©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Pharmacological and Biochemical Profiles of Unique Neurotensin 813 Analogs Exhibiting Species Selectivity, Stereoselectivity, and Superagonism (*)

(Received for publication, January 31, 1995; and in revised form, April 28, 1995)

Bernadette Cusack (1)(§) Daniel J. McCormick (2) Yuan-Ping Pang (3) Terrance Souder (1) Roberto Garcia (1) Abdul Fauq (3) Elliott Richelson (1)

From the  (1)From Neuropsychopharmacology and (2)Neurochemistry Research, Mayo Foundation for Medical Education and Research, Jacksonville, Florida 32224 and the (3)Mayo Protein Core Facility, Mayo Clinic, Rochester, Minnesota 55595

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

After it was first isolated(1) , the tridecapeptide neurotensin (NT), (^1)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) .

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) 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).

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^8 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 alpha 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.

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 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.


MATERIALS AND METHODS

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.

For use in binding assays, crude membranal preparations were prepared by centrifugation of the cellular pellet at 35,600 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 [^3H]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(1) 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-[^3H]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 [^3H]inositol 1-phosphate ([^3H]IP(1)) 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 10^5. 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.


RESULTS

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.



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).



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 Kvalues 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).


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^8 to L-Lys^9), 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^8versus DAB^9) 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.

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 = 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).


Figure 2: Correlation between Kand 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 [^3H]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.



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^9 (NT29) brought about a 200-300-fold decrease in affinity relative to NT25(L-Orn^9). 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).

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 = 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.


Figure 3: Competition binding between [^3H]NT and NT19 in hNTR(leu) (A) and rNTR (B) containing cells. Assays were performed on membranal preparations using 1 nM [^3H]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.




Figure 4: Dose-response curves for NT and NT19 stimulated [^3H]IP(1) 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 [^3H]IP(1), in disintegrations/min/1.5 10^6 cells was 510). The data presented are representative results from one of three independent experiments.



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 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).

In a comparison of K 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.


Figure 5: Correlation between Kvalues 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 bullet 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.



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 Kvalues 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.


Figure 6: Correlation between Kvalues 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).


Figure 7: Correlation between Kvalues 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.




DISCUSSION

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^8 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^8 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^9) as compared to NT27 (D-Orn^9) 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.

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^9) 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.

Possibly the most significant and serendipitous discovery in this study was the finding that [L-Nal]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.

A review of the K 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.

From the correlation of all K 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.

[L-Nal]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(2) 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(2) 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.

For all the compounds with position 11 modifications, the K 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.

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. (^2)

The strong correlation between K 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.

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.


FOOTNOTES

*
This work was supported by the Mayo Foundation and by United States Public Health Service Grant MH27692 (to E. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Neuropsychopharmacology Research, Mayo Foundation for Medical Education and Research, 4500 San Pablo Road, Jacksonville, FL 32224. Tel.: 904-953-2439; Fax: 904-953-7117; cusack{at}mayo.edu.

^1
The abbreviations used are: NT, neurotensin; cGMP, cyclic guanosine-3,5-monophosphate; cAMP, cyclic adenosine-3,5-monophosphate; CHO-K1, Chinese hamster ovary cells; PI, phosphatidylinositol; NTR, neurotensin receptor; HPLC, high performance liquid chromatography; DAB, diaminobutyric acid.

^2
Y.-P. Pang, B. Cusack, and E. Richelson, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. Peter O'Brien for help in statistical analysis and Margaret Peterson for her expert clerical assistance.


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