From the Torrey Pines Institute for Molecular Studies,
San Diego, California 92121 and the
Department of
Pharmacology and Physiology, University of Rochester Medical Center,
Rochester, New York 14642
A combinatorial library of 6,250,000 tetrapeptides in the mixture based positional scanning format was
screened in binding assays for the three opioid receptors, µ,
,
and
. Three different binding profiles were found. Individual
peptides were synthesized representing all possible combinations of the
active amino acids identified from the screening data. New, highly
active peptides selective for each of the three receptors were chosen.
This study demonstrates the power of mixture-based combinatorial
libraries to identify distinctly different ligands for closely related
receptors.
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INTRODUCTION |
Synthetic combinatorial libraries have gained widespread
acceptance for the rapid identification of new drug leads. Complete combinatorial libraries contain all possible arrangements of building blocks used in their synthesis. Peptides were the first compound class
to be used in constructing such libraries (1-3). This laboratory first
demonstrated the application of soluble mixture based combinatorial libraries through the identification of the peptide
Ac-DVPDYA-NH2 from a library of 52 million
N-acetylated hexapeptides. The sequence corresponds to the
antigenic determinant recognized by the antibody 17DO9 (1).
Concurrently, a second combinatorial library containing 52 million
non-acetylated hexapeptides was subsequently used to identify ligands
for the opioid receptors, which were closely related to the natural
ligands methionine- and leucine-enkephalin (YGGFM, YGGFL) (4). In later
studies, six distinctly hexapeptide sequences that specifically bound
to the µ opioid receptor were identified from the acetylated and
nonacetylated libraries (5).
The combinatorial libraries described above are all mixture-based. Many
questions have arisen concerning the use of mixture-based libraries.
For example, how many different families of compounds for a particular
target exist and can they be identified in a given library? What are
the chances of missing the most active compound? What other compounds
are present or could be identified from the data that are not
immediately obvious? Are similar building blocks universally
replaceable? Peptides, because of their ease of synthesis, represent
the most convenient compound class for use in the study of
mixture-based combinatorial libraries. By achieving an appreciation for
the behavior of large mixtures through the use of peptide libraries, we
believe these same principles can be applied to virtually all other
compound classes (i.e. heterocycles and other small
molecules).
The opioid receptors represent a convenient system to investigate the
power of combinatorial libraries to identify distinctly different
ligands for related receptors. There are three primary opioid
receptors: mu (µ), delta (
), and kappa (
). All three receptors
have recently been cloned, and they belong to the seven-transmembrane G-protein-coupled family of receptors and have approximately 60% amino
acid sequence homology. Screening of the same combinatorial library in
separate assays selective for each of the three receptors provides not
only new ligands for these receptors but yields insights into the
ability of combinatorial libraries to discriminate between closely
related receptors.
A combinatorial library of 6,250,000 tetrapeptides, made using 50 different amino acids, was prepared in the positional scanning format
(6, 7). The use of positional scanning synthetic combinatorial
libraries (PS-SCLs)1 enables
the most active amino acids at each position of a peptide or
non-peptide to be determined directly from the initial screening data.
This information can then be used to synthesize highly active individual compounds. A PS-SCL of tetrapeptide amides used in the
current study consists of four separate sublibraries, each having a
single defined position (O) and three mixture positions (X) as follows:
O1XXX-NH2,
XO2XX-NH2,
XXO3X-NH2, and
XXXO4-NH2. The defined positions of
the mixtures making up each of the four separate sublibraries address a
single position in the tetrapeptide. It should be noted that each of
the four positional sublibraries are made up of the same 6,250,000 tetrapeptides. Screening the four sets of mixtures in the three
separate opiate specific assays yielded information about the most
important amino acids of each position in the tetrapeptide and led to
the identification of three different series of active individual
tetrapeptides selective for the µ,
, and
receptors.
