity-based probes,
or ABPs, are used to screen for one or more desired biological
activities or target proteins.
Inventors: |
Cravatt, Benjamin F.; (La
Jolla, CA) ; Sorensen, Erik; (San Diego, CA) ;
Patricelli, Matthew P.; (San Diego, CA) ; Lovato,
Martha; (San Diego, CA) ; Adam, Gregory; (San
Diego, CA) |
Correspondence
Address: |
Lisa A. Haile, Ph.D.
Gray Cary Ware & Freidenrich LLP
Suite 1600
4365 Executive Drive
San Diego
CA
92121-2189
US
|
Assignee: |
The Scripps Research Institute of
an Assignment
|
Family ID: |
27393542 |
Appl. No.: |
09/836145 |
Filed: |
April 16, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09836145 |
Apr 16, 2001 |
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09738271 |
Dec 15, 2000 |
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60195954 |
Apr 10, 2000 |
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60212891 |
Jun 20, 2000 |
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60222532 |
Aug 2, 2000 |
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Current U.S.
Class: |
435/7.1 ;
546/339; 548/570; 568/25 |
Current CPC
Class: |
Y10T 436/16 20150115;
C40B 30/04 20130101; G01N 2500/20 20130101; C12Q 1/26 20130101;
G01N 33/535 20130101; G16B 20/00 20190201; G01N 33/557 20130101;
G01N 33/6842 20130101; C40B 40/04 20130101; G01N 33/6845 20130101;
Y10T 436/18 20150115; C12Q 1/32 20130101; G16B 20/30 20190201; C12Q
1/37 20130101 |
Class at
Publication: |
435/7.1 ;
546/339; 548/570; 568/25 |
International
Class: |
G01N 033/53; C07C
317/10; C07D 213/44; C07D 207/04 |
Goverment Interests
[0002] This invention was made with Government support under
Contract No. MH58542 and CA87660, awarded by the National
Institutes of Health. The government may have certain rights in the
invention.
Claims
What is claimed is:
1. A composition comprising at least one covalent product of a
target enzyme of a complex protein composition and at least one
activity based probe member of a combinatorial chemical library
comprising a plurality of members of the formula R (F-L)-X wherein:
X is a ligand, said ligand having the same chemical structure for
each of said members of said library; L is a bond or linking group,
which is the same in each of the members of said library, and said
linking group is of from 1 to 20 carbon atoms; F is a sulphonyl
group reactive at an active site of a target enzyme; and R is an
organic group of less than 1 kDal, that is different in each of the
members of the library and is bonded to F; and wherein members of
the library have different on rates with said target enzyme.
2. A composition according to claim 1, wherein R is selected from
the group consisting of alkyl, cycloalkyl, heterocycle and aryl and
substituted members thereof.
3. A composition according to claim 1, wherein said target enzyme
is an aldehyde dehyrogenase.
4. A composition according to claim 1, wherein said linking group
is an aliphatic chain.
5. A composition according to claim 1, wherein said linking group
is an alkyleneoxy chain of from one to 6 alkyleneoxy groups,
wherein said alkyleneoxy is of from 2 to 3 carbon atoms.
6. A compound of the formula: R(F-L)-X wherein: X is biotin; L is
alkylene or an alkyleneoxy chain of from 1 to 6 alkyleneoxy groups,
wherein said alkyleneoxy groups are of from 2 to 3 carbon atoms; F
is a sulphonyl group; R is heteroaryl or aryl.
7. A compound according to claim 6, wherein R is pyridyl.
8. A compound according to claim 6, wherein R is thiophenyl.
9. A combinatorial chemical library comprising a plurality of
members of the formula R(F-L)-X wherein: X is a ligand for binding
to a reciprocal receptor; L is alkylene or an alkyleneoxy chain of
from 1 to 6 alkyleneoxy groups, wherein said alkyleneoxy groups are
of from 2 to 3 carbon atoms; F is a sulphonyl group reactive at an
active site of a target enzyme; and R is an organic group of less
than 1 kDal, that is different in each of the members of the
library and is bonded to R; and wherein members of the library have
different on rates with said target enzyme.
10. A combinatorial library according to claim 9, wherein R in one
member of said library is pyridyl or thiophenyl.
11. A combinatorial library according to claim 9, wherein said
ligand is biotin.
12. A method for screening for molecules having an affinity for an
active protein in a complex mixture of proteins from a biological
source, employing a combinatorial chemical library comprising a
plurality of members of the formula R*(F-L)-X wherein: X is a
ligand having the same chemical structure for each of said members
of said library; L is a bond or alkylene or an alkyleneoxy chain
linking group of from 1 to 6 alkyleneoxy groups, wherein said
alkyleneoxy groups are of from 2 to 3 carbon atoms, which is the
same in each of the members of said library; F is a sulfonyl
functional group reactive at an active site of a protein member,
which functional group comprises the same reactive functionality in
each of the members of said library; and R is a group of less than
1 kDal, that is different in each of the members of the library;
the * intends that R is a part of F or L; and wherein members of
said library have different on rates with said protein member; said
method comprising: (1) combining with said complex mixture, in an
active form and an inactivated form, said combinatorial chemical
library under conditions for reaction of said functional group with
active proteins to form a conjugate; (2) isolating conjugates from
said active and inactivated complex mixture; and (3) comparing
conjugates formed with said active and inactivated complex
mixtures; whereby conjugates in said active complex mixture absent
in said inactivated complex mixture are comprised only of active
proteins reactive with members of said combinatorial library.
13. A method according to claim 12, wherein each of said members of
said combinatorial library is isotopically individually labeled,
said method including the additional steps of: isolating conjugates
from said active complex mixtures; and analyzing said conjugates
for the composition of said probe by means of said isotopic
individual label and for the composition of said protein by at
least partial sequencing.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of Ser. No. 09/738,271,
filed Dec. 15, 2000, which claims priority under 35 U.S.C.
.sctn.119(e)(1) to U.S. provisional applications serial Nos.
60/195,954, filed Apr. 10, 2000; 60/212,891, filed Jun. 20, 2000;
and 60/222,532, filed Aug. 2, 2000, all of which are herein
incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0003] The field of this invention is analyzing portions of a
proteome.
BACKGROUND OF THE INVENTION
[0004] Determination of the genomic sequence of higher organisms,
including humans, is now an attainable goal. However, this analysis
represents only one aspect of the information encoded by the
genome. Genes are expressed in an ordered and timely manner, and
are also exhibit a precise spatial and temporal expression pattern.
Consequently, knowing the sequence of the genome is insufficient to
explain biology and to understand disease.
[0005] More significantly, genes are transcribed to messenger RNA,
which is then translated to protein. It is the protein, or gene
product, that exhibits activity, and carries out the work of the
cell. With the post-genome era rapidly approaching, new strategies
for the analysis of proteins are being developed. Most conventional
approaches focus on recording variations in protein level. These
approaches are commonly referred to as "proteomics". In general,
proteomics seeks to measure the abundance of broad profiles of
proteins from complex biological mixtures. In the most common
embodiments, proteomics involves separating the proteins within a
sample by two-dimensional SDS-PAGE. Then, the individual protein
spots patterns of these gels can be compared to get indications as
to the relative abundance of a particular protein in two
comparative samples. The approach can even be extended to determine
the molecular identity of the individual protein spots by excising
the spots and subjecting them to peptide mass fingerprinting. More
recently, methods have been described for eliminating the
electrophoresis steps and performing proteomics by directly
analyzing the complex mixture by mass spectrometry. For example,
methods currently described in the art provide chemically reactive
probes that can be reacted with a protein mixture to label many
proteins in that mixture in a non-specific, or non-directed, manner
providing only a quantitative analysis of proteins (see Aebersold,
PCT/US99/19415). Such methods teach that there are many chemically
reactive amino acid residues within a protein which are
individually reactive and which can be conjugated to chemical
probes, whereby protein conjugates can be subsequently quantified
to yield an indication of protein abundance. Similarly, Wells et
al. (PCT/US99/14267; PCT/US98/21759) describe methods for
identifying small organic molecule ligands that bind to biological
target molecules without the requirement that the ligand bind to an
active site on a target molecule. These methods do not describe
selectively detecting active versus inactive proteins within a
sample.
[0006] The need to devise methods of measuring protein activity, as
opposed to abundance are best illustrated by an important subset of
proteins called enzymes. Many classes of enzymes are encoded by the
genome. Enzymes are key to almost every biologic process, including
blood coagulation, inflammation, angiogenesis, neural plasticity,
peptide hormone processing and T-lymphocyte-mediated cytotoxicity.
Several human diseases are associated with dysfunctions in enzymes.
These include, but are not limited to, hemorrhagic disorders,
emphysema, arthritis and even to cancer.
[0007] Although current proteomic approaches, such as those
described above, could theoretically provide information on the
abundance of an enzyme, these methods fail to report on enzyme
activity. This is a key limitation because the activity of enzymes,
and even other proteins, is often regulated by post-translational
modification. Importantly, the active site represents only a small
portion of the entire surface of the protein. The chemical nature
and reactivity of this active site is governed by the local
environment of the site, which is conferred by its amino acid
compositions and its three dimensional structure. The shape and/or
exposure of the active site of an enzyme can be modulated by any
number of biological events. In many cases, the active site of an
enzyme can be masked by natural inhibitors. Alternatively, the
shape of the active site can be made more favorable for activity by
the action of allosteric cofactors.
[0008] In many cases a library of compounds is screened to identify
those compounds with desired biological effect. Once such compounds
("leads") are identified, an iterative process is undertaken to
refine their chemical and biochemical properties so that they can
be used as drugs. A key step in this iterative process is the
identification of the biological target molecule that is inhibited
by the lead compound. Knowing the identity of the biological target
molecule allows one to streamline the development process by
devising simplified, high-throughput assays to test additional
compounds based on the structure of the lead compound for enhanced
potency. In addition, it is vital to know the identity of the
biological target so that one can interpret studies aimed at
testing such compounds for effect in animals and in human
trials.
[0009] One of the inherent difficulties with the entire development
process is that it is often difficult to identify the biological
target molecule for lead compounds. For example, one might
establish a screen to identify leads that block cell division. If
successful, such a screen might identify a number of leads, all
with varying ability to block cell division. Cell division is a
complex process involving numerous biochemical pathways and
hundreds of proteins. The lead compounds might therefore, bind to
and inhibit any one of these proteins.
[0010] There is no simple way of determining what the biological
target molecule is for lead compounds identified from such screens.
Nor, is there a way of knowing if multiple lead compounds interact
with the same, or with different, biological target molecules.
Consequently, the identification of the biological target molecule
relies on conventional fractionation and purification strategies,
which are cumbersome, time consuming and expensive. Moreover,
without knowledge of the identity of the biological target
molecule, and an understanding of its precise biochemical activity,
one may be unable to devise assays to track its purification during
these steps. Consequently, the identity of such biological target
molecules is often impossible to determine using current
approaches.
SUMMARY OF THE INVENTION
[0011] Systems are provided for identifying portions of proteomes,
where the proteome activity is determined for a plurality of active
proteins and the effect of agents on the activity of such proteome
portion. The system includes methods for identifying probes useful
for reacting with active proteins, probes for reacting with active
proteins and methods for identifying the proteins with which the
probes have reacted. The probes comprise a reactive moiety that as
part of the probe is limited in its reactivity to the target active
proteins, a ligand for sequestering the conjugate of the probe and
the target protein and optionally an identifier that can be
released and analyzed, and will be referred to as "activity based
probes" ("ABPs").
[0012] The system includes identifying groups having specific
affinities for a protein conformation, identifying target proteins
in a proteome of biological interest related to the status of a
biological system, producing probes for binding to groups of target
proteins, assaying proteomic profiles for groups of proteins and
analyzing the resulting data. The systems can also be used for
screening for bioactivity profiles of candidate compounds.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1 is a schematic diagram of integrated cell-based
screening for bioactive compounds (i.e., the non-labeled or
untagged library) with target identification (i.e., using the
corresponding labeled or tagged library).
[0014] FIG. 2 shows a protein profile from a testis extract on a
reducing SDS-PAGE with a pyridyl sulfonate ABP, with (-) and
without heating, and a methyl sulfonate ABP, with
[0015] (-) and without heating.
[0016] FIG. 3 shows the results of a non-directed tagged library of
sulfonates (aryl) as ABPs for the identification of a novel ADH
enzyme.
[0017] FIG. 4 shows an illustration of a strategy for synthesis of
biotinylated sulfonate esters for use as ABPs and a strategy for
stereocontrolled synthesis of conformationally well-defined
spiroepoxides of type VI (see also, Somensen, et al., Angew. Chem.
Int. Ed., 1999, 38:971-974, herein incorporated by reference in its
entirety).
[0018] FIG. 5 shows identification of FP-biotin reactive proteins
from rat brain. Intensity of labeling of FP-biotin as ABP was
compared with that of serial dilution of a FAAH sample reacted to
completion with the inhibitor.
[0019] FIG. 6 shows protein profiles for FP-PEG-biotin compared
with protein profiles for FP-biotin.
[0020] FIG. 7 shows parameters that affect the proteome reactivity
of pyridylsulfonate 1. Time-dependence of 1-proteome reaction (5
.mu.M of 1, 0.5 .mu.g/.mu.L protein, 50 mM Tris.HCl, pH 8.0). B,
Concentration-dependence of 1-proteome reaction. Left panel, 1,
2.5, and 5 .mu.M concentrations of 1 were reacted with the testis
proteome (0.5 .mu.g/.mu.L protein, 50 mM Tris.HCl, pH 8.0, 30 min
reaction). Right panel, 5, 10, 20, and 50 .mu.M concentrations of 1
were reacted with the testis proteome (0.5 .mu.g/.mu.L protein, 50
mM Tris.HCl, pH 8.0, 30 min reaction). Short (upper right panel)
and long (lower right panel) film exposures of these reactions are
shown. Note the presence of an endogenous 80 kDa avidin-reactive
protein in the lane containing an untreated testis proteome. C,
pH-dependence of 1 -proteome reaction (5 .mu.M of 1, 0.5
.mu.g/.mu.L protein, 30 min reaction). Left panel, reactions
conducted from pH 6.0-8.0. Right panel, reactions conducted from pH
8.0-10.0. D, Thiol-dependence of 1-proteome reaction. Control
reactions were conducted under standard conditions. Each thiol (2
mM) was added to the proteome prior to the addition of 1.
[0021] FIG. 8 is a flow diagram of the preparation of an
activity-based probe using a polyethylene linker.
[0022] FIG. 9 is an exemplary synthetic route for the generation of
biotinylated sulfonate esters.
[0023] FIG. 10, A, General structure of an activity-based probe
(ABP), highlighting the probe's four main components: a binding
group (BG), a reactive group (RG), a linker (L), and a tag (T). B,
Structures of members of an ABP library, where the BG is varied,
the RG is a sulfonate ester, the L is an extended alkyl chain, and
the T is biotin.
[0024] FIG. 11 shows specific and nonspecific proteome reactivities
of sulfonates 1-7 (A) and 8-11 (B). For A and B, each sulfonate's
reactivity with both a heated and unheated version of a rat testis
proteome is shown (standard reaction conditions: 5 .mu.M sulfonate,
0.5 .mu.g/.mu.L testis protein, 50 mM Tris.HCl, pH 8.0, 100 mM
NaCl; 30 min reaction, 25.degree. C.). Sulfonate-labeled proteins
were detected by SDS-PAGE (7.5 .mu.g protein/lane) and avidin
blotting. Highlighted with arrowheads and dots are proteins that
reacted with sulfonates in a heat-sensitive manner. The proteins
labeled in the lanes containing preheated proteomes (D) were all
considered "nonspecific" sulfonate reactivities, except an 80 kDa
protein which represented an endogenous avidin-reactive protein
(i.e., also observed in proteomes not treated with sulfonates; see
FIG. 13B). Different film exposure times are presented for the high
(45-100 kDa, 1.times. time exposure) and low (27-45 kDa, 4.times.
time exposure) molecular mass proteins to permit the signals of
labeled proteins to be shown prior to film saturation.
[0025] FIG. 12 shows side-by-side comparisons of the proteome
reactivities of sulfonates 1, 5, and 9. A Left panel, proteome
reactivities of sulfonates 1 and 5. Heat-sensitive protein
reactivities selective for 1 and 5 are highlighted (double and
single arrowheads, respectively). Right panel, Coomassie blue
stained protein gel of samples treated with 1 and 5. Arrowheads
highlight abundant proteins correlating in molecular mass with
proteins labeled by 5 in the preheated proteome. B, Left panel,
proteome reactivities of sulfonates 1 and 9. Heat-sensitive protein
reactivities selective for 1 and 9 are highlighted (double and
single arrowheads, respectively). Right panel, Coomassie blue
stained protein gel of samples treated with 1 and 9. For A and B,
different film exposure times are presented for the high (45-100
kDa, 1.times. time exposure) and low (27-45 kDa, 4.times. time
exposure) molecular mass proteins to permit the signals of labeled
proteins to be shown prior to film saturation.
[0026] FIG. 13 shows the identification of a 55 kDa
specifically-labeled sulfonate target as cytosolic class I aldehyde
dehydrogenase (cALDH-I). A, Avidin-based affinity isolation of the
55 kDa 1-labeled protein. Shown is an avidin blot of samples
containing the partially purified 55 kDa protein (Q Flow Through)
and the affinity-isolated 55 kDa protein (Avidin Beads). Also shown
are the tryptic peptides from this protein that identified it as
cALDH-I. B, Sulfonate 1 labels recombinant cALDH-I in eukaroytic
expression systems. Protein samples from COS-7 (left panel) and
MCF-7 (right panel) cells transfected with the cALDH-I cDNA or
empty vector (mock) were reacted with 1 and resolved by SDS-PAGE
and avidin blotting. A strongly labeled 55 kDa protein was
identified only in the cALDH-I-tranfected cells. C, Sulfonate 1
labels recombinant cALDH-I in prokaryotic expression systems. A
protein sample of E. coli BL-21 cells transformed with a His-tagged
version of cALDH-I was reacted with 1 and resolved by SDS-PAGE and
avidin blotting. A strongly labeled 60 kDa protein was identified,
corresponding to the predicted molecular mass of cALDH-I with an
appended N-terminal histidine tag.
[0027] FIG. 14 demonstrates that pyridylsulfonates are
time-dependent inhibitors of cALDH-I. A, Time-dependent
inactivation of cALDH-I as a function of sulfonate 15
concentration. Recombinant, purified cALDH-I was incubated with
different concentrations of 15 and at the time points shown,
aliquots of the reaction were removed and assayed for enzyme
activity using 1 mM propionaldehyde and 0.5 mM NAD.sup.+ as
substrate and cofactor, respectively. Concentrations of 15 were:
solid diamonds, 2.5 .mu.M; hollow diamonds, 5 .mu.M; solid circles,
7.5 .mu.M; hollow circles, 10 .mu.M; solid squares, 15 .mu.M. B,
Structure-activity relationship for sulfonate 15-cALDH-I reaction.
Sulfonate 15 effectively blocked the labeling of cALDH-I by 1 in
the testis proteome. Analogs of 15 in which this sulfonate's
pyridyl and octyl groups were replaced with methyl and ethyl
groups, respectively (16 and 17, respectively) did not block the
labeling of cALDH-I by 1.
[0028] FIG. 15 shows that multiplexing ABPs increases the number of
protein activities detected in a single proteome. Shown is a
comparison of the heat-sensitive labeling patterns of a testis
proteome treated with pyridylsulfonate 1 (2.5 .mu.M), FP-biotin (4
.mu.M), or a mixture of pyridylsulfonate 1 (2.5 .mu.M) and
FP-biotin (4 .mu.M). The mixture-treated proteome exhibited a
labeling profile similar to that predicted from merging the
profiles of the proteome treated with each ABP alone.
[0029] FIG. 16: FP-peg-biotin labels serine proteases but not their
respective zymogens. Samples of each protein (100 nM) were treated
with FP-peg-biotin (4 .mu.M) for 1 hour (50 mM Tris, pH 7.2),
quenched with 2.times. SDS-PAGE loading buffer (reducing), and
analyzed by SDS-PAGE and blotting with avidin (45 ng protein/lane,
bottom two panels). 50.times. protein stocks were also run on an
SDS-PAGE gel and stained with Coomassie blue (2.2 .mu.g/lane, top
panels). Note that the weak avidin signal seen in the
chymotrypsinogen sample upon longer exposures may represent
FP-peg-biotin reactivity with trace amounts of contaminating
chymotrypsin, which is reported to compose up to 2% of the
purchased proenzyme.
[0030] FIG. 17: Comparing the proteome reactivities of FP-biotin
and FP-peg-biotin. For all of the experiments depicted in this
figure, protein samples represent soluble fractions of rat testis
(1 .mu.g protein/.mu.L). (A) Protein samples were treated with 4
.mu.M of either FP-biotin or FP-peg-biotin for 1 hour (50 mM Tris,
pH 7.2) and analyzed by SDS-PAGE and blotting with avidin. A pair
of 48 kDa serine hydrolases that were robustly labeled by
FP-peg-biotin, but not FP-biotin are highlighted (arrowhead). (B-D)
Concentration dependence of FP--proteome reactions. Protein samples
were treated with 0.5, 1, 2, 4, or 8 .mu.M of either FP-biotin (B)
or FP-peg-biotin (C) for 1 hour (50 mM Tris, pH 7.2) and analyzed
by SDS-PAGE and blotting with avidin. The upper and lower panels
represent 1 and 10 minute film exposures respectively. Several
serine hydrolases appearing to show no FP-peg-biotin concentration
dependence in a one hour reaction (C, arrowheads), exhibited strong
probe concentration dependence in a one minute reaction (D,
arrowheads).
[0031] FIG. 18: pH-dependence of FP-proteome reactions. Protein
samples were treated with either FP-biotin (left panel) or
FP-peg-biotin (right panel) for 1 hour (50 mM Triso50 mM CAPSo50 mM
sodium citrate) at pH 6.0, 7.0, 8.0, or 9.0 and analyzed by
SDS-PAGE and blotting with avidin. Serine hydrolases displaying pH
optima of 8 and 9 are highlighted by triple and double arrowheads,
respectively. Proteins exhibiting heat-insensitive FP-reactivity at
pH 9 are highlighted by single arrowheads.
[0032] FIG. 19: The serine hydrolase activity profile of
Triton-solubilized brain membranes is similar to that of
unsolubilized brain membranes (reaction conditions: 1 .mu.g/.mu.L
protein, 2 .mu.M FP-biotin, 30 min reaction, 50 mM Tris, pH 8.0,
with or without 0.2% Triton X-100).
[0033] FIG. 20: (A) Kinetic analysis of FP-proteome reactions.
Triton-solubilized brain membranes (1 .mu.g protein/.mu.L) was
treated with 4 .mu.M of either FP-biotin (left panel) or
FP-peg-biotin (right panel) for the indicated reaction times, after
which the assays were quenched with 2.times. SDS-PAGE loading
buffer and analyzed by SDS-PAGE-avidin blotting. Serine hydrolase
activities that reacted at a faster rate with FP-biotin or
FP-peg-biotin are highlighted (single and double arrowheads,
respectively). The top and bottom panels represent 1 and 5 minute
film exposures, respectively. (B) Multiplexing of biotinylated FPs
enhances coverage of active serine hydrolases in complex proteomes.
Solubilized brain membrane proteins were treated with the indicated
concentrations of biotinylated FPs (30 min reaction). Highlighted
are three serine hydrolases that are collectively visualized more
clearly in an FP-proteome reaction containing a mixture of
FP-biotin and FP-peg-biotin than in reactions conducted with each
probe alone (bracket).
[0034] FIG. 21. A comparison of membrane-associated serine
hydrolase activities expressed in a panel of rat tissues. Protein
samples (1 .mu.g/.mu.L) were treated with FP-biotin (4 .mu.M) for 1
hour (50 mM Tris, pH 8.0, 0.2% Triton), quenched with 2.times.
SDS-PAGE loading buffer, and analyzed by SDS-PAGE/avidin blotting
on either a 10% (left panels) or 8% (right panels) polyacrylamide
gel. Highlighted are brain-enriched (single arrowheads),
heart-enriched (double arrowhead), and testis-enriched (triple
arrowhead) serine hydrolase activities.
[0035] FIG. 22. Evaluating the target selectivity of a noncovalent
serine hydrolase inhibitor in complex proteomes. Competition
reactions between varying concentrations of oleoyl trifluoromethyl
ketone (OTFMK) and biotinylated FPs identifies several brain
membrane serine hydrolases targeted by OTFMK. The arrowhead
highlights the brain enzyme fatty acid amide hydrolase (FAAH), a
known OTFMK target, and the bracket highlights two additional brain
membrane serine hydrolases sensitive to OTFMK.
[0036] FIG. 23. Affinity isolation and molecular characterization
of FP-labeled serine hydrolase activities. (A) Avidin-agarose beads
effectively bind denatured, but not native FP-labeled proteins. A
sample of FP-biotin-labeled proteins (Pre-Avidin Beads) was mixed
with avidin agarose beads with or without a predenaturation step
(heating in 1% SDS for 10 at 85.degree. C.) for 1 hour at room
temperature. Visualization of the supernatants by SDS-PAGE/avidin
blotting (Post-Beads) identified several proteins (arrowheads) that
only bound the avidin beads in the SDS-treated sample (+SDS) and
were strongly enriched in the corresponding elution fraction
(Elution) relative to a control sample (-SDS). Note that the left
panel represents a 3 min. exposure, while the right panel
represents a 1 sec. exposure. The different exposure times reflect
the high degree of concentration of FP-biotin-labeled proteins
achieved by this method. (B) Molecular identification
FP-biotin-labeled proteins. Eight of the avidin-enriched FP-labeled
proteins were excised from an SDS-PAGE gel, digested with trypsin,
and the resulting peptides analyzed by MALDI and/or electrospray
mass spectrometry. Mass information identified all of these
proteins as serine hydrolases (see text for GenBank accession
numbers).
DETAILED DESCRIPTION OF THE INVENTION
[0037] In accordance with the subject invention systems are
provided for assaying proteomes as to a specific protein or group
of related proteins and to determine the effects of agents on such
proteins in a proteome. The systems include methods for identifying
probes for use in the assay, the probes, the methods of reacting
the probes with the proteomic mixture, and methods for analyzing
the data from the assay for at least semi-quantitatively
determining the target members of the proteome and the effect of
the agents on the activity of a portion of the proteome. Kits are
also provided of combinatorial libraries for screening proteomes to
identify members having specific affinities and for screening
proteomes, where the kits comprise different probes for different
proteins or related groups of proteins. As part of the system for
discovering probes for any related group of proteins, combinatorial
libraries are employed having a common reactive functionality as
part of a functional group and usually a common linker, linking the
functional group to a ligand for which a receptor is available or a
chemically reactive functionality reacting with a reciprocal
functionality for adding a ligand. The results with an active
proteome are repeated with an inactive proteome to determine the
degree of activity of the total target protein as compared to
active protein. Depending on the nature of the functional group,
part of linker or part of the functional group may comprise the
variable component.
