Design and synthesis of somatostatin analogs with topographical properties that lead to highly potent and specific .mu. opioid receptor antagonists with greatly reduced binding at somatostatin receptors

Abstract

A series of conformationally restricted, cyclic octapeptides containing a conformationally stable tetrapeptide sequence related to somatostatin, -Tyr-D-Trp-Lys-Thr-, as a template, were designed and synthesized with the goal of developing highly potent and selective mu opioid antagonists with minimal or no somatostatin-like activity. Three distinct structures of the peptide became targets of chemical modifications and constraints; the N- and C-terminal amino acids and the cyclic 20-membered ring moiety. Based on the conformational analysis of active and inactive analogues of the parent peptide D-Phe1-Cys2-Tyr3-D-Trp4-Lys5-Thr6-Pen7+ ++-Thr8-NH2, CTP (Kazmierski, W.; Hruby, V. J. Tetrahedron 1988, 44, 697-710), we designed analogues to include the tetrahydroisoquinolinecarboxylate (Tic) moiety as the N-terminal amino acid instead of D-Phe, since Tic can exist only as a gauche (-) or a gauche (+) conformer. In this series, the following peptides were synthesized and pharmacologically evaluated: D-Tic-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (TCTP), D-Tic-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (TCTOP), and D-Tic-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (TCTAP). In rat brain membrane opioid radioligand binding assays, all three peptides displayed high affinity for mu opioid receptors (IC50 = 1.2, 1.4, 1.2 nM, respectively), and exceptional mu vs delta opioid receptor selectivity: 7770, 11,396, and 1060, respectively. TCTOP and TCTAP also possess exceptional mu vs somatostatin receptor selectivity: 14,574 and 28,613, respectively. In the peripheral in vitro GPI bioassay, TCTP, TCTOP, and TCTAP were highly effective antagonists of the potent mu opioid receptor agonist PL017, with pA2 = 8.69 for TCTAP, 8.10 for TCTP, and 7.38 for TCTOP. Our results show that a 10-fold higher affinity and selectivity for mu opioid receptors (in both central and peripheral studies) over delta and somatostatin receptor was gained as a result of the D-Tic1 substitution. These three peptides, TCTP, TCTOP, and TCTAP, are the most potent and selective mu opioid antagonists known. CTP has been shown to possess prolonged biological action, much longer than that of naloxone. This renders these analogues potentially useful ligands for investigating the physiological functions of the mu opioid receptor. Analogues of TCTP in which the 20-membered disulfide ring was contracted by deletion of D-Trp4, and/or Lys5, and/or Thr6 led to compounds with greatly reduced potency at the mu opioid receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Potency Differences ford-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2as an Antagonist of Peptide and Alkaloid μ-Agonists in an Antinociception Assay

“…Similarly, CTAP was approximately equipotent as an antagonist of -agonists morphine and PL017 in the present study as in other antinociception procedures in rats and mice (Kramer et al, 1989;Adams et al, 1994;Takasuna et al, 1994). Taken together with radioligand binding and guinea pig ileum studies (Pelton et al, 1986;Kazmierski et al, 1988;Kramer et al, 1989), the data from the present experiment indicate CTAP is a potent -antagonist.…”

“…However, the identification of selective -opioid receptor antagonists has eluded researchers until recently. D-Phe-CysTyr-D-Trp-Arg-Thr-Pen-Thr-NH 2 (CTAP), a peptide antagonist derived from somatostatin analogs, exhibits high affinity and selectivity for -opioid receptors in radioligand binding experiments (Pelton et al, 1986;Kazmierski et al, 1988) ]-morphiceptin (PL017) in various assays, including cold-water tail-flick (Adams et al, 1994) and complete Freund's adjuvant in rats (Hurley and Hammond, 2001) as well as tail-flick (He and Lee, 1998) and hot-plate in mice (Kramer et al, 1989). CTAP lacks -agonist activity and fails to antagonize -agonists in a number of assays (Kazmierski et al, 1988;Kramer et al, 1989;Grider and Maklouf, 1991;Adams et al, 1994).…”

Novel [D-Arg2]dermorphin(1-4) Analogs with .MU.-Opioid Receptor Antagonist Activity.

“…2, analog 4 counteracted the inhibitory effects of YRBF in a dose-dependently manner. The antagonistic effect of 4 (10 mM) was also tested against the d agonist DPDPE in the mouse vas deferens assay and against the k agonist dynorphin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13) in the presence of the m antagonist CTAP 14,15) in the GPI assay. Analog 4 exhibited no antagonist activity against either the d or k receptor, thus, 4 appears to be a specific m receptor antagonist.…”

Pharmacological selectivity of CTAP in a warm water tail-withdrawal antinociception assay in rats

“…In binding assays, CTP, CTOP and CTAP show high affinity and exceptional selectivity for mu receptors Kazmierski et al 1988). In functional assays in guinea pig ileum (GPI) and mouse vas deferens (MVD), CTOP and CTAP exert potent antagonist actions selective for mu receptors (Kramer et al 1989;Mulder et al 1991).…”

“…In vivo, all three octapeptides display little agonist activity but rather act as competitive mu antagonists in noxious thermal antinociception assays (Guyla et al 1988;Kramer et al 1989). Of the three, CTAP shows the most promise as a useful selective competitive mu-antagonist devoid of intrinsic activity, displaying potent and highly selective mu receptor binding in vitro and in vivo (Gulya et al 1986;Kazmierski et al 1988) and functional antagonism in bioassays (Kramer et al 1989).…”