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EXPERIMENTAL PROCEDURES |
Preparation of the Tetrapeptide PS-SCL
The PS-SCL used in this study is composed of 6,250,000 tetrapeptides and contains four sublibraries, in which one of the four positions is defined with a single amino acid (O) and the
three remaining positions are a mixture of 50 different L-,
D-, and unnatural amino acids (X). The
tetrapeptides were synthesized using the solid phase simultaneous
multiple peptide synthesis approach (8) on methylbenzhydrylamine
polystyrene resin using t-butoxycarbonyl-protected amino
acids. Mixture resins (X) were prepared using mixtures of
t-butoxycarbonyl-protected amino acids at each coupling
step. Each of the amino acids was present in a concentration that
yielded close to equimolar coupling of each amino acid. The ratio of
the concentrations of the individual amino acids used to yield this
approximate equimolar coupling was pre-determined using reverse
phase-high pressure liquid chromatography to compare mixture profiles
relative to standard mixtures synthesized using the divide, couple, and
recombine method (1) as detailed in Ref. 9. Coupling completion was
determined using Kaiser's ninhydrin test (10). Side chain deprotection
and cleavage from the resin support were achieved using low hydrogen
fluoride (11) and high hydrogen fluoride (12) procedures. The 200 peptide mixtures were individually extracted with water, lyophilized, and resuspended in water at a final concentration of 10 mg/ml.
Synthesis of Individual Peptides
Peptides were synthesized using Fmoc
(N-(9-fluorenyl)methoxycarbonyl)/t-butyl
chemistry on a COMPAS 242 multiple peptide synthesizer (Spyder
Instruments, San Diego). The peptides were synthesized on
TentaGel resin in polypropylene mesh packets using inclusion volume
synthesis. Reagent and wash solutions were removed by centrifugation (13, 14).
Receptor Binding Assays
µ Receptor Assay--
Membrane homogenates were prepared from
rat brains. Brains were homogenized in 40 ml of Tris-HCl buffer, 50 mM, pH 7.4, 4 °C (buffer A), and centrifuged (Beckman
J2-HC, 35,300 × g) for 10 min. The pellets were
resuspended in fresh buffer and incubated at 37 °C for 40 min.
Following incubation, the suspensions were centrifuged as before, the
resulting pellets resuspended in 100 volumes of Tris buffer, and the
suspensions combined. Membrane suspensions were prepared and used on
the same day. Protein content of the crude homogenates was determined
by the method described by Bradford (15). Each assay tube contained 0.5 ml of membrane suspension, 3 nM
[3H-D-Ala2,MePhe4,Gly5-ol]enkephalin
(DAMGO) and 0.08 mg/ml mixture, and 50 mM Tris-HCl in a
final total volume of 0.65 ml. Assay tubes were incubated for 1 h
at 25 °C. Unlabeled DAMGO was used as a competitor to generate a
standard curve and determine nonspecific binding. The reaction was
terminated by filtration through GF-B filters on a Tomtec harvester
(Orange, CT). The filters were subsequently washed with 6 ml of 50 mM Tris-HCl, pH 7.4 (buffer B), at 4 °C. Bound
radioactivity was counted on a Wallac Beta-plate Liquid Scintillation
Counter (Piscataway, NJ).
Receptor Assay--
Rat brains were homogenized as described
above using 40 ml of 50 mM Tris-HCl buffer, 100 µM phenylmethylsulfonyl fluoride, 5 mM
MgCl2, 100 nM Ac-rfwink-NH2 (16), 1 mg/ml bovine serum albumin, pH 7.4, 4 °C (buffer A). Homogenates
were centrifuged and incubated as above. Each assay tube contained 0.5 ml of membrane suspension 3 nM tritiated
[D-Ser2,Leu5,Thr6]enkephalin
(DSLET) in a total volume of 0.65 ml. Assay tubes were incubated for
2.5 h at 25 °C. The assay was terminated, filtered, and counted
as above. Unlabeled DSLET was used as a competitor to generate a
standard curve and determine nonspecific binding.
Receptor Assay--
Guinea pig cortices and cerebella were
homogenized in 40 ml of buffer A. Homogenates were centrifuged and
incubated as above. Each assay tube contained 0.5 ml of membrane
suspension, 3 nM tritiated U69,593 in a total volume of
0.65 ml. Assay tubes were incubated for 2.5 h at 25 °C. The
assay was terminated, filtered, and counted as above. Unlabeled U50,488
was used as a competitor to generate a standard curve and determine
nonspecific binding.
Adenylyl Cyclase Assay
The human SH-SY5Y neuroblastoma cell line was a generous gift
from Dr. David K. Grandy (Vollum Institute for Advanced Biomedical Research, Portland, OR). The R1.G1 mouse thymoma cell line was obtained
from ATCC (Rockville, MD) and has been shown to express the
opioid
receptor but not the µ or
receptors (17). The cells were cultured
in RPMI 1640 medium, buffered with 12.5 mM HEPES, pH 7.2, and containing 300 µg/ml L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, 50 µM
2-mercaptoethanol, 60 µM 2-ethanolamine, and 10%
iron-supplemented bovine calf serum in 5% CO2 at 37 °C.