[0038] The system uses probes specific for a specific or group of
related proteins and combines one or a mixture of probes, depending
on the specificity of the probes and the variety in the group or
groups of related proteins to be assayed. The reaction mixture
provides conditions under which the probes react substantially
preferentially with active target proteins. By "active" is intended
that the protein is in its native conformation and is able to
interact with an entity that it normally interacts with, e.g.
enzyme with substrate and cofactor, receptor with ligand, etc.
Using the ligand, target conjugated probes are sequestered by means
of the ligand and different protocols may be employed to determine
the amount of the target proteins present in the medium as a group
or individually. Optionally the sequestered proteins may be further
assayed to identify the specific proteins to which the probes
bound.
[0039] The combinatorial aspect of the present invention will be
described first. There are provided combinatorial chemical
libraries containing a plurality of activity-based probes (ABPs),
where the individual members may be correspondingly tagged or
labeled. The methods of the invention employ ABP-containing
libraries for identifying bioactive compounds and for identifying
target proteins in a mixture of proteins. It will be appreciated
that the ABPs of the invention are class-selective and activity
based. Therefore, the present invention allows for rapid target
identification and isolation. (see U.S. Serial Nos. 60/195,954,
filed Apr. 10, 2000 and 60/212,891, filed Jun. 20, 2000, both of
which are incorporated by reference in their entirety herein).
[0040] The combinatorial chemical libraries of the present
invention are useful as screening tools for discovering new lead
structures through evaluation of the compounds in the library
across an array of biological assays, including the discovery of
selective inhibition patterns across isozymes and related enzymes,
where the enzymes share a common functionality at the active site,
allelic proteins, binding to a family of ligands, etc. Thus, the
library is useful as a tool for drug discovery, i.e., it is a means
to discover novel lead compounds by screening the library against a
variety of biological targets, and also as a tool for the
development of structure-activity relationships in large families
of related compounds. The combinatorial libraries after reacting
with a proteome provide compositions of conjugates between members
of the library and target proteins. Such compositions are useful
for producing antibodies for sequestering the target proteins from
the proteomic mixture, digestion and identification of the target
protein using mass spectrometric analysis and data banks, as
standards for measuring the amount of conjugate formed in other
analyses with the same or different probes, and the like.
[0041] The inventive methods employ affinity-labeled or target
protein directed protein reactive reagents, ABPs that allow for the
selective detection and subsequent isolation of active proteins
from complex mixtures. The isolated proteins are characteristic of
the presence of a protein function, e.g., an enzymatic activity,
protein complex formation, protein-nucleic acid interactions, etc.,
in those mixtures. Isolated proteins are optionally characterized
by mass spectrometric (MS) techniques. In particular, the sequence
of isolated proteins can be determined using tandem MS (MS.sup.n)
techniques, and by application of sequence database searching
techniques, the protein from which a sequenced peptide originated
can be identified. The ABPs also provide for differential labeling,
e.g. isotopic or atomic (different atomic weight elements), of the
isolated proteins, which facilitates quantitative determination by
mass spectrometry of the relative amounts of active proteins in
different samples.
[0042] A "combinatorial library" is a collection of compounds in
which the compounds comprising the collection are composed of one
or more types of sub-units. The library will have at least 2
members, rarely less than about 5 members, usually at least about
10 members, frequently will have about 50 members or more, usually
fewer than about 1,000 members, more usually fewer than about 500
members. The sub-units may be selected from natural or unnatural
moieties, including a variety of chemical moieties, such as
synthetic compounds, naturally occurring compounds, e.g. amino
acids, nucleotides, sugars, lipids, and carbohydrates, and
synthetic analogs thereof, which are readily available commercially
in a large variety of compounds. The compounds of the combinatorial
library differ in one or more ways with respect to the number,
order, type or types of or modifications made to one or more of the
sub-units comprising the compounds. Alternatively, a combinatorial
library may refer to a collection of "core molecular organization"
which vary as to the conformation, size and charge distribution as
a result of the presence of other moieties or differences in the
way the core molecular organization is organized. The collection of
compounds is generated in a systematic way. Any method of
systematically generating a collection of sub-units differing from
each other in one or more of the ways set forth above is a
combinatorial library.
[0043] For understanding of the terms used in the subject
application, a number of the generic terms is illustrated with
examples coming within the genus.
[0044] A "chemical group" is an atom or assemblage of atoms and
organic chemical groups include but are not limited to alkyl,
alkenyl, alkynyl, alkoxy, aryl, alkylaryl, heterocycle including
heteroaryl, amide, thioamide, ester, amine, ether, thioether, halo,
imine, cyano, nitro, carboxy, keto, aldehydo, and combinations
thereof.
[0045] "Alkyl" is intended to include aliphatically saturated
linear or branched, hydrocarbon structures and combinations thereof
"Lower alkyl" means alkyl groups of from 1 to 8 carbon atoms.
Examples of lower alkyl groups include methyl, ethyl, propyl,
isopropyl, butyl, s- and t-butyl, pentyl, hexyl, octyl, and the
like. Preferred alkyl groups are those of C20 or below,
particularly C.sub.10 or below.
[0046] "Cycloalkyl" includes cyclic hydrocarbon groups of from 3 to
8 carbon atoms. Examples of lower cycloalkyl groups include
c-propyl, c-butyl, c-pentyl, norbornyl, decalin, and the like, and
may be aliphatically saturated or unsaturated.
[0047] "Alkenyl" includes C2 -C8 unsaturated hydrocarbons of a
linear or branched configuration and combinations thereof. Examples
of alkenyl groups include vinyl, allyl, isopropenyl, pentenyl,
hexenyl, 1-propenyl, 2-butenyl, 2-methyl-2-butenyl, 2,4-hexadienyl
and the like.
[0048] "Alkynyl" includes C2 -C8 hydrocarbons of a linear or
branched configuration and combinations thereof containing at least
one carbon-carbon triple bond. Examples of alkynyl groups include
ethyne, propyne, butyne, pentyne, 3-methyl-1-butyne,
3,3-dimethyl-1-butyne and the like.
[0049] "Alkoxy" refers to groups of from 1 to 8 carbon atoms of a
straight, branched, cyclic configuration and combinations thereof.
Examples include methoxy, ethoxy, propoxy, isopropoxy,
cyclopropyloxy, cyclohexyloxy and the like.
[0050] "Acylamino" refers to acylamino groups of from 1 to 8 carbon
atoms of a straight, branched or cyclic configuration and
combinations thereof. Examples include acetylamino, butyrylamino,
cyclohexylanoylamino, and the like.
[0051] "Hydrocarbylamino" refers to a moiety consisting of hydrogen
and carbon bonded to nitrogen and of from about 1 to 8 carbon atoms
for each hydrocarbyl group, there being up to 4, usually 3,
hydrocarbyl groups. By "hydrocarbyl is intended any molecule or
core of a molecule composed solely of hydrogen and carbon.
[0052] "Halogen" includes F, Cl, Br, and I.
[0053] "Halophenyl" means phenyl substituted with 1-5 halogen
atoms. Examples include pentachlorophenyl, pentafluorophenyl and
2,4,6-trichlorophenyl.
[0054] "Aryl" and "heteroaryl" mean a 5- or 6-membered aromatic or
heteroaromatic ring containing 0-3 heteroatoms selected from O, N,
or S; a bicyclic 9- or 10-membered aromatic or heteroaromatic ring
system containing 0-3 heteroatoms selected from O, N, or S; or a
tricyclic 13- or 14-membered aromatic or heteroaromatic ring system
containing 0-3 heteroatoms selected from O, N, or S; each of which
rings is optionally substituted with 1-3 lower alkyl, substituted
alkyl, substituted alkynyl, .dbd.O, --NO.sub.2, halogen, hydroxy,
alkoxy, OCH(COOH).sub.2, cyano, --NZZ, acylamino, phenyl, benzyl,
phenoxy, benzyloxy, heteroaryl, or heteroaryloxy; each of said
phenyl, benzyl, phenoxy, benzyloxy, heteroaryl, and heteroaryloxy
is optionally substituted with 1-3 substituents selected from lower
alkyl, alkenyl, alkynyl, halogen, hydroxy, alkoxy, cyano, phenyl,
benzyl, benzyloxy, carboxamido, heteroaryl, heteroaryloxy,
--NO.sub.2 or --NZZ (wherein Z is independently H, lower alkyl or
cycloalkyl, and -ZZ may be fused to form a cyclic ring with
nitrogen).
[0055] The aromatic 6- to 14-membered carbocyclic rings include,
e.g., benzene, naphthalene, indane, tetralin, and fluorene and the
5- to 10-membered aromatic heterocyclic rings include, e.g.,
imidazole, pyridine, indole, thiophene, benzopyranone, thiazole,
furan, benzimidazole, quinoline, isoquinoline, quinoxaline,
pyrimidine, pyrazine, tetrazole and pyrazole.
[0056] "Arylalkyl" means an alkyl residue attached to an aryl ring.
Examples are benzyl, phenethyl and the like.
[0057] "Heteroarylalkyl" means an alkyl residue attached to a
heteroaryl ring. Examples include, e.g., pyridinylmethyl,
pyrimidinylethyl and the like.
[0058] "Heterocycloalkyl" means a cycloalkyl where one to two of
the methylene (CH.sub.2) groups is replaced by a heteroatom such as
O, NZ' (wherein Z is H or alkyl), S or the like; with the proviso
that except for nitrogen when two heteroatoms are present, they
must be separated by at least one carbon atom. Examples of
heterocycloalkyl include tetrahydrofuranyl, piperidine, dioxanyl
and the like.
[0059] "Alkylcarbonyl" means --C(O)R", wherein R" is alkyl.
[0060] "Substituted" alkyl, alkenyl, alkynyl, cycloalkyl, or
heterocycloalkyl means alkyl, alkenyl, alkynyl, cycloalkyl, or
heterocycloalkyl wherein up to three H atoms on each C atom therein
are replaced with halogen, hydroxy, loweralkoxy, carboxy,
carboalkoxy, carboxamido, cyano, carbonyl, --NO.sub.2, --NZZ;
alkylthio, sulfoxide, sulfone, acylamino, amidino, phenyl, benzyl,
heteroaryl, phenoxy, benzyloxy, heteroaryloxy, or substituted
phenyl, benzyl, heteroaryl, phenoxy, benzyloxy, or
heteroaryloxy.
[0061] "Aa" represents an amino acid, naturally occurring or
synthetic, and is intended to include the racemates and all optical
isomers thereof. The amino acid side chains of Aa include, e.g.,
methyl (alanine), hydroxymethyl (serine), phenylmethyl
(phenylalanine), thiomethyl (cysteine), carboxyethyl (glutamic
acid), etc. Primary and secondary amino acids are intended to
include alanine, asparagine, N-.beta.-trityl-asparagine, aspartic
acid, aspartic acid-beta-t-butyl ester, arginine,
N.sup.g-Mtr-arginine, cysteine, S-trityl-cysteine, glutamic acid,
glutamic acid-.gamma.-t-butyl ester, glutamine,
N.sup..gamma.-trityl-glutamine, glycine, histidine,
N.sup.1m-trityl-histidine, isoleucine, leucine, lysine,
N.sup..epsilon.-Boc-lysine, methionine, phenylalanine, proline,
serine, O-t-butyl-serine, threonine, tryptophan,
N.sup.m-Boc-tryptophan, tyrosine, valine, sarcosine, L-alanine,
chloro-L-alanine, 2-aminoisobutyric acid, 2-(methylamino)isobutyric
acid, D,L-3-aminoisobutyric acid, (R)-(-)-2 aminoisobutyric acid,
(S)-(+)-2-aminoisobutyric acid, D-leucine, L-leucine, D-norvaline,
L-norvaline, L-2-amino-4-pentenoic acid, D-isoleucine,
L-isoleucine, D-norleucine, 2,3-diaminopropionic acid,
L-norleucine, D,L-2-aminocaprylic acid, .beta.-alanine,
D,L-3-aminobutyric acid, 4-aminobutyric acid,
4-(methylamino)butyric acid, 5-aminovaleric acid, 5-aminocaproic
acid, 7-aminoheptanoic acid, 8-aminocaprylic acid, 11-aminodecanoic
acid, 12-aminododecanoic acid, carboxymethoxylamine, D-serine,
D-homoserine, L-homoserine, D-allothreonine, L-allothreonine,
D-threonine, L-threonine, D,L-4-amino-3-hydroxybutyric acid,
D-,L-3-hydroxynorvaline, (3 S,4S)-(-)-statine,
5-hydroxy-D,L-lysine, 1-amino-1-cyclopropanecarboxylic acid,
1-amino-1-cyclopentanecarboxylic acid,
1-amino-1-cyclohexanecarboxylic acid,
5-amino-1,3-cyclohexadiene-1-- carboxylic acid,
2-amino-2-norbomanecarboxylic acid, (S)-(-)-2-azetidinecarboxylic
acid, cis-4-hydroxy-D-proline, cis-4-hydroxy-L-proline,
trans-4-hydroxy-L-proline, 3,4-dehydro-D,L-proline,
3,4-dehydro-L-proline, D-pipecolinic acid, L-pipecolinic acid,
nipecotic acid, isonipecotic acid, mimosine, 2,3-diaminopropionic
acid, D,L-2,4-diaminobutyric acid, (S)-(+)-diaminobutyric acid,
D-ornithine, L-ornithine, 2-methylornithine,
N-.epsilon.-methyl-L-lysine, N-methyl-D-aspartic acid,
D,L-2-methylglutamic acid, D,L-2-aminoadipic acid, D-2-aminoadipic
acid, L-2-aminoadipic acid, (+/-)-3-aminoadipic acid, D-cysteine,
D-penicillamine, L-penicillamine, D,L-homocysteine,
S-methyl-L-cysteine, L-methionine, D-ethionine, L-ethionine,
S-carboxymethyl-L-cysteine, (S)-(+)-2-phenylglycine,
(R)-(-)-2-phenylglycine, N-phenylglycine,
N-(4-hydroxyphenyl)glycine, D-phenylalanine,
(S)-(-)indoline-2-carboxylic acid,
.alpha.-methyl,D,L-phenylalanine, .alpha.-methyl-D,L-phenylalanine,
D-homophenylalanine, L-homophenylalanine,
D,L-2-fluorophenylglycine, D,L-2-fluorophenylalanine,
D,L-3-fluorophenylalanine, D,L-4-fluorophenylalanine,
D,L-4-chlorophenylalanine, L-4-chlorophenylalanine,
4-bromo-D,L-phenylalanine, 4-iodo-D-phenylalanine,
3,3',5-triiodo-L-thyronine, (+)-3,3',5-triiodo-L-thyronine,
D-thyronine, L-thyronine, D,L-tyrosine, D-4-hydroxyphenylglycine,
D-tyrosine, L-tyrosine, O-methyl-L-tyrosine, 3-fluoro-D,L-tyrosine,
3-iodo-L-tyrosine, 3-nitro-L-tyrosine, 3,5-diiodo-L-tyrosine,
D,L-dopa, L-dopa, 2,4,5-trihydroxyphenyl-D,L-alani- ne,
3-amino-L-tyrosine, 4-amino-D-phenylalanine,
4-amino-L-phenylalnine, 4-amino-D,L-phenylalanine,
4-nitro-L-phenylalanine, 4-nitro-D,L-phenylalanine,
3,5-dinitro-L-tyrosine, D,L-.alpha.-methyltyrosine,
L-.alpha.-methyltyrosine,
(-)-3-(3,4-dihydroxyphenyl)-2-methyl-L-alanine,
D,L-threo-3-phenylserine, trans-4-minomethyl)cyclohexane carboxylic
acid, 4-(aminomethyl)benzoic acid, D,L-3-aminobutyric acid,
3-aminocyclohexane carboxylic acid, cis-2-amino-1-cyclohexane
carboxylic acid, gamma-amino-beta-(p-chlorophen- yl)butyric acid
(Baclofen), D,L-3-aminophenylpropionic acid,
3-amino-3-(4-chlorophenyl)propionic acid,
3-amino-3-(2-nitrophenyl)propio- nic acid, and
3-amino-4,4,4-trifluorobutyric acid.
[0062] An "alkylaryl group" refers to an alkyl (as described
above), covalently joined to an aryl group (as described
above).
[0063] "Carbocyclic aryl groups" are groups wherein the ring atoms
on the aromatic ring are all carbon atoms. The carbon atoms are
optionally substituted.
[0064] "Heterocyclic aryl groups" are groups having from 1 to 3
heteroatoms as ring atoms in the aromatic ring and the remainder of
the ring atoms are carbon atoms. Suitable heteroatoms include
oxygen, sulfur, and nitrogen, and include furanyl, thienyl,
pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl,
imidazolyl and the like, all optionally substituted.
[0065] An "amide" refers to an --C(O)--NH--, where Z is either
alkyl, aryl, alklyaryl or hydrogen.
[0066] A "thioamide" refers to --C(S)--NH--Z, where Z is either
alkyl, aryl, alklyaryl or hydrogen.
[0067] An "ester" refers to an --C(O)--OZ', where Z' is either
alkyl, aryl, or alklyaryl.
[0068] An "amine" refers to a --N(Z")Z"', where Z" and Z"', is
independently either hydrogen, alkyl, aryl, or alklyaryl, provided
that Z" and Z"' are not both hydrogen.
[0069] An "ether" refers to Z-O-Z, where Z is either alkyl, aryl,
or alkylaryl.
[0070] A "thioether" refers to Z-S-Z, where Z is either alkyl,
aryl, or alkylaryl.
[0071] A "cyclic molecule" is a molecule which has at least one
chemical moiety which forms a ring. The ring may contain three
atoms or more. The molecule may contain more than one cyclic
moiety, the cyclic moieties may be the same or different.
[0072] A "linear molecule" does not contain a ring structure.
However, the molecule may be straight or branched.
[0073] An "active site" of a protein refers to the specific area on
the surface of a protein, e.g., an enzyme molecule or surface
membrane receptor, to which a binding molecule, e.g. substrate or
reciprocal ligand is bound and results in a change in the ligand,
e.g. substrate or complex formation with the protein as a result of
ligand binding. For a receptor, the conformation may change, the
protein may become susceptible to phosphorylation or
dephosphorylation or other processing. For the most part, the
active site will be the site(s) of an enzyme where the substrate
and/or a cofactor bind, where the substrate and cofactor undergo a
catalytic reaction, where two proteins form a complex, e.g. the
site at which a G protein binds to a surface membrane receptor, two
kringle structures bind, sites at which transcription factors bind
to other proteins, sites at which proteins bind to specific nucleic
acid sequences, etc. In the case of a membrane receptor, the active
site will be the external site to a membrane, where the ligand
binds and causes transduction of a signal.
[0074] The "activity-based probes" or "ABP"s of the invention are
chemical reagents that are polyfunctional molecules for
non-competitive or substantially irreversible binding to a target
protein and inhibiting the action of the target protein. The ABPs
will comprise at least a reactive functionality and a ligand and
have an affinity for a related group of proteins, whereby the ABP
will bind to the target protein and substantially inactivate the
protein, and the ligand will permit detection and/or isolation.
[0075] In referring to affinity for an ABP to a target protein, one
is concerned with the on-rate of the ABP with the target protein,
since there is no off-rate, where the ABP covalently bonds to the
target protein. One can determine relative on-rates between ABPs by
having less than a stoichiometric amount of the target protein as
compared to the total amount of a plurality of ABPs and then
measuring the relative amounts of the conjugates for each of the
ABPs. In this way one can obtain a measure of the relative activity
of each of the ABPs toward the target protein, which for the
purposes of this invention may be considered the affinity, if not
the binding affinity, of the ABP for the target protein.
[0076] Exemplary protein targets described herein include enzymes,
included in the groups oxidoreductases, hydrolases, ligases,
isomerases, transferases, and lyases and include such enzymes or
enzyme groups as serine hydrolases, metallo-hydrolases,
dehydrogenases, e.g. alcohol and aldehyde dehydrogenases, and
nucleotide triphosphate (NT)-dependent enzymes, although, the
invention envisions ABPs which recognize any protein, e.g., enzyme,
family. Other proteins include proteins that bind to each other or
to nucleic acids, such as transcription factors, kringle structure
containing proteins, nucleic acid binding proteins, G-protein
binding receptors, cAMP binding proteins, etc. The structure of
ABPs of the invention is described more fully below.
[0077] An "active protein" of the invention refers to a protein,
e.g., enzyme, in its normal wild-type conformation, e.g. a
catalytically active state, as opposed to an inactive state. The
active state allows the protein, to function normally. An inactive
state may be as a result of denaturation, inhibitor binding, either
covalently or non-covalently, mutation, secondary processing, e.g.
phosphorylation or dephosphorylation, etc. Functional states of
proteins or enzymes as described herein may be distinct from the
level of abundance of the same proteins or enzymes. An active site
is an available wild-type conformation at a site that has
biological activity, such as the catalytic site of an enzyme, a
cofactor-binding site, the binding site of a receptor for its
ligand, and the binding site for protein complexes, for example. In
many instances, one is interested in knowing the level of
availability of such sites. Targets of interest will be
particularly enzymes, other proteins include receptors,
transcription factors, G-proteins, and the like.
[0078] The subject systems are useful for, among other things,
developing new drugs and identifying new drug targets. One
embodiment of the subject invention is especially useful for
rapidly generating and developing large numbers of drug candidate
molecules. The invention is useful for systematically synthesizing
a large number of molecules that may vary greatly in their chemical
structure or composition, or that may vary in minor aspects of
their chemical structure or composition. The invention is also
useful for randomly generating a large number of drug candidates,
and later optimizing those candidates that show the most medicinal
promise. The combinatorial libraries of the present invention may
also be screened for diagnostically useful compounds. By
diagnostically useful is meant that the compound can be used to
indicate the presence of a particular disease in a human or
animal.
[0079] The combinatorial libraries of the present invention may be
screened for pharmacologically active compounds, including analogs,
that is compounds that can affect the biological status of a
biological system, usually a cellular system. The biological system
will depend on the use of a biological source that will include
cells and/or viruses. By pharmacologically active is meant that a
compound may effect the function of a protein, e.g, an enzyme,
including physiological process such as signal transduction by a
cellular receptor, initiation, cessation or modulation of an immune
response, modulation of heart function, nervous system function, or
any other organ or organ system. A pharmacologically active
compound may also stimulate or inhibit the activity of a bacteria,
virus, fungus, or other infectious agent. A pharmacologically
active compound may modulate the effects of a disease, that is
prevent or decrease the severity of or cure a disease such as
cancer, diabetes, atherosclerosis, high blood pressure, Parkinson's
disease and other disease states. Screening for pharmacological
activity may be performed by assays as would be known in the art,
depending on the function or activity to be assessed. Compounds
which have been shown to be pharmacologically active compounds may
be formulated for therapeutic administration by methods known in
the art. Methods have been reported in the literature by which
individual members of combinatorial libraries may be encoded by
"tagging molecules" ("tags" or "labels"). See, for example, U.S.
Pat. Nos. 5,721,099 and 6,001,579. Thus, a single molecular
structure synthesized on a resin bead, for instance, is uniquely
defined by a series of other, readily detectable molecules also
bound to a bead. Individual beads are treated to release their
library member, by a process which does not displace the tag, and
following identification of this compound as an "active" in a
biological screen, the tags are released and analyzed to deduce the
identity of the "hit". To allow for maximum diversity in a library
it is critical that the chemistry used to introduce the tags is
tolerated by a wide range of functionality. Thereby, introduction
of the tagging molecule does not lead to undesired elaboration of
the library structure, or alternatively, place limits upon the
chemistry used to construct the library. Similarly, if the tag is
removed prior to the library member, the conditions for removal of
the tag does not destroy or react in some manner with the designed
molecule.
[0080] The material upon which the combinatorial syntheses of the
present invention are performed are referred to as solid supports,
beads or resins. These terms are intended to include beads,
pellets, disks, fibers, gels, or particles such as cellulose beads,
pore-glass beads, silica gels, polystyrene beads optionally
cross-linked with divinylbenzene and optionally grafted with
polyethylene glycol and optionally functionalized with amino,
hydroxy, carboxy, or halo groups, grafted co-poly beads,
poly-acrylamide beads, latex beads, dimethylacrylamide beads
optionally cross-linked with N,N'-bis-acryloyl ethylene diamine,
glass particles coated with hydrophobic polymer, and the like,
e.g., material having a rigid or semi-rigid surface; and soluble
supports such as low molecular weight non-cross-linked
polystyrene.
[0081] By "biological status" is intended to include mRNA profile,
protein profile, total and/or active, spatial distribution profile
of the proteins and mRNA, maturity of cells, population of surface
membrane proteins, amount and spatial distribution of complexes,
amount of ligands present, bound and unbound, lipid population,
processing of proteins, such as glycosylation, methylation,
acylation, phosphorylation, ubiquination, farnesylation, etc.,
those differences that distinguish cellular populations.
[0082] Some of the compounds described herein contain one or more
asymmetric centers and may thus give rise to enantiomers,
diastereomers, and other stereoisometric forms that may be defined,
in terms of absolute stereochemistry, as (R)- or (S)- or, as (D)-
or (L)- for amino acids. The present invention is meant to include
all such possible diastereomers, as well as, their racemic and
optically pure forms. Optically active (R)- and (S)-, or (D)- and
(L)- isomers may be prepared using chiral synthons or chiral
reagents, or resolved using conventional techniques. When the
compounds described herein contain olefinic bonds or other centers
of geometric asymmetry, and unless specified otherwise, it is
intended that the compounds include both E and Z geometric isomers.
Likewise, all tautomeric forms are also intended to be
included.
[0083] Activity-based probes (ABPs) are provided for specific
reaction with the active site of one or more target proteins, where
the target protein is a member of a class of proteins, particularly
enzymes, for detection of the presence and quantitation of one or
more active members. The ABPs have a common electrophile whose
environment is changed in each of the ABPs to provide a different
reactivity with different target proteins. The probes may be
divided into four general regions: (1) a functional group (F) that
specifically and covalently bonds to the active site of a protein;
(2) a label or ligand (hereinafter collectively referred to as
"ligand") for sequestering and detecting the conjugate of the ABP
and the active protein (X) 3) a linker L, between the F and the L;
and 4) binding moiety or affinity label, that may be associated
with or part of the linker region and/or the functional group (R).
A linker is a bond or chemical group used to link one moiety to
another, serving as a divalent bridge, where it provides a group
between two other chemical moieties. Binding or affinity moiety
refers to a chemical group, which may be a single atom, that is
conjugated to the reactive functional group or associated with the
linker, as a side chain or in the chain of the linker, and provides
enhanced binding affinity for protein targets. A ligand refers to a
molecule that can be used to detect and/or capture the ABP in
combination with any other moieties that are bound strongly to the
ligand so as to be retained in the process of the reaction of the
functional group with the target active protein. The ABP may
include a chemically reactive functionality, not found in proteins,
that will react with a reciprocal functionality, e.g. vic.-diols
with boronic acid, aldehydes and ketones, etc. These reactive
functionalities may be used to bind to a ligand after reaction with
the target protein. In some embodiments described herein, the ABP
may be truncated and lack the ligand, but will always have a
functional group, F, a linker, L and an R group (binding moiety),
but no ligand, X (see FIG. 1).
