The discriminatory power of differential receptor arrays is improved by prescreening - A demonstration in the analysis of tachykinins and similar peptides

Article abstract of DOI:10.1002/anie.200701236

(Graph Presented) Patterning pain: Six strong-binding receptors for the decapeptide tachykinin neurotransmitter α-neurokinin were identified by screening a solid-supported library. Solution-phase studies with these six receptors metalated with three diffe

Full text of DOI:10.1002/anie.200701236

Communications
DOI: 10.1002/anie.200701236
Supramolecular Chemistry
The Discriminatory Power of Differential Receptor Arrays Is Improved
by Prescreening—A Demonstration in the Analysis of Tachykinins and
Similar Peptides**
Aaron T. Wright, Nicola Y. Edwards, Eric V. Anslyn,* and John T. McDevitt*
Complex odors and flavors are discriminated by cross-
reactive receptor proteins that act in tandem to provide
diagnostic signals.[1] This strategy has provided inspiration for
the creation of differential sensing arrays.[2,3] These arrays
have the potential to discriminate challenging analytes such as
proteins and peptides. Peptide recognition has been the
subject of a tremendous body of work, and recently systems
capable of short peptide recognition, as well as sequence-
selective and stereoselective peptide recognition, have been
developed.[4]
Tachykinins, exemplified by a-neurokinin and substan-
ce P, provide a particularly challenging group of biomolecule
analytes. They are neurotransmitter peptides which are
implicated in pain transmission in the mammalian brain.[5]
Toward the goal of detecting tachykinins, we targeted a-
neurokinin analogues His-Lys-Thr (1) and His-Lys-Thr-Asp
(3).[3d] The tripeptide His-Glu-Thr (2) was also targeted for
comparison. Peptides 1 and 2 differ only by a single amino
acid, while peptides 1 and 3 differ in kind by one amino acid.
a-Neurokinin (His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-
C(O)-NH2; 4) and substance P (Arg-Pro-Lys-Pro-Gln-Gln-
Phe-Phe-Gly-Leu-Met-C(O)-NH2; 5) completed the set of
targeted analytes. These tachykinins share a C-terminal
sequence of Phe-X-Gly-Leu-Met-C(O)-NH2 which is con-
served throughout this family of peptides. We report here the
use of library 6 as differential receptors for discriminating
these five peptides, and the discovery that the discriminatory
capabilities of the receptors in an array are significantly
improved by prescreening this library for the desired targets.
Library 6 has a metal-binding ligand with two appended
peptides.[3d,6] The metal center imparts selectivity toward
peptides terminating in His, such as a-neurokinin.[6] One
peptide arm was consistently Lys-Gly-Asp, while the second
was added through combinatorial chemistry and provides the
differential recognition character.[3d] We previously showed
that 30 randomly chosen members of library 6 (incubated with
Cu(OTf)2)[7] successfully differentiated similar tripeptides.
Slopes at a preselected region of indicator-uptake kinetic
traces were calculated for the receptors and the dimension-
ality of the data set reduced using principal component
analysis (PCA). However, the majority of the variance in the
data (91%) was on a single PC axis, and the PC factor loading
values for all 30 receptors were similar. This indicated that the
library members did not have significant selectivity between,
or cross-reactivity for, the targets.
To target a peptide family as large and complex as the
tachykinins in an array setting, the discriminatory capabilities
of the potential receptors had to be improved. We hypothe-
sized that by prescreening library 6 we would find receptors
possessing affinity for the tachykinins, but also likely possess-
ing differential reactivity between members of this class of
analytes, thereby leading to an array of receptors which would
serve our purpose. Screening libraries of receptors has been
done previously to find the single best receptor for a specific
target,[8] but screening to uncover a series of receptors for
discrimination of structurally similar members within a class
of analytes is a new concept.