SH-SY5Y neuroblastoma cells were cultured in media containing 10 µM retinoic acid for 6 days before harvesting in order to
differentiate the cells as described previously (18). Cell membranes
were prepared for use in the adenylyl cyclase assays as described
previously (19). After the initial centrifugation at 200 × g for 15 min at 4 °C, the cells were resuspended in
sucrose buffer (0.32 M sucrose, 40 mM HEPES, 2 mM EGTA, pH 7.6). Cells were centrifuged again at 200 × g and then homogenized in sucrose buffer with five
strokes of a Dounce homogenizer. Membranes were centrifuged at
22,000 × g for 20 min at 4 °C, followed by
resuspension in sucrose buffer. The protein concentration was
determined by the method of Bradford (15) using bovine serum albumin as
standard. Cell membranes at a protein concentration of 1-4 mg/ml were
stored at
80 °C until use.
Membranes were incubated in a final volume of 100 µl of 40 mM HEPES, containing 15 units of creatine phosphokinase, 20 mM phosphocreatine, 1 mM
1,10-o-phenanthroline, 60 µM
isobutylmethylxanthine, 50 µM ATP, 50 µM
GTP, 3 mM MgCl2, and 100 mM NaCl.
Agonists and antagonists were included at final concentrations as
stated in the text. Naloxone, ICI 174,864, and nor-binaltorphimine were used to block µ,
, and
opioid receptors, respectively. The reaction was initiated by the addition of 36 µg of membrane protein. After 15 min at 30 °C, the reaction was stopped by the addition of
40 µl of cold 30% potassium bicarbonate, and then the membranes were
centrifuged at 12,00 × g for 4 min at 4 °C in a
microcentrifuge.
The amount of cyclic AMP present in 100 µl of the supernatant,
equivalent to the cyclic AMP produced by 15 µg of membrane protein,
was determined by the use of a cyclic AMP kit (Diagnostic Products
Corp., Los Angeles, CA). This procedure, which uses a cyclic
AMP-binding protein in a competitive protein binding assay, is based on
the method of Tovey et al. (20) and was used with the
following modification. [3H]Cyclic AMP (specific activity
31.4 Ci/mmol), obtained from Amersham Pharmacia Biotech, was used
instead of the [3H]cyclic AMP included in the assay kit.
[3H]Cyclic AMP, 0.9 µCi, was added into 6 ml of
H2O, and 100 µl of the diluted [3H]cyclic
AMP was added to the assay tubes. The final supernatants were counted
in 10 ml of Ecolite (+) scintillation fluid (ICN Pharmaceuticals,
Covina, CA).
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RESULTS |
The tetrapeptide PS-SCL was screened in each of the three separate
opioid receptor binding assays described above. Each mixture contained
125,000 tetrapeptides (503). The mixtures were initially
screened at a fixed concentration (0.08 mg/ml) at this mixture
concentration each peptide was present at a concentration of .06 nM. Each of the 200 mixtures (50 for each of the four
positions) making up the library was screened for its ability to
inhibit binding of the tritiated ligand to brain homogenates.
µ Receptor--
The library was screened again at a 10-fold
lower concentration (0.008 mg/ml) (Fig.
1), since too many mixtures inhibited >90% of [3H]DAMGO binding in the initial screening.
IC50 values were subsequently calculated for all mixtures
that inhibited >90% of radioligand binding from each of the four
positions (Fig. 2). The most active mixture from the series in which the first position was defined was
YXXX-NH2 (IC50 = 638 nM). This mixture was 3-fold more active than the second
most active mixture (L-Nal)XXX-NH2
(IC50 = 1946 nM) and 7-fold more active than
the third most active mixture FXXX-NH2
(IC50 = 4526 nM). There was a 25-fold
difference in activity between the most active and least active mixture
tested.

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Fig. 1.
Screening of the non-acetylated tetrapeptide
PS-SCL for the ability to inhibit the binding of selective radiolabels
to the µ, , and receptors. The µ receptor was labeled
using [3H]DAMGO, and the receptor was labeled using
[3H]DSLET. Both assays were carried out using rat brain
homogenates. The receptor was labeled using
[3H]U69,593 and guinea big brain homogenates. Each panel
represents one of the four positional SCLs (i.e. position
one SCL is O1XXX-NH2).