[0084] The ABP will have an affinity for an active site, which may
be specific for a particular active site or generally shared by a
plurality of related proteins. The affinity may result from the
functional group, the linker, or the binding moiety or combination
thereof. For drug discovery, one may be interested in specificity
for a single target, while for proteome analysis, one will usually
be interested in binding to a plurality of targets that are
related.
[0085] Exemplary Fs as used in an ABP of the invention include an
alkylating agent, acylating agent, ketone, aldehyde, sulphonate or
a phosphorylating agent. Examples of particular Fs include, but are
not limited to fluorophosphonyl, fluorophosphoryl, fluorosulfonyl,
alpha-haloketones or aldehydes or their ketals or acetals,
respectively, alpha-haloacyls, nitriles, sulfonated alkyl or aryl
thiols, iodoacetylamide group, maleimides, sulfonyl halides and
esters, isocyanates, isothiocyanantes, tetrafluorophenyl esters,
N-hydroxysuccinimidyl esters, acid halides, acid anhydrides,
unsaturated carbonyls, alkynes, hydroxamates,
alpha-halomethylhydroxamates, aziridines, epoxides, or arsenates
and their oxides. Sulfonyl groups may include sulfonates, sulfates,
sulfinates, sulfamates, etc., in effect, any reactive functionality
having a sulfur group bonded to two oxygen atoms. Epoxides may
include aliphatic, aralkyl, cycloaliphatic and spiro epoxides, the
latter exemplified by fumagillin, which is specific for
metalloproteases.
[0086] The ABPs of the subject invention may be illustrated by the
following formula:
R* (F-L)-X
[0087] where the symbols are as defined previously and the asterisk
intends that R may be included in F or L and X is bonded to L, more
specifically:
[0088] wherein:
[0089] X is a ligand present prior to formation of said product or
added to a reactive functionality to provide said ligand, said
ligand having the same chemical structure for each of said members
of said library;
[0090] L is a bond or linking group, which is the same in each of
the members of said library;
[0091] F is a functional group reactive at an active site of a
protein member, which functional group comprises the same reactive
functionality in each of the members of said library; and
[0092] R is a group of less than 1kDal, that is different in each
of the members of the library; the * intends that R is a part of F
or L; and
[0093] wherein members of said library have different on rates with
said protein member.
[0094] For example, when X is biotin or any ligand, L is any linker
of varied composition and length, F is a sulfonate and R is a
pyridyl group, a distinct protein profile is observed as compared
with the same ABP wherein the R group is methyl (FIG. 2). Thus by
varying R when bonded to a sulfonyl group, different binding
profiles will be obtained, so that one can look for specificity,
which may lead to designing a drug based on the structure of R or
look for binding to related target proteins for proteome
analysis.
[0095] Illustrative of the method is the use of a reactive
functionality having a leaving group that is varied to provide the
combinatorial library for identifying a chemical compound which
affects the activity of a protein. The method includes contacting a
combinatorial chemical library with a biological sample, where the
library comprises a plurality of differing functional groups (F-R)
reactive with an active protein, wherein, for example, a sulfonate
ester can have R as any group, such as alkyl, heterocyclic, such as
pyridyl, substituted pyridyl, imidazole, pyrrole, thiophene, furan,
azole, oxazole, aziridine, etc., aryl, substituted aryl, amino acid
or peptidyl, oligonucleotide or carbohydrate group; and detecting
an effect on a biological activity in a biological sample (e.g.,
inhibition of cell proliferation or inhibition of an enzyme
activity). One can take an ABP identified by screening the
libraries as described herein and confirm the specificity of the
ABP for the protein. For example, as exemplified in FIG. 3, one can
take an individual ABP identified from a library of sulfonates and
identify through MALDI mapping, a target enzyme, as shown in the
Experimental section, aldehyde dehydrogenase (ADH). An ABP lacking
X, or a label (e.g., biotin) is utilized to confirm the inhibition
of ADH activity by the specific ABP identified. To further
characterize the bond between the sulfonate ABP and the ADH, for
example, one can include modifying agents in an assay for detection
of inhibition of ADH activity. For example, in FIG. 7, addition of
a variety of free thiols, including glutathione (GSH or glut),
dithiothreitol (DTT), or .alpha.-mercaptoethanol (BME), did not
quench the bonding of the ABP/ADH, and provides further information
as to the bond between the ABP and newly identified ADH. Other
protein effectors, thiols, metals, chelators (e.g., EDTA), ATP,
calcium and the like can be added to reactions between an ABP and
protein target to provide further characterization.
[0096] The ligand portion permits capture of the conjugate of the
target protein and the probe. The ligand may be displaced from the
capture reagent by addition of a displacing ligand, which may be
free ligand or a derivative of the ligand, or by changing solvent
(e.g., solvent type or pH) or temperature conditions or the linker
may be cleaved chemically, enzymatically, thermally or
photochemically to release the isolated materials (see discussion
of the linker moiety, below).
[0097] Examples of ligands (including labels), X, include, but are
not limited to, biotin, deiminobiotin, dethiobiotin, vicinal diols,
such as 1,2-dihydroxyethane, 1,2-dihydroxycyclohexane, etc.,
digoxigenin, maltose, oligohistidine, glutathione,
2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, a peptide of
polypeptide, a metal chelate, a saccharide, rhodamine or
fluorescein, or any hapten to which an antibody can be generated.
Examples of ligands and their capture reagents include but are not
limited to: dethiobiotin or structurally modified biotin-based
reagents, including deiminobiotin, which bind to proteins of the
avidin/streptavidin family, which may, for example, be used in the
forms of strepavidin-Agarose, oligomeric-avidin-Agarose, or
monomeric-avidin-Agarose; any 1,2-diol, such as 1,2-dihydroxyethane
(HO--CH.sub.2--CH.sub.2--OH), and other 1,2-dihyroxyalkanes
including those of cyclic alkanes, e.g., 1,2-dihydroxycyclohexane
which bind to an alkyl or aryl boronic acid or boronic acid esters,
such as phenyl-B(OH).sub.2 or hexyl-B(OEthyl).sub.2 which may be
attached via the alkyl or aryl group to a solid support material,
such as Agarose; maltose which binds to maltose binding protein (as
well as any other sugar/sugar binding protein pair or more
generally to any ligand/ligand binding protein pairs that has
properties discussed above); a hapten, such as the dinitrophenyl
group, for any antibody where the hapten binds to an anti-hapten
antibody that recognizes the hapten, for example the dinitrophenyl
group will bind to an anti-dinitrophenyl-lgG; a ligand which binds
to a transition metal, for example, an oligomeric histidine will
bind to Ni(II), the transition metal capture reagent may be used in
the form of a resin bound chelated transition metal, such as
nitrilotriacetic acid-chelated Ni(II) or iminodiacetic
acid-chelated Ni(II); glutathione which binds to
glutathione-S-transferase.
[0098] In general, any affinity label-capture reagent commonly used
for affinity enrichment which meets the suitability criteria
discussed above can be used in the method of the invention. Biotin
and biotin-based affinity tags are particularly illustrated herein.
Of particular interest are structurally modified biotins, such as
deiminobiotin or dethiobiotin, which will elute from avidin or
streptavidin (strept/avidin) columns with biotin or under solvent
conditions compatible with ESI-MS analysis, such as dilute acids
containing 10-20% organic solvent. It is expected that
deiminobiotin tagged compounds will elute in solvents below about
pH 4.
[0099] The linker group, while potentially can be a bond, is
preferred to be other than a bond. The linker can be a cleavable
linker that is cleaved, for example, by thermal, chemical or
photochemical reaction. The choice of linker for the label and the
functional group will be part of the synthetic strategy, since
depending on the synthetic strategy, the linking group can result
in a residual functionality on the product upon release from the
support. It will usually be difficult, but feasible, to further
modify the product after detachment from the bead. In designing the
synthetic strategy, one can use a functionality to be retained in
the product as the point of attachment for the linking group.
Alternatively, when permitted by the nature of the product, one
could use a cleavage or detachment method which removes the linking
functionality, e.g., an arylthioether or silyl with a metal hydride
or acid. Since in many cases, the synthetic strategy will be able
to include a functionalized site for linking, the functionality can
be taken advantage of in choosing the linking group. In some
instances it may be desirable to have different functionalities at
the site of linking the product to the support, which may
necessitate using different modes of linking, which modes must
accommodate either the same detachment method or different
detachment methods which may be carried out concurrently or
consecutively, e.g., irradiation with light and acid hydrolysis.
The choice of linker, as with the choice of an R group, has been
shown to alter the specificity of an ABP. For example, a linker for
FP biotin, described in the Examples herein (see also FIG. 1), and
a linker comprising PEG, have distinct specificities and provide
distinct protein profiles (see FIG. 6). Thus, one of skill in the
art can select the linker portion of the ABP in order to provide
additional specificity of the ABP for a particular protein or
protein class.
[0100] Photocleavable groups in the linker may include the
1-(2-nitrophenyl)ethyl group. Thermally labile linkers may include
a double-stranded duplex formed from two complementary strands of
nucleic acid, a strand of a nucleic acid with a complementary
strand of a peptide nucleic acid, or two complementary peptide
nucleic acid strands which will dissociate upon heating. Cleavable
linkers also include those that have disulfide bonds, acid or base
labile groups, including diarylmethyl or trimethylarylmethyl
groups, silyl ethers, carbamates, oxyesters, thioesters,
thionoesters and alpha-fluorinated amides and esters. Enzymatically
cleavable linkers can contain protease-sensitive amides or esters,
beta-lactamase-sensitive beta-lactam analogs and linkers that are
nuclease-cleavable or glycosidase cleavable.
[0101] Linker groups include among others, ethers, polyethers,
diamines, ether diamines, polyether diamines, amides, polyamides,
polythioethers, disulfides, silyl ethers, alkyl or alkenyl chains
(straight chain or branched and portions of which may be cyclic)
aryl, diaryl or alkyl-aryl groups. While normally amino acids and
oligopeptides are not preferred, when used they will normally
employ amino acids of from 2-3 carbon atoms, i.e. glycine and
alanine. Aryl groups in linkers can contain one or more heteroatoms
(e.g., N, O or S atoms). Linkages also include substituted benzyl
ethers, esters, acetals or ketals, diols, and the like (See, U.S.
Pat. No. 5,789,172 for a list of useful functionalities and manner
of cleavage, herein incorporated by reference). The linkers, when
other than a bond, will have from about 1 to 60 atoms, usually 1 to
30 atoms, where the atoms include C, N, O, S, P, etc., particularly
C, N and O, and will generally have from about 1 to 12 carbon atoms
and from about 0 to 8, usually 0 to 6 heteroatoms. The atoms are
exclusive of hydrogen in referring to the number of atoms in a
group, unless indicated otherwise.
[0102] The linker and/or the ligand may be isotopically labeled,
for example by substitution of one or more atoms in the linker with
a stable isotope. For example, .sup.1H can be substituted with
.sup.2H or .sup.12C can be substituted with .sup.13C. Alternatively
one may substitute one atom for another, such as H with F, use
unsaturation to provide a different mass, or other known means.
While ligands or linking groups may have different isotopic
distributions, for the purposes of this invention they will be
considered to be of the same chemical composition, where the atomic
numbers of the atoms and their organization in the ligands or
linking groups is the same. Therefore, in one aspect, the method of
the invention provides for labeling of the ligand and/or linker to
facilitate quantitative analysis by mass spectrometry of the
amounts of proteins in different samples or in samples subjected to
different conditions (e.g., in the presence and absence of a drug).
Further, the label or linker can be non-radioisotopically labeled,
e.g, with a fluorophore. In one aspect, the label produces an
electromagnetic signal.
[0103] The process and compositions described in WO00/11208, which
is specifically incorporated by reference herein, may be used with
the subject invention. In the application, one uses an affinity
tagged, substantially chemically identical and differentially
isotopically labeled probe, where the conjugates or fragments
thereof are identified by mass spectrometry. The ratio of the
different isotopic probes for each of the proteins with which the
probes have reacted provides for the relative quantities of the
individual proteins.
[0104] Linkers may be varied widely depending on their function,
including alkyleneoxy and polyalkyleneoxy groups, where alkylene is
of from 2-3 carbon atoms, methylene and polymethylene, polyamide,
polyester, and the like, where individual monomers will generally
be of from 1 to 6, more usually 1 to 4 carbon atoms. The oligomers
will generally have from about 1 to 10, more usually 1 to 8
monomeric units. The monomeric units may be amino acids, both
naturally occurring and synthetic, oligonucleotides, both naturally
occurring and synthetic, condensation polymer monomeric units and
combinations thereof. Alteration in the linker region has been
shown to alter the specificity of the ABP for a target protein or
class of proteins (e.g., enzymes).
[0105] Since the combinatorial production of probes is intended to
find probes specific for target proteins and/or probes that provide
information about the active site of a protein, these probes will
usually be modified so as to be identifiable. Also, there can be a
cleavable site between the target protein and at least a portion of
the probe, where the identifiable moiety of the probe can be
released from the target protein. Once the probe has been
identified, for example, by mass spectrometry, fluorometry,
electrochemically, etc., or combination thereof, the single probe
may then be used with the same proteome mixture. At this stage, the
protein target(s) can then be determined by conventional ways,
using immunoassays, if available, sequencing, mass spectrometry and
the like. The affinity label will then provide a basis for the
design of a drug specific for the target protein.
[0106] With the ABP compounds described herein, screening assays
such as FACS sorting and cell lawn assays may be used. When ligand,
X, is detached prior to evaluation, its relationship to its solid
support can be maintained, e.g., by location within the grid of a
standard 96-well plate or by location of activity on a lawn of
cells. Whether the compounds are tested attached to or detached
from, the solid supports, the tags attached to the solid support
associated with bioactivity may then be decoded to reveal the
structural or synthetic history of the active compound (see for
example, Ohlmeyer et al., Proc. Natl. Acad. Sci. USA, 90,
10922-10926, 1993). The usefulness of such libraries as screening
tools is demonstrated by Burbaum et al., (Proc. Natl. Acad. Sci.
USA, 92, 6027-6031, 1995), who describe the assaying of encoded
combinatorial libraries for, e.g., carbonic anhydrase inhibition.
Even when none of the compounds in a particular assay are found to
be active for a given screen, such lack of activity often, however,
provides useful structure-activity information.
[0107] In one embodiment, the invention employs two different
compositions, intact ABPs and truncated ABPs lacking the ligand.
See FIG. 1. In most cases, the combination will employ
combinatorial libraries that are being screened for bioactivity.
For most intact ABPs containing ligands, it is difficult to obtain
entry into an intact cell. Using electroporation, permeabilizing
agents, or the like will change the status of the cell and could
interfere with the assay. To use convenient ligands, such as
biotin, one prepares two libraries, intact ABPs and truncated ABPs
lacking the ligand. One then introduces the truncated ABPs under
conditions that allow the truncated ABPs to enter the cell without
significant disruption of the cell membrane. Any change in
phenotype may be determined, such as apoptosis, proliferation,
change in surface membrane proteins, inability to respond to
ligands for cellular receptors, etc. If the change in phenotype is
of interest, the cells may then be lysed and the lysate treated
with the intact ABPs. In addition, one would use an inactivated
lysate, to compare and select only those proteins that were
conjugated in the active lysate and not conjugated in the
inactivated lysate. The proteins to which the ABPs are conjugated
can then be characterized in manners discussed in the specification
and in the Experimental section. Alternatively, one may establish
conditions for transport of the ABP into the cell without modifying
the cell to change its response or choose lipophilic ligands or
provide conditions where the ABPs will be entrained with another
compound that provides permeability. In this way, one can determine
conditions that will allow the screening of libraries where the
ABPs will enter the cells without an undesirable change in the
character of the cell. These approaches allow for the determination
of ABPs that can serve as precursors in the design of drugs for
specific binding to target proteins, while at the same time
determining the effect on the phenotype of modifying the activity
of the target protein. Of course, depending on the nature and size
of the combinatorial library, one or more libraries may be
used.
[0108] The above described method may also find application in
determining whether the cellular environment affects the reaction
of the ABP with a protein. One can use a radioactive label with the
truncated ABP to identify whether the protein that reacted in the
lysate also reacted in the cell. Alternatively, one may prepare
monoclonal antibodies to the protein-ABP conjugates obtained in the
lysate and use them to fish out the analogous protein from a cell
that has been treated with the truncated ABP. By establishing that
the truncated ABP bound to the same protein as the analogous ABP,
one would establish that the specific affinity of the ABP provided
the same intracellular activity or affinity as observed in the
lysate.
[0109] Finally, one of skill in the art can identify the biological
target in more detail by standard methods including SDS-PAGE or
Western Blot analysis. As an illustrative example, the following
protocol can be used to identify biological targets in a sample.
After incubation of protein sample (0.5-2.5 mL, 0.5-1.0 mg/mL) with
the ABPs, the sample is diluted to 2.5 mL in Tris or phosphate
buffer and passed over a PD-10 size exclusion column to remove
excess unreacted ABP. The protein is collected from the column in
3.5 mL of buffer, treated with SDS (final concentration of 0.5%),
and heated for 10 min at 80.degree. C. The sample is then diluted
2.5.times. and incubated with 100 .mu.L of avidin agarose beads
(Sigma) for 1-4 hours at room temp. The beads are then washed with
several volumes of buffer to remove unbound protein and the
ABP-labeled proteins are eluted with 100 .mu.L of standard SDS-PAGE
loading buffer by heating at 90.degree. C. for 5 minutes. The
eluted proteins are run on an SDS-PAGE gel and ABP-labeled proteins
identified by staining and/or avidin blotting, excised from the
gel, digested with trypsin, and the resulting peptide mixture
characterized by MALDI and/or electrospray mass spectrometry. The
mass spectrometric information is used in database searches to
identify the ABP-labeled proteins.
[0110] Once one has established a probe, by combinatorial or other
means, generally the probe may then be used to analyze a proteome
for active protein(s). The probe may be specific for a single
protein or more usually a related group of proteins. By related
group of proteins is intended proteins that perform the same
activity, as with enzymes that belong to the same group and
catalyze the same reaction, e.g. hydrolysis, phosphorylation,
oxidation, etc., and usually having one or more of the following
characteristics: the same functionality at the active site; the
same spatial orientation of functional groups that bind to the
ligand; similar spatial structure and conformation; similar
molecular weight; the same or similar cofactors or complexing
proteins; and similar function. To enhance the distinction between
active proteins and inactive proteins, special chemically reactive
groups are employed.
[0111] A "chemically reactive group" is a moiety including a
reactive functionality that does not react efficiently with the
generally available functional groups of proteins, e.g. amino,
hydroxy, carboxy, and thiol, but will react with a functionality
present in a particular conformation on a surface. In some
situations the reactive functionality will serve to distinguish
between an active and an inactive protein. In other situations, the
conformation of the chemically reactive group will bind to the
specific conformation of the target protein(s), whereby with a
slowly reactive functionality or one that requires activation, the
predominant reaction will be at the active site. For example a
photoactivatable group may be used such as a diazoketone,
arylazide, psoralen, arylketone, arylmethylhalide, etc. any of
which can bind non-selectively to the target protein, while the
probe is bound to the active site. Olefins and acetylenes to which
are attached electron withdrawing groups such as a sulfone,
carbonyl, or nitro group may be used to couple to sulfhydryl
groups.
[0112] A detectable label is a group that is detectable at low
concentrations, usually less than micromolar, preferably less than
nanomolar, that can be readily distinguished from other analogous
molecules, due to differences in molecular weight, redox potential,
electromagnetic properties, binding properties, and the like. The
detectable label may be a hapten, such as biotin, or a fluorescer,
or an oligonucleotide, capable of non-covalent binding to a
complementary receptor other than the active protein; a mass tag
comprising a stable isotope; a radioisotope; a metal chelate or
other group having a heteroatom not usually found in biological
samples; a fluorescent or chemiluminescent group preferably having
a quantum yield greater than 0.1; an electroactive group having a
lower oxidation or reduction potential than groups commonly present
in proteins; a catalyst such as a coenzyme, organometallic
catalyst, photosensitizer, or electron transfer agent; a group that
affects catalytic activity such as an enzyme activator or inhibitor
or a coenzyme.
[0113] Detectable labels may be detected directly by mass
spectroscopy, detection of electromagnetic radiation, measurement
of catalytic activity, potentiometric titration, cyclic voltametry,
and the like. Alternatively labels may be detected by their ability
to bind to a receptor thereby causing the conjugate to bind to the
receptor. Binding of the conjugate to a receptor can be detected by
any standard method such as ellipsometry, acoustic wave
spectroscopy, surface plasmon resonance, evanescent wave
spectroscopy, etc. when binding is to a surface, or by an
immunoassay such as ELISA, FRET, SPA, RIA, in which the receptor
may carry a label and an antibody to the active protein can be
employed which may optionally carry a second label. Detectable
labels may also be detected by use of separation methods such as
HPLC, capillary or gel electrophoresis, chromatography,
immunosorption, etc. In these methods the conjugate can be caused
to bind to a member of an array of specific binding substances such
as an array of antibodies where each member is an antibody for a
specific active protein.
[0114] One aspect of the method of the invention is subjecting a
portion of the sample to conditions that inactivate proteins in the
sample. Significantly, information from studies with ABPs is
preferable if one compares the level of protein conjugates in a
portion of the sample that has been treated with inactivating
conditions to a portion of the sample that contains active,
wild-type or untreated proteins. Active wild-type proteins intend
proteins with their natural conformation that are capable of
carrying out binding and other functions, as appropriate. Other
functions include enzymatic activity, ability to be modified, e.g.
phosphorylation or dephosphorylation, acylation, etc., binding to
cofactors or other proteins, functions that are necessary for
biological activity. Differences between the protein profile in
each of the active and the inactive portion are detected in order
to identify active proteins, e.g. enzymes, in the sample.
Inactivating conditions include chemical or physical means for
inactivating, normally by denaturing the protein. For example,
chemical means include denaturants such as organic solvents, harsh
detergents, e.g. SDS, chaotropic.agents, e.g. urea, guanidinium
chloride or isocyanate, etc., and other denaturing agents. Physical
means include heat, freezing, electromagnetic radiation, shearing,
drying, electrical discharge and the like. Inactivating agents that
bind to the active site or an allosteric site affecting activity
may bind covalently or non-covalently, with non-covalent binding
being preferable. In a preferred embodiment, proteins in a sample
are inactivated by heating, although other agents will be preferred
if heating results in precipitation of the protein making it
unavailable for reaction with an ABP.
[0115] Samples that can be analyzed by methods of the invention
include biological samples, such as cell lysates, microsomal
fractions, cell fractions, tissues, organelles, etc., and
biological fluids including urine, sputum, saliva, blood,
cerebrospinal fluid, tears, ejaculate, serum, pleural fluid,
ascites fluid, stool, or a biopsy sample.
[0116] If the sample is impure (e.g., plasma, serum, stool,
ejaculate, sputum, saliva, cerebrospinal fluid, or blood or a
sample embedded in paraffin), it may be treated prior to employing
a method of the invention, frequently to remove contaminants of the
components of interest. Procedures include, for example,
filtration, extraction, centrifugation, affinity sequestering, etc.
Where the probes do not readily pass through a cellular membrane,
intact or permeabilized, or where a lysate is desirable, the cells
are treated with a reagent effective for lysing the cells contained
in the fluids, tissues, or animal cell membranes of the sample, and
for exposing the proteins contained therein and, as appropriate,
partially separating the proteins from other aggregates or
molecules such as microsomes, lipids, carbohydrates and nucleic
acids in the sample. Methods for purifying or partially purifying
proteins from a sample are well known in the art (e.g., Sambrook et
al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Press, 1989, herein incorporated by reference).
[0117] The samples may come from different sources and be used for
different purposes. In many instances, the ABPs will be used to
analyze a protein sample for active enzyme. This may include a
relatively pure sample of the enzyme to determine the activity in
relation to total protein of the sample. The sample may be a single
cell or a mixture of cells, a neoplastic sample or other biopsy or
tissue comprising a single cell type or a mixture of cell types,
such as tissue from an organ, e.g. heart, lung, esophagus, kidney,
brain, blood, etc., diseased tissue or healthy tissue, etc. The
cells may be prokaryotic or eukaryotic, vertebrate or
non-vertebrate, particularly mammalian and more particularly human.
The cells or tissues, or lysates thereof may be prepared in a
variety of ways, including fractionation, using chromatography,
centrifugation, fluorescence activated cell sorting, dilution,
dialysis, concentration, etc. The sample will usually be treated so
as to preserve the activity of the target enzyme(s), so that the
manner of treatment will be mild, ambient or lower temperatures
will be used, particularly below 37.degree. C., and other
denaturing conditions will be avoided, such as organic solvents, or
high salts.
[0118] Usually, a proteome will be analyzed. By a proteome is
intended at least about 20% of total protein coming from a
biological sample source, usually at least about 40%, more usually
at least about 75%, and generally 90% or more, up to and including
all of the protein obtainable from the source. Thus the proteome
may be present in an intact cell, a lysate, a microsomal fraction,
an organelle, a partially extracted lysate, biological fluid, and
the like. The proteome will be a mixture of proteins, generally
having at least about 20 different proteins, usually at least about
50 different proteins and in most cases 100 different proteins or
more. In effect, the proteome is a complex mixture of proteins from
a natural source and will usually involve having the potential of
having 10, usually 20, or more proteins that are target proteins
for the ABPs that are used to analyze the proteome profile. The
sample will be representative of the target proteins of
interest.
[0119] Generally, the sample will have at least about 0.05 mg of
protein, usually at least about 1 mg of protein and may have 10 mg
of protein or more, conveniently at a concentration in the range of
about 0.1-10 mg/ml. The sample may be adjusted to the appropriate
buffer concentration and pH, if desired. One or more ABPs may then
be added, each at a concentration in the range of about 0.001 mM to
20 mM. After incubating the reaction, generally for a time for the
reaction to go substantially to completion, generally for about
1-60 min, at a temperature in the range of about 20-40.degree. C.,
the reaction may be quenched. The sample may now be assayed in
different ways, depending upon the reagents to be used.
[0120] In one aspect of the invention, the method provides for
quantitative measurement of specific active proteins in biological
fluids, cells or tissues. Target identification can be applied to
determine the global protein activity profiles in different cells
and tissues. The same general strategy can be broadened to achieve
the proteome-wide, qualitative and quantitative analysis of the
state of activity of proteins, by employing ABPs or libraries of
ABPs with differing specificity for reaction with proteins. The
method and reagents of this invention can be used to identify
active proteins of low abundance, active in complex mixtures and
can be used to selectively analyze specific groups or classes of
proteins, such as membrane or cell surface proteins, or proteins
contained with organelles, sub-cellular fractions, or biochemical
fractions such as immunoprecipitates. Further, these methods can be
applied to analyze differences in expressed proteins in different
cell states. For example, the methods and reagents herein can be
employed in diagnostic assays for the detection of the presence or
the absence of one or more active proteins indicative of a disease
state, such as cancer, particularly profiles. The ABPs may be a
single ABP that usually binds to at least 5, more usually at least
about 10, different target proteins or may be a mixture of ABPs
that bind to the same number or fewer proteins and may bind to
related or unrelated proteins. Usually the mixture will have from
about 2-20, more usually 2-15 different ABPs, where the profile
will include a multiplicity of target proteins, encompassing
individual or groups of related proteins. Usually, there will be
the capability of binding to at least 10 different proteins, more
usually at least about 15 different proteins and the number of
proteins may be 20 or more, one at least one ABP will be capable of
bonding to at least about 5 different target proteins.
