From the Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461-1602
Received for publication, December 13, 2000, and in revised form, January 12, 2001
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ABSTRACT |
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A wide variety of proteins have been shown to
recognize and bind to specific amino acid sequences on other proteins.
These sequences can be readily identified using combinatorial peptide libraries. However, peptides containing these preferred sequences ("consensus sequence peptides") typically display only modest affinities for the consensus sequence-binding site on the intact protein. In this report, we describe a parallel synthesis strategy that
transforms consensus sequence peptides into high affinity ligands. The
work described herein has focused on the Lck SH2 domain, which binds
the consensus peptide acetyl-Tyr(P)-Glu-Glu-Ile-amide with a
KD of 1.3 µM. We employed a strategy
that creates a series of spatially focused libraries that challenge
specific subsites on the target protein with a diverse array of
functionality. The final lead compound identified in this study
displayed a 3300-fold higher affinity for the Lck SH2 domain than the
starting consensus sequence peptide.
There exist an impressive array of biological phenomena that are
regulated by protein-protein interactions. Perhaps nowhere is this more
evident than in the formation of coherent signal transducing cascades,
which are required to drive such diverse processes as cell motility (1)
and division (2), the immune response (3), apoptosis (4), and neuronal
activity (5). The dependence of these and many other phenomena on
highly specific protein-protein interactions has generated considerable
interest in elucidating the molecular basis of these interactions. Much of the initial work in this field focused on the ability of proteolyzed peptide fragments from one component of a known protein-protein pair to
interact with the intact binding partner. Such studies have led, for
example, to the acquisition of a number of peptide-based protein kinase
substrates and inhibitors (6, 7). More recent work has relied on the
use of peptide libraries (8-10) for the identification of "consensus
sequences" (11) for a wide variety of protein-interacting species,
including the SH2 (Src homology 2)
domain (12).
The SH2 module consists of ~100 amino acids and has thus far been
identified in >100 different proteins (13-15). SH2 domains recognize
and bind to amino acid sequences that encompass a Tyr(P) moiety.
Although Tyr(P) is required for SH2 recognition, specificity is
conferred by neighboring residues. In general, simple SH2-directed peptide ligands recapitulate these properties. Peptides lacking a
phosphorylated Tyr moiety display little or no affinity for SH2
domains, and SH2 selectivity can be realized by the simple expediency
of incorporating Tyr(P) into the appropriate amino acid sequence.
However, the use of peptides as ligands for SH2 domains in particular
and as agents that can modulate protein-protein interactions in general
is fraught with limitations. First and foremost is the general
phenomenon that the affinity of peptides for specific binding sites on
proteins tends to be weak by comparison with small low molecular
species. For example, although a number of ATP analogs have been
described for tyrosine-specific protein kinases with affinities in the
pM/nM range, peptides that target the
protein-binding site of these enzymes typically display affinities in
the high µM/low mM range (16). One possible
explanation for this dichotomy is the fact that peptides are limited to
an array of 20 standard amino acid residues, whereas the inherent
structural diversity possible with low molecular weight compounds, such
as ATP analogs, is virtually limitless. High diversity allows one to
identify synthetic compounds that are able to engage in an assortment
of productive interactions with the target protein, interactions that
are otherwise unavailable to conventional peptides.
The use of combinatorial chemistry to generate libraries of high
molecular diversity has had a profound impact on the fashion by which
biologically useful compounds are synthesized and identified. Indeed, a
high premium has been placed on the importance of diversity since
seemingly trivial modifications in molecular structure can alter the
potency of small molecule enzyme inhibitors by as much as 3-4 orders
of magnitude (17, 18). Unfortunately, conventional combinatorial
peptide libraries are not designed to identify these comparatively
subtle structural factors. Although a variety of synthetic methods
(e.g. the one bead/one peptide approach) have been used to
generate million member peptide libraries, the "local" diversity
associated with these libraries is limited to the 20 standard amino
acids employed at each position along the peptide chain. One approach
that has been used to enhance local diversity in peptide libraries is
to employ additional unnatural amino acid residues, which necessitates
the use of molecular encoding (19). Although parallel synthesis
(i.e. spatially separated peptides (20)) obviates the need
for encoding, the size of these libraries is, by necessity,
significantly smaller than what is feasible using the split-and-pool
and related methods.
In this report, we describe a strategy to transform consensus sequence
peptides, with modest affinities for target proteins, into high
affinity ligands. This approach employs peptide libraries with two key
attributes: moderate size (~103 members each), yet high
structural diversity. Due to their small size, these libraries can be
synthesized in parallel, which allows each library member to be
individually evaluated and eliminates the requirement for subsequent
structural deconvolution. Furthermore, since these libraries possess a
high structural diversity focused within narrow spatial windows on the
target protein, small regions of the protein can be challenged with a
multitude of functionality containing structural differences that vary
from subtle to gross. We have targeted the peptide-binding region of
the Lck (lymphoid T-cell tyrosine
kinase) SH2 domain to illustrate the utility of this strategy.