[*] Dr. A. T. Wright, Dr. N. Y. Edwards, Prof. E. V. Anslyn,
Prof. J. T. McDevitt
Department of Chemistry and Biochemistry
University of Texas at Austin
1 University Station A5300, Austin, TX 78712 (USA)
Fax: (+1)512-471-7791
Bead-supported library 6 was screened with a colorimetric
variant of a-neurokinin (7) to identify strong binding
receptors. The a-neurokinin variant was a conjugate of the
first four amino acid residues of a-neurokinin with an aspartic
acid modified with Disperse Red 1. Library 6 (5 mg) was
preincubated with CuCl2 (1 mm, 200 mL), followed by addi-
tion of 7 (70 mm, in 2-[4-(2-hydroxyethyl)-1-piperazinyl]eth-
anesulfonic acid (HEPES) 10 mm, pH 7.4).
E-mail: anslyn@ccwf.cc.utexas.edu
[**] We thank Dr. AndrewEllington for the use of his 96-well plate reader.
This work was supported by the National Institutes of Health
(EB0059).
Supporting information for this article (including full experimental
and data processing details) is available on the WWW under http://
www.angewandte.org or from the author.
Library members which displayed the strongest binding
affinity for 7 were identified from images obtained using an
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(267 mm). The use of six ligands, two metals, and two different
counterions for the Cu2+ ions, led to an array of 18 members.
Peptides 1–5 (267 mm) were then added to each ligand/metal/
counterion complex in HEPES (10 mm, pH 7.4). UV/Vis
spectra were taken of each well before and after the addition
of peptides 1–5. An example is given in Figure 2 for 6d/
Olympus stereoscope equipped with a charge-coupled device
and a video capture card (Figure 1). Only 0.5% of the beads
were intensely colored, thus indicating selectivity among the
Figure 2. UV/Vis absorbance spectra obtained before and after addi-
tion of 4 to 6d/CuOTf.
CuOTf2 and 4. Absorbance data at 315, 321, and 333 nm
(wavelengths of peaks or large changes) with and without
peptide were subtracted to yield the difference (DA values).
This created a data set of 54 DA values for each peptide, and
DA values as large as 0.3 absorbance units were observed.
Peptides 1–3 were tested three times, while 4 and 5 were
tested four times.
The UV/Vis data indicated that there were differences in
the extent of the binding between the peptides and the
metalated receptors, such that a unique pattern of absorbance
changes was created for each peptide. A representative
histogram showing peptide patterns created by complexes
formed between receptors (6c, 6e, and 6 f) with metal salts
(CuCl2, CuOTf2, and CdOAc2) at 315 nm is shown in Figure 3.
Figure 1. Image of 1/8 of the screened library. Only one bead displays
good association with 7 as evidenced from the strong coloration.
beads. To verify that the affinity was not due to binding of the
azo-dye portion of 7, library 6 was mixed with CuCl2 and
incubated with an ester variant of Disperse Red 1. Not a
single bead displayed binding to the Disperse Red 1 variant,
thereby indicating that binding was due to interactions
between 6 and the peptide portion of 7.
Six intensely colored beads were selected and sequenced
by Edman degradation. This revealed sequences 6a–6 f. It is
evident that a number of hydrophobic groups (Met, Trp, Phe,
Ala, and Ile) were important for binding 7, in addition to
acidic (Asp) and polar residues (Ser and Thr).
In this study, an equilibrium assay was developed to assess
the binding of the peptides to the receptors as monitored in a
96-well plate, rather than an “on-bead” kinetic assay in a
silicon chip, which was previously employed.[3d] This required
the synthesis of 6a–6 f for use in solution, and a combination
of solid- and solution-phase methods was used (see the
Supporting Information). The expected variations in the
binding constants of the peptide–receptor complexes were
based on a previous study from our research group performed
on an analogous copper receptor with amino acids and
tripeptides; binding constants obtained by spectroscopy
varied from 106 to less than 102 mÀ1 in 1:1 methanol/buffered
water at pH 7.4.[6] The optimal array was created using
receptors 6a–6 f (267 mm), each metalated in separate wells
with CuCl2 (534 mm), Cu(OTf)2 (267 mm), and Cd(OAc)2
Figure 3. Fingerprints created for peptides 1–5 by receptors 6c, 6e,
and 6 f, and CuCl2, CuOTf2, and CdOAc2 at 315 nm.
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These patterns facilitated the evaluation of the data set by
PCA.