[0121] The ABPs of the invention may be used to isolate and
identify members of a class from the same or different species.
With a neutral ABP (does not significantly discriminate between
more than half of the members of the class of a single species,
where the class has at least about 15 members, more usually at
least about 20 members, usually being able to bind to at least 10
members or more), one can determine the available binding activity
in a physiological sample of the members that bind, one can isolate
new members, and one can inhibit the activity of members of the
class, where such inhibition is of interest. In the case of
affinity labels, one can determine the available activity in a
protein composition of the target proteins, one can differentiate
the activity between the target protein and other members of the
class on the properties of the protein composition, e.g. cell(s) or
lysate, one can obtain a protein activity profile for tissue, cells
or lysate in response to various stimuli and one can screen
compounds for their binding affinity to the target protein, e.g.
drug screening. (It should be understood that enzymes are
particularly exemplary of the target proteins and classes of
enzymes will be the primary targets. To that extent, enzymes are
paradigmatic of the class of target proteins and will be referred
to in the future as exemplary and not limiting of the targets).
[0122] In this way the probes can be used in research, isolation
and identification of proteins of a target class, diagnosis and
developing therapies, and with combinatorial libraries designing
target compounds having affinity for the target site of the target
protein. For enzymes, because of their roles in regulation,
cellular activity, response to external stimuli, and the like,
there is a particular interest in being able to determine the
enzyme activity in a composition, e.g. cell, as distinct from total
enzyme, which could include enzyme that is not active, and how the
enzyme activity varies in relation to external stimuli and/or
changes in the status of the cell. For reacting with the active
form of the enzyme or other protein, it is desirable that one
employs a functionality that is at least relatively specific for
the target enzyme genus. By relatively specific is intended that
less than 20%, usually less than 10%, of the proteins other than
the target enzymes in the genus will react with the functionality.
Equally important is that the functionality optimally does not
react, desirably less than 25%, at other than the wild-type active
site, particularly with the inactive protein. Methods as those
described in this application are employed to distinguish this
non-active site labeling from activity-dependent labeling of the
active site.
[0123] For many of the enzyme genera, functionalities are known
that do not significantly react with enzymes of other genera,
particularly non-enzymatic proteins and enzymes that have different
reactive sites. It is also desirable that the functionality does
not react with inactive target enzyme. Examples of inactive states
include: 1) proenzymes, e.g. requiring cleavage of the protein; 2)
enzymes bound by endogenous inhibitors (either covalent or
non-covalent); 3) enzymes in an inactive conformation (e.g. enzymes
that require the bindng of another protein, a conformational
change, covalent modification by phosphorylation,/reductio-
n/oxidation/methylation/acylation (e.g. formic or acetic acid) for
conversion to an active state; 4) denatured enzymes; 5) mutant
enzymes; 6) enzymes bound by either reversible or irreversible
exogenous inhibitors; and 7) enzymes requiring a cofactor for
activity. The enzymes of interest will usually have at least one of
serine, threonine, cysteine, histidine, lysine, arginine, aspartate
or glutamate as a member of the active site involved in the
catalysis of the enzyme reaction. One or more of the
functionalities of these amino acids may be the target of the ABP.
The manner in which the inactive enzyme is inactivated is chosen to
emphasize the differences in bonding of the ABP between the active
and inactive state. However, if through the course of implementing
the subject methodology, an exogenous inactivator is added to the
protein sample and the effects of this treatment on the target
protein activity profile of the sample relative to a control
(absence of the exogenous inactivator in the sample) are
determined, knowledge will be gained as to the form, quantity, and
identity of the targeted protein (i.e. inactivated) by this
inhibitor.
[0124] Enzymes typically fall within six main classes including
oxidoreductases, transferases, hydrolases, lyases, isomerases and
ligases. In a particular embodiment illustrated herein, an enzyme
group of interest includes the class of hydrolases. One genus of
the class is serine hydrolases, which includes sub-genera such as
proteases, e.g. trypsins, chymotrypsins, esterases, such as
acetylcholinesterases, thioesterases, amidases, such as FAAH, and
acylpeptide hydrolases, lipases, transacylases, such as
lecithin:cholesterol acyltransferase. Another sub-genus is cysteine
hydrolases, such as caspases, cathepsins, and palmitoyl
acyltransferases. Another sub-genus is metallohydrolases, including
matrix metalloproteinases ("MMPs"), e.g. MMP1-13, membrane type
metalloproteinases, aminopeptidases, and ADAMalysins. In addition,
are phosphatases, such as alkaline phosphatases, acid phosphatases,
protein tyrosine phosphatases, and serine/threonine phosphatases.
Further included are the GTPases and ATPases. Besides hydrolases
are kinases, which include enzymes such as tyrosine kinases, e.g.
src, abl, and lck, serine/threonine kinases, e.g. MAP kinases, MAPK
kinases, CAM kinases, protein kinase C, and casein kinases. Also of
interest are oxidoreductases, such as cytochrome P450s, amine
oxidases, alcohol dehydrogenases, aldehyde dehydrogenases, such as
ALDH1, ALDH2, ALDH3, desaturases, etc. Other proteins that are of
interest include receptors, such as HLA antigens, hormone
receptors, G-proteins coupled receptors, ion channels,
transcription factors, protein inhibitors and the like.
[0125] The enzymes and/or the sites to which the ABPs bind may be
identified in a variety of conventional ways, such as isolating the
enzyme, e.g. using an affinity matrix, and characterizing it by
mass spectrometry, isolating and sequencing the enzyme or
proteolytically fragmenting the enzyme and determining the
fractions as a profile for a specific enzyme, electrophoretic
separation and Western blotting, or immunoassays employing labeled
antibodies specific for the enzyme. The conditions under which the
binding is determined will generally be mild conditions,
conveniently ambient, using a buffer solution, where the buffer
concentration will generally be in the range of about 50-200 mM and
the concentration of each active enzyme will generally be about
0.01 pg (picograms)/ml to 0.1 mg (milligrams)/ml. After sufficient
time for enzyme binding, non-specifically bound enzyme may be
washed away. One may wish to use conditions of increasing
stringency, by increasing salt concentration, organic solvent,
temperature, etc., to determine levels of binding affinity. By
comparison of the sequences at different levels of affinity, one
may readily optimize the affinity sequence. In this way one or more
libraries of affinity moieties are developed and can be used in
conjunction with the other members of the ABP. By having a
repertoire of affinity moieties, a specific affinity moiety can be
selected that provides the least amount of background in a
particular environment. For example, one affinity moiety may be
preferred for a target enzyme in a particular background of other
enzymes of the same genus.
[0126] The conjugates between ABPs and active proteins can be
detected and analyzed by a number of different methods. For Western
blotting analysis, conventional conditions are employed; quenching
can be performed with conventional quenching media, e.g. 2.times.
SDS-PAGE loading buffer (reducing), heated for 5-10 min at
80.degree. C. and then run on an SDS-PAGE gel (8-14% acrylamide).
After transferring the protein from the gel to a nitrocellulose
paper by electroblotting, the blot is: 1) blocked for 15-60 min
with 3% non-fat dry milk in TBS-Tween; 2) incubated with
avidin-enzyme conjugate, e.g. horse radish peroxidase (where biotin
has been employed as the ligand or other receptor-enzyme conjugate
for a different ligand) for sufficient time for complex formation
(1-2 hrs); 3) washed with TBS-Tween to remove non-specifically
bound receptor-enzyme conjugate; 4) treated with an appropriate
enzyme substrate for production of a detectable signal; and 5)
detecting the site on the blot of the ABP bound to target.
Quantification of differentially expressed enzyme activities among
different protein samples is conducted by film densitometry using
an AlphaImager 2000 (Alphalnnotech). Alternatively, one may analyze
blots using a chemiluminescence detection system, such as the
Lumi-Imager (Roche).
[0127] Other analytical techniques include binding of the conjugate
to a surface by means of the ligand. Conjugated monoclonal
antibodies conjugated with a label and specific for one or group of
enzymes are added, with the antibodies binding to any target enzyme
that is bound to the surface. The presence and amount of the enzyme
may then be determined by the label, where the label may be a
fluorescent label or an enzyme label, where the enzyme product
provides a detectable signal, e.g. fluorescence. Other techniques
include releasing the conjugate from the receptor, adding
fluorescent receptor and using capillary electrophoresis to
quantitate the enzyme.
[0128] One may also determine minimal or partial activity. One can
do this by comparing the biotin (or other compound binding to a
receptor) signals of protein activities found in crude samples to
those produced by a fully biotinylated protein standard. For
example, take a purified active serine hydrolase and conjugate it
to completion with an appropriate ABP, so that there is no further
enzyme activity. Then use this conjugated enzyme to generate a
standard curve of signals on a gel blot that also contains crude
proteomes conjugated with the same ABP. A protein activity in the
crude proteome whose signal intensity matches the signal intensity
of, for example 10 ng, of the standard enzyme would be considered
to represent minimally 10 ng of the active enzyme in the proteome.
By performing kinetics and probe concentration dependence assays,
one can further determine the average partial activity, where the
enzyme is only partially active.
[0129] For two samples in which the active proteins of a given
family present in these samples are to be quantitatively compared,
the following method can be used. A portion of each sample is
treated so that the active proteins in the one portion are
inactivated. Protein portions of the active and inactive samples
are then treated with isotopic variants of the same ABP (e.g., one
variant contains 5-10 hydrogens (light probe) and is applied to the
inactive portions, the second variant has these 5-10 hydrogens
substituted with deuteriums (heavy probe) and is applied to the
active portions). After sufficient reaction time, the inactive and
active portions of each sample are then separated from their
respective ABPs (e.g., by gel filtration chromatography), combined
to form a mixed sample, and this mixed sample is digested with a
protease (e.g., trypsin) to create a mixture of peptides. These
peptides are then treated with an affinity support to selectively
isolate peptides covalently tagged with an ABP (e.g., avidin is the
affinity support if the probe's tag is biotin). The isolated
peptides are then optionally separated by a liquid chromatography
step (e.g. HPLC) and characterized by mass spectrometry. ABP-tagged
peptides representing active proteins are defined as those found in
significantly greater excess (e.g., at least three-fold greater in
mass ion abundance) bonded to the heavy probe than to the light
probe. The molecular sequence of these peptides can be determined
by Tandem Mass Spectrometry to provide the identity of the active
proteins from which the ABP-labeled peptides are derived. This
first procedure will thereby determine the members of a given
protein family that are both present and active in the sample. Two
protein portions of the active sample are then treated with the
heavy and light probes and processed as described above. The levels
of active protein activities will be quantitatively compared across
the two samples by ratioing the mass ion abundances corresponding
to heavy and light probe-bonded versions of individual peptides.
Only those peptides that were determined in the first procedure to
represent active proteins will be compared in this manner. To
analyze simultaneously more than two samples, the same method may
be followed, but an additional, unique isotopic variant of the
activity-based probe will be required for each additional
sample.
[0130] Of particular interest as ABPs are labeled
fluorophosphonates, such as biotin-linked fluorophosphonates.
[0131] For the most part, the compounds come within the following
formula:
X-L-R-(O).sub.mP(O)(OT)-F
[0132] wherein:
[0133] F, P and O have their normal meaning of fluoro, phospho and
oxy;
[0134] X is a ligand (including detectable label);
[0135] L is a linking group;
[0136] R is an aliphatic group of at least 2 carbon atoms, usually
at least 4 carbon atoms and not more than about 16 carbon atoms,
usually not more than about 12 carbon atoms, usually being straight
chain alkylene or alkyleneoxy (wherein the alkylene groups are of
from 2-3 carbon atoms), saturated or unsaturated, usually having
not more than 2 sites of unsaturation;
[0137] m is 0 or 1; and
[0138] T is alkyl of from 1 to 6, usually 1 to 3 carbon atoms.
[0139] For the most part the ligand will be biotin or derivative
thereof, e.g. deiminobiotin or dethiobiotin, and the detectable
label may be a fluorescent compound, e.g. fluorescein, rhodamine,
Texas red, etc.
[0140] Also of interest are compounds come within the following
formula:
X-L-SO.sub.2-R
[0141] wherein:
[0142] X is a ligand (including detectable label);
[0143] L is a linking group;
[0144] R is an aryl or heterocyclic group of from 5 to 12 carbon
atoms having from 1-2 nitrogen atoms, which may be substituted or
unsubstituted, where substituents may be halo, nitro, cyano, oxy,
thio, amino, etc. In some instances, alkyl and substituted alkyl of
from 1 to 20, usually 1-12 carbon atoms can find use.
[0145] Of particular interest are compounds where the linking group
includes a dicarboxamido-.alpha.,.alpha.-alkylene, particularly
where biotin (including derivatives thereof) is used which includes
a carboxyl group naturally. The alkylene will generally be of about
2-6 carbon atoms, the length will be desirably related to not
interfering with the binding of the ligand to its respective
receptor and reaction of the sulfonate.
[0146] The ligand can be any ligand that does not interfere with
the binding of the subject compounds to the serine hydrolases,
relatively small, less than about 1 kDal, frequently less than
about 500 Dal, has an appropriate receptor and is synthetically
accessible. There are a number of popular ligands, such as biotin,
dethiobiotin, deiminobiotin, digoxin, 2,4-dinitrophenyl, and
derivatives thereof, fluorescein, etc. These ligands have strongly
binding natural receptors, such as strept/avidin for biotin and
dethio- or deiminobiotin, and antibodies for the remaining listed
ligands. In some instances it will be desirable to release the
serine hydrolase bonded to the inhibitor of this invention from the
receptor. A useful pair is dethiobiotin or deiminobiotin, which can
be replaced by biotin.
[0147] The subject compounds can be prepared using an
.alpha.,.omega.-(halo or pseudohalo)alkene, where the halo or
pseudohalo group is displaced with a trialkylphosphite, followed by
selective oxidation of the olefin to a carboxy or aldehyde. The
activated carboxy, e.g. N-succinimidyl ester or carbodiimide
anhydride, may be reacted with the ligand or detectable label
bonded to a linking group terminating in an amino group to form an
amide. The aldehyde may be bonded to an amine by forming an imine
or Schiff's base or by reductive amination, forming an alkylated
amine. Other than the ligand, the subject compounds will have fewer
than 30 carbon atoms, usually fewer than 25 carbon atoms. There may
be and preferably is 1 or more functionalities in the chain joining
the ligand or detectable label to the alkylfluorolphosphoric acid
group, generally neutral functionalities, such as amido, oxy, thio,
urea, thiourea, etc.
[0148] When the subject compound is bound to the serine hydroxyl of
a serine hydrolase, the resulting inhibited enzyme may have the
following formula:
X-L-R-(O).sub.m-P(O)(OT)-SH
[0149] where all of the symbols have been defined previously,
except for SH, which is a serine hydrolase bonded at the serine
group of the active site to the phosphate.
[0150] Known serine hydrolases include fatty acid amide hydrolases
(FAAH), kallikreins, acylpeptide hydrolases, prostate specific
antigen, cholinesterases, trypsins, chymotrypsins, plasmin,
thrombin, phospholipases, signal peptidases, amidase signature
enzymes, plasminogen activators, prohormone convertases, granzymes,
seprase, dipeptidyl and tripeptidyl peptidases, usually being
derived from mammalian sources, particularly human, but may come
from other sources, both prokaryotic and eukaryotic, including
plants, birds, invertebrates, fungi, etc.
[0151] The subject inhibitors may be used in a variety of ways. One
application is to determine the enzyme, e.g. serine hydrolase,
activity of a physiological sample. The sample may be blood, cells,
tissue, or other physiological sample of interest. In some
situations, samples that are suspected of having one serine
hydrolase may be monitored, as in the genetic engineering of serine
hydrolase proteins, where the efficiency of synthesis would be of
interest. In the case of tissue or cells, the cells may be lysed in
accordance with conventional conditions, using a homogenizer,
blender, pellets, centrifuge or other convenient device. The
resulting lysed cellular composition may be centrifuged and the
supernatant adjusted for protein content. Depending on the nature
of the ligand, the supernatant fraction may be freed of naturally
occurring ligand and/or receptor. The supernatant may be further
treated, as appropriate, adding buffer, further dilution,
fractionation by chromatography, etc. Where fractionated,
individual fractions will be used in the assay.
[0152] Candidate compounds to be used as therapeutics associated
with indications involving enzyme dysfunction, particularly for
inhibiting specific or groups of related enzymes, may be monitored
by preparing a reaction mixture with one or more related enzymes
and monitoring the effect on the rate of inhibition. One would add
one or more subject compounds and a candidate compound and then
monitor the rate of inhibition, by isolating aliquots and analyzing
the aliquot for enzyme activity or isolating bound enzymes and
analyzing the bound serine hydrolases.
[0153] The sample will generally be adjusted to have between about
0.1 to 5 .mu.g/ml, more usually 0.5 to 2 .mu.g/ml of protein,
larger amounts being unnecessary. The sample may be added in
conventional buffers, such as Tris, HEPES, phosphate, etc. Most
chemically and biologically inert buffers providing the desired pH
may be used. The ABP for serine hydrolase will be a
fluorophosphoric acid compound, which can be combined dry or
preferably as a water-miscible polar organic solution that is
chemically and biologically inert under the conditions of the
assay. Ethanol, DMSO, DMF, etc. may be used. Usually, an excess of
the ABP will be employed, generally at least about 2 fold excess,
usually at least about 5 fold excess, based on actual enzyme
concentration, if known, or estimated enzyme, based on total
protein, to ensure that substantially all of the available target
enzyme has reacted. The mixture will ordinarily be incubated for
about 10 min or more, usually not more than about 1 h. The reaction
may then be quenched with any convenient quenching agent,
particularly elevated temperatures. The resulting denatured
composition may then be analyzed, for example, using capillary
electrophoresis (microfluidic device), gel electrophoresis, HPLC,
mass spectrometry (MALDI), Western blotting, or the like, where the
fractions may be observed by virtue of the ligand. Either the
ligand provides a detectable signal, usually fluorescence, although
electrochemical and chemiluminescence may also be employed, or the
ligand may be reacted with a labeled receptor, and detected as the
ligand-receptor complex. The receptor may be conjugated with an
enzyme that produces a colored or fluorescent product. There are
many enzymes known for this purpose. Illustrative enzymes include
horse radish peroxidase, .alpha.-galactosidase, G6PDH, MDH,
alkaline phosphatase, lysozyme, etc. In many cases commercially
available substrates can be used for detection.
[0154] As indicated, the proteins conjugated to the subject
compounds may be separated by electrophoresis or other technique,
which provides for independent fractions. One can do time courses,
to see how the amount of the different fractions changes with time
by quantitating the signal. One can also determine the specific
member of the group of related enzymes by using antibodies specific
for one or more epitopes of each of the enzyme members of interest.
After separating the protein into distinct bands, the
electrophoretic bands may be reacted with labeled antibodies, where
the labels are different from the labels of the subject compounds,
so as provide a distinguishable signal, e.g. fluorescence at a
different wavelength, electrochemical detection as compared to
fluorescence, chemiluminescence, etc.
[0155] Of particular interest is to perform kinetics of inhibition
by candidate compounds of the rate of reaction of one or more
probes with a proteome of target proteins, e.g. a family of
proteins. One can select conditions, whereby at a selected time,
the amount of conjugate formation is still changing, so that the
amount of conjugate varies with the amount of candidate compound
present. In this way one can obtain the binding affinity of the
candidate compound, where the target proteins can be saturated with
the candidate compound. One combines the candidate compound at a
predetermined concentration with a fixed amount of the proteome and
the probe(s) under conditions whereby the amount of conjugate
formation is related to the amount of candidate compound present.
The conditions selected may be as close to physiological as the
determination may allow or other conditions may be selected, that
provide broader differentiation between the reactions of the
different target proteins with the conjugate in the presence of the
candidate compound. Changes, such as temperature, pH, additives,
etc., may be evaluated for their effect on the bioactivity of the
candidate compound. One can select conditions that avoid
indiscriminate binding of the candidate compound to the proteins
present in the protein mixture. Conditions can be chosen where the
reactivity of the candidate compound is minimized and then varying
the appropriate parameter(s) to enhance activity of the candidate
compound. By determining the rates of reaction of the probe at
different concentrations of the candidate compound, the K.sub.I of
the candidate compound for each of the target proteins can be
determined.
[0156] One can follow the reaction with time by taking aliquots
from the reaction mixture and quenching the aliquots, so that no
further reaction occurs. Alternatively, one can select a specific
time at which the entire reaction is quenched. Quenching can be by
heat, reactive reagent that reacts with the probe, separation of
the protein from the reagents, changing another condition that
terminates the reaction, or the like. The amount of conjugate for
each target protein is then determined. By using probes that
provide a detectable signal and isolating the target proteins
individually, e.g. using antisera or monoclonal antibodies specific
for an individual target protein, one can determine the amount of
conjugate for each of the target proteins at at least two different
concentrations of the candidate compound. With a plurality of
determinations at different concentrations of the candidate
compound, one can graph the variation in conjugate formation for
each of the target proteins with variation in concentration of the
candidate compound. This provides a three dimensional analysis of
the variation in inhibitory effect of the candidate compound on a
plurality of proteins, particularly related proteins of a protein
family. In this way one obtains with a few kinetic determinations a
profile of the inhibitory activity of a candidate compound against
a plurality of target proteins and conveniently in the presence of
a complex proteome, such as a lysate. The analysis not only
provides the inhibitory effect of the candidate compound, but the
effect of other proteins on the inhibitory effect. In this way, for
each concentration of candidate compound, one obtains a result for
each of a plurality of target proteins in a complex environment. By
having an antibody chip, with each of the antibodies for a target
protein at a predetermined region, one contacts the quenched
reaction mixture with the chip and can determine the amount of
conjugate at each site by means of the detectable ligand.
[0157] The information concerning the nature of the protein target,
the degree to which the probes bind at a given target and probe
concentration under one or more different sets of conditions, the
effect of agents on the binding profile and a comparison of the
different structures that bind can all be given to a data processor
to allow for analysis and comparison of the data. Thus, the subject
system in evaluating changes in proteome profile with one or a
group of probes or the effect of an agent on probe binding can be
analyzed with a data processor and a data bank developed for
comparison with known compounds having physiological activity and
candidate compounds that have been screened in accordance with the
subject methods. The results of the assays can serve as a basis for
designing new compounds, modifying existing compounds and
predicting the effects of drugs on the proteome profile of
cells.
[0158] Depending upon the information desired, different data banks
will be employed. A data bank concerned with the development of
drugs will include the effect of candidate compounds on the ABP
profile of the target protein, as well as the other related
proteins. In addition, the data bank includes the effect of
variation in structure and composition of different compounds on
the ABP profile. Also included in the data bank is common
cross-reactivity with non-related proteins for compounds that
affect the binding to the target protein.
[0159] The information that is developed can be used in a variety
of ways using data processing. The combinatorial library allows for
designing drugs for binding to the active site of a target protein.
By determining which variations in structure enhance or reduce the
binding affinity of the ABPs, one can employ algorithms that define
spatial forms of the active affinity groups and identify
modifications that reduce the affinity. Introducing this
information into a data processor will provide for the spatial
conformation that fits the active site and can also be used to
define the charge distribution on the surface. One or more
optimized conformations and charge distributions may then be used
as the affinity portion of a probe and the affinity determined. The
optimized probes can be used individually or in competitive modes
to determine their relative affinities for the active site. This
information may then be analyzed by a data processor, whereby the
desired structure may be further refined. In this way drugs may be
rapidly designed for optimum binding affinity.
[0160] The information developed with the probes in analyzing
proteomes can also be organized in relation to cellular status. The
distribution of active related enzymes is related to particular
cellular status, so that one obtains a profile of the amount of
each of the active proteins to which the ABPs bind. The information
is useful in comparisons between cellular status and the protein
profile, where the status is established by other means. A data
bank then serves to relate the profile to a basis for treatment of
a diseased state. Cancers are exemplary. There is great interest in
knowing how aggressive a cancer is, what stage it is in becoming
metastatic and how the cancer will respond to different treatments,
e.g. hormone treatment, chemotherapy, radiotherapy, etc. Having a
correlation between protein profiles and cellular status, even when
the proteins have not been shown to be directly involved with the
disease pathway permits a rapid determination of the cellular
status by obtaining the protein profile. In addition, one may
follow the course of the disease by monitoring changes in the
protein profile. The information concerning the profile is
introduced into a data processor having a data bank of protein
profiles, status and outcomes. By comparing the profile with the
existing bank of profiles, particularly comparing the changes in
profile with time and treatment, the effectiveness of the treatment
can be evaluated and a more certain evaluation of the outcome
achieved.
[0161] In employing the system, the method described above for
determining one or more profiles from different related groups of
proteins is employed to obtain the initial results or raw data.
Based on the conjugation of the ABP to the related group of
proteins, one may obtain the amount of each individual target
protein and, optionally, the total amount of protein in the related
group bound to the target protein providing for relative abundance.
If one gets the individual amount and the total amount, one can
obtain a relative abundance for each target protein, which will
diminish effects that are consistent with the entire group of
related proteins and affect the observed value for an individual
protein. This information is then processed in a programmed data
processor, where the data processor can be programmed with
information related to the purpose for the measurement. One employs
a program that compares the absolute amount of a target protein
and/or the relative abundance to results obtained in other
experiments, where standards are devised. The standards may include
the effect of known agents on the target protein and/or the
relative abundance, the difference between two cell types, e.g.
normal or neoplastic, differentiated and non-differentiated, two
different differentiated cells, such as T-cells and B-cells,
Th.sub.1 and Th.sub.2, myoblast and fibroblast, genetically
modified cell and a wild-type cell, etc. Therefore, the standard
may be the results from a single test or plurality of the same test
on the same or analogous sample or may be a composite of compiled
data related to the purpose of the determination.
[0162] Various methods and algorithms have been used for analyzing
results and presenting the results in useful form for
interpretation. Eisen, et al., Proc. Natl. Acad. Sci. USA (1998)
95, 14863-14868, (specifically incorporated herein by reference)
describes using cluster analysis using standard statistical
algorithms with graphic display of the results. By employing a
dendrogram that assembles all elements into a single tree, where
for any set of n genes, an upper diagonal similarity matrix is
computed by using the described metric, which contains similarity
scores for all pairs of genes. The matrix is scanned to identify
the highest value (representing the most similar pair of genes). A
node is created joining these two genes, and a gene expression
profile is computed for the node by averaging observation for the
joined elements (missing values are omitted and the two joined
elements are weighted by the number of genes they contain). The
similarity matrix is updated with this new node replacing the two
joined elements, and the process is repeated n-1 times until only a
single element remains. Software implementation of this algorithm
can be obtained from the authors at http://rana.stanford.edu/cl-
ustering.
[0163] Ordering was based on weighting genes, such as average
expression level, time of maximal induction, or chromosomal
position with the element with the lower average weight placed
earlier in the final ordering. The display is represented
graphically with unchanged genes colored black and increasingly
positive log ratios with reds of increasing intensity and
increasingly negative log ratios with greens of increasing
intensity.