Materials--
Chemicals were obtained from Aldrich, except for
piperidine, protected amino acids, amino acid derivatives,
1-hydroxybenzotriazole (HOBt),1
benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), and TentaGel resin, which were obtained from Advanced Chemtech and Bachem California. Biotinyl- Peptide Synthesis--
All peptides were synthesized on an
automated peptide synthesizer using a standard Fmoc solid-phase peptide
synthesis protocol. Crude peptides were purified on a preparative HPLC
column using three Waters radial compression modules (25 × 10 cm) connected in series. Purified peptides were further
characterized by mass spectrometry.
Synthesis of Libraries 10and 11--
Cystamine
dihydrochloride (10 eq, 2.25 g) was added to a mixture of TentaGel
S COOH resin (90 µm, 5 g, 0.2 mmol/g), BOP (1.2 eq, 0.53 g), HOBt (1.2 eq, 0.184 g), and N-methylmorpholine (30 eq,
3.3 ml) in 20 ml of DMF and subsequently shaken overnight at ambient
temperature. The free amine substitution level was determined to be
0.01 mmol/g. This low substitution level is ideal for our purposes
since this not only ensures a higher coupling yield, but in addition,
larger quantities of resin (with greater weight accuracy) can be
subsequently introduced into the 96-well plates. The
coumarin-NH-pYXXI peptide was synthesized on the
cystamine-substituted TentaGel resin using an Fmoc solid-phase peptide
synthesis protocol. After deprotection of the
NH-t-butyloxycarbonyl group, the resin was extensively
washed and subsequently dried in vacuum. The peptide-bound resin was
distributed in 5-mg quantities into each well of solvent-resistant 96-well filter plates. In addition, each well contained a carboxylic acid-containing compound (400 eq, 20 µmol),
benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (200 eq), HOBt (200 eq), and N-methylmorpholine (1000 eq) in 100 µl of DMF. A total of 900 different carboxylic acids were employed.
The plates were shaken overnight, and then each well was subjected to a
series of wash steps (3 × 200 µl of DMF, 3 × 200 µl of
water, 3 × 200 µl of DMF, 3 × 200 µl of CH2Cl2, 2 × 200 µl of MeOH, and 2 × 200 µl of 50 mM Tris (pH 7.5)). The peptide-nonpeptide
conjugates were cleaved from the disulfide-containing resin with 10 mM dithiothreitol (DTT) in Tris buffer (1 × 200 µl
for 1 h and 2 × 150 µl for 1 h each) and filtered
into a receiving set of 96-well plates using the vacuum manifold (final
volume of 500 µl). The efficiency of acid coupling, peptide cleavage from the resin with DTT solution, and purity of the peptide-nonpeptide conjugates were assessed with several ligands
(7-hydroxycoumarin-4-acetic acid, 3-nitrocinnamic acid,
2-phenoxypropionic acid, and 3,5-dibromo-4-hydroxybenzoic acid). No
free N-terminal peptide was detected, and >90% of total ligand was
cleaved from the resin with first the DTT wash step. The final
two DTT washings removed the residual resin-bound peptide. Compound
purity was >90% as assessed by HPLC, and the HPLC-purified compounds
(i.e. removal of Tris buffer and DTT) were characterized by
matrix-assisted laser desorption ionization mass spectrometry.
Synthesis of Consensus Sequence
Peptides--
Coumarin-Tyr(P)-Dap(R2)-Dap(R3)-ICONHCH2CH2SH
and
Tyr(P)-Dap(R2)-Dap(R4)-ICONHCH2CH2SH
were synthesized as described above, except that Fmoc-Dap(1-(1'-adamantyl)-1-methylethoxycarbonyl) was employed at the
P+1 position. The 1-(1'-adamantyl)-1-methylethoxycarbonyl protecting
group was removed by shaking the resin with 3% trifluoroacetic acid in
CH2Cl2 for 3 min. This step was repeated three
times or until the protecting group was completely removed. The resin
was washed thoroughly using CH2Cl2, MeOH, DMF,
and 10% piperidine/DMF. 5-Sulfosalicylic acid was then coupled to the
peptide using the benzotriazol-1-yloxytripyrrolidinophosphonium
hexafluorophosphate/HOBt method. After the washing step, the
t-butyloxycarbonyl protecting group was removed by using
50% trifluoroacetic acid/CH2Cl2 (with 5%
H2O, 30 min). The washing steps mentioned above were
applied to the resin before the appropriate carboxylic acid was coupled to the peptide. The final washing and cleaving steps were the same as
described above. The collected mixture was purified by HPLC. The
structures were confirmed using matrix-assisted laser desorption
ionization mass spectrometry and NMR. 1H NMR
(Me2SO-d6) for peptide
17: Determination of Peptide Concentration--
Peptide
concentrations were determined by the intensity of coumarin absorption
( Screening of the Peptide-Nonpeptide Conjugate Library--
An
ELISA was employed to screen the library for SH2 affinity (21). 100 µl of biotinyl- Determination of KD Values--
With the exception
of compounds 12 and 15-17, peptides of the
general structure
coumarin-Tyr(P)-Gln-Dap(R)-ICONH2CH2CH2SH are highly fluorescent and exhibit little or no change in fluorescence upon coordination to the SH2 domains of Lck. Therefore, the
KD values for the SH2 complexes of these species
were determined via equilibrium dialysis (21). All samples were
prepared in buffer containing TBS and 1 mM DTT at pH 7.5. Slide-A-Lyzer dialysis slide cassettes (0.1-0.5-ml capacity) were
employed and contained 3-5 nM Lck SH2-GST fusion proteins.
