(Received for publication, January 31, 1995; and in revised form, April 28, 1995)
From the
Recently, the rat neurotensin receptor and the two human
neurotensin receptor clones (differing by one amino acid residue) have
been isolated. We present results with 33 newly synthesized neurotensin
analogs. We have evaluated their binding potency at the three
neurotensin receptor clones by determining equilibrium dissociation
constants and coupling to phosphatidylinositol turnover. Our work
focused on position 8 and 9 substitutions as well as position 11 of the
neurotensin hexamer NT8-13. The results presented include: 1) the
development of a compound that is species selective, with a binding
potency at the rat receptor that is 20-fold more potent than at the
human receptor; 2) the development of a pair of stereoselective
compounds with the L-isomer exhibiting 190-700-fold more
potency than the D-isomer; and 3) the development of an
agonist that has a K
After it was first isolated(1) , the tridecapeptide
neurotensin (NT), ( Recently, the rat NT receptor was molecularly
cloned from adult rat brain(18) . It consists of 424 amino
acids. Subsequently, the human NT receptor was molecularly cloned (19) from a human tumor cell line that originated in the colon.
Our laboratory also cloned the human NT receptor; however, the source
of the receptor was the substantia nigra of the brain(20) . For
purposes of this paper, we have designated the clone from Vita's
group as hNTR(Leu), while the respective human clone from our
laboratory will be referred to as hNTR(Phe). This reflects the one
amino acid difference (AA Early in the study of NT,
reseachers observed that only the last 6 amino acid residues of the
peptide were needed for function(21) . For the present study,
we synthesized a series of 33 NT 8-13 peptide analogs. We have
divided the compounds into two groups based on their substitutions.
Previous results in our laboratory indicated that we could replace
Arg We report here the
results of our findings: 1) the development of a compound that is
species selective, exhibiting a binding potency at the rat receptor
that is 20 fold more potent than at the human receptor; 2) the
development of a pair of stereoselective compounds with the L-isomer exhibiting 190 fold and 700 fold more potency than
the D-isomer at the human NTR and rat NTR respectively; and 3)
the development of an agonist that has a K
For use
in binding assays, crude membranal preparations were prepared by
centrifugation of the cellular pellet at 35,600
NT(8-13) derivatives, other than those involving
substitutions with ornithine, are listed in Table 1. The
structures of the ornithine derivatives of NT(8-13) are found in Table 2. We have numbered the compounds for easy reference.
We tested the effect of substituting
cyclohexylalanine (Fig. 1B) in position 11 (NT21). This
residue is similar to L-Phe except that there are no pi
electrons on the 6-membered ring. A comparison of K
Figure 1:
A,
chemical structures of position 8 and 9 substitutions. B,
chemical structures of position 11
substitutions.
For the most potent
analogs synthesized, NT2 and NT3, changes in potency occurred when the
substitutions were switched from position 8 to position 9 of the
NT(8-13) parent peptide. Thus, moving L-Lys from
position 8 to position 9 (NT2 versus NT10, i.e.L-Lys Next, we tested the
ability of these compounds to stimulate neurotensin receptors by
measuring the release of inositol phosphates with intact cells
incubated with these compounds. All peptides tested were full agonists (Table 3). NT3 was clearly the most potent at stimulating PI
turnover of all the compounds tested (EC
Figure 2:
Correlation between K
In the doubly substituted hexamers NT25, NT26, NT29,
and NT31, the L-isomers in position 9 again showed a much
higher affinity than did the D-isomers. Introduction of D-Orn Selected peptides were tested for their ability to stimulate PI
turnover (Table 4). All the hexamers tested were full agonists.