[0164] See also, U.S. Pat. Nos. 6,114,114 and 6,132,969, for
additional methods of analyzing results and reporting data. These
techniques are readily adapted to the present systems in providing
for comparisons of the levels of connugation with different ABPs,
the variations in profiles of conjugate formation in the presence
of candidate drugs or other reagents and the relationship between
conjugate profile and disease indications.
[0165] The invention will now be described in greater detail by
reference to the following non-limiting examples.
EXPERIMENTAL
EXAMPLES
[0166] The following examples provide an illustrative synthetic
scheme for a fluorophosphonates-based ABP which includes a biotin
label or tag. This "FP-biotin" ABP is in no way limiting and is
merely illustrative. One of skill in the art can use standard
methods to design other ABPs as described herein.
Example 1
[0167] Compound 1 is the starting material tetraethyleneoxy
(3,6,9-oxa-1,11-diolundecane) as depicted in the flow chart in FIG.
8.
[0168] Compound 2. A solution of 1 (3.9 g, 20.0 mmol, 3.0 equiv) in
DMF (8.0 mL) was treated with TBDMSCl (1.0 g, 6.64 mmol, 1.0 equiv)
and imidazole (0.9 g, 13.3 mmol, 2.0 equiv) and the reaction
mixture was stirred for 12 h at room temperature. The reaction
mixture was then quenched with saturated aqueous NaHCO3 and
partitioned between ethyl acetate (200 mL) and water (200 mL). The
organic layer was washed with dried (Na2SO4) and concentrated under
reduced pressure. Chromatography (SiO2, 5.times.15 cm, 50-100%
ethyl acetate-hexanes) afforded 2 (1.1 g, 2.0 g theoretical, 55%)
as a colorless oil: 1H NMR (CDCl3, 400 MHz) .delta. 3.8-3.5 (m,
16H, CH2OR), 0.88 (s, 9H, CH3C), 0.0 (s, 6H, CH3Si).
[0169] Compound 3. A solution of 2 (0.61 g, 2.0 mmol, 1.0 equiv) in
benzene (15 mL, 0.13 M) was treated sequentially with PPh3 (2.6 g,
10.0 mmol, 5 equiv), I2 (2.3 g, 9.0 mmol, 4.5 equiv), and imidazole
(0.7 g, 10.3 mmol, 5.2 equiv) and the reaction mixture was stirred
at room temperature for 30 min, producing a yellow-orange
heterogeneous solution. The soluble portion of the reaction mixture
was removed and the insoluble portion washed several times with
ethyl acetate. The combined reaction and washes were then
partitioned between ethyl acetate (200 mL) and saturated aqueous
Na2S2O3 (200 mL). The organic layer was washed sequentially with
H2O (100 mL) and saturated aqueous NaCl (100 mL), dried (Na2SO4),
and concentrated under reduced pressure. Chromatography (SiO2,
5.times.15 cm, 5-25% ethyl acetate-hexanes) afforded 3 (0.54 g,
0.82 g theoretical, 66%) as a colorless oil:1H NMR (CDCl3, 400 MHz)
.delta. 3.85-3.60 (m, 12H, CH2OR), 3.54 (t, J=5.6, 2H, CH2OTBDMS),
3.23 (t, J=7.0 Hz, 2H, CH2I), 0.88 (s, 9H, CH3C), 0.0 (s, 6H,
CH3Si).
[0170] Compound 4. Triethylphosphite (1.2 mL, 7.0 mmol, 5.4 equiv)
was added to 3 (0.53 g, 1.29 mmol, 1.0 equiv) and the mixture was
stirred at 150.degree. C. for 1 h. The reaction mixture was cooled
to room temperature and directly submitted to flash chromatography
(SiO2, 5.times.15 cm, 100% ethyl acetate) to afford 4 (0.43 g, 0.54
g theoretical, 80%) as a colorless oil: 1H NMR (CDCl3, 400 MHz)
.delta. 4.20-4.05 (m, 4H, CH3CH2OP), 3.80-3.55 (m, 14H, CH2OR),
2.15 (m, 2H, CH2P), 1.31 (t, J=6.0 Hz, 6H, CH3CH2OP), 0.88 (s, 9H,
CH3C), 0.0 (s, 6H, CH3Si).
[0171] Compound 5. A solution of compound 4 (0.21 g, 0.5 mmol, 1.0
equiv) in CH2Cl2 (2.8 mL, 0.18 M) was treated with HF-pyridine
(0.084 mL, .about.0.84 mmol, .about.1.7 equiv). The reaction was
stirred at 25.degree. C. for 30 min and then partitioned between
ethyl acetate (100 mL) and water (100 mL). The organic layer was
dried (Na2SO4) and concentrated under reduced pressure.
Chromatography (SiO2, 2.times.8 cm, 3-10% CH3OH--CH2Cl2) afforded 5
(0.050 g, 0.28 g theoretical, 32.5%) as a clear oil: 1H NMR (CDCl3,
400 MHz) .delta. 4.20-4.05 (m, 4H, CH3CH2OP), 3.80-3.55 (m, 14H,
CH2OR), 2.15 (m, 2H, CH2P), 1.31 (t, J=6.0 Hz, 6H, CH3CH2OP);
MALDI-FTMS m/z 337.1377 (C12H27O7P+Na+requires 337.1387).
[0172] Compound 6. A solution of 5 (0.030 g, 0.096 mmol, 1.0 equiv)
in DMF (0.28 mL, 0.34 M) was treated sequentially with
N,N-disuccinimidyl carbonate (0.058 g, 0.22 mmol, 2.2 equiv) and
triethylamine (0.035 .mu.L, 0.25 mmol., 2.5 equiv). The reaction
mixture was stirred at room temperature for 12 h and then
partitioned between CH2Cl2 (100 mL) and H2O (100 mL). The organic
layer was washed with saturated aqueous NaCl (100 mL), dried
(Na2SO4), and concentrated under reduced pressure. Chromatography
(SiO2, 2.times.8 cm, 1-10% CH3OH--CH2Cl2) afforded 50.035 g, 0.043
g theoretical, 81%) as a clear oil: 1H NMR (CDCl3, 400 MHz) .delta.
4.45 (m, 2H, CH2OC(O)OR), 4.20-4.05 (m, 4H, CH3CH2OP), 3.80-3.55
(m, 12H, CH2OR), 2.84 (s, 4H, CH2C(O)N), 2.15 (m, 2H, CH2P), 1.31
(t, J=6.0 Hz, 6H, CH3CH2OP). MALDI-FTMS m/z 478.1456
(C17H30NO11P+Na+requires 478.1449).
[0173] Compound 7. A solution of 6 (0.020 g, 0.044 mmol, 1.0 equiv)
in CH2Cl2 (0.14 mL, 0.40 M) was cooled to 0.degree. C. and treated
with oxalyl chloride (0.082 mL, 2M in CH2Cl2, 0.164 mM 3.7 equiv).
The reaction mixture was allowed to warm to room temperature and
stirred for 18 h. The reaction mixture was then concentrated under
a stream of gaseous nitrogen and the remaining residue treated with
H2O (0.1 mL) for 5 min. The H2O was evaporated under a stream of
gaseous nitrogen and the remaining residue dried by vacuum to
provide 7 (0.015 mg, 0.019 mg theoretical, 80%) as a clear
oil/film: 1H NMR (CDCl3, 400 MHz) .delta. 4.45 (m, 2H, CH2OC(O)OR),
4.10 (m, 2H, CH3CH2OP), 3.80-3.55 (m, 12H, CH2OR), 2.84 (s, 4H,
CH2C(O)N), 2.15 (m, 2H, CH2P), 1.31 (t, J=6.0 Hz, 3H,
CH3CH2OP).
[0174] Compound 8. A solution of 7 (0.007 g, 0.016 mmol, 1.0 equiv)
in CH2Cl2 (0.22 mL, 0.075 M) at -78.degree. C. was treated with
(diethylamino)sulfur trifluoride (DAST, 0.007 mL, 0.048 mmol, 3.0
equiv) and the reaction mixture was stirred for 10 min. The
reaction mixture was then partitioned between ethyl acetate (100
mL) and H2O (100 mL) and the organic layer was washed with
saturated aqueous NaCl (100 mL), dried (Na2SO4), and concentrated
under reduced pressure. Chromatography (SiO2, Pasteur pipette, 100%
ethyl acetate) afforded 8 (0.003 g, 0.007 g theoretical, 42%) as a
clear oil: 1H NMR (CDCl3, 400 MHz) .delta. 4.45 (m, 2H,
CH2OC(O)OR), 4.27 (m, 2H, CH3CH2OP), 3.80-3.55 (m, 12H, CH2OR),
2.84 (s, 4H, CH2C(O)N), 2.32-2.26 (m, 2H, CH2P), 1.31 (t, J=6.0 Hz,
3H, CH3CH2OP).
[0175] FP-peg-biotin (9). A solution of 8 (0.003 g, 0.007 mmol, 1.0
equiv) in DMF (0.1 mL, 0.07 M) was added to
5-(biotinamido)-pentylamine (Pierce, 0.0035 g, 0.011 mmol, 1.5
equiv) and the reaction mixture was stirred for 4 h. The solvent
was evaporated under a stream of gaseous nitrogen and the remaining
residue was washed sequentially with diethyl ether and ethyl
acetate, solubilized in a minimal volume of chloroform, transferred
to a clean glass vial, and the solvent evaporated. This process was
repeated twice more to rid the desired biotinylated product of
excess reagents and byproducts, affording 9 as a white film (0.0017
g, 0.0045 g theoretical, 38%): 1H NMR (CDCl3, 400 MHz) .delta. 6.04
(b s, 1H, NH), 5.77 (b s, 1H, NH), 5.24 (b s, 1H, NH), 5.09 (b s,
1H, NH), 4.51 (m, 1H), 4.40-4.21 (m, 5H, including, CH2OC(O)NR and
CH3CH2OP), 3.90-3.55 (m, 12H, CH2OR), 3.30-3.11 (m, 5H, including
CH2NHC(O)), 2.92 (dd, J=4.9 and 12.9 Hz, 1H), 2.74 (d, J=12.9 Hz,
1H), 2.35-2.18 (m, 4H, CH2CONHR and CH2P), 1.85-1.40 (m, 12H); 1.31
(t, J=6.0 Hz, 3H, CH3CH2OP); MALDI-FTMS m/z 665.2742
(C26FH48N4O9PS+Na+requires 665.2756). 1-[(p-Toluenesulfonyl)oxy]-
-10-undecene (2). A solution of 1 (2.0 g, 11.8 mmol, 1.0 equiv) in
pyridine (14.0 mL, 177 mmol, 15 equiv) was cooled to 0.degree. C.
and treated with pTsCl (4.5 g, 23.6 mmol, 2.0 equiv). The reaction
mixture was kept at 0.degree. C. for 10 h and then partitioned
between ethyl acetate (200 mL) and water (200 mL). The organic
layer was washed with 10% aqueous HCl (2.times.200 mL), saturated
aqueous NaCl (200 mL), dried (Na2SO4), and concentrated under
reduced pressure. Chromatography (SiO2, 5.times.15 cm, 2% ethyl
acetatehexanes) afforded 2 (3.6 g, 3.8 g theoretical, 94%) as a
colorless oil: 1H NMR (CDCl3, 250 MHz) .delta. 7.76 (d, J=6.5 Hz,
2H, ArH), 7.32 (d, J=7.3 Hz, 2H, ArH), 5.95-5.75 (m, 1H,
RCH.dbd.CH2), 5.03-4.90 (m, 2H, RCH.dbd.CH2), 3.98 (t, J.dbd.6.5
Hz, 2H, CH2OTs), 2.42 (s, 3H, ArCH3), 2.02 (m, 2H, CH2CH.dbd.CH2),
1.65 (p, J=6.9 Hz, 2H, CH2CH2OTs), 1.50-1.20 (m, 12H); MALDI-FTMS
(DHB) m/z 347.1657 (C18H28O3S+Na+requires 347.1658).
Example 2
[0176] Preparation of FP-Biotin
[0177] FP-Biotin was prepared as described by Liu et al. (Proc.
Natl. Acad. Sci. 96(26):14694, 1999) and in U.S. Serial Nos.
60/195,954 and 60/212,891, herein incorporated by reference in
their entirety. 1-Iodo-10-undecene (3). A solution of 2 (3.4 g,
10.5 mmol, 1.0 equiv) in acetone (21 mL, 0.5 M) was treated with
NaI (3.2 g, 21 mmol, 2.0 equiv) and the reaction mixture was
stirred at reflux for 2 h, producing a yellow-orange solution. The
reaction mixture was then partitioned between ethyl acetate (200
mL) and water (200 mL). The organic layer was washed sequentially
with saturated aqueous Na2S2O3 (100 mL) and saturated aqueous NaCl
(100 mL), dried (Na2SO4), and concentrated under reduced pressure.
Chromatography (SiO2, 5.times.15 cm, 1-2% ethyl acetatehexanes)
afforded 3 (2.3 g, 2.9 g theoretical, 78%) as a colorless oil: 1H
NMR (CDCl3, 250 MHz) .delta. 5.95-5.75 (m, 1H, RCH.dbd.CH2),
5.03-4.90 (m, 2H, RCH.dbd.CH2), 3.16 (t, J=7.0 Hz, 2H, CH2I), 2.02
(m, 2H, CH2CH.dbd.CH2), 1.80 (p, J=6.9 Hz, 2H, CH2CH2I), 1.50-1.20
(m, 12H).
[0178] 1-[Bis(ethoxy)phosphinyl]-10-undecene (4). Triethylphosphite
(12.2 mL, 71 mmol, 10 equiv) was added to 3 (2.0 g, 7.1 mmol, 1.0
equiv) and the mixture was stirred at reflux for 15 h. The excess
triethylphosphite was removed by distillation and the remaining
residue submitted to flash chromatography (SiO2, 5.times.15 cm,
25-50% ethyl acetate-hexanes gradient elution) to afford 4 (1.30 g,
2.1 g theoretical, 62%) as a colorless oil: 1H NMR (CDCl3, 250 MHz)
.delta. 5.95-5.75 (m, 1H, RCH.dbd.CH2), 5.03-4.90 (m, 2H,
RCH.dbd.CH2), 4.05 (m, 4H, CH3CH2OP), 2.02 (m, 2H, CH2CH.dbd.CH2),
1.80-1.20 (m, 20H); MALDI-FTMS (DHB) m/z 291.2088
(C15H31O3P+H+requires 291.2089).
[0179] 1-(Ethoxyhydroxyphosphinyl)-10-undecene (5). A solution of
compound 4 (0.31 g, 1.07 mmol, 1.0 equiv) in CH2Cl2 (4.0 mL, 0.3 M)
was treated dropwise with trimethylsilyl bromide (TMSBr, 0.17 mL,
1.28 mmol, 1.2 equiv). The reaction was stirred at 25.degree. C.
for 1 h, quenched with 5 mL of 5% [w/v] KHSO4, and stirred
vigorously for 5 minutes. The reaction mixture was then partitioned
between ethyl acetate (100 mL) and water (100 mL), and the organic
layer was washed with saturated aqueous NaCl (200 mL), dried
(Na2SO4), and concentrated under reduced pressure. Chromatography
(SiO2, 2.times.8 cm, 12-20% CH3OH--CHCl3 with 1% aqueous NH4OH)
afforded 5 (0.10 g, 0.28 g theoretical, 36.2.%; most of the
remaining mass was recovered as starting material) as a clear oil:
1H NMR (CDCl3, 250 MHz) .delta. 5.95-5.75 (m, 1H, RCH.dbd.CH2),
5.03-4.90 (m, 2H, RCH.dbd.CH2), 4.05 (m, 2H, CH3CH2OP), 2.02 (m,
2H, CH2CH.dbd.CH2), 1.80-1.20 (m, 20H). MALDI-FTMS (DHB) m/z
285.1589 (C13H27O3P+Na+requires 285.1596).
[0180] 10-(Ethoxyhydroxyphosphinyl)-decanoic acid (6). Compound 5
(0.10 g, 0.38 mmol, 1.0 equiv) in a biphasic solution composed of
CCl4-CH3CN-H2O (1.0 mL-1.0 mL-1.5 mL; total volume of 3.5 mL, 0.11
M) was treated sequentially with sodium periodate (0.31 g, 1.56
mmol, 4.1 equiv) and ruthenium trichloride hydrate (0.002 g, 0.009
mmol, 0.022 equiv). The reaction mixture was stirred at 25.degree.
C. for 2 h and then partitioned between CH2Cl2 (50 mL) and 1 N
aqueous HCl (50 mL). The organic layer was washed with saturated
aqueous NaCl (25 mL), dried (Na2SO4), and concentrated under
reduced pressure. The resulting residue was resuspended in 40 mL of
diethyl ether, filtered through a Celite pad, and concentrated
under reduced pressure to afford 6 (0.09 g, 0.11 g theoretical,
83%) as a colorless semisolid: 1H NMR (CDCl3, 250 MHz) .delta. 4.05
(m, 2H, CH3CH2OP), 2.32 (t, J=7.5 Hz, 2H, CH2COOH), 1.80-1.20 (m,
16H); FABHRMS (NBA-NaI) m/z 303.1340 (C12H25O5P+Na+requires
303.1337).
[0181] FP-biotin, or
10-(fluoroethoxyphosphinyl)-N-(biotinamidopentyl)-dec- anamide (7).
A solution of 6 (0.007 g, 0.025 mmol, 4.0 equiv) in CH2Cl2 (0.4 mL,
0.06 M) at -78.degree. C. was treated dropwise with
(diethylamino)sulfur trifluoride (DAST, 0.021 mL, 0.10 mmol, 4.0
equiv), brought to 25.degree. C., and stirred for 5 min. The
reaction mixture was then treated with one-half reaction volume of
dimethyl formamide containing N-hydroxysuccinimide (0.05 g, 0.25
mmol, 10 equiv) and stirred for an additional 10 min at 25.degree.
C. The reaction mixture was then partitioned between ethyl acetate
(50 mL) and water (50 mL), and the organic layer was washed with
saturated aqueous NaCl (200 mL), dried (Na2SO4), and concentrated
under reduced pressure to afford
10-(fluoroethoxyphosphinyl)-N-(hydroxysuccinyl)-decanamide (as
judged by crude 1H NMR;). Without further purification, this
compound was treated with 5-(biotinamido)-pentylamine (Pierce,
0.0021 g, 0.062 mmol, 1.0 equiv) in MeOH (0.02 mL) and stirred for
10 min. The solvent was evaporated under a stream of gaseous
nitrogen and the remaining residue was washed sequentially with
diethyl ether and ethyl acetate, solubilized in a minimal volume of
chloroform, transferred to a clean glass vial, and the solvent
evaporated. This process was repeated once more to rid the desired
biotinylated product of excess reagents and byproducts, affording 7
as a white film (0.0011 g, 0.0038 g theoretical, 29%): 1H NMR
(CDCl3, 400 MHz) .delta. 5.98 (b s, 1H, NH), 5.83 (b s, 1H, NH),
5.60 (b s, 1H, NH), 4.90 (b s, 1H, NH), 4.51 (m, 1H), 4.32 (m, 1H),
4.27 (m, 2H, CH3CH2OP), 3.22 (m, 4H, CH2NHCOR), 3.15 (m, 1H), 2.92
(dd, J=4.9 and 12.9 Hz, 1H), 2.72 (d, J=12.9 Hz, 1H), 2.20 (m, 4H,
CH2CONHR), 1.85-1.24 (m, 31H); FABHRMS (NBA-NaI) m/z 593.3319
(C27FH50N4O5PS+H+requires 593.3302).
[0182] Preparation of Tissue Samples for Reaction with FP-Biotin.
Rat tissues were Dounce-homogenized in Tris buffer (50 mM Tris-HCl
buffer, pH 8.0, with 0.32 M sucrose). Tissue extracts were
centrifuged sequentially at 1,100.times.g (5 min), 22,000.times.g
(30 min), and 105,000.times.g (60 min). The final supernatant
(cytosolic fraction) was adjusted to 1 mg protein/mL and then
incubated for 30 min at 4.degree. C. with one-tenth volume of
avidin-agarose (Sigma) to deplete endogenous avidin-binding
proteins. The resulting supernatant after a brief spin to pellet
the avidin-beads (2 min at 10,000.times.g) was removed and treated
with FP-biotin as describe below.
[0183] Reaction of Protein Samples with FP-Biotin. Unless otherwise
indicated, reactions between protein samples and FP-biotin were
conducted as follows: FP-biotin (0.4 nmol) in CHCl3 was added to a
glass vial and the solvent evaporated under a stream of gaseous
nitrogen. Ethanol (7.5 .mu.L) was added to the vial, followed
immediately by 192.5 .mu.L of a 1 .mu.g/.mu.L protein stock in Tris
buffer, and the reaction mixture was incubated at 25.degree. C. for
30 min (final concentration of FP-biotin was 2 .mu.M). The reaction
mixture was quenched by adding one volume equivalent of standard
2.times. SDS-PAGE loading buffer (reducing) and heating the sample
at 80.degree. C. for 5 min. Reactions conducted for longer times (1
hr) or with higher concentrations of FP-biotin (20 .mu.M) did not
produce significant increases in the labeling intensity of most
proteins, indicating that the majority of proteins had reacted to
completion under the reported conditions. However, reactions with
higher concentrations of FP-biotin did begin to show significant
levels of nonspecific labeling (defined as the appearance of new
protein bands that reacted with FP-biotin in both preheated and
unheated samples).
[0184] Detection of FP-Biotin Reactive Proteins by SDS-PAGE-Western
Blotting. Quenched FP-biotin reactions were run on SDS-PAGE (10
.mu.g protein/gel lane) and transferred by electroblotting onto
nitrocellulose membranes, which were blocked in TBS with 1% Tween
(TBS-Tween) and 3% (w/v) nonfat dry milk for either 1 h at
25.degree. C. or overnight at 4.degree. C. Blots were then treated
with an avidin-horseradish peroxidase (HRP) conjugate (BioRad,
1:2000 dilution) in TBS-Tween with 1% nonfat dry milk for 30 min at
25.degree. C. The blot was washed with TBS-Tween three times (10
minutes/wash), treated with SuperSignal chemiluminescence reagents
(Bio-Rad), and exposed to film for 0.1 to 8 minutes prior to
development. For the comparison of STI-treated versus untreated
Charotein samples, the relative amounts of FP-biotin labeling were
estimated by film densitometry using an AlphaImager 2000
(AlphaInnotech).
[0185] Identification of target proteins by avidin affinity
purification. After incubation of protein sample (0.5-2.5 mL,
0.5-1.0 mg/mL) with the ABPs, the sample is diluted to 2.5 mL in
Tris or phosphate buffer and passed over a PD-10 size exclusion
column to remove excess unreacted ABP. The protein is collected
from the column in 3.5 mL of buffer, treated with SDS (final
concentration of 0.5%), and heated for 10 min at 80.degree. C. The
sample is then diluted 2.5.times. and incubated with 100
microliters of avidin agarose beads (Sigma) for 1-4 hours at room
temp. The beads are then washed with several volumes of buffer to
remove unbound protein and the ABP-labeled proteins are eluted with
100 microliters of standard SDS-PAGE loading buffer by heating at
90.degree. C. for 5 minutes. The eluted proteins are run on an
SDS-PAGE gel and ABP-labeled proteins identified by staining and/or
avidin blotting, excised from the gel, digested with trypsin, and
the resulting peptide mixture characterized by MALDI and/or
electrospray mass spectrometry. The mass spec information is used
in database searches to identify the ABP-labeled proteins.
Example 3
[0186] Molecular Characterization of FP-Biotin Reactive
Proteins
[0187] Brain soluble extracts were run over a Q sepharose column
using an AKTA FPLC (Amersham Pharmacia Biotech) and eluted with a
linear gradient of 0-500 mM NaCl. Samples of the elution fractions
(10.times.2.5 mL fractions) were labeled with FP-biotin as
described above, and those fractions containing the 75 kDa and 85
kDa labeled proteins were pooled and passed over a Mono-Q sepharose
column. Proteins were eluted from the Mono-Q column with a linear
gradient of 200-500 mM NaCl and those elution fractions enriched in
the two labeled proteins were then run on SDS-PAGE and transferred
to polyvinylidine difluoride (PVDF) membranes by electroblotting.
Regions of the PVDF membranes containing the 75 and 85 kDa
FP-biotin reactive proteins were excised, digested with trypsin,
and the resulting peptides analyzed by matrix-assisted laser
desorption ionization (MALDI) and MALDI-post-source decay
time-of-flight mass spectrometry (Chaurand, et al. (1999) J. Am.
Soc. Mass. Spectrom. 10, 91-103) on a Kratos Kompact Seq Instrument
equipped with a curved field reflectron. The MALDI peptide data
were used in MS-Fit and MS-Tag searches of the ProteinProspector
databases (http://falcon.ludwig.ucl.ac.- uk/mshome3.2.htm), which
identified the 75 kDa protein as the rat orthologue of a human
protein sequence KIAA0436 and the 85 kDa protein as acylpeptide
hydrolase (APH). (see FIG. 5).
[0188] Expression of Serine Hydrolases in HEK-293 Cells. The rat
APH cDNA was cloned as follows. Primers were designed based on the
enzyme's cDNA sequence (Kobayashi, et al. (1989) J. Biol. Chem.
264, 8892-8899)and used in polymerase chain reaction experiments to
amplify a 1.4 kb partial cDNA clone from a rat liver 5' Stretch
Plus cDNA library (Clontech). This amplified cDNA was used as a
probe to isolate a full length APH cDNA from a liver library. The
APH cDNA was subcloned into the eukaryotic expression vector,
pcDNA3, and transiently transfected into HEK-293 cells as described
previously. Transfected cells were harvested by trypsinization,
washed with Hepes buffer (125 mM Hepes, pH 8.0, 100 mM NaCl) and
Dounce-homogenized in Hepes buffer. Cytosolic and membrane
fractions were isolated as described previously (Giang and Cravatt
(1997) Proc. Natl. Acad. Sci. USA 94, 2238-2242) and labeled with
FP-biotin as detailed above.
[0189] Design and Synthesis of a Biotinylated Fluorophosphonate,
FP-Biotin. For the generation of a tagged, activity-based probe for
the serine hydrolase family of enzymes, we considered several
possible reactive groups and labeling strategies. Previous work by
Glynn and colleagues had demonstrated that a saligenin
phosphoramidate was a potent inhibitor of neuropathy target
esterase (NTE) and could be synthesized with a biotin tag to
identify this protein in tissue extracts (Glynn, et al. (1994) 301,
551-556). However, this inhibitor displayed remarkable specificity
for NTE in these experiments, and thus appeared too selective to be
useful as a general probe for serine hydrolases. Powers and
colleagues had generated isocoumarin inhibitors coupled to biotin
as serine hydrolase inhibitors (Kam, et al. (1993) Bioconjugate
Chem. 4, 560-567; Winkler, et al. (1996) Mol. Immunol. 33,
516-623). While these isocoumarins reacted with a greater range of
serine hydrolases than the aforementioned salgenin phosphoramidate,
the requirement that these compounds alkylate a second functional
group in the enzyme active site to achieve stable irreversible
inhibition suggested that a significant number of serine hydrolases
might remain insensitive to such reagents. In contrast, FP
inhibitors seemed to satisfy the dual requirement of displaying 1)
reactivity against the majority of serine hydrolases, and 2)
selectivity for this enzyme family among the various classes of
hydrolytic enzymes. While radiolabeled FPs were available
commercially and through our own synthetic efforts (Patricelli, et
al. (1999) Biochemistry 38, 9804-9812), the detection of such
agents by fluorography requires several days to weeks (Patricelli,
et al., supra; Keshavarz-Shokri, et al. (1999) Anal. Biochem. 267,
406-411), greatly limiting their general utility as rapid and
high-sensitivity probes for profiling serine hydrolase expression
and function.