The cassettes (400-µl final volume) were placed in a beaker
containing a volume of buffer solution (TBS and 1 mM DTT at
pH 7.5) that was at least 250-fold greater than that of the sample
volume in the dialysis cassette. As a consequence, concentrations of
non-SH2-bound peptide were held constant in the dialysis slide cassette
over the course of the experiment. Equilibrium dialysis experiments
were performed over a period of 12 h and maintained at 4 °C.
Differences in fluorescence between the solution in the slide cassette
and that in the beaker were measured. The excitation wavelength
employed for the peptides was 330 nm. Emission was monitored at 460 nm.
Equation 1 was used for the determination of
KD,
Combinatorial peptide libraries offer a relatively straightforward
method for identifying consensus recognition sequences of proteins that
interact with other proteins. As an initial first step, a consensus
sequence furnishes invaluable information concerning potential
endogenous substrates and/or binding partners and serves as a starting
point for the generation of synthetic species that can modulate
protein-protein interactions. Although it is relatively straightforward
to acquire consensus sequences via the application of peptide
libraries, the subsequent conversion of peptide templates into species
that display high affinities (i.e. KD
values in the nM range) for the desired protein target
often requires a more tortuous route.
Once a consensus sequence has been acquired, the binding contributions
of individual residues in the sequence can be assessed via the
"alanine scan" method (22, 23). This approach employs a series of
peptides containing an Ala residue positioned at each site along the
peptide chain. Analogous "phenylalanine scans," "glycine
scans," and other variations (24) have also been reported. We
describe herein a "library scan" that replaces specific residues in
a consensus sequence peptide with ~103 different amino
acid moieties. The overall strategy is outlined in Fig.
1. One or more peptides are synthesized
that contain a Day moiety at specific sites along the peptide chain.
Once the Dap-containing peptides have been prepared, the side chain Dap amine is deprotected, and the resin-appended peptide is transferred in
equal amounts to individual wells of multiwell plates. The free
amine-containing Dap functionality on the peptide-bound resin in each
well is subsequently condensed with one of ~103 different
carboxylic acids (which vary by molecular weight, charge, polarity,
hydrophobicity, sterics, etc.). Following removal of any remaining
protecting groups, the peptide is released from the resin and delivered
to a receiving multiwell plate in an assay-ready form. These libraries
can be constructed in a linear, iterative fashion (Fig.
2a) or in parallel (Fig.
2b). The former strategy requires the identification of a
lead residue (Dap-COR1) from an initial library scan, which
can then be used as a biasing agent in the construction of subsequent
sublibraries. This approach is useful if the residues to be replaced on
the peptide bind to the target protein in an energetically coupled
fashion. Alternatively, a series of libraries can be synthesized
simultaneously (Fig. 2b), an approach that is synthetically
more expedient since the nonbiased libraries prepared via this
method are not dependent upon the acquisition of lead residues from any
other library(ies) (Fig. 2a).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-aminocaproyl-EPQpYEEIPIYL was purchased from Bachem California. The SH2-GST fusion protein, Lck-(120-226), and polyclonal rabbit anti-GST antibody were
purchased from Santa Cruz Biotechnology. Horseradish
peroxidase-conjugated goat anti-rabbit antibody, peroxidase substrate
(1-Step Turbo trimethylbenzidine ELISA), streptavidin- or
NeutrAvidin-coated 96-well plates, SuperBlock blocking buffer, and
Slide-A-Lyzer dialysis slide cassettes (Mr
10,000 cutoff) were purchased from Pierce. Solvent-resistant
MultiScreen 96-well filter plates and the MultiScreen 96-well filter
plate vacuum manifold were purchased from Millipore Corp.