Dose-response curves for the stereoisomers NT24 and NT27 and PI
turnover yielded EC
Figure 3:
Competition binding between
[
Figure 4:
Dose-response curves for NT and NT19
stimulated [
The potencies of NT13, which has L-Trp in
position 11, decreased in relation to NT1 by 23-fold at the hNTR(Leu)
and only 2-fold at the rNTR. The K In a
comparison of K
Figure 5:
Correlation between K
With regard
to the ornithine NT(8-13)-substituted analogs, exchanging the D or L form of ornithine in position 8 was equally
effective and showed little difference at either the human or the rat
NTR (see Table 4, NT22 and NT23; and NT25 and NT26). However,
when the same substitutions were made in position 9, binding potency
changed markedly with the addition of the D-isomer. Thus, at
the hNTR, NT24 had an affinity that was 190-fold more potent than that
of its stereoisomer, NT27 (Table 4). At the rNTR, the steric
effect was more substantial, with NT24 exhibiting an affinity that was
700-fold greater than that for NT27. In Fig. 6we have
compared the K
Figure 6:
Correlation
between K
Figure 7:
Correlation between K
Our research is aimed at developing potential new compounds
for the treatment of certain neuropsychiatric diseases. To that end, we
synthesized and tested for activity a series of 33 peptides based on a
fragment of the neurotensin molecule, namely, NT(8-13). Our
studies were aided by the availability of the molecularly cloned rat
neurotensin receptor and two forms of the molecularly cloned human
neurotensin receptor. Many of the compounds synthesized had
substitutions in position 8 and 9. Previously, our group showed that
substitution of D-Lys for L-Arg We were interested to
see the effect on binding of further shortening of the side chains for
substitutions at positions 8 and 9. Therefore, we synthesized another
set of NT analogs with the compound L-2,4-DAB replacing the
native L-Arg in positions 8 and 9. The side chain on DAB is
one methyl group shorter than on ornithine (see Fig. 1A). Our results showed that residue 9 was more
affected by side chain length than was residue 8. For the novel
substitutions at position 9, the rank order of potency at both the
human and rat receptors was NT3 > NT24 > NT10 > NT27. This
order corresponds to the increasing chain length of the side group on
residue 9. NT3 (DAB Possibly the most significant and
serendipitous discovery in this study was the finding that
[L-Nal A review of the K From the
correlation of all K [L-Nal For
all the compounds with position 11 modifications, the K We were interested to determine the binding effect of
pi electron density on the side chain of residue 11. The rank order for
the compounds with similar position 11 substitutions according to
electron density of the aromatic ring is NT1 > NT14 > NT11 >
NT21 (see Fig. 1B). Considering that the binding
potencies of NT14 and NT11 are not significantly different, this rank
order also defines the rank order of binding potency at the hNTR(Leu) (Table 3). With no pi electrons on the side chain of residue 11,
NT21 had an extraordinarily low binding affinity compared to NT1 or to
NT14 (a decrease of 5000- and 200-fold, respectively, at the
hNTR(Leu)). At the rNTR the rank order of potency of this same set of
compounds was the same, that is, NT1 > NT14 > NT11 > NT21 (Table 3). However, the magnitude of change in binding potency
was not as striking for NT1 versus NT21 (1800-fold lower
affinity) at the rat receptor. These results suggest that the presence
of pi electrons on the side chain of residue 11 are important for
binding at the NTR. Additionally, electronic density may be more
critical to binding at the hNTR(Leu) than at the rat receptor. Finally,
we and others have noted the importance of position 11 modifications on in vivo analgesic effects(36, 37) . Since
this is the first report to highlight the importance of pi electrons,
it will be interesting to examine the correlation between pi electron
density and analgesia. Some information relevant to the
three-dimensional structures of the NT receptors may be inferred from
the structure activity relationships presented here. Clearly, the side
chain length at position 9 was important for binding affinity and may
reveal significant information concerning the volume of the NT
receptors' binding sites. Also, the side chain of the residue at
position 11 should provide insights into the topography of the NT
receptors' binding sites. Our results demonstrated the importance
at the 11 position of pi electron density and the effects of steric
bulk on binding affinity. In this regard, our results also showed that
the rat receptor was more tolerant of changes in pi electron density
and steric bulk than was the human receptor. These experimental
findings provide necessary structural constraints for molecular
modeling studies to elucidate the binding sites of the human and rat NT
receptors. They also provide pivotal criteria to assess the
hypothetical three-dimensional models of the NT binding sites derived
from ``ab initio'' calculations. It is beyond the scope of
this paper to discuss fully the possible three-dimensional structures
of the NT receptors. We are currently doing further three-dimensional
modeling studies involving more detailed analyses of receptor-ligand
interactions to establish a NT receptor model, which takes into
consideration the results presented here. ( The strong
correlation between K Finally, from the binding results obtained with hNTR(Leu)
and hNTR(Phe), it appears that they have a high degree of correlation
in structure-activity relationships as demonstrated by a correlation
coefficient of 0.99 (Fig. 7). At least for the compounds tested
here, the 1 residue difference in the amino acid sequence of the two
human NTR clones does not appear to affect the binding potency of these
peptides. In conclusion, the development of the compounds presented
here represent several novel findings, specifically: 1) a highly
species-selective compound (NT19); 2) a pair of stereoselective
compounds (NT24 and NT27); and 3) a novel, highly potent
(``superagonist'') and efficacious NT agonist (NT3). These
newly characterized peptides may provide important tools for studying
NT receptor function at the molecular level (in mapping the
ligand-binding site on the NT receptor), for exploring their in
vivo effects, and for characterizing a possible low affinity NT
subtype. Additionally, any one of these compounds may also prove to be
important in defining the therapeutic roles suggested for neurotensin.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
of 0.3 and 0.2
nM at the human and rat neurotensin receptor, respectively,
ranking it as among the most potent tested. Also, we present the first
evidence that 1) the effect of pi electrons at position 11 (L-Tyr) are important for binding to the neurotensin receptor,
and 2) the length of the side chain on position 9 (L-Arg)
changes binding potency.