[0190] 10-undecen-1-ol (1)(numbering from Scheme 1) was converted
to iodinated compound 3 through a tosylate intermediate (2).
Reaction of 3 with excess triethylphosphite under reflux conditions
afforded the diethoxy phosphonate 4, which was converted to the
ethoxyhydroxy phosphonate 5 by treatment with trimethylsilylbromide
(TMSBr). The double bond of 6 was oxidatively cleaved with
ruthenium trichloride and sodium periodate (Carlsen, et al. (1981)
J. Org. Chem. 46, 3k936-3938) to yield the terminal carboxylic acid
product 6. Treatment of 6 with excess diethylaminosulfur
trifluoride (DAST) and N-hydoxysuccinimide (NHS) afforded an
N-succinyl fluorophosphonate intermediate which was reacted with
5-(biotinamido) pentylamine (NH2-biotin) to generate FP-biotin (7).
This synthetic route also allowed for the facile coupling of 6 to
other reporter groups, including fluorescein cadaverine, which
generated a fluorescent fluorophosphonate, FP-fluorescein [8;
MALDI-FTMS (DHB) m/z 778.2671 (C38H47FN3O8PS+Na+requires
778.2703)]. A similar scheme was utilized to synthesize
biotinylated sulfonate esters for use as ABPs as shown in FIG. 4.
Similarly, FIG. 4 shows a strategy for stereocontrolled synthesis
of conformationally well-defined spiroepoxides of type VI (see
also, Somensen, et al., Angew. Chem. Int. Ed., 1999, 38:971-974,
herein incorporated by reference in its entirety). Compounds of
type VI are analogs of the metalloprotease (MetAp-2) inhibitor
fumagillin and are used as ABPs herein.
Example 4
[0191] FP-Biotin is an Activity-Based Probe for Serine
Hydrolases
[0192] To initially test FP-biotin's utility as an activity-based
probe for serine hydrolases, we reacted this agent with the
mammalian serine amidase, fatty acid amide hydrolase (FAAH)
(Cravatt, et al., (1996) Nature (London) 384k, 83-87). FP-biotin
behaved as a potent irreversible inhibitor of FAAH (data not
shown), displaying properties similar to those of other FP
inhibitors of the enzyme (Patricelli, et al., supra; Deutsch, et
al. (1997) Biochem. Pharmacol. 53, 255-260). We have shown that
serine residue 241 serves as FAAH's catalytic nucleophile and
mutation of this residue to alanine (S241A) generates an inactive
enzyme (Patricelli, et al., supra). Therefore, FP-biotin (2 .mu.M)
was reacted with both FAAH and the S241A mutant (80 nM) for 10
minutes, after which the proteins were subjected to standard
SDS-PAGE-Western blotting procedures using either anti-FAAH
antibodies or avidin as detection reagents (FIG. 1A). While
anti-FAAH antibodies identified both FAAH and the S241A mutant,
avidin detected only FAAH in the FP-biotin reactions, demonstrating
that this inhibitor exclusively reacted with the active form of the
enzyme.
[0193] To further explore FP-biotin's reactivity with serine
hydrolases, we incubated soluble fractions of rat testis with this
inhibitor. Consistent with the abundance of proteases found in this
tissue (Monsees, et al. ((1997) Adv. Exp. Med. Biol. 424, 111-123),
FP-biotin labeled more than ten testicular proteins. Phosphonylated
proteins of a variety of molecular masses were observed, ranging
from 20-100 kDa, with a high concentration of labeled proteins
found between 25-40 kDa, possibly representing members of the
kallikrein clan of serine proteases (MacDonald, et al. (1996) J.
Biol. Chem. 271, 13864-13690). Importantly, boiling the protein
sample prior to treatment with FP-biotin blocked nearly all protein
labeling, further supporting that this tagged inhibitor reacts with
serine hydrolases in an activity-dependent manner. Kinetic analyses
revealed that the identified serine hydrolases displayed remarkably
different rates of FP-biotin reactivity, with two of the larger
proteins labeling to apparent completion within one minute and most
of the smaller proteins reacting more slowly over the course of
several minutes.
[0194] Considering that many serine proteases exist in vivo as
inactive complexes with endogenous inhibitory proteins (Kato (1999)
Hum Mutat. 13, 87-98; Declerck, et al. (1997) Adv. Exp. Med. Biol.
425, 89-97; Monsees, et al., supra), we compared the ability of
FP-biotin to react with both free and inhibitor-bound proteases.
While FP-biotin reacted strongly with free trypsin, the tagged
inhibitor did not label a trypsin sample that was preincubated with
the Kunitz-type serine protease inhibitor, soybean trypsin
inhibitor (STI), despite the presence in the latter reaction of
significantly greater amounts of trypsin. Soluble fractions of rat
testis were also exposed to STI and then treated with FP-biotin.
Consistent with the relatively broad specificity of this protease
inhibitor, several, but not all FP-biotin reactive proteins showed
significantly lower labeling intensities in the STI-treated
sample.
[0195] Collectively, these results highlight that FP-biotin can
detect differences in the functional state of a serine hydrolase,
even in the special cases where enzyme activity varies without
correlation to enzyme quantity. Such observations gain particular
significance when one considers the complexity and diversity of
serine proteases and inhibitors typically present in whole cell and
tissue samples (Kato, supra; Declerck, supra; Deutsch, et al.,
supra). Without an activity-based probe like FP-biotin, standard
genomics and/or proteomics studies would have difficulty
distinguishing free (active) from inhibitor-bound (inactive)
proteases in these samples. Finally, the ability to monitor rates
of FP-biotin labeling can greatly assist in the identification of
even quite subtle changes in serine hydrolase activities.
[0196] Molecular Characterization of FP-Biotin Reactive Proteins.
In order to verify that the proteins labeled by FP-biotin in crude
tissue extracts were indeed serine hydrolases, two phosphonylated
proteins were isolated from rat brain cytosol. The most strongly
labeled brain proteins ranged from 75-85 kDa in size and eluted
from a Q sepharose column between 300 and 450 mM NaCl. To estimate
the abundance of these proteins in the Q elutions, the intensity of
their labeling with FP-biotin was compared to that of a serial
dilution of a FAAH sample reacted to completion with the inhibitor.
Both the 75 and 85 kDa FP-biotin reactive proteins displayed
labeling intensities similar to that of a 20 ng sample of FAAH
(0.35 pmoles), setting a lower limit for the quantity of these
proteins that was well within the range needed to obtain protein
sequence information. The 85 and 75 kDa proteins were identified by
standard protein chemistry techniques as acylpeptide hydrolase
(APH) (Kobayashi, et al. (1989) J. Biol. Chem. 264, 8892-8899), a
serine peptidase that has been shown to react with DIFP (Scaloni,
et al. (1992) J. Biol. Chem. 267, 3811-3818), and the rat
orthologue of a human protein sequence KIAA0436 (Ishikawa, et al.
(1997) DNA Res. 4, 307-313). Interestingly, a homology search
revealed that the KIAA0436 protein shares 30% identity with the
prokaryotic enzyme Protease II, also an established serine
hydrolase that reacts with DIFP (Yoshimoto, et al. (1995) J.
Biochem. 117, 654-660). The Ser-His-Asp catalytic triad residues of
Protease II were conserved in the KIAA0436 protein, supporting that
this mammalian protein is a novel member of the protease II family
of serine proteases. Finally, FP-biotin also labeled a 100 kDa
brain protein that appeared to be expressed at much lower levels
(equivalent to 15 fmoles, or .about.1 ng, of FAAH), demonstrating
that this tagged inhibitor can readily detect subnanomolar
concentrations of serine hydrolases (15 fmol/20 .mu.L per gel
lane). (see FIG. 5).
[0197] To test whether FP-biotin could record changes in the
expression level of serine hydrolases in crude cellular extracts,
we transfected cDNAs for both APH and FAAH into HEK-293 cells.
Treatment of the cytosolic and membrane fractions of these cells
with FP-biotin identified a strongly phosphonylated 85 kDa protein
in the APH-transfected cells, but not in control cells transfected
with either empty vector or the FAAH cDNA. In contrast to this
labeling pattern, an abundant 65 kDa phosphonylated protein was
identified exclusively in the membrane fraction of FAAH-transfected
cells, consistent with previous characterizations of this serine
hydrolase as an integral membrane protein (Giang and Cravatt,
supra, Patricelli, et al. (1998) Biochemistry 37, 15177-15187).
Longer exposures of the cytosol blot identified in the mock and
FAAH-transfected HEK cells a weak 85 kDa signal that may represent
endogenous levels of APH in this cell type.
[0198] In summary, the data presented demonstrate that FP-biotin
can: 1) react with numerous serine hydrolases in crude cell and
tissue samples, 2) detect subnanomolar concentrations of serine
hydrolases, and 3) record differences in both the functional state
and expression level of these enzymes. It is also important to
highlight that the identification of FP-biotin labeled proteins
using standard avidin-HRP chemiluminescence assays is extremely
rapid (requiring exposure times of only seconds to minutes), making
this chemical agent particularly well suited for high throughput
proteomics investigations. Additionally, the covalent attachment of
a biotin molecule to phosphonylated serine hydrolases should assist
in the subsequent biochemical characterization of these enzymes.
For example, Schriemer and colleagues have recently developed a
method that combines immobilized avidin beads with MALDI mass
spectrometry to facilitate the chemical analysis of biotinylated
proteins and peptides (Schreimer, et al. (1998) Anal. Chem. 70,
1569-1575). If integrated with FP-biotin, this technique allows for
the molecular identification of serine hydrolases (as well as their
respective catalytic nucleophiles) directly from whole cell and
tissue samples.
Example 5
[0199] Profiling Serine Hydrolases in Rat Tissues with
FP-Biotin
[0200] To test FP-biotin's ability to resolve complex patterns of
serine hydrolase expression, we compared the profiles of
phosphonylated proteins from soluble extracts of rat brain, liver,
testis, and prostate. In the lower molecular mass range, clear
tissue-specific and tissue-restricted FP-biotin reactive proteins
were identified. Interestingly, a strongly labeled 33 kDa protein
was identified exclusively in prostate (III). While the molecular
size of this phosophonylated protein is consistent in mass with
human prostate specific antigen (PSA) (Bei, et al. (1995) J. Clin.
Lab. Anal. 9, 261-268), a serine protease expressed primarily in
this tissue, orthologues of PSA are not thought to exist in rodents
based on previous molecular (Southern blots) and cell biological
(immunocytochemistry) studies (Karr, et al. (1995) Cancer Res. 55,
2455-2462). The identification of an FP-biotin reactive protein
abundantly and selectively expressed in rat prostate suggests that
this organism may indeed possess functional (but not necessarily
high sequence-related) homologues of human PSA, an observation that
merits further investigation considering PSA's status as a
principal marker for prostate cancer (Polascik, et al. (1999) J.
Urol. 162, 293-306). Several other FP-biotin reactive proteins also
displayed tissue-restricted patterns of expression, including a
testis-specific 42 kDa protein (I) and two 38 kDa proteins, one of
which was found in brain and testis, and the other in brain and
liver (II). In the larger molecular mass range, most of the
FP-biotin reactive proteins appeared to display broad tissue
distributions. However, a labeled 65 kDa protein was found in
highest relative abundance in liver, at lower levels in testis and
prostate, and was not detected in brain (I). Similarly, a
phosphonylated 70 kDa protein was found exclusively in liver
(II).
[0201] The subject invention provides reagents that can be used for
identifying enzyme activity for a class, sub-class or individual
members of the class in an enzyme sample. The reagent(s) comprise a
functionality reactive with an amino acid at the active site, a
linker that, depending on the purpose of the determination, will
bind to all or most members of the class, a sub-group of the class
or individual members of the class, and a ligand for sequestering
the reagent-conjugated-enzyme. In this way tissue or cells can be
assayed for enzyme activity of the different groups indicated
above, to determine the response to external stimuli, such as
drugs, heat, infection, surgical intervention, neoplasia, etc. In
addition, changes in the nature of a cell may be determined with
aging, transformation, gene therapy, etc. The reagents specific for
a member of a class are prepared using combinatorial synthesis of
the linker and screening for compounds with high affinity for a
specific enzyme. The ligand allows for isolation and identification
of the enzyme with which the reagent has reacted, so as to identify
the enzyme for which the reagent is specific. Once identified, the
linker may be further modified for optimization.
Example 6
[0202] Comparison of Serine Hydrolase Profile of FP-Biotin and
FP-Peg-Biotin
[0203] Results
[0204] Synthesis of FP-Peg-Biotin (9). A first generation FP probe,
FP-biotin, possessed a linear decamethylene chain connecting its FP
reactive group to 5-(biotinamido)-pentylamine through an amide bond
(15). A variant of FP-biotin in which the agent's decamethylene
chain was replaced by a more hydrophilic tetraethylene glycol
linker was synthesized. Briefly, tetraethyleneglycol (1) was
monosilyated to provide compound 2, which following conversion to
alkyl iodide 3, was reacted with triethylphosphite to give
phosphonate 4. Desilyation of 4 with HF-pyridine, followed by
treatment with N,N-disuccinimidyl carbonate, provided NHS-carbonate
6. Compound 6 was converted to monoethoxy phosphonate acid 7 by
treatment with oxalyl chloride followed by hydrolytic workup.
Compound 7 was treated with DAST to give fluorophosphonate 8, which
was then coupled to 5-(biotinamido) pentylamine (Pierce) to provide
FP-peg-biotin (9).
[0205] FP-Peg-Biotin Reacts with Serine Proteases in an
Activity-Dependent Manner. To demonstrate that biotinylated FPs
could distinguish active proteases from their inactive zymogens,
FP-peg-biotin was incubated with equal amounts of trypsin,
trypsinogen, chymotrypsin, and chymotrypsinogen. The products of
each reaction were then compared by SDS-PAGE and blotting with
avidin (FIG. 16). FP-peg-biotin strongly labeled both trypsin and
chymotrypsin, but exhibited little or no reactivity with their
respective zymogens. Protein staining revealed that a significant
fraction of the chymotrypsin sample had been degraded during the
course of setting up the reaction. Nonetheless, FP-peg-biotin still
showed a much stronger labeling intensity with the chymotrypsin
sample than with its chymotrysinogen counterpart, despite the
substantially lower quantity of protein present in the former
reaction. Indeed, the low level of FP-peg-biotin labeling observed
in the chymotrypsinogen sample (seen only in the 5.times. exposure
panel) may have actually represented reactivity with trace amounts
of chymotrypsin rather than with the zymogen itself, as the
purchased proenzyme is reported to possess up to 2% active
protease. Comparing the Serine Hydrolase Activity Profiles
Generated with FP-Biotin and FP-peg-Biotin. Soluble fractions of
rat testis (1 .mu.g/.mu.L) were treated with either FP-biotin or
FP-peg-biotin (4 .mu.M) for 1 h at room temperature (50 mM Tris, pH
7.2, 150 mM NaCl) and the labeled serine hydrolase activities
detected by SDS-PAGE and blotting with avidin. Control reactions in
which the proteome was heat-denatured prior to treatment with
biotinylated FPs were also analyzed to distinguish specific
(heat-sensitive) from nonspecific (heat-insensitive) protein
reactivity. The serine hydrolase activity profiles produced by each
biotinylated FP were strikingly similar to one another with a
single notable exception (FIG. 17A). A pair of 50 kDa serine
hydrolase activities were strongly labeled by FP-peg-biotin, but
displayed very low reactivity with FP-biotin (arrowhead).
FP-proteome reactions conducted for longer times (2 hours to
overnight) increased the labeling intensity of some serine
hydrolases, but did not result in the detection of any new
heat-sensitive protein reactivities. Therefore, one hour
FP-proteome reactions provided profiles that were considered to
represent "maximal coverage" of serine hydrolase activities, and
unless otherwise noted, subsequently described reactions were
conducted for this length of time.
[0206] The Probe Concentration Dependence of FP-Proteome Reactions.
Samples of the rat testis proteome were treated for one hour with
either FP-biotin or FP-peg-biotin over a probe concentration range
of 0.5-8.0 .mu.M (FIGS. 17B and C). Most FP-biotin-labeled proteins
displayed enhanced signal intensities with increasing amounts of
probe, indicating that their reactivities were not saturated at low
micromolar FP-biotin concentrations. In contrast, several of the
FP-peg-biotin-labeled proteins showed no detectable change in their
signal intensities with increasing concentrations of probe (FIG.
17C, arrowheads). These enzymes had either reacted to completion,
or were saturated in their rates of labeling at all of the
FP-peg-biotin concentrations tested. Kinetic experiments supported
the former explanation, as clear concentration-dependent labeling
for all of these proteins could be observed in reactions conducted
for a shorter time (1 min; FIG. 17D, arrowheads). Notably, in
reactions conducted for one hour with concentrations of 4-8 .mu.M
FP-peg-biotin, at least 18 distinct serine hydrolase activities
could be resolved on a single lane of a one-dimensional SDS-PAGE
gel (FIG. 17C).
[0207] An even higher concentration of FP-peg-biotin (16 .mu.M)
produced a qualitatively similar serine hydrolase activity profile
to that observed with 4 and 8 .mu.M probe (i.e., no new
heat-sensitive protein reactivities were detected at 16 .mu.M), but
also generated a high level of nonspecific protein reactivity.
Therefore, it was concluded that biotinylated FPs displayed maximum
ratios of specific to nonspecific proteome reactivity at
concentrations ranging from 4 to 8 .mu.M.
[0208] The pH-Dependence of FP-Proteome Reactions. A single mixed
buffer assay was used to evaluate the pH-dependence of FP-proteome
reactions over a pH range of 6.0-9.0 (conditions: 1 .mu.g/.mu.L
soluble testis protein, 4 .mu.M biotinylated FP, 50 mM Bis-Tris
Propane, 50 mM CAPS, 50 mM citrate, 150 mM NaCl; one hour
reaction). Several types of pH-dependence were observed among the
FP-labeled proteins, with some serine hydrolases exhibiting an
optimal FP reactivity at pH 8.0 (FIG. 18, triple arrowheads) and
others showing an FP reactivity that continued to increase in
intensity as the pH was raised to 9.0 (double arrowheads). Although
multiple serine hydrolases appeared to show "pH-independent" FP
reactivities, kinetic analyses indicated that most of these enzymes
had labeled to completion during the time course of the reactions
(see above). Thus, these enzymes likely react with FPs in a
pH-dependent manner that might be visualized by modifying the
parameters of the reaction to slow their rates of labeling (e.g.,
lowering the probe concentration and/or reducing the incubation
time). Finally, background FP reactivity increased significantly at
pH 9.0, with several labeled proteins appearing in the preheated
control lane (single arrowheads). Coomassie blue staining revealed
that these labeled proteins were all high abundance constituents of
the testis proteome, indicating that heat-insensitive labeling
represents a nonspecific form of FP reactivity. Considering further
that the majority of serine hydrolases displayed similar (or
greater) FP reactivities at pH 8.0 relative to pH 9.0, the former
pH appears better suited for the functional analysis of this enzyme
family in complex proteomes.
[0209] Kinetic Analysis of FP-Proteome Reactions. Although single
time point measurements offer a simple and rapid means to obtain a
general profile of the active serine hydrolases present in complex
proteomes, kinetic analyses are required to decipher more intricate
changes in enzyme activity. For example, alterations in serine
hydrolase activity that take place in the absence of changes in
enzyme abundance may remain undetected with biotinylated FPs unless
rates of reactivity are measured. To explore the kinetics of
FP-proteome reactions, rat brain membrane fractions were treated
with biotinylated FPs and the time course of protein labeling
followed by SDS-PAGE and blotting with avidin The goal of these
studies was two-fold: 1) to confirm that membrane-associated serine
hydrolases could be profiled with biotinylated FPs, and 2) to test
whether individual serine hydrolases showed significant differences
in their respective rates of reactivity with FP-biotin and
FP-peg-biotin.
[0210] Initially, brain membrane proteins were treated with
FP-biotin both prior to and after solubilization with Triton X-100
to examine the effects of detergent on the FP-membrane proteome
reaction. Similar serine hydrolase activity profiles were observed
with membrane-associated and Triton-solubilized brain protein
samples (FIG. 19), indicating that detergent solubilization
maintained most of the membrane-bound serine hydrolases in a
catalytically active state. Therefore, subsequent labeling
experiments were conducted with Triton-solubilized brain membrane
proteins. Most brain membrane serine hydrolases exhibited rates of
FP reactivity that could be monitored over a time course of 1-60
minutes under the following set of reaction conditions: 4 .mu.M
biotinylated FP, 1 .mu.g/.mu.L protein, 50 mM Tris buffer, pH 8.0,
with 0.2% Triton X-100. Interestingly, examples were observed for
each of the three potential types of serine hydrolase reactivity
profiles: 1) enzymes that labeled with FP-biotin at faster rates
than FP-peg-biotin (FIG. 20A, single arrowhead), 2) enzymes that
labeled with FP-peg-biotin at faster rates than FP-biotin (double
arrowheads), and 3) enzymes that labeled with FP-biotin and
FP-peg-biotin at similar rates (triple arrowheads). In particular,
a 65 kDa SH activity was apparently labeled to completion with
FP-biotin within one minute (FIG. 20A, left panel), but reacted at
a much slower rate with FP-peg-biotin (its signal intensity still
increasing from 30 to 60 minutes; FIG. 20A, right panel). We
suspected that To demonstrate that this serine hydrolase represents
fatty acid amide hydrolase (FAAH), a brain integral membrane enzyme
that displays a strong preference for long aliphatic amide
substrates, we treated brain membrane proteins with each
biotinylated FP and measured FAAH catalytic activity at two time
points during the reaction. No FAAH activity could be detected in
the FP-biotin treated samples after incubations for either 10 or 60
minutes. In contrast, significant FAAH activity was observed in the
FP-peg-biotin treated samples at both time points, with the 10 and
60 minute incubations displaying 60% and 30% FAAH activity,
respectively (relative to an untreated control sample). Thus, FAAH
was inactivated at a much faster rate by FP-biotin than
FP-peg-biotin, consistent with this enzyme's substrate
selectivity.
[0211] Considering that several brain membrane serine hydrolases
reacted preferentially with one biotinylated FP over the other, we
showed that by treating a single proteome with a mixture of both
probes, a more complete serine hydrolase activity profile is
obtained. Treating brain membrane proteins with 2 .mu.M each of
FP-biotin and FP-peg-biotin provided a serine hydrolase activity
profile that resembled closely the predicted merger of profiles
generated with each FP individually (FIG. 20B). These data indicate
that adding multiple activity-based probes to a single proteome
("multiplexing") can enhance the coverage of active enzymes present
in that sample.
[0212] Comparing the Membrane-Associated Serine Hydrolase Activity
Profiles from Different Rat Tissues. Having observed the large
number of serine hydrolases associated with brain membranes, we
compared the serine hydrolase activity profiles of membrane
fractions from a panel of rat tissues. Each membrane fraction was
first washed with 1 M NaCl prior to solubilzation of its protein
content with Triton X-100. This protocol was selected to enrich for
integral membrane proteins, a class of proteins notoriously
resistant to analysis by standard proteomics methods.
Interestingly, each of the tissues examined possessed a unique and
complex profile of membrane-associated serine hydrolase activities
(FIG. 21). Notably, a set of three 48-52 kDa serine hydrolase
activities were enriched in brain membranes (single arrowhead).
Likewise, heart-enriched and testis-enriched serine hydrolase
activities were also observed (double and triple arrowheads,
respectively).
[0213] Evaluating the Target Selectivity of Noncovalent Serine
Hydrolase Inhibitors. Biotinylated FPs not only distinguish active
serine hydrolases from zymogens (FIG. 16), but also from
inhibitor-bound enzymes. To demonstrate the target selectivity of
serine hydrolase inhibitors directly in complex proteomes, each
biotinylated FP was added to a brain membrane proteome treated with
varying concentrations of oleoyl trifluoromethyl ketone (OTFMK), a
previously described noncovalent FAAH inhibitor. After a 30 minute
incubation, the reactions were quenched and analyzed by SDS-PAGE
and blotting with avidin. Several serine hydrolases displayed
reduced FP-reactivity in the presence of increasing concentrations
of OTFMK (FIG. 22) indicating that this electrophilic ketone was
not only an effective inhibitor of FAAH, but of other brain
membrane serine hydrolases as well. Film densitometry was used to
estimate OTFMK's potency as an inhibitor of each of its serine
hydrolase targets. A pair of 50 kDa serine hydrolases displayed
approximately 40 and 80% reductions in both their FP-biotin and
FP-peg-biotin reactivities in the presence of 50 and 200 .mu.M
OTFMK, respectively (bracket). In contrast, FAAH's FP-peg-biotin
reactivity was reduced by approximately 30, 90, and 95% in the
presence of 5, 50, and 200 .mu.M OTFMK, respectively (arrowhead).
These data reveal that OTFMK displays a relatively modest
(approximately one order of magnitude) selectivity for FAAH among
other brain membrane serine hydrolases. Although both 50 and 200
.mu.M concentrations of OTFMK blocked greater than 90% of FAAH's
FP-peg-biotin reactivity, this enzyme's FP-biotin reactivity was
only weakly affected (.about.20% reduction) and partially affected
(.about.70% reduction) by 50 .mu.M and 200 .mu.M OTFMK,
respectively. These apparently conflicting data can be rationalized
as follows. Of the three observed OTFMK-sensitive serine
hydrolases, exclusively FAAH reacted with FP-biotin at a rate that
was too fast to monitor under the reaction conditions employed
(FIG. 19A, left panel). In such a case, the binding of a reversible
inhibitor will only be detected if the inhibitor reduces its
protein target's rate of FP reactivity to the extent that the
protein no longer labels to completion during the time course of
the reaction. Thus, FAAH's sensitivity to OTFMK was likely
underestimated in the 30 minute FP-biotin competition assay. On the
other hand, FP-peg-biotin labeled FAAH at a rate slow enough to be
followed over a 60 minute time course (FIG. 19A, right panel), and
therefore reactions conducted with this probe provided a more
accurate assessment of the sensitivity of FAAH to competitive
active site-directed agents. Collectively, these results
demonstrate that biotinylated FPs can, in cases where they display
discernible labeling kinetics, identify the serine hydrolase
targets of noncovalent inhibitors directly in complex
proteomes.