9.07-9.12 (m, 2H), 8.92 (d, J = 7.7 Hz, 1H), 8.82 (d, J = 1.4 Hz, 2H), 8.72 (d,
J = 1.4 Hz, 1H), 8.54 (d, J = 6.9 Hz,
1H), 8.38-8.44 (m, 2H), 8.35 (d, J = 1.7 Hz, 1H), 8.21 (d, J = 7.8 Hz, 1H), 7.91 (bs, 1H), 7.82 (dd,
J = 1.7, 8.5 Hz, 1H), 7.31 (d, J = 8.5 Hz, 2H), 7.26 (d, J = 8.5 Hz, 2H), 7.21 (d,
J = 8.7 Hz, 1H), 7.04 (d, J = 7.04 Hz, 1H), 6.82 (dd, J = 8.7, 2 Hz, 1H), 6.69 (d,
J = 2 Hz, 1H), 6.22 (s, 1H), 4.70-4.74 (m, 3H), 4.40 (m, 1H), 3.66-3.97 (m, 6H), 3.32-3.45 (m, 2H), 3.10-3.25 (m, 1H),
2.85-2.95 (m, 1H), 2.57-2.75 (m, 2H), 2.45 (bs, 1H), 1.85-1.95 (m,
1H), 1.50-1.70 (m, 1H), 1.10-1.30 (m, 1H), 0.97 (d, J = 6.6 Hz, 3H), and 0.91 (t, J = 7.3 Hz, 3H).
= 1.187 × 104
M
1
cm
1). The protein-tyrosine
phosphatase-catalyzed hydrolysis reaction of phosphotyrosine was also
performed to determine the peptide concentration by monitoring the
increase in absorbance at 282 nm (
= 970 M
1 cm
1)
at pH 7.0 in buffer containing 50 mM 3,3-dimethylglutarate, 1 mM EDTA, and 1 mM DTT (I = 0.15 M) at 30 °C. The concentrations obtained via the
phosphatase method were close or identical to concentrations obtained
from UV.
-aminocaproyl-EPQpYEEIPIYL (10 ng/ml in
Tris-buffered saline (TBS; 50 mM Tris and 150 mM NaCl (pH 7.5))) was added to each well of
NeutrAvidin-coated 96-well microtiter plates. The plates were shaken
overnight at 4 °C and rinsed with TBS (2 × 200 µl) followed
by BSA-T-TBS (0.2% BSA, 0.1% Tween 20, and TBS, 2 × 200 µl).
Each well was then blocked with 100 µl of SuperBlock blocking buffer
(30 min at ambient temperature). Two wells in each plate were reserved
for standards (one well contained the starting peptide (i.e.
either peptide 8 or 9) that had not been
acylated, and the other well contained the starting peptide that had
been acetylated). A 50-µl solution of each member of the P+1 and P+2
libraries (50 nM in TBS) and a 50-µl solution of the Lck
SH2-GST fusion protein (3.2 ng/ml in BSA-T-TBS) were added to
individual wells of 96-well plates, and the plates were subsequently
shaken for 1 h at room temperature. The solutions were removed,
and each well was rinsed with 4 × 200 µl of BSA-T-TBS. 100 µl
of polyclonal rabbit anti-GST antibody (100 ng/ml in BSA-T-TBS) was
then added to each well, and the plates were incubated for 1 h at
room temperature. Following subsequent washing steps with BSA-T-TBS
(4 × 200 µl), 100 µl of horse radish peroxidase-conjugated goat anti-rabbit antibody (200 ng/ml in BSA-T-TBS) was added to each
well, and the plates were subsequently incubated for 1 h at room
temperature. After a series of final wash steps (2 × 200 µl of
BSA-T-TBS and 2 × 200 µl of TBS), 100 µl of peroxidase
substrate (1-Step Turbo trimethylbenzidine ELISA) was added to each
well and incubated for 5-15 min. 100 µl of 1 M sulfuric
acid solution was introduced to stop the peroxidase reaction, and
absorbance was measured at 450 nm with a plate reader. IC50
values were determined using the ELISA screening method around a
130-fold range of ligand concentrations.
where [E]T = total SH2 domain
concentration, [L] = total ligand concentration, and
[E·L] = SH2/ligand concentration.
(Eq. 1)
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (20K):
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Fig. 1.
General library scan strategy:
preparation of the P+1 (10) and P+2 (11) modified
sublibraries. An Fmoc solid-phase peptide synthesis
protocol is conducted on a novel disulfide-containing resin (peptide
1). Peptides contain a Dap residue, which is deprotected and
individually acylated with a structurally diverse array (hydrophobic,
negatively and positively charged, polar, aromatic and aliphatic,
acyclic, cyclic, and multicyclic) of 900 carboxylic acids. The acylated
peptides are then cleaved from the resin using the subsequent assay
buffer containing DTT. tBu, tert butyl; pTyr,
Tyr(P); TFA, trifluoroacetic acid.