)was found in many areas of the mammalian
central nervous system. Among the central nervous system effects that
can be attributed to NT are hypothermia(2) , potentiation of
barbiturate- and ethanol-induced (3) sedation, muscle
relaxation (4) , antinociception(5) ,
catalepsy(6) , and decreased locomotor activity(7) .
Additionally, it is a potent antinociceptive agent (8) that is
naloxone insensitive. From a clinical standpoint, studies with NT have
led to implications of its involvement in schizophrenia(9) ,
Parkinson's disease(10) , and Alzheimer's
disease(11, 12) . At the cellular level, it is coupled
to the production of cGMP(13) , phosphatidylinositol
turnover(14) , calcium mobilization(15) , and the
production of cAMP(16) . Both NT-binding sites and function are
regulated with respect to cell division in a neuronal cell
type(17) .
) among the two clones. An
examination of the rat and human amino acid sequence indicates an 84%
sequence similarity. The human receptor has six fewer amino acids than
does that of the rat. All three NT receptor types were stably expressed
in Chinese hamster ovary cells (CHO-K1).
with D-Lys with the resulting peptide
exhibiting more potency at the NT receptor than NT and almost equally
effective at cGMP stimulation as NT(8-13)(22) . Based on
the structure of lysine, we decided to test other amino acids that were
positively charged and substitute them in the 8 and 9 positions of
NT(8-13). Additionally, at position 11 L-Tyr has been
shown to be important to binding to the NT receptor(23) . We
explored permutations of this amino acid by substitutions that changed
steric bulk, pi electron density, and stereogeometry of the
carbon. We evaluated binding potency at the hNTR(Leu), rNTR, and
hNTR(Phe) by determining equilibrium dissociation constants (K
values) from radioligand binding
assays. In vitro functional assays were then carried out to
determine agonism or antagonism of compounds that had binding potency
at the NTR. For this purpose we measured the turnover of
phosphatidylinositol (PI) in intact CHO-K1 cells.
of 0.3 nM and 0.2 nM at the hNTR(Leu) and
rNTR respectively, ranking it as among the most potent tested, and
clearly the most potent at stimulation of PI turnover. Finally, we
present here the first evidence that 1) the effect of pi electrons at
position 11 (L-Tyr) are important for binding to the NT
receptor; and 2) the length of the side chain on position 9 (L-Arg) changes binding potency.
Peptide Analogs
The peptides and pseudopeptides
used here are listed in Tables I and Table 2. Peptides were
synthesized by Doctor Daniel J. McCormick in the Mayo Protein Core
Facility, (Mayo Clinic, Rochester, MN) as described by Morbeck et
al.(24) . Briefly, peptides were synthesized using Fmoc
chemistry with t-butyl-protected side chains, either
individually on automated peptide synthesizers (ABI 430A or 431A) or
simultaneously on a multiple peptide synthesizer (ACT350, Advanced
Chemtech, Louisville, KY). Protocols concerning activation coupling
times, amino acid dissolution, coupling solvents, and synthesis scale
were followed according to the manufacturer's instructions. All
peptides were purified by reverse-phase HPLC using a C18 column (2.2
25 cm, Vydac, Hesperia, CA) in 0.1% trifluoroacetic acid/water
and a gradient of 10%-60% acetonitrile in 0.1% trifluoroacetic
acid/water. A combination of analytical HPLC and mass spectrometry was
used to analyze peptide purity.