Each bar within a panel represents percent inhibition by a
peptide mixture defined in the O position with one of 50 amino acids. Amino acids are listed in the footnote to Table II. While
these graphs illustrate active peptide mixtures, the choice of amino
acids for the synthesis of individual peptides was based on
IC50 values.
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Fig. 2.
IC50 values of selected mixtures
found to be the most active in the initial screeing of the tetrapeptide
PS-SCL at the µ receptor. Each panel represents one of the
four positional SCLs (i.e. position one SCL is
O1XXX-NH2). Each
bar represents the affinity of the mixture for the µ receptor expressed as log of the IC50 value
(M) ± S.E. Amino acids defined in the mixtures
(O) are represented on the x axis.
Arrows indicate the mixtures from which amino acids were
chosen to make individual compounds.
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IC50 values were calculated for seven mixtures in which the
second position was defined (XOXX-NH2). The most
active mixtures found were all defined with D-amino acids.
These mixtures exhibited a smaller range between the most and least
active than for the first position.
X(D-Nve)XX-NH2
(IC50 = 690 nM) was the most active mixture
found. The second, third, and fourth most active mixtures were within a
2-fold difference in activity:
X(D-Nle)XX-NH2
(IC50 = 1112 nM),
yXXX-NH2 (IC50 = 1378 nM), and rXXX-NH2 (IC50 = 1507 nM) (where y indicates D-tyrosine and r
indicates D-arginine).
IC50 values were calculated for seven mixtures in which the
third position was defined (XXOX-NH2). Six of
the mixtures exhibited activities below 2,000 nM, and all
six contained L-amino acids in the defined position. The
three most active mixtures found were
XXFX-NH2 (IC50 = 824 nM), XXGX-NH2
(IC50 = 1119 nM), and XXWX-NH2 (IC50 = 1227 nM).
IC50 values were calculated for seven mixtures in which the
fourth position was defined (XXXO-NH2). The most
active mixture found was
XXX(L-Nal)-NH2 (IC50 = 279 nM). The second most active mixture was 3-fold less
active (XXXW-NH2; IC50 = 850 nM) than the most active mixture, and the third most active
mixture found (XXXF-NH2; IC50 = 1545 nM) was over 5-fold less active than the most active
mixture. The amino acids chosen to make 32 (1 × 4 × 4 × 2) individual peptides are listed in Table
I. Ki values obtained
for these peptides in the µ receptor binding assay are given in Table
II. All 32 of the peptides were found to
have high affinity for the µ receptor (Ki values
were < 15 nM). Three of the amino acids in the most
active peptide
Tyr-(D-Nve)Gly(L-Nal)-NH2 (Ki = 0.4 nM) were the most active amino
acids found for their particular position, whereas glycine in the third
position was the second most active amino acid found for that position. The four D-amino acids chosen for the second position were
found to be replaceable (i.e. peptides which differed only
by the amino acid at this position had very similar activities).
Whereas all four amino acids chosen in the third position yielded
active peptides, those with small amino acid side chains (glycine and
alanine) were more active than those with aromatic side chains.
L-Naphthylalanine and L-tryptophan were found
to be replaceable at the fourth position. The general motif of active
peptides identified at the µ receptor was Tyr-(D-amino
acid)(L-amino acid with small side chain)
(L-aromatic)-NH2. Since all of the peptides
synthesized were found to be active, additional µ-selective peptides
are likely to be identified from this library. The selectivity ratios
(Ki of peptide at
or
receptor/Ki value of peptide at the µ receptor) of
the 32 peptides are illustrated in Fig.
3A. All 32 peptides were found
to be µ selective; none of the ratios were less than 1. The most
µ-selective peptide found was YrAW-NH2 (Rank 13 in Table
II). All peptides with D-arginine at the second position exhibited excellent µ selectivity. These µ-selective peptides were
generally more active at the
receptor than at the
receptor.
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Table I
Amino acids chosen for synthesis of individual compounds for the µ receptor
Number of individual tetrapeptides, 1 × 4 × 4 × 2 = 32.
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Table II
Ki values for individual peptides identified from the
tetrapeptide PS-SCL in an assay selective for the µ receptor
The affinities at the µ receptor of 32 tetrapeptides, representing
all possible combinations of the amino acids chosen, are given. Binding
conditions are detailed under "Experimental Procedures." Peptides
are ranked by activity
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Fig. 3.