View larger version (11K):
[in a new window]
Fig. 2.
Iterative (a) and parallel
(b) strategies for the preparation of Dap-containing
peptide libraries. The iterative strategy (a) employs
the stepwise identification of optimal Dap-containing residues at each
position along the peptide chain. This approach holds the advantage
that side chain residues that are "energetically coupled" can be
readily identified. The parallel strategy (b) ignores the
latter issue since the acylated Dap residues are acquired independently
of each other. However, the parallel approach is synthetically more
expedient. AA, amino acid.
The design of the libraries in this study was based upon the following considerations. (a) The consensus peptide, acetyl-Tyr(P)-Glu-Glu-Ile-amide (peptide 2), displays a KD of 1.3 ± 0.2 µM for the Lck SH2 domain (21), a value consistent with those obtained for Src SH2 domain-targeted peptides in general. We have recently found that the coumarin-appended peptide 3 (Fig. 3 and Table I) displays a 37-fold higher affinity (KD = 35 ± 7 nM) for the Lck SH2 domain than peptide 2 (21). Consequently, the coumarin moiety was employed as a biasing element in the construction of the SH2 domain-targeted libraries.
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(b) The Glu residues in Tyr(P)-Glu-Glu-Ile, which only modestly interact with the SH2 domain, were substituted with Dap-based libraries (14, 25). The Tyr(P) and Ile residues were not replaced because they participate in high affinity interactions with residues on the SH2 domain (14). Our decision to employ Dap rather than some other diamine-containing species (e.g. lysine) was based on the comparatively short Dap side chain, which limits the conformational mobility (i.e. entropy) of substituents attached to the side chain amine moiety. Under these circumstances, the amine-bound substituents should be more favorably positioned to engage SH2 functionality that encompasses the peptide-binding region of the SH2 domain.
(c) Our initial objective required the preparation of libraries of moderate size (~103) in an assay-ready form. We have recently described the preparation of a cystamine-linked TentaGel resin (peptide 1) and its use in peptide library synthesis (21, 26). The disulfide linkage between the peptide and the TentaGel resin is stable to the conditions of Fmoc-based solid-phase peptide synthesis. Furthermore, the disulfide moiety is cleaved in essentially quantitative yield by conditions (TBS and 10 mM dithiothreitol at pH 7.5) that are virtually the same as and therefore compatible with the subsequent ELISA screen (TBS and 1 mM dithiothreitol at pH 7.5).
(d) Dap-based moieties will ultimately replace both Glu residues in -Tyr(P)-Glu-Glu-Ile-. However, the initial libraries employ only a single Dap insertion, thereby requiring a transient "caretaker" residue at Xaa (i.e. -Tyr(P)-Dap-Xaa-Ile- and -Tyr(P)-Xaa-Dap-Ile-). We chose Gln for this purpose since its non-nucleophilic character will not interfere with the subsequent Dap acylation step. Furthermore, we found that the Gln-for-Glu substitutions in the parent peptide (coumarin-Tyr(P)-Glu-Glu-Ile-amide (peptide 2; KD = 35 ± 7 nM)) furnishes peptides that display only slightly reduced affinities for the Lck SH2 domain (coumarin-Tyr(P)-Glu-Gln-Ile-amide (peptide 4; KD = 49 ± 14 nM) and coumarin-Tyr(P)-Gln-Glu-Ile-amide (peptide 5; KD = 116 ± 24 nM)). Consequently, the TentaGel-appended peptides coumarin-Tyr(P)-Dap-Gln-Ile-cystamine-TentaGel (peptide 6) and coumarin-Tyr(P)-Gln-Dap-Ile-cystamine-TentaGel (peptide 7) served as the starting species for the synthesis of the P+1 and P+2 libraries, respectively (Fig. 1).
The TentaGel-appended peptides 6 and 7 were prepared using a standard Fmoc protocol (Fig. 1). The t-butyloxycarbonyl-protected Dap side chain in these resin-bound peptides was subsequently removed with 50% trifluoroacetic acid in CH2Cl2 to furnish peptides 8 and 9, respectively. These TentaGel-appended peptides were then introduced, in 5-mg quantities, into the individual wells of solvent-resistant 96-well filter plates. One of 900 different carboxylic acids (20 µmol dissolved in 50 µl of DMF) was subsequently added to each well in an ~500-fold molar excess relative to peptide to ensure complete acylation of the free Dap amine moiety. The acylation reaction was initiated by the addition of 200 eq of BOP, 200 eq of HOBt, and 1000 eq of N-methylmorpholine in 50 µl of DMF to each well. The plates were gently shaken for 12 h, and each well was subsequently subjected to a series of wash steps to remove excess reagents (see "Experimental Procedures"). All plate washings were conducted via vacuum filtration using a 96-well filter plate manifold. Libraries 10 and 11 were then released from the resin with three washings with a DTT-based solution (TBS and 10 mM DTT at pH 7.5). These washings were filtered into receiving 96-well plates, and these peptide-containing solutions were directly used, without purification, in the subsequent ELISA-based assay.