Cell Culture
CHO-K1 cells that had been stably
transfected with the hNTR(leu), rNTR, and hNTR(phe) gene were cultured
in 150-mm Petri plates containing 35 ml of Dulbecco's modified
Eagle's medium containing 100 µM minimal essential
medium nonessential amino acids (Life Technologies, Inc.) supplemented
with 5% (v/v) FetalClone II bovine serum product (Hyclone Labs, Logan,
UT). CHO cells (subculture 9-19) were harvested at confluence by
aspiration of the medium, followed by a wash with 50 mM
Tris-HCl, pH = 7.4 (6 ml), which was discarded, resuspension in
5-10 ml of Tris-HCl, scraping the cells with a rubber spatula
into a centrifuge tube and collection of cells by centrifugation at 300
g for 5 min at 4 °C, in a GPR centrifuge (Beckman
Instruments, Fullerton, CA). The cellular pellet (in 50 mM Tris-HCl, 1 mM EDTA, pH = 7.4) was stored at
-180 °C until radioligand binding was performed.
g for
10 min. The supernatant was decanted and discarded, and the cellular
pellet was resuspended in 2 ml of Tris-HCl + 1 mM EDTA
(pH = 7.4) followed by homogenization with a Brinkmann Polytron
at setting 6 for 10 s. Centrifugation was repeated as above, the
supernatant was decanted and discarded, and the final cellular pellet
was resuspended in 50 mM Tris-HCl, 1 mM EDTA, 0.1%
bovine serum albumin, and 0.2 mM bacitracin. Protein
concentration of the membranal preparation was estimated by the method
of Lowry et al.(25) using bovine serum albumin as a
standard.
Radioligand Binding Assays
We used a Biomek 1000
robotic workstation for all pipetting steps in the radioligand binding
assays as described previously by our group(26) . Competition
binding assays with [H]NT (1 nM) and
varying concentrations of unlabeled NT, and peptide analogs were
carried out with membranal preparations from the appropriate cell
lines. Nonspecific binding was determined with 1 µM
unlabeled NT in assay tubes with a total volume of 1 ml. Incubation was
at 20 °C for 30 min. The assay was routinely terminated by addition
of cold 0.9% NaCl (5
1.5 ml), followed by rapid filtration
through a GF/B filter strip that had been pretreated with 0.2%
polyethylenimine. Details of binding assays have been described before (27) . The data were analyzed using the LIGAND
program(28) .
PI Turnover Assays
Intact CHO-K1 cells were
harvested for PI turnover analysis at about 80% confluence. Cells were
detached from the Petri plates by removal of culture medium and
followed by incubation of the cellular monolayer for 20 min at 37
°C with gentle shaking in a modified Puck's D solution containing 2 mM EGTA. We have described
elsewhere the details of assaying in intact cells the relative changes
in PI turnover by using a radioactively labeled precursor(29) .
Briefly, intact CHO cells were prelabeled with D-myo-[
H]inositol (18.3
Ci/mmol) in the presence of lithium chloride (final concentration, 10
mM). Cells were then stimulated with NT or the appropriate NT
analogs. The amount of [
H]inositol 1-phosphate
([
H]IP
) produced by the cells was
isolated chromatographically on Dowex 1-X8 (200-400 mesh). For
the experiments described here the stimulation time was 30 min. The
number of CHO cells/assay tube was 1.5
10
. Some
compounds were tested at one final concentration of 0.1 mM to
determine agonism or antagonism. No EC
values were derived
for these compounds. Compounds designated ``agonists''
exhibited a maximum response that was comparable to that derived with 1
µM NT.
Statistical Analysis
The values presented for K and EC
are expressed as
the geometric means ± S.E.(30, 31) .
Statistical analysis of the correlation of the groups of data were
evaluated by analysis of the regression line (procedure 5) as described
by Tallarida and Murray (32) . p values less than
<0.05 were considered statistically significant.