Selectivity ratios of Ki
values for peptides found from screening the library in the µ receptor assay (A), the receptor assay (B),
and the receptor assay (C). The selectivity ratio
was determined using the following formula: Ki value
at receptor A/Ki at receptor
B, where A is either of the two alternate
receptors, and B is the receptor for which the peptides were
synthesized. The ratios are expressed in log scale; the higher the
value the greater the selectivity of the compound for the particular
receptor. Values less than 0 represent ratios of less than 1, which
indicates that the peptides bind preferentially to another receptor. An
asterisk (*) indicates that the peptide was inactive at the
highest concentration tested, and Ki values could
not be determined.
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Receptor--
After an initial screening (0.08 mg/ml) in a
-selective receptor binding assay using [3H]DSLET as
radioligand, the tetrapeptide PS-SCL was screened again at a 10-fold
lower concentration (Fig. 1). IC50 values were calculated for those mixtures that inhibited >60% of [3H]DSLET
binding for positions 1 and 2 and >70% of [3H]DSLET
binding for positions 3 and 4 (Fig. 4).
The most active of the 10 mixtures tested at position 1 included
YXXX-NH2 (IC50 = 2468 nM), WXXX-NH2 (IC50 = 6250 nM), wXXX-NH2 (IC50 = 7906 nM; w indicates D-tryptophan). The most
active mixture was approximately 3-fold more active than the second
most active mixture. Although the active mixtures identified were
similar to those found in the µ receptor assay, they were less
active, e.g. the mixture YXXX-NH2 was
4-fold more active in the µ versus the
receptor assay.

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Fig. 4.
IC50 values of selected mixtures
found to be the most active in the initial screening of the
tetrapeptide PS-SCL at the receptor. Each panel
represents one of the four positional SCLs (i.e. position
one SCL is O1XXX-NH2).
Each bar represents the affinity of the mixture for the µ receptor expressed as log of the IC50 value
(M) ± S.E. Amino acids defined in the mixtures
(O) are represented on the x axis.
Arrows indicate the mixtures from which amino acids were
chosen to make individual compounds.
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Active mixtures with defined amino acids in the second position were
also found to be similar to those identified in the µ assay.
IC50 values were determined for 11 mixtures. The five most active mixtures found contained D-amino acids:
XyXX-NH2 (IC50 = 4990 nM), XfXX-NH2
(IC50 = 7830 nM),
XwXX-NH2 (IC50 = 8099 nM) X(D-Nve)XX-NH2
(IC50 = 9970 nM), and
X(D-Nal)XX-NH2
(IC50 = 11795 nM).
The 10 mixtures tested in which the third position was defined were
found to be less active than those of the remaining three positions,
and no mixture was found to have an IC50 value lower than
5,000 nM. The four most active mixtures were
XX(aAba)X-NH2 (IC50 = 6474 nM), XXGX-NH2
(IC50 = 7115 nM),
XX(L-Cha)X-NH2
(IC50 = 8462 nM), and
XXMX-NH2 (IC50 = 9085 nM). There was very little difference in affinity between
the most active mixtures found at the third position; the 10 mixtures
had IC50 values between 6,000 and 10,000 nM.
IC50 values were calculated for nine mixtures in which the
fourth position was defined. Unlike the results found in the µ receptor assay, the two most active amino acids found at the fourth position were positively charged: XXXR-NH2
(IC50 = 4529 nM) and XXXK-NH2 (IC50 = 9026 nM). Mixtures ranked third and fourth at this position were
aromatics, as was also found in the µ receptor assay
[XXXW-NH2 (IC50 = 6966 nM) and XXXF-NH 2 (IC50 = 7395 nM)]. The amino acid combinations chosen for the
synthesis of 60 individual peptides are listed in Table
III. Peptides that had
Ki values below 500 nM in the
-selective assay are shown in Table IV. Only three of the peptides were found
to have activity under 10 nM. The
selectivity of the
peptides is shown in Fig. 3B. Twelve of the peptides had
greater activity in the µ assay than in the
-selective assay
(ratio of less than 1). This is not altogether surprising as many of
the mixtures with defined amino acids chosen for the
peptides were
more active in the µ receptor assay. It is also not surprising that
the most
-selective peptide found, Wy(aAba)R-NH2, (Rank
2 in Table III) contained L-arginine in the fourth
position, since this amino acid was ranked first in the
receptor
screening and ranked 17 in the µ receptor screening. Many of the
peptides tested were virtually inactive at the
receptor at the
highest concentration tested (10,000 nM). These data points are indicated by an asterisk in Fig. 3B.