We selected four compounds from each library (i.e. peptides derivatized at the P+1 and P+2 Dap positions with 7-hydroxycoumarin-4-acetic acid, 3-nitrocinnamic acid, 2-phenoxypropioic acid, and 3,5-dibromo-4-hydroxybenzoic acid) to examine the extent of amine acylation and the efficiency of DTT cleavage. In all cases, we were unable to detect the presence of any free amine following treatment of the peptide-bound resin with the 500-fold molar excess of carboxylic acid. In addition, >90% of the peptide was cleaved from the resin with the first DTT wash step. The final two DTT washings removed the residual resin-bound material. Finally, all compounds were found to be >90% pure by HPLC, and the HPLC-purified compounds (i.e. removal of Tris buffer and DTT) furnished the expected mass profiles by mass spectrometry.
The P+1 library ELISA-based screen identified peptide 12 as
the single lead, whereas three clear leads (peptides
13-15) were obtained from the P+2 library (Fig. 3
and Table I). We employed the ELISA to obtain an initial assessment of
the affinity of these compounds for the Lck SH2 domain (i.e.
IC50 = ligand concentration that blocks 50% of the ELISA
readout at 450 nm). Values in the low nM range were
obtained for all four peptides (Fig. 3 and Table I). We noted that, in
the ELISA, the modified peptides created in libraries 10 and
11 directly competed for the Lck SH2 domain with a known
SH2-directed standard (biotinyl--aminocaproyl-EPQpYEEIPIYL) bound to
the wells of the 96-well plates. Consequently, the lead peptides
depicted in Table I most likely associate with the same site on the SH2
domain as biotinyl-
-aminocaproyl-EPQpYEEIPIYL.
The inherent fluorescence associated with the N terminus-appended coumarin moiety was unaltered in the presence of the Lck SH2 domain. This behavior allowed us to acquire dissociation constants on select compounds via equilibrium dialysis using Slide-A-Lyzer cassettes. Unfortunately, the coumarin fluorescence was partially quenched in peptides 12 and 15. Consequently, we were unable to acquire KD values for these species. However, as is apparent from Table I, the ELISA-based IC50 values correlated well (within 2-fold) with the experimentally derived dissociation constants. Clearly, the P+2 library derivatives 13-15 displayed a substantial enhancement in Lck SH2 domain affinity relative to the parent peptide 5 (~50-100-fold). By contrast, the lead compound from the P+1 library (peptide 12) exhibited only an 8-fold higher affinity for the SH2 domain compared with its parent peptide 4.
Based on the results described above, we prepared the double-substituted Dap-derivatized peptides 16 and 17 (see "Experimental Procedures"). Since both peptides 16 and 17 contain the Dap(R2) residue present in peptide 12, we were not surprised to find that, like peptide 12, the coumarin fluorescence was partially quenched in the double-substituted Dap-derivatized peptides. Consequently, we were able to obtain IC50 values for only peptides 16 and 17. Peptide 16, which contains the acylated Dap residues present in peptides 12 and 14, displayed an IC50 of 3.0 ± 0.6 nM. The similar Lck SH2 affinities of the monosubstituted (peptide 14) and disubstituted (peptide 16) ligands clearly indicate that the separate SH2 energies of interaction of the Dap(R2) and Dap(R4) moieties are not additive when contained within the same peptide. This result illustrates one of the potential hazards associated with combining lead substituents from separate libraries (Fig. 2b), a hazard common to many combinatorial peptide library strategies: lead residues obtained independently of one another are not necessarily energetically additive in the final consensus sequence (27). Despite this potential difficulty, peptide 17, which contains the acylated Dap residues present in peptides 12 and 13, did display an enhanced affinity for the Lck SH2 domain (IC50 = 200 ± 20 pM). The latter result implies that the two acylated Dap residues in peptide 17 do bind independently of one another to the Lck SH2 domain. Insertion of Dap(R3) into the P+2 position of peptide 5 furnished peptide 13 and resulted in a 30-fold enhancement of SH2 affinity. By comparison, incorporation of Dap(R3) into the same position of peptide 12 (to provide peptide 17) resulted in a 65-fold greater affinity for the Lck SH2 domain. In an analogous vein, Dap(R2) insertion at P+1 in peptide 4 generated a 4-fold improvement in affinity (cf. peptides 5 and 12), whereas the incorporation of Dap(R2) into the same position in peptide 14 furnished a 20-fold binding enhancement. The IC50 value of 200 ± 20 pM displayed by peptide 17 for the Lck SH2 domain represents a 3300-fold enhanced affinity relative to the simple consensus peptide 2.