NT(8-13) Substitutions: Binding and Biological
Activity
The results of radioligand binding studies with
NT(8-13) substituted analogs at the hNTR(Leu), rNTR, and
hNTR(Phe) receptors are presented in Table 3. The peptides are
listed in rank order of potency at the hNTR(Leu). All the peptides
tested had Hill coefficients close to unity (data not included),
indicating binding to a single class of receptors. NT(8-13) was
the most potent in this series at all the receptor types studied. Of
the new peptides synthesized, NT2 and NT3 were the most potent with K values in the range of 0.22-0.61
nM. The least potent compound was NT21 with K
values of 280-700 nM (Table 3).
values for NT21 versus NT14
revealed a 200-fold decrease in potency at hNTR(Leu) and a 380-fold
decrease in potency at rNTR (Table 3).
to L-Lys
), caused
a 6.8-fold decrease in potency at the hNTR(Leu) and a 4.1 decrease at
the rNTR. However, moving DAB from the 8 to the 9 position (NT5 versus NT3, i.e. DAB
versus DAB
) caused an increase in affinity of about
3-5-fold at these receptors. None of the remaining doubly
substituted peptides exhibited any further increase in potency as
compared to their singly substituted congeners.
= 0.84
± 0.09 nM: geometric mean ± S.E.). A comparison
of K
values with EC
values
for the hNTR(Leu) showed a strong correlation, p < 0.025 (Fig. 2).
and EC
in the hNTR(leu)
cell line. Comparison of K
for binding
(membranal preparations) and EC
of PI turnover (intact
cells) in the hNTR(leu) cell line were derived from data presented in Table 3. The points were fitted by linear regression analysis.
The correlation coefficient (r) is given together with the
slope and the statistical significance (p). Compound reference
numbers are given in Table 1.
Ornithine Substitutions: Binding and Biological
Activity
In Table 4we present the results of competitive
binding studies with the ornithine-substituted analogs in competition
with [H]NT in membrane preparations from cell
lines expressing hNTR(Leu) and rNTR, respectively. The compounds are
listed in rank order of potency at the hNTR(Leu). The most potent
compound was NT22 with a K
of 0.26
± 0.02 and 1.2 ± 0.2 nM at the hNTR(Leu) and the
rNTR, respectively. The least potent were NT32 and NT33. All of the
peptides tested had Hill coefficients equal to 1, indicating binding
followed the laws of mass action and involved one class of receptors.
We calculated the K
ratios of the human versus the rat receptor to evaluate any significant
differences.
(NT29) brought about a 200-300-fold
decrease in affinity relative to NT25(L-Orn
). For
NT26 versus NT31, the D-isomer in position 9 resulted
in a reduced affinity of 170-200-fold. For the pentapeptides NT28
and NT30, the D-Orn-substituted compound was likewise less
potent than the L-isomer by approximately 2-3-fold.
Finally, the replacement of L-Tyr
with D-Tyr
resulted in the most dramatic loss of
binding potency for the two compounds tested (NT32 and NT33).
= 3.2 ± 0.6 and 690
± 50 nM (geometric mean ± S.E.), respectively.
This represents a 215-fold difference in potency, which compares well
with the 190-fold difference in binding affinities for the same
compounds at the hNTR(Leu).
hNTR(Leu) Versus rNTR
Among the NT(8-13)
analogs studied, substitutions at position 11 were systematically
evaluated, first by changing the size of the ring structure at position
11. At the hNTR(Leu), replacement of L-Try with L-Phe
(and the subsequent loss of an OH group, Fig. 1B, NT14) resulted
in a 24-fold loss in potency. However, at the rNTR the same
substitution resulted in only a 4.6-fold decrease (see NT1 versus NT14, Table 3). Replacement of the OH with a fluorine atom
on the aromatic ring of position 11 (NT11) resulted in similar changes
in potency. Specifically, for NT1 versus NT11, at the
hNTR(Leu) there was a 21-fold decrease, while at the rNTR, there was a
14-fold decrease (Table 3). Most notable was NT19 with a Nal
substitution, which, compared to L-Tyr, has a larger ring
structure (Fig. 1B). With a K at hNTR(Leu) of 89 ± 9 nM and at rNTR of 3.9
± 0.2 nM (geometric mean ± S.E.), the ratio of
human to rat K
was 23. In Fig. 3, A and B, we present representative competitive
binding curves for NT and NT19 at the hNTR(Leu) and the rNTR,
respectively. Clearly, NT19 was binding to these two receptors with
significantly different potencies (p < 0.001). A
representative dose-response curve for stimulation of PI turnover by
the species-selective NT19 in hNTR(Leu) intact cells is shown in Fig. 4.