PCA was performed on a data matrix consisting of DA
values at the wavelengths of choice for receptors 6a–6 f
(columns: 54 variables = receptors ” metal complexes ”
wavelengths) and analytes (rows: 18 variables = peptides ”
experimental trials) to discern patterns for the recognition
and the discrimination of the peptides. Four principal
components were extracted which accounted for approxi-
mately 98% of the variance. A plot of the factor scores of the
analytes for the first three principal components (PC1, PC2,
and PC3) is shown in Figure 4 A. Excellent classification of
the analytes and clustering of the experimental trials within
analyte groups, the desirable attributes of a PCA, are the
noteworthy features of this plot. Therefore, although the
bead-supported library 6/CuCl2 was screened with only a
single analogue of the target peptides, the resulting array gave
excellent pattern-based disrimination of all the peptides. The
power of the protocol is demonstrated by the ability to
differentiate the subtly different small peptides (1, 2, and 3) as
well the large tachykinins (4 and 5). Additionally, a significant
amount of the variance was distributed throughout PCs 1–3,
thus indicating a high degree of cross-reactivity between the
receptors in this array. This is a marked improvement over our
previous study in which the maximum amount of the variance
(91%) was carried by PC 1.
Closer inspection of this PCA data set by examination of
plots of PC 2 versus PC 1, PC 3 versus PC 1, and PC 3 versus
PC 2 (Figure 4B–D) revealed discernable trends with respect
to sequence properties (see Table 1 in the Supporting
Information) and analyte PC axes dependencies of the
peptides. With the exception of 2, the peptides are generally
distributed along PC 1 in accordance with their hydropho-
bicity. Plots of PC 2 versus PC 1 and PC 3 versus PC 1 reveal
that PC 2 is able to discriminate the tripeptides whereas PC 3
is able to discriminate the tripeptides as well as a-neurokinin
from its analogues (peptides 1 and 3). No correlation or axis
dependency relating to the theoretical pI value of a peptide
was ascertained. This finding meant that the response of the
array was not based solely on electrostatic interactions
between the receptors and peptides.
The most influential variables in the discrimination of
peptides 1–5, as measured by factor loading values along the
PC axes, were determined to be complexes formed from
receptors 6b and 6d with Cu(OTf)2 and CuCl2. This result is
consistent with findings that Asp residues form hydrogen
bonds and/or ion pairs with the N-H groups on the main chain
of peptides and lysine side chains, respectively. We postulate
that the polar Ser and Thr residues also participate in
hydrogen-bonding interactions with these peptides, which
are key to the recognition of these analytes. The presence of
the Phe residue also suggests that hydrophobic contacts play a
role. The preference for the copper salts is undoubtedly due to
their histidine-binding properties; four of the five peptides
terminate with a histidine residue.
Figure 4. A) PCA scores plot showing PC 1 versus PC 2 versus PC 3 for
peptides 1–5. PCA scores plots of B) PC 2 versus PC 1, C) PC 3 versus
PC 2, and D) PC 3 versus PC 1.
We speculated that in accordance with array-sensing
principles, a reduced library consisting of receptors 6a–6 f and
a single metal salt would be a less competent discriminating
agent than the full array of receptors and metal complexes. To
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2425; d) A. T. Wright, E. V. Anslyn, J. T. McDevitt, J. Am. Chem.
test this hypothesis, PCA was performed on these reduced
libraries (Supporting Information). In the case of Cd(OAc)2
and CuCl2, classification of all peptides was achieved, but with
reduced spatial separation between peptide groups and
clustering of experimental trials within each peptide group.
Interestingly for CuOTf2, there was a dramatic reduction in
both spatial separation and clustering, such that tachycinins 4
and 5 were not discriminated.
In summary, the implemention of a prescreening process,
and the addition of diversity through metal salts, created an
array of differential receptors with extraordinary discrimina-
tory capabilities. Short peptides, considered to be one of the
most challenging set of analytes, which varied in length and
kind by only a single amino acid, as well as tachykinins, which
share a high degree of sequence identity, were all successfully
classified. It is not yet clear if prescreening improves differ-
ential sensing schemes in general, but this is currently under
investigation in our laboratories with schemes that monitor or
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Received: March 20, 2007
Revised: July 30, 2007
Published online: September 26, 2007
Keywords: arrays · pattern recognition · peptides · sensors ·
.
supramolecular chemistry
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