Akihiro Ambo

[D-ALA2]deltorphin II analogs with high affinity and selectivity for delta-opioid receptor

Nine [D-Ala2]deltorphin II (DL-II:Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2) analogs having various aliphatic amino acids at positions 5 and 6 were synthesized to gain more information about the role of hydrophobic Val5,6 residues for the delta-opioid receptor selectivity. Binding assays of analogs replaced by Ala demonstrated the importance of hydrophobic Val5,6 residues in DL-II for delta-affinity and selectivity, and especially critical importance of Val5 residue for higher delta-selectivity. By enhancing the hydrophobicity of residues at positions 5 and 6, we have developed analogs with very high delta-affinity and selectivity over those of DL-II, e.g., [Ile5,6], [norleucine5,6] and [gamma-methyl-leucine5,6]DL-II, which will be useful as delta-selective ligands for investigation of the physiological role of opioid receptors.

Possible involvement of β-endorphin in docosahexaenoic acid-induced antinociception.

We have previously demonstrated that the n-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) has an antinociceptive effect on various pain stimuli in a naloxone-reversible manner. In the present study, the role of the endogenous opioid peptide β-endorphin in DHA-induced antinociception was examined. DHA-induced antinociception was abolished when mice were pretreated with the μ-opioid receptor antagonist β-funaltrexamine (β-FNA) and the δ-opioid receptor antagonist naltrindole, but not by the κ-opioid receptor antagonist nor-binaltorphimine (nor-BNI) in the acetic acid-induced writhing test. In the radioligand binding assay, DHA itself did not have affinity for μ- , δ- or κ-opioid receptors. On the other hand, the pretreatment of anti-β-endorphin antiserum inhibited DHA-induced antinociception. Furthermore, the intracerebroventricular injection of DHA dose-dependently reduced writhing behavior, and this effect was inhibited by d-Phe-Cys-Tyr-Orn-Thr-Pen-Thr-NH(2) (CTOP) and naltrindole, but not nor-BNI. β-endorphin-induced antinociception was inhibited by the pretreatment of β-FNA, but not naltrindole or nor-BNI, and its levels in plasma were increased by DHA treatment. These findings suggest that the induction of antinociception by DHA may partially involve the μ-opioid receptor via the release of β-endorphin.

Long‐lasting antinociceptive effects of a novel dynorphin analogue, Tyr‐D‐Ala‐Phe‐Leu‐Arg ψ (CH2NH) Arg‐NH2, in mice

Tyr‐D‐Ala‐Phe‐Leu‐Arg ψ (CH2NH) Arg‐NH2 (SK‐9709) is a dynorphin derivative in which the peptide bond was replaced with a ψ (CH2NH) bond. In the present study, the antinociceptive effects of SK‐9709 were determined in an acetic acid‐induced writhing test and a hot‐plate test. In the acetic acid‐induced writhing test, significant antinociceptive effects were observed after subcutaneous (s.c.), intracerebroventricular (i.c.v.) and intrathecal (i.t.) injection of SK‐9709, with maximal effects at 120, 30 and 15 min, respectively. The antinociceptive effects were dose‐dependent and ED50 values (range of 95% confidence limits) after s.c., i.c.v. and i.t. injection were 1.36 (0.61 – 3.02) μmol kg−1, 2.11 (1.18 – 3.79) and 0.79 (0.61 – 1.03) nmol per mouse, respectively. The effects of SK‐9709 (s.c., i.c.v. and i.t.) were reversed by the opioid receptor antagonist naloxone (1.36 μmol kg−1, s.c.). The effects of SK‐9709 (s.c.) were also reversed by the selective μ‐opioid receptor antagonist β‐funaltrexamine (4.7 nmol per mouse, i.c.v.), and κ‐opioid receptor antagonist nor‐binaltorphimine (4.9 nmol per mouse, i.t.). In the hot‐plate test, the antinociceptive effect of SK‐9709 (s.c., i.c.v. and i.t.) was also dose‐dependent with the maximal peak effect at 120, 15 and 15 min similarly to the acetic acid‐induced writhing test. The antinociceptive effects were dose‐dependent and ED50 values (range of 95% confidence limits) after s.c., i.c.v. and i.t. injection were 39.1 (5.4 – 283.0) μmol kg−1, 6.5 (4.0 – 10.7) and 7.4 (5.0 – 11.0) nmol per mouse, respectively. These findings indicated that systemically administered SK‐9709 produced long‐lasting antinociceptive effects and these effects were mediated by both supra‐spinal μ‐ and spinal κ‐opioid receptors.

Potent in vivo antinociception and opioid receptor preference of the novel analogue [Dmt1]endomorphin-1.

[Dmt1]Endomorphin-1 is a novel analogue of the potent mu-opioid agonist endomorphin-1. Given the physiological role of endomorphin-1 in vivo, this compound was investigated to determine if the antinociception occurred through systemic, supraspinal or in a combination of both neuronal pathways. This compound exhibited a potent dose-dependent effect intracerebroventricularly in both spinal and supraspinal regions, and was blocked by opioid antagonist naloxone, which verified the involvement of opioid receptors. Specific opioid antagonists characterized the apparent receptor type: beta-funaltrexamine (mu1/mu2-irreversible antagonist) equally inhibited spinal- and central-mediated antinociception; on the other hand, naloxonazine (mu1-subtype) was ineffective in both neural pathways and naltrindole (delta-selective antagonist) partially (26%), though not significantly, blocked only the spinal-mediated antinociception. Therefore, spinal antinociception was primarily triggered by mu2-subtypes without involvement of mu1-opioid receptors; however, although a slight enhancement of antinociception by delta-receptors cannot be completely ruled out since functional bioactivity indicated mixed mu-agonism/delta-antagonism. In terms of the CNS action, [Dmt1]endomorphin-1 appears to act through mu2-opioid receptor subtypes.