H]NT and NT19 in hNTR(leu) (A) and rNTR (B) containing cells. Assays were performed on membranal
preparations using 1 nM [
H]NT and
varying concentrations of drugs as described in the text. Curves were
generated using the LIGAND program. Data points are the means of
duplicate determinations and are representative results from one of 38
(NT) or one of three (NT19) independent
experiments.
H]IP
formation in intact
hNTR(leu) containing cells. Data are means of triplicate determinations
from which the average of triplicate basal values has been substracted
(basal levels of [
H]IP
, in
disintegrations/min/1.5
10
cells was 510). The data
presented are representative results from one of three independent
experiments.
ratio
of hNTR(Leu)/rNTR in this case was 9.6, less than that found for NT19
at the two receptors. For NT16 the additional substitutions at position
8 and 9 increased the human to rat ratio to 12 (Table 3).
values for the hNTR(Leu)
with those for the rNTR, we found a stronger correlation among the
peptides when the compounds were separated into two groups (Fig. 5). The smaller subset included peptides with position 11
substitutions, while the remaining peptides comprised the second set.
There was a strong correlation for both sets as indicated by the
correlation coefficients of 0.97 and 0.92, respectively.
values for hNTR(leu) and rNTR:
NT8-13 substituted analogs. K
values were derived from data presented in Table 3using membranal preparations from the indicated cell
lines. The points were fitted by linear regression analysis. ▪
indicates a subset of compounds with position 11 substitutions, while
indicates the remaining peptides from Table 3. The
correlation coefficient (r) is given together with the slope
and the statistical significance (p) for each set. Compound
reference numbers are given in Table 1.
values for ornithine
substituted compounds at the human and rat receptors. The correlation
coefficient of 0.96 indicates similar structure-activity relationships
between both receptor sources and the compounds tested.
values for hNTR(leu) and rNTR:
ornithine-substituted analogs. K
values
were derived from data presented in Table 4using membranal
preparations from the indicated cell lines. The points were fitted by
linear regression analysis. The correlation coefficient (r) is
given together with the slope and the statistical significance (p). Compound reference numbers are given in Table 2.
hNTR(Leu) Versus hNTR(Phe)
A comparison of the K values derived for the two human clones
indicated that except for NT3 the values for both sets of data were
almost identical. The correlation between the two sets of data for the
two types of human receptors was significant (Fig. 7).
values for hNTR(leu) and hNTR(phe):
NT8-13 substituted analogs. K
values were derived from data presented in Table 3using membranal preparations from the indicated cell
lines. The points were fitted by linear regression analysis. The
correlation coefficient (r) is given together with the slope
and the statistical significance (p). Compound reference
numbers are given in Table 1.
results
in a compound with significant potency at the NT receptor in murine
neuroblastoma clone N1E-115 cells(33) . To study the effect of
the chain length of the Lys residue on binding (Fig. 1A), we decided to replace it with ornithine.
This amino acid has the same charge as Lys but has one less methyl
group on its side chain. Additionally, we explored stereoselectivity
with the use of its L- and D-isomers. Of all the
ornithine substitutions examined, NT22 with D-Orn
was the most potent (see Table 4). Interestingly, the L-isomer in the same position was almost equally potent at
both the human and the rat NT receptors. This result suggests that
stereo configuration of these residues is not important for binding
potency. However, the same substitution at position 9 revealed a much
different result. NT24 (L-Orn
) as compared to NT27 (D-Orn
) had a 190- and 700-fold increase in
binding potency at the human and rat receptors, respectively. This
stereoselectivity was also shown for PI turnover. That is, based on the
EC
for NT24 and NT27 at the hNTR(Leu), NT24 was 220-fold
more potent (Table 4). All of the ornithine-substituted compounds
were full agonists at the human NT receptor.
