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A. STRUCTURE–ACTIVITY STUDIES OF TIP(P) PEPTIDES

A. STRUCTURE–ACTIVITY STUDIES OF TIP(P) PEPTIDES

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Table 2 Antagonist Potencies and Opioid Receptor Affinities of TIPP Analogues

Compound

H-Tyr-Tic-Phe-Phe-OH (TIPP)

H-Tyr-Tic-Phe-OH (TIP)

Tyr(NMe)-Tic-Phe-Phe-OH

H-Dmt-Tic-Phe-Phe-OH

H-Tyr-TicC[CH2-NH]Phe-Phe-OH

(TIPP[C])

H-Tyr-TicC[CH2-NH]Phe-OH (TIP[C])

H-Tyr-TicC[CH2-NCH3]Phe-Phe-OH

H-Tyr(3V-I)-Tic-Phe-Phe-OH

H-Tyr(3V-I)-Tic-Phe-OH

H-Tyr(3V-I)-TicC[CH2-NH]Phe-Phe-OH

H-Tyr-Tic-Leu-Phe-OH

H-Tyr-Tic-Ile-Phe-OH

H-Tyr-Tic-Cha-Phe-OH (TICP)

H-Tyr-TicC[CH2-NH]Cha-Phe-OH

(TICP[C])

H-Tyr(3V-I)-Tic-Cha-Phe-OH

H-Dmt-Tic-OH

Naltrindole

DPDPE

[D-Ala2]deltorphin II

a

b



Ke

(nM)a

5.86

11.7

1.22

0.196

2.89



K Ai

(nM)b



K yi

(nM)b



K Ai /K yi



1,720

1,280

13,400

141

3,230



1.22

1,410

9.07

141

1.29 10,400

0.248

569

0.308 10,500



9.06

10,800

4.76

13,400

Agonist 5,230

141

12,100

19.2

2,660

7.32

904

12.7

6,460

0.438

3,600

0.219

1,050



1.94

5,570

0.842 15,900

24.8

211

60.0

202

2.08

1,280

2.84

318

4.37

1,480

0.611 5,890

0.259 4,050



12.7

6.55

0.636

Agonist

Agonist



4,010

3.33

1,360

1.84

3.86 0.182

943

16.4

3,930

6.43



1,200

739

21.2

57.5

611



Determined against DPDPE in the MVD assay.

Binding assay based on displacement of [3H]DAMGO (A-selective) and [3H]DSLET

(y-selective) from rat brain membrane binding sites.



and shows even slightly higher y-receptor selectivity than the TIPP[C]

parent peptide.

For the purpose of opioid receptor binding studies, TIPP was also

radioiodinated. Surprisingly, [125I]TIPP binding to y receptors in N4TG1

neuroblastoma cells was substantially reduced in the presence of Na+ and

Gpp(NH)p [42]. These results indicated that substitution of an iodine atom

at the 3V position of Tyr1 in TIPP had turned the y antagonist into a y

agonist. The corresponding ‘‘cold’’ analogue, H-Tyr(3’-I)-Tic-Phe-PheOH, was then synthesized and shown to be a full agonist in the MVD

assay (IC50 = 97 nM). This agonist effect was antagonized by TIPP (Ke =

11 nM) [38]. Corresponding iodination of the Tyr residue in TIP and

TIPP[C] did not result in agonism, but somewhat reduced antagonist



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



potency was observed (Table 2). It therefore appears that the astonishing

conversion observed with the tetrapeptide TIPP may be due to an overall

conformational effect rather than to a direct, local effect of the iodine

substituent. Interestingly, substitution of a bromine or chlorine atom at the

3V position of Tyr1 in TIPP produced partial agonists with respective

intrinsic efficacies of 0.16 and 0.12, whereas the Tyr(3V-F)-analogue was

again a pure antagonist (Ke = 13.0 nM) [38]. Thus, systematic substitution

of halogen atoms beginning with iodine and in the order of the periodic

table produced a progressive decrease in intrinsic activity and a concomitant increase in affinity at the y receptor (K yi = 24.2, 3.62, 3.00 and 1.62

nM, respectively).

Replacement of the Phe3 residue in TIPP with the aliphatic amino

acid residues Leu or Ile resulted in analogues that retained high yantagonist potency and considerable y selectivity (Table 2). This result is

in agreement with the weak y-antagonist activity that had been reported for

the tripeptide H-Tyr-Tic-Ala-OH [43]. Obviously, an aromatic residue at

the 3 position of the peptide sequence of TIP(P) peptides is not absolutely

required for y antagonist activity. Most interestingly, saturation of the

Phe3 aromatic ring in TIPP, as achieved through substitution of cyclohexylalanine (Cha), led to H-Tyr-Tic-Cha-Phe-OH [TICP], a compound

showing substantially increased y-antagonist potency and higher y selectivity than the parent peptide [44]. The corresponding pseudopeptide,

H-Tyr-TicC[CH 2 -NH]Cha-Phe-OH (TICP[C]), showed a further

improvement in y-antagonist activity. Its y-antagonist potency is comparable to that of the analogue H-Dmt-Tic-Phe-Phe-OH but, in comparison

with the latter peptide, it is seven times more y selective (K Ai /K yi = 4050)

[44]. Both TIPP[C] and TICP[C] were prepared in tritiated form [45,46]

and should turn out to be valuable new radioligands for y receptor labeling

studies in vitro and in vivo. The analogue H-Tyr(3V-I)-Tic-Cha-Phe-OH

was an antagonist in the MVD assay with a potency about 30 times lower

than that of TICP. Thus, unlike in the case of TIPP, introduction of an

iodine substituent at the 3V position of Tyr1 in TICP did not produce a y

agonist. This result demonstrates once again how a relatively subtle

structural modification, such as the saturation of an aromatic ring, can

have a determinant effect on agonist versus antagonist behavior.