The individual members of the Src kinase family are generally limited
to specific cell types. For example, the Lck protein-tyrosine kinase
expression is restricted to T-cells. However, a number of SH2
domain-containing proteins are ubiquitously expressed, including
phospholipase C, phosphatidylinositol 3-kinase, and Grb2
(growth factor receptor-bound
protein-2). Consequently, we examined the issue of Lck SH2
selectivity using the SH2 domains from three universally expressed
proteins as controls. The coumarin-substituted peptide 3 displayed a >2 orders of magnitude selectivity for the Lck SH2 domain
(KD = 35 ± 7 nM) versus
the SH2 domains of phospholipase C1 (KD = 4.9 ± 0.7 µM), Grb2 (KD = 11.3 ± 3.1 µM), and the p85
subunit of
phosphatidylinositol 3-kinase (KD = 9.3 ± 0.9 µM). As noted above, we were unable to acquire
dissociation constants for peptide 17 due to the partial
quenching of fluorescence of the coumarin moiety in this compound.
However, the IC50 values obtained from the ELISA screen
revealed that peptide 17 was even more selective than
peptide 3 for the SH2 domain of Lck (IC50 = 200 ± 20 pM) versus those of phospholipase
C
1 (IC50 = 7.4 ± 2.5 µM), Grb2
(IC50 = 0.90 ± 0.09 µM), and
phosphatidylinositol 3-kinase (IC50 > 30 µM).
With the advent of combinatorial peptide libraries, it is now a
relatively straightforward matter to obtain consensus recognition sequences for proteins that bind to and/or process other proteins. Unfortunately, a not uncommon trait among consensus sequence peptides is their comparatively low affinity for protein targets. Typically, these peptides contain only a few residues that participate in key
interactions with their protein-binding partners. Many if not the
majority of residues in any given consensus peptide can often be
replaced with little or no impact on overall binding affinity. We have
described the use of a parallel synthesis strategy to identify high
affinity replacements for these noncritical residues. The attributes of
this strategy include the generation of a high diversity library
(i.e. 50-fold diversity greater than what is available using
standard amino acid residues) in an individual well format. The latter
allows one to separately assess the efficacy of each library member
without the need to resort to subsequent structural deconvolution.
Furthermore, the synthetic methodology delivers the library in an
assay-ready solution format, which provides a seamless transition
between library synthesis and the subsequent library screen. We have
applied this strategy to the SH2 domain of the Lck protein-tyrosine
kinase, an enzyme that plays a key role in T-cell activation. The lead
ligand 17, which was identified via a series of
sublibraries, displayed 3 orders of magnitude higher affinity for the
Lck SH2 than the standard consensus peptide 2. Indeed, with
an IC50 of 200 ± 20 pM, peptide
17 is among the tightest binding peptide-based ligands
described for any protein-protein interaction site.
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FOOTNOTES |
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* This work was supported by the National Institutes of Health and the Comprehensive Cancer Center of the Albert Einstein College of Medicine.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry,
Albert Einstein College of Medicine of Yeshiva University, 1300 Morris
Park Ave., Bronx, NY 10461-1602; Tel.: 718-430-8641; Fax: 718-430-8565;
E-mail: dlawrenc@aecom.yu.edu.
Published, JBC Papers in Press, January 16, 2001, DOI 10.1074/jbc.M011232200
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ABBREVIATIONS |
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The abbreviations used are: HOBt, 1-hydroxybenzotriazole; BOP, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate; GST, glutathione S-transferase; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HPLC, high performance liquid chromatography; DMF, N,N-dimethylformamide; DTT, dithiothreitol; Dap, L-2,3-diaminopropanoic acid; ELISA, enzyme-linked immunosorbent assay; TBS, Tris-buffered saline; BSA, bovine serum albumin.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Chan, A. Y.,
Bailly, M.,
Zebda, N.,
Segall, J. E.,
and Condeelis, J. S.