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Table III
Amino acids chosen for synthesis of individual compounds for the
receptor
Number of individual tetrapeptides, 2 × 3 × 5 × 2 = 60.
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Table IV
Ki values for selected individual tetrapeptides derived
from PS-SCL using a selective assay
Sixty peptides, representing all possible combinations of the amino
acids chosen, were synthesized. Ki values were
determined. Binding conditions are detailed under "Experimental
Procedures." The 20 most active peptides are ranked by affinity.
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Receptor--
For the
receptor, the library was screened
at an initial concentration of 0.08 mg/ml in guinea pig brain
homogenates using [3H]U69,593 as radioligand (Fig. 1).
IC50 values were subsequently calculated for those mixtures
which inhibited >90% of [3H]U69,593 binding for
positions 1-4 (Fig. 5). The most active mixtures found in the
-selective assay do not bear any resemblance to those found in the µ and
receptor assays. The 12 mixtures tested in which the first position was defined ranged in
IC50 value from 3,000 to 22,000 nM. The four
most active mixtures contained D-amino acids at this
position (fXXX-NH2 (IC50 = 3615 nM); (D-Cha)XXX-NH2 (IC50 = 4045 nM);
(D-Nle)XXX-NH2 (IC50 = 5936 nM); and iXXX-NH2 (IC50 = 6910 nM)).

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Fig. 5.
IC50 values of selected mixtures
found to be the most active in the initial screening of the
tetrapeptide PS-SCL at the receptor. Each panel
represents one of the four positional SCLs (i.e. position
one SCL is O1XXX-NH2).
Each bar represents the affinity of the mixture for the µ receptor expressed as log of the IC50 value
(M) ± S.E. Amino acids defined in the mixtures
(O) are represented on the x axis.
Arrows indicate the mixtures from which amino acids were
chosen to make individual compounds.
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Eleven mixtures were tested in which the second position was defined.
As observed for the first position, D-amino acids were also
favored. The most active mixture,
X(D-Nal)XX-NH2
(IC50 = 1545 nM), was 4-fold more active than
the second most active mixture XfXX-NH2 (IC50 = 4115 nM).
The most active mixtures found for the third position were
XX(D-Nle)X-NH2
(IC50 = 2346 nM) and
XXlX-NH2 (IC50 = 3019 nM). Eight of the nine mixtures tested at this position
were found to have IC50 values of less than 10,000 nM. The mixture ranked third at this position contained
L-tryptophan, XXWX-NH2
(IC50 = 5115 nM), whereas all other mixtures
contained D-amino acids.
Seven mixtures were tested for the fourth position. As was found in the
receptor assay, positively charged amino acids were found to have
the greatest activity in the fourth position
[XXXr-NH2 (IC50 = 1526 nM) and XXXXk-NH2 (IC50 = 2013 nM)]. The third most active mixture was
XXX(D-Cha)-NH2 (IC50 = 2929 nM). The amino acids chosen for inclusion in the
synthesis of individual peptides are shown in Table
V. Twenty four peptides were synthesized, and their Ki values are given in Table
VI. Fourteen of the 24 peptides had
Ki values below 50 nM, 11 of which were
below 10 nM. The most active peptide was found to be
ff(D-Nle)r-NH2 (Ki = 1.2 nM). D-Phenylalanine and
D-norleucine were replaceable at the first position.
Surprisingly, D-phenylalanine and
D-naphthylalanine were replaceable at the second position. D-Norleucine and D-isoleucine were replaceable
at the third position; however, none of the peptides containing
L-tryptophan in the third position had a
Ki value below 1000 nM. This suggests the existence of another family of
ligands containing
L-tryptophan in the third position. The active peptides
(those ranked 1-16) were also highly selective for the
receptor
(Fig. 3C). The most
selective peptides found had µ/
and µ/
ratios of greater than 5,000.
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Table V
Amino acids chosen for synthesis of individual compounds for the
receptor
Number of individual tetrapeptides, 2 × 2 × 3 × 2 = 24.
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Table VI
Ki values for individual tetrapeptides derived from the
PS-SCL using a selective assay
The affinities at the receptor of 24 peptides, representing all
possible combinations of the amino acids chosen, are presented. Binding
conditions are detailed under "Experimental Procedures." Peptides
are ranked by affinity.