) had a K
that was among the most potent of all the compounds tested
at the three receptor types (Table 4). Most notable was the
potency of NT3 at stimulation of PI turnover. It was the most potent of
all the compounds tested and almost 2-fold more potent than
NT(8-13) at the human NT receptor. These in vitro results for NT3 as a very potent and effective NT agonist with
subnanomolar affinity make it a strong candidate for in vivo testing. Further experiments are needed to determine its possible
analgesic and neuroleptic properties. Regardless of the in vivo effects to be evaluated for NT3, the results presented here
provide the first evidence for a relationship between side chain length
at position 9 and binding potency.
]NT(8-13) (NT19) showed
species-selective binding affinity. This compound had a 23-fold greater
affinity for the rat receptor than for the human NTR(Leu). A comparison
of this peptide in relation to the others with position 11
substitutions suggests that the steric bulk of the L-Nal side
chain was important in its species selectivity. L-Nal is a
widely employed derivative that was originally synthesized as a
replacement for tryptophan in the design of new peptide analogs.
Indeed, NT16 which has L-Trp
exhibits some
species selectivity as evidenced by the human to rat ratio of 12. L-Trp has more steric bulk than does L-Tyr or L-Phe, but less so than L-Nal.
values for the 11-position
substitutions suggests that the rat NTR is more able to accommodate the
steric bulk than is the human NTR (Table 3). That is, the change
in K
values for binding to the rat
receptor is not as dramatic as that for the human receptor for the
analogs with changes at position 11. In addition, in all cases of these
11-position compounds, the affinity for the human receptor was less
than that for respective values at the rat receptor.
data (Fig. 5), it appears that the peptides that displayed the
largest differences in potency between the human and rat NT receptors
defined a subset with a correlation different from the rest of the
compounds. Exactly what the variable is that is responsible for these
differences cannot be defined at this time. However, it is remarkable
that this difference does occur when there is such high sequence
similarity of the amino acid arrangement (84%) between the human and
the rat NT receptors.
]NT(8-13) could next be
used to help define the ligand-binding site. Through the construction
of rat-human chimeric receptors, the binding domain may be localized.
With site-directed mutagenesis, it may be possible to determine
specific amino acid residues that are responsible for such a
species-selective effect. Others have reported for the 5HT
receptor that a single residue defines the binding properties of
this serotonin receptor subtype as that of rat or that of
human(34) . Specifically, Ser
in the putative
fifth transmembrane domain of the 5HT
receptor is
responsible for the change in pharmacology observed with mesulergine at
this receptor. This residue is not present in either the rat (18) or human (19, 20) NT receptors. Further
studies involving molecular techniques along with NT19 may lead to the
identification of the ligand-binding site of the NT receptor.
values changed more dramatically for
the human than for the rat receptor (Table 3). We noted that a
change in position 11 from L-Tyr to L-Phe resulted in
a significant loss in binding potency. Similar results were found in
studies with rat brain synaptic membranes and a human colon carcinoma
cell line(35) . The difference between these 2 amino acid
residues is an OH group (Fig. 1B). Interestingly, the
loss of binding potency with the substitution of L-Phe
(NT14, Table 3) was more dramatic at the hNTR(Leu) than at
the rNTR, i.e. a 24-fold versus a 4.6-fold decrease,
respectively.
)
values derived in
human and rat NT receptors for all the compounds tested ( Fig. 5and Fig. 6) indicates that these receptors are
similar and most likely of the high-affinity type. Recently, there has
been more evidence to support the existence of a subtype of the NT
receptor(36, 37) . Additionally, the development of a
potent nonpeptide NT antagonist (SR48962) provides another tool for
defining the binding and functional characteristics of the NT
receptor(38) . It has been suggested that this antagonist is
selective for the high affinity NT receptor, i.e. the cloned
receptors that were used for the studies we report here(39) .
While SR48962 will antagonize the hypolocomotor effect of ICV NT
injection, it failed to inhibit the hypothermic and analgesic responses
in both the rat and mouse(39) . These findings, along with the
evidence that position 11 substitutions appear to yield compounds with
selectivity for in vivo analgesic and hypothermic
effects(36, 37) , suggest other experiments with the
compounds presented here. It will be interesting to evaluate the
antinociceptive and hypothermic effects of these position 11
substituted analogs. Also, further experiments will be needed to define
the in vivo effects of the stereoselective analog pair, NT24
and NT27.
We thank Dr. Peter O'Brien for help in
statistical analysis and Margaret Peterson for her expert clerical
assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.