(2000)
J. Cell Biol.
148,
531-542 |
2. | Shackney, S. E., and Shankey, T. V. (1999) Cytometry 35, 97-116[CrossRef][Medline] [Order article via Infotrieve] |
3. | Kelly, M. E., and Chan, A. C. (2000) Curr. Opin. Immunol. 12, 267-275[CrossRef][Medline] [Order article via Infotrieve] |
4. | Wang, E., Marcotte, R., and Petroulakis, E. (1999) J. Cell. Biochem. Suppl. 75, 95-102[CrossRef] |
5. | Grewal, S. S., York, R. D., and Stork, P. J. (1999) Curr. Opin. Neurobiol. 9, 544-553[CrossRef][Medline] [Order article via Infotrieve] |
6. | Cheng, H. C., van Patten, S. M., Smith, A. J., and Walsh, D. A. (1985) Biochem. J. 231, 655-661[Medline] [Order article via Infotrieve] |
7. | Scott, J. D., Fischer, E. H., Demaille, J. G., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 4379-4383[Abstract] |
8. | Pinilla, C., Appel, J., Blondelle, S., Dooley, C., Dorner, B., Eichler, J., Ostresh, J., and Houghten, R. A. (1995) Biopolymers 37, 221-240[Medline] [Order article via Infotrieve] |
9. | Lebl, M., Krchnak, V., Sepetov, N. F., Seligmann, B., Strop, P., Felder, S., and Lam, K. S. (1995) Biopolymers 37, 177-198[Medline] [Order article via Infotrieve] |
10. | Zwick, M. B., Shen, J., and Scott, J. K. (2000) J. Mol. Biol. 300, 307-320[CrossRef][Medline] [Order article via Infotrieve] |
11. | Aitken, A. (1999) Mol. Biotechnol. 12, 241-253[Medline] [Order article via Infotrieve] |
12. | Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S., Lechleider, R. J., Neel, B. G., Birge, R. B., Fajardo, J. E., Chou, M. M., Hanafusa, H., Schaffausen, B., and Cantley, L. C. (1993) Cell 72, 767-778[Medline] [Order article via Infotrieve] |
13. | Mayer, B. J., and Gupta, R. (1998) Curr. Top. Microbiol. Immunol. 228, 1-22[Medline] [Order article via Infotrieve] |
14. | Kuriyan, J., and Cowburn, D. (1997) Annu. Rev. Biophys. Biomol. Struct. 26, 259-288[CrossRef][Medline] [Order article via Infotrieve] |
15. | Schaffhausen, B. (1995) Biochim. Biophys. Acta 1242, 61-75[CrossRef][Medline] [Order article via Infotrieve] |
16. | Lawrence, D. S., and Niu, J. (1998) Pharmacol. Ther. 77, 81-114[CrossRef][Medline] [Order article via Infotrieve] |
17. | Bridges, A. J., Cody, D. R., Zhou, H., McMichael, A., and Fry, D. W. (1995) Bioorg. Med. Chem. Lett. 3, 1651-1656[CrossRef] |
18. | Fry, D. W., Kraker, A. J., McMichael, A., Ambroso, L. A., Nelson, J. M., Leopold, W. R., Connors, R. W., and Bridges, A. J. (1994) Science 265, 1093-1095[Medline] [Order article via Infotrieve] |
19. | Combs, A. P., Kapoor, T. M., Feng, S., Chen, J. K., Daude-Snow, L. F., and Schreiber, S. L. (1996) J. Am. Chem. Soc. 118, 287-288[CrossRef] |
20. | Emili, A. Q., and Cagney, G. (2000) Nat. Biotechnol. 18, 393-397[CrossRef][Medline] [Order article via Infotrieve] |
21. | Lee, T. R., and Lawrence, D. S. (1999) J. Med. Chem. 42, 784-787[CrossRef][Medline] [Order article via Infotrieve] |
22. | Beck-Sickinger, A. G., Wieland, H. A., Wittneben, H., Willim, K. D., Rudolf, K., and Jung, G. (1994) Eur. J. Biochem. 225, 947-958[Abstract] |
23. | Tam, J. P., Liu, W., Zhang, J. W., Galantino, M., Bertolero, F., Cristiani, C., Vaghi, F., and de Castiglione, R. (1994) Peptides (Elmsford) 15, 703-708[CrossRef][Medline] [Order article via Infotrieve] |
24. | Garcia-Echeverria, C., Gay, B., Rahuel, J., and Furet, P. (1999) Bioorg. Med. Chem. Lett. 9, 2915-2920[CrossRef][Medline] [Order article via Infotrieve] |
25. | Bradshaw, J. M., and Waksman, G. (1999) Biochemistry 38, 5147-5154[CrossRef][Medline] [Order article via Infotrieve] |
26. | Lee, T. R., and Lawrence, D. S. (2000) J. Med. Chem. 43, 1173-1179[CrossRef][Medline] [Order article via Infotrieve] |
27. | Fauchere, J.-L., Boutin, J. A., Henlin, J.-M., Kucharczyk, N., and Ortuno, J. C. (1998) Chemometrics Intelligent Lab. Syst. 43, 43-68[CrossRef] |