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Adenylyl Cyclase Assay--
Opioid agonists inhibit adenylyl
cyclase activity, resulting in reduced levels of cyclic AMP (cAMP) (21,
22). An adenylyl cyclase assay using SH-SY5Y neuroblastoma or R1G1
thymoma cell line membranes was used to rapidly determine whether a
peptide was an opioid agonist or antagonist. The opioid receptors
expressed on the SH-SY5Y cell line after culturing in the presence of
10 µM retinoic acid are approximately 85% of the µ type and 15% of the
type
(18).2 The R1G1 thymoma cell
line contains only the
receptor subtype. A reduction of cyclic AMP
levels to less than 70% of the basal cAMP levels was regarded as being
indicative of an opioid agonist effect, provided that the inhibition of
cAMP was blocked by an opioid antagonist. Naloxone was used for µ receptors and nor-binaltorphimine was used for
receptors. Their
ability to inhibit the accumulation of cAMP was similar to that of
DAMGO. Inhibition of cAMP was antagonized by naloxone but not the
-specific antagonist ICI 174,864, indicating that the reduction in
cAMP was mediated by µ receptors. The most active peptides found for
the µ and
assays have been tested for ability to inhibit cAMP
accumulation (Table VII). All peptides tested were found to be agonists at their respective receptor. The
percent inhibition of cAMP was similar to the standard ligands, indicating that their efficacy at the receptor is similar to standard ligands.
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Table VII
Inhibition of cyclic AMP levels in SH-SY5Y human neuroblastoma cell
membranes or R1G1 thymoma cell membranes
Tetrapeptides with high selectivity for µ and receptors were
tested for agonist activity, as measured by inhibition of cyclic AMP,
in cell lines containing large populations of µ (SH-SY5Y) or (R1G1) receptors. Assay conditions are detailed under "Experimental
Procedures." Peptides were tested at a final concentration of 100 µM. A µ-selective agonist, DAMGO (1 µM),
and a -selective agonist, ( )-U50,488 (10 µM), were
used as standards. Inhibition of cyclic AMP was found to be reversed by
the opioid antagonist Naloxone (500 µM) or the
-selective antagonist nor-binaltorphimine (500 µM),
indicating that the cyclic AMP inhibition was mediated by opioid
receptors.
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DISCUSSION |
The opiate receptor systems used in the current study have been
described in our earlier work (4, 7, 16, 23). In this laboratory
studies involving combinatorial libraries for the identification of
opioid ligands have focused on the µ receptor. These studies have a
dual purpose as follows: first to find new ligands and expand our
knowledge of the opioid receptors, and second to explore the use of
combinatorial libraries made up of large mixtures. The ability to
identify highly active individual ligands using iterative and
positional scanning deconvolution strategies was demonstrated for the µ receptor (4, 7), from which novel µ-selective antagonists, the
acetalins (23) and an all D-amino acid agonist
(Ac-rfwink-NH2) (16), were found. More recently we have
used the µ receptor assay to demonstrate how a number of different
sequences could be identified from such libraries. In the present
study, the same PS-SCL containing 6,250,000 tetrapeptides was screened
in receptor assays for the µ,
, and
opioid receptors. The
screening data obtained using this same library in three closely
related receptors yielded three different binding profiles. The
mixtures screened were found to have the greatest activity in the µ receptor relative to their activities at the
and
receptors. The
general motif of active peptides identified for the µ receptor was
Tyr-(D-amino acid)(L-amino acid)(aromatic side
chain)-NH2. Tyrosine was the only amino acid chosen for the first position due to the excellent specificity found for this amino
acid. The L-amino acids at the third position were either glycine or the aromatic amino acids phenylalanine or tryptophan. This
peptide motif is similar to the truncation analogs of dermenkephalin and deltorphin described previously, YOFG where O = D-methionine, D-alanine, or
D-tyrosine (24). The peptide YmFG-NH2 was
previously identified from a tetrapeptide library using an iterative
deconvolution process (25) but was not identified here because
D-methionine was not included in this library. The most
µ-selective peptides found in this study contain
D-arginine at the second position and are similar to
peptides reported previously (YrFK-NH2 (DALDA) (26)). There
are other reported tetrapeptides with high affinity for the µ receptor that were not identified in this report (e.g. YPWF-NH2 (27) and WWPR-NH2 (5)). YPWF-NH2 (27)
would have been identified using a tetrapeptide library made up only of
20 L-amino acids or if more amino acids were chosen at each
position. We are currently working on deconvolution strategies that
would minimize the peptides required to be synthesized while maximizing the number of amino acids chosen at each position. In all three assays,
the number of amino acids chosen for the synthesis of individual
peptides was restricted since the number of combinations rises
exponentially with the number of amino acids at each position (i.e. 81 tetrapeptides for 3 amino acids are chosen at each
position, 256 tetrapeptides if 4 amino acids are chosen at each
position, and 625 tetrapeptides if 5 amino acids are chosen at each
position). This restriction clearly results in a limitation in the
identification of additional active sequences. This can be seen in the
current report in which active sequences for the µ receptor were
identified when the same library was screened against the
receptor.