In 1995 the dipeptide H-Dmt-Tic-OH was reported to be a y-opioid

antagonist with unprecedented y-receptor affinity (K yi = 0.022 nM) and y

receptor selectivity (K Ai /K yi = 150,000) [47]. However, in a direct comparison under identical assay conditions, this compound showed about 30

times lower y-antagonist potency and 6 times lower y-receptor selectivity



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



than TICP[C] [48] (Table 2). Similar results were obtained in a more recent

study [49], which confirmed that H-Dmt-Tic-OH had much lower y

receptor affinity [IC50(y) = 1.6 nM] and much lower y selectivity [IC50(A)/

IC50(y) = 558] than had originally been reported. Furthermore, H-DmtTic-OH was found to be unstable in organic solvents owing to diketopiperazine formation (P. W. Schiller and T. M.-D. Nguyen, unpublished

results).



B. Conformational Studies of TIP and TIPP

A molecular mechanics study (grid search and energy minimization) of the

tripeptide y-antagonist TIP resulted in several low energy conformers

having energies within about 2 kcal/mol of that of the lowest energy

structure [50]. The centrally located Tic residue imposes a number of

conformational constraints on the N-terminal dipeptide segment; however, the results of molecular dynamics simulations indicate that this

tripeptide still shows some structural flexibility at the Phe3 residue.

Attempts to demonstrate spatial overlap between the pharmacophoric

moieties of low-energy conformers of TIP and the structurally rigid nonpeptide y antagonist naltrindole were made by superimposing either the

Tyr1 and Phe3 aromatic rings and the N-terminal amino group or the Tyr1

and Tic2 aromatic rings and the N-terminal amino group of the peptide

with the corresponding aromatic rings and nitrogen atom in the alkaloid

structure. In each case the investigators found a conformer of TIP with an

energy very close to that of the lowest energy structure (2.1 kcal/mol

higher). However, the low-energy conformer showing spatial overlap of its

Tic2 aromatic ring with the six-membered aromatic ring of the indole

moiety in naltrindole (Fig. 3) appears to be a more plausible candidate

structure of the y-receptor-bound conformation for two reasons:

1.

2.



The Tic2 aromatic ring has been shown to be of crucial

importance for y antagonist activity [51].

The y-antagonist properties are maintained upon replacement of

the Phe3 residue in the peptide with an aliphatic amino acid

residue (see earlier).



This model of the receptor-bound conformation of TIP is characterized by

a clustered configuration of the three aromatic moieties with the Phe3

aromatic ring sandwiched between the Tyr1 and Tic2 aromatic rings.

A molecular mechanics study of TIPP and TIPP[C] produced about

70 structures within 3 kcal/mol of the lowest energy conformation in each



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



Figure 3 Superimposition of a low energy conformer of TIP (heavy lines) with

the minimized structure of naltrindole (light lines). The Tyr1 and Tic2 aromatic

rings and the N-terminal amino group of the peptide are superimposed with the

corresponding moieties in the alkaloid structure. The superimposed molecules

are shown in two different orientations.



case [52]. The lowest energy conformers of both TIPP and TIPP[C]

showed good overlap of their Tyr1 and Tic2 aromatic rings and N-terminal

amino group with the corresponding pharmacophoric moieties of naltrindole. Thus, these results are in agreement with the model of the

receptor-bound conformation of TIP proposed earlier. This model is

characterized by all-trans peptide bonds and was definitely confirmed by

conformational analyses of two TIPP analogues (y antagonists) in which a

cis peptide bond between the Tyr1 and Tic2 residues is sterically forbidden

[53]. Both TIPP and TIPP[C] are very hydrophobic peptides, and the

results of the theoretical conformational analyses clearly indicated that

they enjoy considerable structural flexibility, particularly in their Cterminal dipeptide segment. There is no doubt that their conformations

are quite dependent on the environment. According to our theoretical

analysis, a crystal structure of TIPP published in 1994 [54] is about 3 kcal/

mol higher in energy than the lowest energy structure and shows no

similarity to any of the calculated low energy structures [52,53]. The

crystal structure of TIPP appears to be stabilized by a large number of

intermolecular hydrophobic contacts between layers of TIPP molecules in

the crystal and by several hydrogen bonds to solvent (AcOH) molecules.

There is no reason to believe that it resembles the y-receptor-bound

conformation of TIPP. In an aqueous environment TIP(P) peptides

may undergo a so-called hydrophobic collapse [55]. It is possible that

subtle structural modifications, such as introduction of an iodine sub-



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



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