In vitro binding and CNS effects of novel neurotensin agonists that cross the blood–brain barrier
Introduction
Neurotensin (p-Glu1,l-Leu2,l-Tyr3,l-Glu4,l-Asn5,l-Lys6,l-Pro7,l-Arg8,l-Arg9,l-Pro10,l-Tyr11,l-Ile12,l-Leu13) is a tridecapeptide found in the central nervous system, as well as in the gastrointestinal tract. Many studies show that neurotensin (NT) is a neurotransmitter or neuromodulator capable of exerting potent effects in animals, including hypothermia and antinociception (Bissette et al., 1976, Clineschmidt et al., 1979, Elliott and Nemeroff, 1986, Al-Rodhan et al., 1991, Kasckow and Nemeroff, 1991). NT receptors (NTRs) are distributed heterogeneously in the central nervous system with both high and low affinity sites (Goedert et al., 1984, Sadoul et al., 1984, Moyse et al., 1987, Kanba et al., 1988). It has been known for many years that the last six amino acids of the parent NT, are sufficient for biological activity at the NT receptor. However, for NT or NT(8-13) to exert these pharmacological and physiological effects, it must be delivered directly into the brain, due in part to its rapid degradation by peptidases upon systemic administration (Clineschmidt and McGuffin, 1977).
Centrally administered NT causes a variety of effects similar to those exhibited by neuroleptics including potentiation of sedatives and hypothermia (Bissette and Nemeroff, 1995). Clinically useful typical antipsychotics (e.g. haloperidol) have been found to cause an increase in NT levels in both the caudate nucleus and nucleus accumbens, while clozapine, an atypical antipsychotic, causes an increase in NT in the nucleus accumbens but not the striatum (Levant and Nemeroff, 1992). Other studies have demonstrated that antipsychotic drugs increase NT mRNA expression in specific dopamine terminal areas (Merchant et al., 1992). In addition, we showed that haloperidol, but not clozapine elevates NTR-1 mRNA levels in the substantia nigra/ventral tegmental region of rat (Bolden-Watson et al., 1993).
In clinical studies, schizophrenic patients with the lowest levels of CSF NT suffered from higher levels of pretreatment psychopathology (Sharma et al., 1997). Improvements in overall psychopathology were correlated with increases in CSF NT concentrations during antipsychotic treatment. Similarly, post-mortem studies of schizophrenic patients show reduced levels of neurotensin receptors in the caudate, cingulate, and prefrontal cortices as compared to normal controls (Lahti et al., 1998).
Because of the potential use of an NT agonist as a novel antipsychotic drug, we have been working on developing an agonist that would be resistant to peptidases and could cross the highly selective blood–brain barrier (BBB). Although peptides generally do not cross the BBB (Pardridge, 1998), a NT agonist has been described that crosses this barrier-a compound developed by Eisai (Machida et al., 1993, Banks et al., 1995) (Table 1). We found that the Eisai compound had much lower affinity for the human NTR-1, compared to its affinity for the rat receptor (Table 1). Thus, we wanted to design a compound with a high affinity for the human receptor.
From our previous structure-activity studies (Cusack et al., 1995, Cusack et al., 1996), we found that the orientation of steric bulk at position 11 of NT(8-13) was important for binding at the human NTR. More specifically, we found that the NT(8-13) binding site of the human NTR is slightly smaller than that of the rat NTR (Pang et al., 1996). Thus, the human receptor preferentially binds molecules that are larger than Tyr and smaller than naphthylalanine at position 11 (Cusack et al., 1996), compared to that for the rat receptor. This led to the design and synthesis of a novel analog of Trp, named 4-indole-tryptophan or neo-tryptophan (neo-Trp) (Fig. 1). We reasoned that the orientation of the aromatic indole side chain of neo-Trp would reorient the steric bulk and thus fit the relatively smaller human NTR better. Additionally, neo-Trp maintained appropriate pi-pi interactions with the receptor binding site that have been shown to be critical to binding. It was also suggested that the Pro10-Tyr11 and Tyr11-Ile12 bonds of NT are susceptible to peptidase degradation by the metalloendopeptidases 24.11 and 24.16 (Checler et al., 1986). Therefore, the addition of the novel amino acid (neo-Trp) to the peptide backbone would provide resistance to peptidase degradation and thus increase its bioavailability in vivo. We have incorporated neo-Trp into NT(8-13) and several analogs of this hexapeptide to determine its effect on receptor binding affinity, stability to degradation by peptidases, and in vivo behavior. Here we report the characterization of several novel NT analogs, and in particular, NT66L, which has high affinity for the rat NTR-1 and potent in vivo effects resulting from its apparent permeability through the BBB.
Section snippets
Synthesis of neurotensin analogs
The synthesis of neo-Trp [(2S)-2-amino-3-(1H-4-indolyl)propanoic acid] (Fig. 1), a novel analog of tryptophan, is described elsewhere (Fauq et al., 1998). Peptides containing neo-Trp were synthesized as previously described (Cusack et al., 1995). Briefly, peptides were synthesized using NαFmoc solid phase 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
Binding studies (Table 1)
In this series of NT(8-13) analogs, merely substituting l-neo-Trp for l-Tyr in position 11 (NT64L) increased the affinity for binding at the rat and human NTR-1 over that for NT(8-13). Marked stereoselectivity of the receptor was seen with, e.g. NT66L {[d-Lys8, l- neo- Trp11, tert-Leu12]NT(8-13)} having about 90- and 30-fold higher affinity than NT66D {[d-Lys8, d-neo-Trp11, tert-Leu12]NT(8-13)} at the rat and human NTR-1, respectively. Except for this analog with the d-isomer of neo-Trp, all
Discussion
Prior to this report, the Eisai compound was the only known NT peptide analog that crossed the BBB. Although the Eisai compound has high affinity for the rat NTR-1 (Kd=5.4 nM), it is only about 1/20th as potent at the human receptor. Thus, for the development of a neurotensin receptor agonist that might be of potential use as an antipsychotic drug, we wanted a compound with a high affinity for the human receptor. Both NT67L and NT66L were found to be much more potent at the human NTR-1 than was
Acknowledgements
This work was supported in part by the Mayo Foundation, U.S.P.H.S. grant MH27692 from NIMH (ER), NINDS grant 1F32 NS 10406-01A1 (BMT), and the Forrest C. Lattner Foundation.
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