It should be noted that these sequences would have been identified from
the µ screening data if a greater number of combinations had been
synthesized. Furthermore, due to limited resources a subjective
choice of amino acids is involved. In choosing the amino acids at
each position, one endeavors to compromise between covering the
greatest range in chemical diversity and avoiding the choice between
two similar amino acids which are not replaceable.
The majority of the combinations identified from the data for the
-selective assay were found to be more active in the µ receptor
assay. A common motif for
-selective peptides was
WOOR-NH2, as can be seen in Table
VIII, which shows the effect on
Ki values at the µ and
receptors of
substitution analogs of the
-selective peptide
Wy(aAba)R-NH2. There is no simple correlation between
chemical similarities in amino acids and selectivity. Replacement of
tryptophan at position 1 by tyrosine retains selectivity, but
replacement of tyrosine by tryptophan at position 2 results in a
substantial reduction of selectivity. Replacement of
L-norvaline at position 3 by
L-cyclohexylalanine results in reduction of selectivity, whereas a double replacement of tyrosine at position 1 and by L-cyclohexylalanine at position 3 completely reverses
the selectivity, yielding a µ-selective peptide. This illustrates the
dangers inherent in making broad statements on the replaceability of
amino acids of similar chemical character.
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Table VIII
Influence of amino acid substitution on µ/ selectivity
Conservative amino acid substitutions on a peptide sequence may affect
peptide affinity, selectivity, or both.
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The similarities between the peptides found to have activity µ and
receptors appear to indicate a closer relationship between the two
receptors, as opposed to the peptides found to be active at the
receptor. DSLET is also known to bind to µ receptors, but we have
included 100 nM Ac-RFWINK-NH2 (16),
a highly selective µ peptide, in the assay buffer in order to
adequately block the radioligand from binding to µ receptors.
Furthermore, the library was also screened using
[3H]Naltrindole as a
-selective radiolabel, and no
significant library profiles differences were observed.
The sequences found from screening the tetrapeptide PS-SCL in an assay
selective for the
receptor are perhaps the most interesting of this
study. They are unlike any peptides reported to have activity at
receptors and would not have been identified by classical structure-activity studies. A general motif is not easily identified. The sequence clearly appears to favor D-amino acids at all
four positions. Also, any peptide synthesized containing
L-tryptophan at position 3 had poor activity. The first
position accepts an aromatic side chain, D-phenylalanine,
or an aliphatic side chain, D-norleucine, but whether this
may be generalized to all similar amino acids is not known. Peptides
with Ki values below 10 nM were found
with D-arginine in the fourth position. Sequences that
differed only in the fourth position were always more active with
D-arginine than with D-cyclohexylalanine.
Active peptides identified for the kappa receptor were highly
selective. A study involving the synthesis of 1,000-3,000 individual
tetrapeptides based on the data presented in this study is
underway.
In this study, highly active individual compounds and highly µ- and
-selective compounds were rapidly identified from a large mixture-based positional scanning combinatorial library. All of the
most active peptides tested were found to be agonists. It has yet to be
determined if these peptides are capable of crossing the blood-brain
barrier, but it is expected that the presence of D-amino
acids in their sequences will prolong their biological half-lives. The
peptides identified may prove useful for pain management, as
compounds have come into focus in recent reports as having greater
efficacy in analgesia for women (28). On the other hand, if these
peptides do not cross the blood-brain barrier, they may be useful in
attenuating the pain and/or progression of adjuvant arthritis (29).
This study illustrates not only the power of the positional scanning
concept for the rapid identification of new ligands but also how
distinct ligands may be rapidly identified for closely related
receptors.
We thank Amy Bower, David Dale, Christa
Schoner, and Kevin Hill for technical assistance, and Eileen Weiler for
editorial assistance.