[0214] Affinity Isolation and Molecular Characterization of
FP-Labeled Serine Hyrdrolases. For activity-based probes like the
biotinylated FPs to be of lasting use to proteomics research, these
reagents must not only serve as tools for protein detection, but
also for protein isolation and identification. In the course of
attempting to affinity purify FP-biotinylated proteins by avidin
agarose chromatography, we noted that several labeled proteins
failed to bind the avidin matrix in their native state (FIG. 23A,
arrowheads). However, if protein samples were denatured prior to
treatment with avidin beads, efficient depletion of all of the
biotinylated proteins was achieved. Subsequent washing of the beads
and elution with SDS-PAGE loading buffer provided a sample greatly
enriched for biotinylated proteins (FIG. 8A, right panel). The
elution sample was run on an SDS-PAGE gel and the biotinylated
proteins excised from the gel, digested with trypsin, and the
resulting peptide mixtures analyzed by MALDI mass spectrometry.
This one-step isolation method identified seven of the labeled
serine hydrolases present in a soluble rat testis proteome as:
acylpeptide hydrolase (82 kDa, accession #CAA33040), prolyl
oligopeptidase (80 kDa, BAA25544), carboxylesterase 1 (80 kDa,
accession #JX0054), carboxylesterase 10 (60 kDa, accession
#P16303), long chain acyl CoA hydrolase (48 kDa, accession
#088267), platelet-activating factor (PAF) acetylhydrolase al
subunit (32 kDa, accession #NP.sub.--032802), and PAF
acetylhydrolase a2 subunit (30 kDa, accession #035264) (FIG. 8B).
An additional 45 kDa serine hydrolase activity provided a MALDI
tryptic peptide map that did not match those of any proteins in the
public databases. Analysis of the electrospray MS fragmentation
pattern of one of the tryptic peptides from this protein (M+H+=1099
Da) provided the following sequence information: GFVVAAIEHR. BLAST
database searches with this peptide sequence identified 80 and 90%
identical sequences in the human and dog versions, respectively, of
a 45 kDa serine hydrolase referred to as plasma PAF acetylhydrolase
or LDL-associated phospholipase A2 (human protein accession
#AAB04170.1). Thus, the isolated 45 kDa FP-biotin-reactive testis
protein was likely a novel rodent member of this family of secreted
serine hydrolases.
Example 7
[0215] Sulfonate Ester Combinatorial Library as ABPS
[0216] Materials and Methods
[0217] Synthesis Alkyl and Aryl Sulfonate Esters
[0218] All reactions were carried out under an atmosphere of argon
unless specified. Methylene chloride (CH.sub.2Cl.sub.2) was dried
by passing through activated alumina columns. Commercial reagents
of high purity were purchased and used without further purification
unless otherwise noted. NMR spectra were obtained on a Bruker
AMX-400 instrument and calibrated to the residual solvent peak. The
multiplicities are abbreviated as follows: s=singlet, d=doublet,
t=triplet, q=quartet, p=pentet, m=multiplet).
[0219] The synthesis of
10-((2-pyridylsulfonyl)oxo)-N-biotinamidopentyldec- anamide (1) is
provided as a representative synthesis of the eleven biotinylated
alkyl and aryl sulfonates (1-11).
[0220] ((2-Pyridylsulfonyl)oxo)-10-undecene (13): A solution of
.alpha.-undecylenyl alcohol (12)(0.50 g, 2.91 mmol, 1.0 equivalents
(equiv.)) in pyridine (4 mL) was cooled to 0.degree. C. and treated
with 2-pyridylsulfonyl chloride (1.04 g, 5.87 mmol, 2.0 equiv.),
prepared according to the procedure of Corey and colleagues [Corey,
et al. (1989). J. Org. Chem. 54, 389-93]. The reaction mixture was
kept at 0.degree. C. for 6 hours, then partitioned between ethyl
acetate (50 mL) and water (25 mL). The organic layer was washed
with 10% aqueous HCl (2.times.50 mL) and saturated aqueous NaCl (50
mL), dried (MgSO.sub.4), and concentrated under reduced pressure.
Column chromatography (2% EtOAc/Hex) afforded 13 as a colorless oil
(98%): .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 8.61 (m, 1H, ArH),
7.89 (m, 2H, ArH), 7.47 (m, 1H, ArH), 5.67-5.60 (m, 1H,
RCH.dbd.CH.sub.2), 4.84-4.74 (m, 2H, RCH.dbd.CH.sub.2), 4.21 (t,
J=6.4 Hz, 2H, CH.sub.2OSO.sub.2Pyr), 1.88 (m, 2H
CH.sub.2CH.dbd.CH.sub.2), 1.55 (p, 2H, J=6.8 Hz, 2H,
CH.sub.2CH.sub.2OSO.sub.2Pyr), 1.20-1.08 (m, 12H); matrix-assisted
laser desorption ionization (MALDI)-FTMS 334.1433
(C.sub.16H.sub.25NO.sub.3S+Na.sup.+ requires 334.1447).
[0221] 10-((2-Pyridylsulfonyl)oxo)-decanoic acid (14): Compound 13
(0.90 g, 2.88 mmol, 1 equiv.) in a biphasic solution composed of
CCl.sub.4--CH.sub.3CN--H.sub.2O (10 mL-10 mL-15 mL) with a total
volume of 35 mL was treated sequentially with sodium periodate
(2.53 g, 11.80 mmol, 4.1 equiv.) and ruthenium trichloride hydrate
(0.005 g, 0.02 mmol, 0.03 equiv.). The reaction was stirred at
25.degree. C. overnight then partitioned between CH.sub.2Cl.sub.2
(100 mL) and 1N aqueous HCl (2.times.100 mL). The organic layer was
washed with saturated aqueous NaCl (100 mL), dried (MgSO.sub.4) and
concentrated under reduced pressure. Column chromatography (40%
EtOAc/Hex) afforded 14 (80%): .sup.1H NMR (CDCl.sub.3, 400 MHz)
.delta. 8.84 (d, J=4.0 Hz, 1H, ArH), 8.11 (d, J=5.9 Hz, 1H, ArH),
8.05 (t, J=6.0 Hz, 1H, ArH), 7.65 (t, J=3.3 Hz, 1H, ArH), 4.37 (t,
J=6.6 Hz, 2H, CH.sub.2OSO.sub.2Pyr), 2.34 (t, J=7.4 Hz, 2H,
CH.sub.2COOH), 1.70 (p, J=8.0 Hz, 2H, CH.sub.2CH.sub.2COOH), 1.61
(p, J=7.3 Hz, 2H, CH.sub.2CH.sub.2OSO.sub.2Py- r), 1.25 (m, 10H):
MALDI-FTMS (DHB) m/z 352.1202 (C.sub.15H.sub.23NO.sub.5- S+Na.sup.+
requires 352.1189).
[0222] 10-((2-Pyridylsulfonyl)oxo)-N-biotinamidopentyldecanamide
(1): A solution of 14 (0.030 g, 0.09 mmol, 10 equiv.) in
CH.sub.2Cl.sub.2 (1.5 mL) at -78.degree. C. was treated dropwise
with (diethylamino)sulfur trifluoride (0.027 mL, 0.21 mmol, 22
equiv.), brought to 25.degree. C., and stirred for 10 minutes. The
reaction was then treated with one-half reaction volume of
dimethylformamide containing N-hydroxysuccinimide (0.05 g, 0.04
mmol, 40 equiv.) and stirred for an additional 15 min at 25.degree.
C. The reaction mixture was partitioned between ethyl acetate (50
mL) and water (50 mL). The organic layer was washed with saturated
aqueous NaCl (200 mL), dried (Na.sub.2SO.sub.4), and concentrated
under reduced pressure to afford
10-((2-pyridylsulfonyl)oxo)-N-(hydroxysuccinyl- )decanamide (as
judged by crude .sup.1H NMR; data not shown). Without further
purification, the intermediate was treated with
5-(biotinamido)-pentylamine (Pierce, 0.003 g, 0.009 mmol, 1.0
equiv.) in MeOH (0.04 mL) and stirred for 30 min. The solvent was
evaporated under a stream of nitrogen, and the remaining residue
was washed with ethyl acetate (2.times.2.5 mL), solubilized in a
minimal volume of chloroform, transferred to a clean glass vial,
and the solvent evaporated. This process was repeated to rid the
desired biotinylated product of excess reagents and byproducts,
affording 4 as a white film (0.004 g, 46%): .sup.1H NMR
(CDCl.sub.3, 400 MHz) .delta. 8.79 (d, J=7.0 Hz, 1H, ArH), 8.06 (m,
2H, ArH), 7.61 (t, J=7.3 Hz, 1H, ArH), 6.05 (b s, 1H, NH), 5.91 (b
s, 1H, NH), 5.60 (b s, 1H, NH), 4.77 (b s, 1H, NH), 4.54 (m, 1H),
4.39 (m, 1H+2H CH.sub.2OSO.sub.2R), 3.22 (m, 4H, CH.sub.2NHCOR),
3.11 (m, 1H), 2.92 (dd, J=4.0 and 12.9 Hz, 1H), 2.76 (d, J=13.3 Hz,
1H), 2.18 (m, 4H, CH.sub.2CONHR), 1.67-1.28 (m, 26H); MALDI-FTMS
(DHB) m/z 640.3209 (C.sub.30H.sub.49N.sub.5O.sub.6S.sub.2+H.sup.+
requires 640.3202).
[0223] 10-((Benzenesulfonyl)oxo)-N-biotinamidopentyldecanamide (2):
.sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 7.93 (d, J=7.0 Hz, 2H,
ArH), 7.67 (t, J=7.4 Hz, 1H, ArH), 7.59 (t, J=7.3 Hz, 2H, ArH),
6.06 (b s, 1H, NH), 5.87 (b s, 1H, NH), 5.63 (b s, 1H, NH), 4.79 (b
s, 1H, NH), 4.53 (m, 1H), 4.37 (m, 1H), 4.07 (t, J=6.4 Hz, 2H,
CH.sub.2OSO.sub.2R), 3.26 (m, 4H, CH.sub.2NHCOR), 3.09 (m, 1H),
2.93 (dd, J=5.0 and 7.9 Hz, 1H), 2.76 (d, J=13.0 Hz, 1H), 2.19 (m,
4H, CH.sub.2CONHR), 1.62-1.26 (m, 26H); MALDI-FTMS (DHB) m/z
639.3244 (C.sub.31H.sub.50N.sub.4O.sub.6S.sub.2+H.su- p.+ requires
639.3245).
[0224] 10-((p-Toluenesulfonyl)oxo)-N-biotinamidopentyldecanamide
(3): .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 7.78 (d, J=8.5 Hz,
2H, ArH), 7.37 (d, J=7.9 Hz, 2H, ArH), 5.91 (b s, 1H, NH), 5.84 (b
s, 1H, NH), 5.49 (b s, 1H, NH), 4.76 (b s, 1H, NH), 4.53 (m, 1H),
4.35 (m, 1H), 4.02 (t, J=6.4 Hz, 2H, CH.sub.2OSO.sub.2R), 3.25 (m,
4H, CH.sub.2NHCOR), 3.18 (m, 1H), 2.93 (dd, J=5.0 and 7.9 Hz, 1H),
2.76 (d, J=12.9 Hz, 1H), 2.46 (s, 3H, CH.sub.3Ar), 2.19 (m, 4H,
CH.sub.2CONHR), 1.70-1.50 (m, 26H); MALDI-FTMS (DHB) m/z 653.3381
(C.sub.32H.sub.52N.sub.4O.sub.6S.sub.2+H.su- p.+ requires
653.3401).
[0225]
10-((4-Methoxybenzenesulfonyl)oxo)-N-biotinamidopentyldecanamide
(4): .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 7.86 (d, J=8.8 Hz,
2H, ArH), 7.03 (d, J=8.8 Hz, 2H, ArH), 5.96 (b s, 1H, NH), 5.85 (b
s, 1H, NH), 5.57 (b s, 1H, NH), 4.84 (b s, 1H, NH), 4.53 (m, 1H),
4.37 (m, 1H), 4.01 (t, J=6.5 Hz, 2H, CH.sub.2OSO.sub.2R), 3.90 (s,
3H, CH.sub.3OAr), 3.25 (m, 4H, CH.sub.2NHCOR), 3.17 (m, 1H), 2.95
(dd, J=4.7 and 7.7 Hz, 1H), 2.73 (d, J=12.9 Hz, 1H), 2.25 (m, 4H,
CH.sub.2CONHR), 1.63-1.26 (m, 26H); MALDI-FTMS (DHB) m/z 669.3381
(C.sub.32H.sub.52N.sub.4O.sub.7S.sub.- 2+H.sup.+ requires
669.335).
[0226] 10-((Methylsulfonyl)oxo)-N-biotinamidopentyldecanamide (5):
.sup.1H NMR (CDCl.sub.3, 400 MHz), .delta. 6.00 (b s, 1H, NH), 5.85
(b s, 1H, NH), 5.60 (b s, 1H, NH), 4.81 (b s, 1H, NH), 4.53 (m,
1H), 4.37 (m, 1H), 4.35 (t, J=6.2 Hz, 2H, CH.sub.2OSO.sub.2R), 3.26
(m, 4H, CH.sub.2NHCOR), 3.18 (m, 1H), 2.93 (dd, J=5.0 and 7.9 Hz,
1H), 2.76 (d, J=12.9 Hz, 1H), 2.21 (m, 4H, CH.sub.2CONHR), 2.05 (s,
3H, H.sub.3CSO.sub.3R), 1.75-1.27 (m, 26H); MALDI-FTMS m/z 577.3105
(C.sub.26H.sub.48N.sub.4O.sub.6S.sub.2+- H.sup.+ requires
577.3088).
[0227] 10-((Butylsulfonyl)oxo)-N-biontinamidopentyldecanamide (6):
.sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 5.93 (b s, 1H, NH), 5.84
(b s, 1H, NH), 5.53 (b s, 1H, NH), 4.82 (b s, 1H, NH), 4.54 (m,
1H), 4.37 (m, 1H), 4.21 (t, J=6.2 Hz, 2H, CH.sub.2OSO.sub.2R), 3.26
(m, 4H, CH.sub.2NHCOR), 3.19 (m, 1H), 3.09 (t, J=4.1 Hz, 2H,
CH.sub.2SO.sub.3R), 2.93 (dd, J=4.6 and 7.4 Hz, 1H), 2.76 (d, J=9.1
Hz, 1H), 2.21 (m, 4H, CH.sub.2CONHR), 2.16-1.31 (m, 30H), 0.97 (t,
J=7.3 Hz, 3H); MALDI-FTMS (DHB) m/z 619.3530
(C.sub.29H.sub.54N.sub.4O.sub.6S.sub.2+H.sup.+ requires 619.3530).
10-((Octylsulfonyl)oxo)-N-biotinamidopentyldecanamide (7): .sup.1H
NMR (CDCl.sub.3, 400 MHz) .delta. 6.05 (b s, 1H, NH), 5.87 (b s,
1H, NH), 5.81 (b s, 1H, NH), 5.00 (b s, 1H, NH), 4.53 (m, 1H), 4.35
(m, 1H), 4.21 (t, J=6.2 Hz, 2H, CH.sub.2OSO.sub.2R), 3.25 (m, 4H,
CH.sub.2NHCOR), 3.17 (m, 1H), 3.09 (t, J=8.5 Hz, 2H,
CH.sub.2SO.sub.3R), 2.93 (dd, J=4.7 and 7.9 Hz, 1H), 2.76 (d,
J=12.9 Hz, 1H), 2.19 (m, 4H, CH.sub.2CONHR), 1.86 (p, J=7.9 Hz, 2H,
CH.sub.2CH.sub.2SO.sub.3R), 1.76-1.31 (m, 36H), 0.89 (t, J=6.4 Hz,
3H); MALDI-FTMS (DHB) m/z 675.4173 (C.sub.33H.sub.62N.sub.4-
O.sub.6S.sub.2+H.sup.+ requires 675.4184).
[0228]
10-((4-Nitrobenzenesulfonyl)oxo)-N-biotinamidopentyldecanamide (8):
.sup.1H NMR (CDCl.sub.3, 400 MHz), .delta. 8.44 (d, J=9.1 Hz, 2H,
ArH). 8.14 (d, J=9.1 Hz, 2H, ArH), 5.90 (b s, 1H, NH), 5.82 (b s,
1H, NH), 5.50 (b s, 1H, NH), 4.86 (b s, 1H, NH), 4.54 (m, 1H), 4.37
(m, 1H), 4.14 (t, J=6.5 Hz, 2H, CH.sub.2OSO.sub.2R), 3.25 (m, 4H,
CH.sub.2NHCOR), 3.18 (m, 1H), 2.96 (dd, J=5.0 and 7.9 Hz, 1H), 2.76
(d, J=15.0 Hz, 1H), 2.21 (m, 4H, CH.sub.2CONHR), 1.72-1.26 (m,
26H); MALDI-FTMS (DHB) m/z 684.3069
(C.sub.31H.sub.49N.sub.5O.sub.8S.sub.2+H.sup.+ requires
684.3095).
[0229] 10-((8-Quinolinesulfonyl)oxo)-N-biotinamidopentyldecanamide
(9): .sup.1H NMR (CDCl.sub.3, 400 MHz), .delta. 9.17 (m, 1H, ArH),
8.53 (d, J=5.8 Hz, 1H, ArH), 8.50 (d, J=6.4 Hz, 1H, ArH), 8.16 (d,
J=6.7 Hz, 1H, ArH), 7.70 (t, J=7.6 Hz, 1H, ArH), 7.61 (q, J=3.9 Hz,
1H, ArH), 6.06 (b s, 1H, NH), 5.93 (b s, 1H, NH), 5.73 (b s, 1H,
NH), 5.08 (b s, 1H, NH), 4.54 (m, 1H), 4.35 (m, 1H), 4.30 (t, J=6.5
Hz, 2H, CH.sub.2OSO.sub.2R), 3.27 (m, 4H, CH.sub.2NHCOR), 3.18 (m,
1H), 2.96 (dd, J=5.0 and 7.9 Hz, 1H), 2.77 (d, J=15 Hz, 1H), 2.21
(m, 4H, CH.sub.2CONHR), 1.68-1.18 (m, 26H); MALDI-FTMS 712.3189
(C.sub.34H.sub.51N.sub.5O.sub.6S.sub.2+Na.sup.+ requires
712.3173).
[0230]
10-((2-Naphthalenesulfonyl)oxo)-N-biotinamidopentyldecanamide (10):
.sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 8.49 (s, 1H, ArH), 7.98
(t, J=8.2 Hz, 2H, ArH), 7.93 (d, J=7.9 Hz, 1H, ArH), 7.87 (d, J=8.8
Hz, 1H, ArH), 7.65 (p, J=7.0 Hz, 2H, ArH), 6.05 (b s, 1H, NH), 5.91
(b s, 1H, NH), 5.60 (b s, 1H, NH), 4.77 (b s, 1H, NH), 4.53 (m,
1H), 4.37 (m, 1H), 4.06 (t, J=6.4 Hz, 2H, CH.sub.2OSO.sub.2R), 3.25
(m, 4H, CH.sub.2CONHR), 3.17 (m, 1H), 2.95 (dd, J=5.0 and 7.6 Hz,
1H), 2.77 (d, J=14.0 Hz), 2.17 (m, 4H, CH.sub.2CONHR), 1.71-1.20
(m, 26H); MALDI-FTMS (DHB) m/z 689.3379
(C.sub.35H.sub.52N.sub.4O.sub.6S.sub.2+H.sup.+ requires
689.3401).
[0231] 10-((2-Thiophenesulfony)oxo)-N-biotinamidopentyl)decanamide
(11): .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 7.74 (t, J=5.0 Hz,
2H, Ar), 7.16 (t, J=3.8 Hz, 1H, ArH), 5.90 (b s, 1H, NH), 5.84 (b
s, 1H, NH), 5.47 (b s, 1H, NH), 4.75 (b s, 1H, NH), 4.53 (m, 1H),
4.35 (m, 1H), 4.12 (t, J=6.4 Hz, 2H, CH.sub.2OSO.sub.2R), 3.26 (m,
4H, CH.sub.2NHCOR), 3.19 (m, 1H), 2.95 (dd, J=5.3 and 7.6 Hz, 1H),
2.76 (d, J=12.9 Hz, 1H), 2.17 (m, 4H, CH.sub.2CONHR), 1.67-1.26 (m,
26H); MALDI-FTMS (DHB) m/z 645.2817
(C.sub.29H.sub.48N.sub.4O.sub.6S.sub.3+H.sup.+ requires
645.2809).
[0232] 1-(2-Pyridylsulfonyl)oxo-octane (15): To 3.0 mL of anhydrous
triethylamine (23.04 mmol, 30 equiv.) at 0.degree. C. was added
1-octanol (0.10 g 0.77 mmol, 1 equiv.) followed by the addition of
2-pyridylsulfonyl chloride in one portion. The mixture was kept at
0.degree. C. for 3 hours followed by the addition of water (5 mL).
The resulting mixture was extracted with diethyl ether (3.times.50
mL), then the organic extracts were combined and washed with
aqueous NaHCO.sub.3 solution (50 mL), dried (MgSO.sub.4), and
concentrated under reduced pressure. Column chromatography (2%
EtOAc/Hex) afforded 15 (99%): .sup.1H NMR (CDCl.sub.3, 400 MHz)
.delta. 8.68 (d, J=7.0 Hz, 1H, ArH), 7.94 (p, J=8.8 Hz, 2H, ArH),
7.54 (t, J=6.4 Hz, 1H, ArH), 4.26 (t, J=6.7 Hz, 2H,
CH.sub.2OSO.sub.2R), 1.61 (p, J=7.9 Hz, 2H), 1.13 (m, 10H), 0.76
(t, J=7.0 Hz, 3H); MALDI-FTMS 294.1130
(C.sub.13H.sub.21NO.sub.3S+Na.sup.+ requires 294.1134).
[0233] 1-(2-Pyridylsulfonyl)oxo-ethane (16): To a solution of
triethylamine (0.86 g, 8.51 mmol, 2.2 equiv.) in dichloromethane (3
mL) at 0.degree. C. was added ethanol (0.18 g, 3.87 mmol, 1 equiv.)
followed by the addition of 2-pyridylsulfonyl chloride (0.83 g,
4.65 mmol, 1.2 equiv.). After stirring for 4 hours at 0.degree. C.,
the solution was concentrated under reduced pressure. The
concentrate was dissolved in aqueous NaHCO.sub.3 solution (50 mL)
and extracted with diethyl ether (3.times.50 mL). The ether
extracts were combined and washed with aqueous NaCl (50 mL), dried
(MgSO.sub.4), and concentrated under reduced pressure. Column
chromatography (5% EtOAc/Hex) afforded 16 (95%): .sup.1H NMR
(CDCl.sub.3, 400 MHz) .delta. 8.70 (d, J=4.7 Hz, 1H, ArH), 7.95 (p,
J=6.4 Hz, 2H, ArH), 7.56 (t, J=5.3 Hz, 1H, ArH), 4.37 (q, J=7.0 Hz,
2H, CH.sub.2OSO.sub.2R), 1.29 (t, J=7.0 Hz, 3H); MALDI-FTMS
188.0000 (C.sub.6H.sub.9NOS+H.sup.+ requires 188.0376).
[0234] 1-(Methanesulfonyl)oxo-octane (17): To a solution of
triethylamine (0.12 g, 1.15 mmol, 1.5 equiv.) in dichloromethane (3
mL) was added octanol (0.10 g, 0.77 mmol, 1.0 equiv.) at 0.degree.
C. followed by the addition of methanesulfonyl chloride (0.10 g,
0.85 mmol, 1.1 equiv.), over a period of 5 minutes. After 30
minutes at 0.degree. C., the reaction mixture was diluted in
dichloromethane (50 mL) and extracted with ice cold water (50 mL),
ice cold 10% aqueous HCl (50 mL), saturated aqueous NaHCO.sub.3 (50
mL), and with saturated aqueous NaCl (50 mL). The organic layer was
dried (MgSO.sub.4) and concentrated under reduced pressure to
afford 17 (97%): .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta. 4.21 (t,
J=6.5 Hz, 2H, CH.sub.2OSO.sub.2Me), 2.99 (s, 3H, CH.sub.3SO.sub.3),
1.77 (p, J=6.7 Hz, 2H), 1.35 (m, 10 H), 0.87 (t, J=6.1 Hz, 3H).
[0235] Proteome Sample Preparation, Labeling, and Detection
[0236] Rat tissues were Dounce-homogenized in Tris buffer (50 mM
Tris-HCl buffer, pH 8.0, 0.32 M sucrose). Tissue extracts were
centrifuged sequentially at 1,100.times.g (10 min), 22,000.times.g
(30 min), and 105,000.times.g (60 min). The final supernatant
(soluble fraction) was adjusted to 0.5 mg protein/mL with Tris
buffer (without sucrose) and kept at 0.degree. C. until utilized.
Unless otherwise indicated, reactions between protein samples and
biotinylated reagents were conducted as follows: all biotinylated
reagents were stored as stock solutions in DMSO at 20.degree. C.
and then added directly to reactions with protein extracts, keeping
the DMSO concentration constant at 1% of the total reaction volume.
The reaction mixture was incubated at 25.degree. C. for 30 min
(final concentration of the probe was 5 .mu.M), then quenched by
the addition of 1 vol. equiv. of standard 2.times. SDS-PAGE loading
buffer (reducing). Quenched reactions were run on SDS-PAGE (7.5
.mu.g protein/gel lane) and transferred by electroblotting onto
nitrocellulose membranes, which were blocked in Tris-buffered
saline (TBS) with 1% Tween (TBS-Tween) and 3% (wt/vol) nonfat dry
milk for either 1 h at 25.degree. C. or overnight at 4.degree. C.
Blots were then treated with an avidin-horseradish peroxidase
conjugate (Bio-Rad, 1: 1,500 dilution) in TBS-Tween for 2 hr at
25.degree. C. The blot was washed with TBS-Tween three times (5
min/wash), treated with SuperSignal chemiluminescence reagents
(Bio-Rad), and exposed to film for 0.1 to 20 min before
development. For the pH-dependence studies, the following reaction
buffers were used; pH 6-8: 50 mM Tris.HCl; pH 8-10: 50 mM Tris.HCl,
50 mM CAPS.
[0237] Enrichment and Molecular Characterization of a 55 kDa
Sulfonate-Reactive Protein
[0238] Rat liver soluble fractions were run over a Q Sepharose
column by using an AKTA FPLC (Amersham Pharmacia Biotech) and
eluted with a linear gradient of 0-500 mM NaCl. Aliquots of the
elution fractions (10.times.2.5 mL fractions) as well as the flow
through (3.times.2.5 mL fractions) were labeled with 1 as described
above to identify the fractions containing the labeled proteins.
The flow-through fractions, which contained the 55 kDa protein,
were concentrated to a volume of 1 mg protein/mL vol. followed by
labeling 2.5 mL of the sample with 1 utilizing the standard
conditions. After incubating the reaction for thirty minutes, it
was applied to a PD-10 size exclusion column and eluted with 3.5 ml
of pH 8, 50 mM Tris-HCl buffer. Sodium dodecyl sulfate (SDS) (0.5%
wt/vol) was added and the labeled samples heated to 90.degree. C.
for 10 min in order to denature the proteins allowing for a more
accessible biotin moiety. The sample was then diluted 2.5 fold
(0.2% SDS) and incubated with 50-100 .mu.L of avidin beads on a
rotator for 1 hour at 25.degree. C. The eluant was then removed
followed by washing with 5 ml of 0.2% SDS and three washes with pH
8, 50 mM Tris-HCl buffer. Standard 2.times. SDS-PAGE loading buffer
was added followed by heating the sample to 90.degree. C. in order
to elute the proteins labeled with 1 from the avidin beads. The
eluant was run on an 8% Novex Tris-Glycine gel and stained with
Coomasie blue stain followed by destaining in a 30% methanol-water
solution. The desired 55 kDa 1-reactive-protein was excised from
the gel and digested with trypsin. The resulting peptides were
analyzed by matrix assisted laser desorption time-of-flight
(MALDI-TOF) mass spectrometry. The MALDI peptide data was utilized
in both the MS-Fit search of the Protein Prospector databases
(falcon.ludwig.ucl.ac.uk/mskome3.2.htm) and the ProFound search of
the Proteometrics databases
(www.proteometrics.com/prowl-cgi/ProFound.exe), which identified
the protein as cytosolic 2 class I rat aldehyde dehydrogenase
(cALDH-I).
[0239] Recombinant Expression and Purification of cALDH-I
[0240] Primers were designed based on cALDH-I's cDNA sequence and
used to amplify the enzyme's cDNA from a liver cDNA library
(Clontech). The cALDH-I cDNA was subcloned into the prokaryotic
expression vector, TrcHisA, followed by transformation and
expression in E. coli BL-21 cells. Expression was induced with 1 mM
isopropyl .alpha.-D thiogalactoside (IPTG) when cultures grew to an
OD.sub.600 of 0.6. After 4 h, the cells were pelleted and the
supernatant removed. The cell pellet was resuspended in Tris buffer
(20 mM Tris-HCl buffer, pH 8.0, 100 mM NaCl), lysed by treatment
with lysozyme (1 mg/ml) for 30 min and then sonication. The soluble
fraction was isolated by centrifugation 39,800.times.g (25 min).
The His-tagged cALDH-I was purified from the soluble fraction by
rotating with Talon cobalt beads for 30 minutes at 4.degree. C.
followed by centrifugation and removal of the lysate. After
washing, the beads were eluted with 80 mM imidazole buffer and the
eluted protein concentrated to 10 mg protein/mL. The concentrated
protein solution was subjected to gel filtration chromatography
(Superose 6 column, AKTA FPLC, Amersham Pharmacia Biotech). Gel
filtration samples containing purified cALDH-I were combined,
concentrated, and stored at -78.degree. C. in Tris buffer
containing 1 mM DTT (final cALDH-I protein concentration, 1.5
mg/mL).
[0241] Expression of cALDH-I in Eukaryotic Cells
[0242] The cALDH-I cDNA was subcloned into the eukaryotic
expression vector pcDNA3 and transiently transfected into COS-7
cells and MCF-7 cells by using methods described previously [Liu,
et al. (1999) Proc. Natl. Acad. Sci. USA 96, 14694-99]. Transfected
cells were harvested by scraping, resuspended in Tris buffer and
their protein concentrations determined (D.sub.c protein assay kit,
Bio-Rad). Whole cell suspensions were labeled with 1 as described
above.
[0243] cALDH-I Enzyme Assay and Inhibition Studies
[0244] cALDH-I activity was determined at 25.degree. C. in Tris
buffer (20 mM Tris-HCl, pH 8, 100 mM NaCl). Purified cALDH-I (0.2
.mu.M) was preincubated with 15 (2.5 .mu.M-15 .mu.M) in DMSO (30
.mu.L, 3% total incubation vol) in a volume of 950 .mu.L for 5 to
45 min. After preincubation of the enzyme with inhibitor, remaining
catalytic activity was measured by adding NAD.sup.+ (500 .mu.M
final concentration) and propionaldehyde (1 mM final concentration)
in 50 .mu.L of buffer. Production of NADH from the oxidation of
propionaldehyde was monitored by measuring the change in absorbance
at 340 nm for 2 min. In substrate competition assays, purified
cALDH-I (0.2 .mu.M) was preincubated with either NAD.sup.+ (50
.mu.M) or propionaldehyde (25 .mu.M) and 10 .mu.M of 15 for 10
minutes at 25.degree. C. in a volume of 950 .mu.L. Remaining
catalytic activity was monitored as described above.
[0245] Results
[0246] Selection and synthesis of biotinylated sulfonate esters as
candidate ABPs A library of candidate ABPs was synthesized based on
the general scaffold outlined in FIG. 9. The structure of an ABP
was conceptually divided into four pieces: a binding group (BG), a
reactive group (RG), a linker (L), and a tag (T). The library's
reactive group was selected as a sulfonate ester, based on the
following criteria.
[0247] A series of biotinylated sulfonates (1-11; FIG. 10) was
synthesized according to the three step sequence outlined in Scheme
1. The corresponding aryl or alkylsulfonyl chloride was added
slowly to a solution of undecene-1-ol (12) dissolved in pyridine at
0.degree. C. to form the sulfonate (13). The procedure of Sharpless
and colleagues was utilized to oxidatively cleave the terminal
olefin, resulting in formation of the corresponding carboxylic acid
(14) [Carlsen, et al (1981) J. Org. Chem. 46, 3936-38]. Treatment
of 14 with diethylaminosulfur trifluoride (DAST) followed by the
addition of N-hydroxysuccinimide afforded the N-hydroxysuccinimidyl
ester intermediate. The latter compound was reacted with
commercially available 5-(biotinamido)pentylamine (NH.sub.2-biotin,
Pierce) in methanol to form the desired biotinylated sulfonate
ester.
[0248] Evaluating the specific proteome reactivity of biotinylated
sulfonates
[0249] To determine whether members of the sulfonate library
specifically targeted proteins in the proteome, a method was
developed to rapidly distinguish a probe's specific and nonspecific
proteome reactivity. Each sulfonate (5 .mu.M) was reacted with two
versions of a rat testis proteome: a native proteome and a
denatured proteome (generated by preheating the protein sample for
5 min at 80.degree. C.). After 30 minutes at 25.degree. C., the
sulfonate-proteome reactions were quenched by adding one reaction
volume of standard SDS-PAGE loading buffer and analyzed by SDS-PAGE
and avidin blotting. A sulfonate's specific and nonspecific
proteome reactivity were defined as those protein targets that
displayed heat-sensitive and heat-insensitive labeling,
respectively. All sulfonates except the octylsulfonate 7 labeled at
least one member of the testis proteome in a heat-sensitive manner
(FIGS. 11A, 11B). Interestingly, four general patterns of specific
proteome reactivity were observed among the sulfonates.
Thep-toluenesulfonate 3, butylsulfonate 6, and naphthylsulfonate 10
each specifically labeled one 55 kDa protein (FIGS. 11A, B; single
arrowhead). The p-methoxybenzenesulfonate 4 and methylsulfonate 5
labeled four members of the proteome in a heat-sensitive manner
[FIG. 11A; 55 kDa (single arrowhead), 42 kDa (double arrowhead), 40
kDa (triple arrowhead), and 32 kDa (single dot)], but also showed
significant additional reactivity with the preheated proteome. The
benzenesulfonate 2 reacted with three of the four proteins labeled
by 4 and 5, failing to specifically label only the 40 kDa protein.
The p-nitrobenzenesulfonate 8, quinolinesulfonate 9, and
thiophenesulfonate 11 each reacted specifically with a 55 kDa
protein, as well as two additional proteins poorly labeled by the
other reagents [FIG. 11B; 36 kDa (single dot) and 30 kDa (triple
dot)]. Finally, the pyridylsulfonate 1 labeled a 55 kDa protein and
a 31 kDa protein (FIGS. 11A and B, single arrowhead and double dot,
respectively), the latter protein appearing uniquely reactive with
1 among the sulfonates that were surveyed. Importantly, most of the
sulfonate probes with the exception of 2, 4, and 5 showed low or
negligible reactivity with the preheated proteome.
[0250] Although several sulfonate probes showed overlapping
patterns of specific proteome reactivity, their relative
reactivities with individual proteins differed considerably. For
example, a 36 kDa protein reacted more strongly with
quinolinesulfonate 9 than with pyridylsulfonate 1,
p-nitrobenzenesulfonate 8 or thiophenesulfonate 11, while a 55 kDa
protein displayed the opposite probe selectivity (FIG. 11B). To
further examine the different proteome reactivities exhibited by
individual sulfonates, the labeling pattern of pyridylsulfonate 1
was compared to that of methylsulfonate 5 and quinolinesulfonate 9
in side-by-side analyses (FIGS. 12A and 3B, respectively). Each
sulfonate labeled a unique set of proteins. The patterns of
specific reactivity of 1 and 5 appeared nearly orthogonal to one
another, with 1 most strongly labeling 55 and 31 kDa proteins (FIG.
12A, left panel, double arrowheads) and 5 most strongly labeling 42
and 32 kDa proteins (FIG. 12A, left panel, single arrowheads). A
comparison of the proteome reactivities of 1 and 9 identified two
proteins that showed preferred reactivity with 1 (FIG. 12B, left
panel, 55 kDa and 31 kDa; double arrowheads) and two proteins that
showed enhanced reactivity with 9 (FIG. 12B, left panel, 36 kDa and
30 kDa; single arrowheads). The greater nonspecific reactivity of 5
was also evident in these side-by-side comparisons, as this agent
labeled several proteins in a heat-insensitive manner that were
unreactive towards 1 and 9. Notably, a Coomassie blue stained
protein gel revealed that the proteins labeled by 5 in the
preheated proteome represented very abundant proteins (FIG. 12A,
right panel, single arrowheads), consistent with the notion that
heat-insensitive labeling reflects a nonspecific form of sulfonate
reactivity. In contrast, the sulfonate library's heat-sensitive
proteome reactivity showed no such bias towards abundant
proteins.
[0251] Parameters that influence a sulfonate's specific proteome
reactivity The following features of the sulfonate-proteome
reaction were varied in order to test their influence on the
observed specific and nonspecific protein labeling patterns: time,
sulfonate concentration, pH, and the presence/absence of scavenging
nucleophiles. For these studies, the reactivity of pyridylsulfonate
1 with the testis proteome was examined. The two testis proteins
specifically targeted by 1 were labeled at similar rates, with
their signal intensities increasing from 5-40 minutes and then
plateauing from 40-120 minutes (FIG. 13A). The absence of
additional reactivity from 40-120 minutes could signify that the
proteins had labeled to completion by 40 minutes, or less likely,
that the concentration of 1 in the reaction was significantly
depleted by these later time.
[0252] Sulfonate 1's specific and nonspecific proteome reactivities
were evaluated over a range of probe concentrations (1-50 .mu.M).
From 1-10 .mu.M, sulfonate 1 showed specific reactivity with the 31
and 55 kDa proteins that increased in intensity with increasing
concentrations of reagent (FIG. 13B). Over this concentration
range, sulfonate 1 displayed very low levels of heat-insensitive
reactivity with the proteome. From 10 to 50 .mu.M of 1, the signal
intensity of the 31 kDa protein continued to increase, while the
intensity of the 55 kDa protein remained relatively constant. Over
this concentration range, sulfonate 1's nonspecific labeling
increased dramatically, especially in the higher molecular mass
range where most of the abundant testis proteins reside.
Importantly, however, no new specifically labeled protein targets
were identified over this concentration range. Thus, a
concentration range of 5-10 .mu.M appeared optimal for maximizing
sulfonate 1's specific versus nonspecific proteome reactivity.
[0253] The nonspecific and specific proteome reactivities of
sulfonate 1 showed different pH-dependencies, with the former
appearing as an inverted bell-shape curve (higher background
labeling at pH 6 and 9 than at pH 7 and 8) and the latter
increasing in intensity from pH 6 to 8 and plateauing from pH 8 to
10 (FIG. 13C, D). Thus, reactions conducted at pH 7 and 8 produced
the highest level of specific reactivity, while at the same time
resulting in the lowest degree of nonspecific reactivity.
[0254] Sulfonate 1's intrinsic reactivity with nucleophiles was
examined by conducting proteome reactions in the presence of
millimolar concentrations of free thiols (glutathione,
.alpha.-mercaptoethanol, or dithiothreitol). If this sulfonate
displayed a high reactivity with generic nucleophiles, then the
probe's effective concentration in thiol-treated proteome reactions
should be greatly reduced, resulting in a significant decrease in
the signal intensity of specifically labeled proteins. However,
none of the tested thiols affected the labeling intensity of the 55
kDa protein, indicating that sulfonate 1's intrinsic reactivity
with nucleophiles is low (FIG. 13D). In contrast, a moderate
decrease in the labeling intensity of the 31 kDa protein was
detected in the presence of free thiols.
[0255] Molecular Identification of a Protein Labeled by
Biotinylated Sulfonates
[0256] The screening method described above was enacted to rapidly
identify protein targets specifically labeled by members of the
sulfonate library. By defining these "specific protein targets" as
ones that displayed heat-sensitive reactivity with sulfonates the
focus was restricted to proteins whose activities would be affected
by sulfonate labeling. The assumption inherent to this strategy was
that heat-sensitive labeling was reflective of an event taking
place within a structured portion of a protein suitable for
small-molecule binding. Such structures were anticipated to often
represent either ligand binding pockets of receptors or active
sites of enzymes. As such, if a sulfonate reacted specifically with
one of these sites on a receptor or an enzyme, its reaction would
be expected to affect the activity of this protein. Accordingly,
the molecular identification of the 55 kDa protein specifically
labeled by several members of the sulfonate library was
investigated.
[0257] A tissue blot with sulfonate 1 revealed that the labeled 55
kDa protein was most abundant in soluble fractions of rat liver,
and therefore the protein was purified from this source. The 55 kDa
protein was partially purified by Q-Sepharose anion exchange
chromatography. Aliquots of the flow-through and elution fractions
of this column were labeled with 1, and the 55 kDa protein was
identified in the flow-through fractions. These fractions were
combined, labeled with 5 .mu.M 1 for 30 min, and the protein
separated from excess sulfonate by size exclusion chromatography.
The protein sample was then treated with avidin agarose beads to
isolate the 1-labeled 55 kDa protein. Elution of the avidin-bound
proteins was achieved by adding one volume of standard SDS-PAGE
loading buffer and heating (90.degree. C., 5 min). This
avidin-based affinity purification procedure provided a highly
concentrated sample of the 55 kDa protein that was separated by
SDS-PAGE and either blotted with avidin (FIG. 14A) or stained with
Coomassie blue. The 55 kDa protein was excised from the stained
gel, treated with trypsin, and the resulting peptide mixture
analyzed by MALDI-TOF mass spectrometry. MS-FIT and Profound
searches of protein databases identified the protein as cytosolic 2
class I aldehyde dehydrogenase (cALDH-I; nine tryptic peptides
ranging from 1189 to 2055 Da matched this enzyme, 50% total
sequence coverage; FIG. 14A), a member of a superfamily of
NAD.sup.+-dependent enzymes responsible for the oxidation of
endogenous and exogenous aldehydes to carboxylic acids [Wang, et
al. (1996) J. Biol. Chem. 271, 16288-93; Yoshida, et al. (1998)
Eur. J. Biochem. 251, 549-57].
[0258] Recombinant Expression of cALDH-I
[0259] In order to confirm the specific reactivity of cALDH-I with
sulfonate 1, this protein was recombinantly expressed in both
eukaryotic and prokaryotic systems. The cALDH-I cDNA was subcloned
into the pcDNA3 mammalian expression vector and then transfected
into COS-7 and MCF-7 cells. cALDH-I-transfected COS-7 and MCF-7
cells both expressed a 55-kDa protein that labeled strongly with
sulfonate 1 (FIG. 14B). In contrast, this sulfonate-reactive
protein was not detected in mock-transfected versions of each cell
type. cALDH-I was also recombinantly expressed in E. coli using the
pTrcHis system. Lysates from cALDH-I-transformed E. coli were
treated with sulfonate 1 and found to express a single reactive
protein of the predicted size for the cALDH-I enzyme bearing an
N-terminal histidine tag (60 kDa; FIG. 14C). The His-tagged cALDH-I
was purified from E. coli lysates by sequential metal affinity and
gel filtration chromatography. This prokaryotic expression system
routinely provided 15 mg/L culture volume of purified cALDH-I
enzyme.
[0260] Pyridylsulfonates are Time-Dependent Inhibitors of cALDH-I
Catalytic Activity
[0261] cALDH-I-catalyzed oxidation of propionaldehyde to propionic
acid was measured by observing the reduction of NAD.sup.+ to NADH
at 340 nm. The observed Michealis constant for propionaldehyde
(Km=4.2 .mu.M) displayed by the His-tagged recombinant cALDH-I was
comparable to the reported literature value for this enzyme (Km=6.5
.mu.M) [Penzes, et al. (1997) Biochim. Biophys. Acta 1342, 175-81].
To examine the effect of sulfonates on cALDH-I's catalytic
activity, the enzyme was treated with varying concentrations of
2-pyridylsulfonyl octanoate (15), a variant of 1 lacking the
probe's biotin tag. Sulfonate 15 inhibited cALDH-I's catalytic
activity in a time-dependent manner that increased in rate from 2.5
to 15 .mu.M inhibitor (FIG. 15A). Concentrations of 15 greater than
15 .mu.M inactivated cALDH-I at a rate that was too fast to measure
under the assay conditions employed. The average K.sub.obs/[I]
value calculated from reactions conducted at 2.5, 5.0, 7.5, 10, and
15 .mu.M 15 was (9.7+1.8).times.10.sup.3 M.sup.-1min.sup.-1.
Although the inactivation of cALDH-I at each concentration of 15
could be fit to psuedo-first order kinetics, extrapolation of these
reactions back to time zero did not predict 100% enzyme activity.
Considering that cALDH-I is a homotetrameric protein, one possible
explanation for this data was that individual cALDH-I subunits
exhibited different rates of reactivity with 15. In general support
of this notion, the kinetics of reactions conducted at lower
concentrations of 15 appeared biphasic in nature, with time points
preceding 50% enzyme inhibition predicting a slightly faster rate
of cALDH-I inactivation than time points at which greater than 50%
cALDH-I activity was inhibited. Finally, incubating 15 in the
reaction buffer (20 mM Tris.HCl, pH 8.0, 100 mM NaCl, 1 mM DTT) for
60 min prior to the addition of cALDH-I did not affect the
inhibitor's potency, indicating that this sulfonate was stable to
the assay conditions employed (including the presence of excess
freel thiols).
[0262] To probe the nature of 15's interaction with cALDH-I,
competition studies were performed with both propionaldehyde and
NAD+. Recombinant cALDH-I was treated with 10 .mu.M 15 for 10
minutes either in the presence or absence of 25 .mu.M
propionaldehyde or 50 mM NAD.sup.+ and the percentage of enzyme
activity remaining was determined (Table 1). Propionaldehyde had no
detectable effect on 15's inactivation kinetics. In contrast, 50
.mu.M NAD.sup.+ significantly reduced 15's inhibition of cALDH-I,
and higher concentrations of NAD.sup.+ completely protected the
enzyme from inactivation.
1TABLE 1 Competition studies for 15 with propionaldehyde and
NAD.sup.+ in cALDH Substrate Sulfonate 15 % Activity Remaining --
-- 100 -- 5 .mu.M 15 50 .mu.M NAD.sup.+ 5 .mu.M 61 25 .mu.M
Propionaldehyde 5 .mu.M 14
[0263] Features of 15 Responsible for cALDH-I Inactivation
[0264] Analogs of 15 were synthesized in which the agent's octyl
and pyridyl substituents were replaced with ethyl (16) and methyl
(17) groups, respectively. cALDH-I was incubated for 60 minutes
with 50 .mu.M of either 15, 16, or 17, and the percentage of
cALDH-I activity remaining was determined (Table 2). While 15
completely inactivated cALDH-I under these conditions, 16 and 17
produced weak and no inhibition, respectively. A K.sub.obs/[I]
value of 0.25 M.sup.-1min.sup.-1 was calculated for 16,
representing a second order inhibition rate constant 40000 times
lower than that determined for 15. 15-17 were also tested for their
ability to block sulfonate 1's reactivity with cALDH-I in the
soluble testis proteome. The testis proteome was preincubated for
30 min with each nonbiotinylated sulfonate at concentrations of 5
or 50 .mu.M. The proteome samples were then treated with 5 .mu.M 1
and the reaction mixtures incubated for 30 minutes prior to
analysis by SDS-PAGE and avidin blotting. Consistent with the
inhibition kinetics described above, only 15 blocked the labeling
of cALDH-I by 1 in the testis proteome (FIG. 15B). Thus, the
structure-activity relationship (SAR) determined for sulfonate
inactivation of purified cALDH-I correlated well with the SAR
observed for sulfonate labeling of this enzyme in complex
proteomes. Collectively, these data highlight that the interaction
of 15 (and by extrapolation 1) with cALDH-I depends on both the
aryl and alkyl chain groups of the inhibitor's structure.
2TABLE 2 Structure-activity relationship for sulfonate
inactivationof purified cALDH Inhibitor % Activity Remaining None
100 15 2 16 42 17 100
[0265] Multiplexing ABPs Increases Proteome Coverage
[0266] The rat testis proteome was treated with either 2.5 .mu.M
sulfonate 1, 5 .mu.M FP-biotin, or a mixture of 2.5 .mu.M 1 and 5
.mu.M FP-biotin [Liu, et al. Proc. Natl. Acad. Sci USA 96,
14694-99], and the resulting heat-sensitive labeling profiles were
visualized by SDS-PAGE and avidin blotting. As can be seen in FIG.
17, applying a mixture of sulfonate 1 and FP-biotin to the testis
proteome effectively detected in a single sample the proteins
labeled by both probes individually. Notably, a preheated sample
treated with the same probe mixture displayed a very low level of
labeling that was comparable to the nonspecific reactivity observed
when each probe was tested alone. These data support that by
multiplexing ABPs, one can significantly increase the coverage of
specific protein reactivities detectable in a single proteome
assay.
Example 8
[0267] To generate chemical probes for the active sites of other
enzymes like metallo-hydrolases and NT-dependent enzymes, we will
develop chemical probes are developed that can profile
metallo-hydrolases and NT-dependent enzymes in an
activity-dependent manner. Experiments for this Specific Aim will
include: i) the design and synthesis of tagged active site-directed
inhibitors of metallo-hydrolases and NT-dependent enzymes, and ii)
the testing of candidate inhibitors for selective and
activity-based reactivity with metallo-hydrolases and NT-dependent
enzymes in whole cell and tissue samples.
[0268] As with serine hydrolases, metallo-hydrolases and
NT-dependent enzymes are often regulated posttranslationally by
either endogenous inhibitory proteins or cis-acting autoinhibitory
sequences. Collectively, these posttranslational regultory
mechanisms make traditional genomics/proteomics approaches
inappropriate for recording dynamics in metallohydrolase and/or
kinase function. The goals of this specific aim are to design,
synthesize, and test candidate activity-based chemical probes that
target metallo-hydrolases and NT-dependent enzymes.
[0269] The design and synthesis of active site-directed chemical
probes that target metallo-hydrolases and NT-dependent enzymes. For
metallo-hydrolase-directed agents, we will create hydroxamic acid
inhibitors coupled to biotin, with the hope that these agents will
bind tightly [either reversibly or irreversibly] to
metallo-hydrolase active sites. The attached biotintag will allow
for the rapid detection and isolation of reactive proteins. We will
synthesize modest-sized combinatorial libraries of tagged
hydroxamates diversified at the R1 and R2 positions and select
those inhibitors that exhibit both 1) broad spectrum reactivity
with defined classes of metallo-hydrolases, and 2) low
crossreactivity with unrelated proteins. Several additional
candidate activity-based probes of metallo-hydrolases will also be
synthesized, founded on the principle that these molecules should
possess a group whose latent reactivity is unveiled on coordination
to a Lewis acidic, active site-bound metal ion. These reagents
include: pyridyl sulfonates and metal-induced vinylnitrosonium and
aziridinium ions. Activity-based probes that are target
NT-dependent enzymes will consist of biotin-tagged, reactive or
non-hydrolyzable chemical analogs of the appropriate NT. These
molecules should take advantage of the fact that many NT-dependent
enzymes do not bind NTs in their autoinhibited states.
[0270] Once each candidate probe is generated, it will be screened
against both commerically available enzymes and whole cell/tissue
samples. An initial assessment of the activity-based nature of the
observed interactions will be determined by comparing the
reactivity of heated versus unheated protein samples. Those
metallo-hydrolase-directed reagents that show selective and
heat-sensitive reactivity with MMPs will be further studied for
their ability to distinguish free MMPs (active) from those bound by
TIMPs (inactive). Those reagents directed against NT-dependent
enzymes that show selective and heat-sensitive reactivity will be
further studied for their ability to distinguish active versus
inactive (e.g., autoinhibited) enzymes. Finally, it is important to
stress a major advantage of our experimental strategy: because each
chemical reagent that we synthesize will be coupled to biotin, we
have an opportunity to rapidly define its specific sites of action
in a complex proteome. Thus, even if a reagent fails to react with
the proposed target (e.g., metallo-hydrolases), we will still be
able to assess whether it shows specific reactivity with other
enzyme families. In this manner, the work proposed may not only
identify class-selective reagents for metallo-hydrolases and
NT-dependent enzymes, but simultaneously reactive chemical groups
that target other enzyme families as well. This form of reverse
drug discovery, in which a postulated chemical inhibitor is mixed
with whole cell/tissue extracts and its target selectivity revealed
by tag recognition should prove quite powerful for the
identification of novel small molecule-enzyme interactions that can
in turn be exploited for activity-based proteomics
investigations.
[0271] The subject inhibitors may be used in a variety of ways. One
application is to determine the serine hydrolase activity of a
physiological sample. The sample may be blood, cells, tissue, or
other physiological sample of interest. In some situations, samples
that are suspected of having one or have serine hydrolases may be
monitored, as in the genetic engineering of serine hydrolase
proteins, where the efficiency of synthesis would be of interest.
In the case of tissue or cells, the cells may be lysed in
accordance with conventional conditions, using a homogenizer,
blender, pellets, centrifuge or other convenient device. The
resulting lysed cellular composition may be centrifuged and the
supernatant adjusted for protein content. Depending on the nature
of the ligand, the supernatant fraction may be freed of naturally
occurring ligand and/or receptor. The supernatant may be further
treated, as appropriate, adding buffer, further dilution,
fractionation by chromatography, etc. Where fractionated,
individual fractions will be used in the assay.
[0272] Candidate compounds to be used as therapeutics associated
with indications involving serine hydrolase dysfunction,
particularly for inhibiting specific or groups of serine
hydrolases, may be monitored by preparing a reaction mixture with
one or more hydrolases and monitoring the effect on the rate of
inhibition. One would add one or more subject compounds and a
candidate compound and then monitor the rate of inhibition, by
isolating aliquots and analyzing the aliquot for serine hydrolase
activity or isolating bound serine hydrolases and analyzing the
bound serine hydrolases.
[0273] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0274] While the invention has been described in detail with
reference to certain preferred embodiments thereof, it will be
understood that modifications and variations are within the spirit
and scope of that which is described and claimed.
* * * * *
References