Cell‐penetrating peptide‐conjugated XIAP‐inhibitory cyclic hexapeptides enter into Jurkat cells and inhibit cell proliferation

X‐Linked inhibitor of apoptosis protein (XIAP) is a member of the inhibitor of apoptosis protein family that is overexpressed in human cancers. There is great interest in the development of XIAP inhibitors, which are predicted to promote apoptosis in cancer cells and thus have therapeutic potential. A cyclic hexapeptide (CH), CPFKQC, which is one of the consensus motifs that can bind to the baculovirus IAP repeat 2 domain of XIAP, has been identified using phage‐displayed combinatorial chemistry techniques [Tamm I, Trepel M, Cardo‐Vila M, Sun Y, Welsh K, Cabezas E, Swatterthwait A, Arap W, Reed JC & Pasqualini R (2003) Peptides targeting caspase inhibitors. J Biol Chem278, 14401–14405]. In this study, we designed and synthesized covalently linked conjugates of CHs, cyclo[Cys‐Pro‐Xaa‐Lys‐Gln‐Glu(‐CO‐)‐NH2] (Xaa = various amino acids; cyclization via a peptide bond between the N‐terminal amino group of Cys1 and the side‐chain carboxylic acid of Glu6), and a cell‐penetrating peptide (CPP), Ac‐Cys‐Trp‐(Arg)8‐Lys‐NH2. CH–CPP conjugates (CHCPPs) with aromatic and hydrophobic Xaa residues, such as Phe (CHCPP 1), 2,6‐dimethyl‐phenylalanine (CHCPP 2) and 3‐(1‐naphthyl)‐alanine/3‐(2‐naphthyl)‐alanine (CHCPPs 3 and 4), potently inhibited the proliferation of Jurkat cells in a dose‐dependent manner, whereas analogues with nonaromatic or less hydrophobic amino acids at the Xaa residue were less potent or caused no inhibition. A morphological study of nuclei after treatment with CHCPPs 1–4 revealed that nuclear fragmentation occurred, suggesting that these conjugates induce apoptosis. A kinetic study of the uptake of fluorescein‐labelled CHCPP 2 into the cells showed that the conjugates were translocated within a few minutes. The cellular uptake of fluorescein isothiocyanate‐labelled CHCPP 1 and CPP was greatly reduced in high‐K+ buffers, suggesting that CPP and its conjugate are translocated by a mechanism associated with cell membrane potential. Competitive binding studies performed using fluorescence correlation spectroscopy demonstrated that CHCPP 1 binds to the baculovirus IAP repeat 2 domain of XIAP via the CH (Xaa = Phe) moiety. CHCPPs 1 and 2 showed the most potent inhibitory activity of the CHCPPs and embelin, a nonpeptide inhibitor of XIAP, suggesting that they are good templates for the design of a new class of anticancer drug.

Further Studies on Lead Compounds Containing the Opioid Pharmacophore Dmt-Tic

Some reference opioids containing the Dmt-Tic pharmacophore, especially the delta agonists H-Dmt-Tic-Gly-NH-Ph (1) and H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid (4) (UFP-512) were evaluated for the influence of the substitution of Gly with aspartic acid, its chirality, and the importance of the -NH-Ph and N(1)H-Bid hydrogens in the inductions of delta agonism. The results provide the following conclusions: (i) Asp increases delta selectivity by lowering the mu affinity; (ii) -NH-Ph and N(1)H-Bid nitrogens methylation transforms the delta agonists into delta antagonists; (iii) the substitution of Gly with L-Asp/D-Asp in the delta agonist H-Dmt-Tic-Gly-NH-Ph gave delta antagonists; the same substitution in the delta agonist H-Dmt-Tic-NH-CH2-Bid yielded more selective agonists, H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid and H-Dmt-Tic-NH-(R)CH(CH2-COOH)-Bid; (iv) L-Asp seems important only in functional bioactivity, not in receptor affinity; (v) H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid(N(1)-Me) (10) evidenced analgesia similar to 4, which was reversed by naltrindole only in the tail flick. 4 and 10 had opposite behaviours in mice; 4 caused agitation, 10 gave sedation and convulsions.

Dermorphin and deltorphin heptapeptide analogues: replacement of Phe residue by Dmp greatly improves opioid receptor affinity and selectivity.

The usefulness of 2,6-dimethylphenylalanine (Dmp) as a Phe surrogate in two opioid peptides, dermorphin (DM) and deltorphin II (DT), was investigated. Compared to DM, [L-Dmp(3)]DM (1) showed a 170-fold increase in mu affinity and only a 4-fold increase in delta affinity, resulting in a 40-fold improvement in mu receptor selectivity. Compared to DT, [L-Dmp(3)]DT (3) showed a 22-fold increase in delta affinity and somewhat of a loss in mu affinity, and consequently a marked (75-fold) improvement in delta receptor selectivity. The D-Dmp replacement, however, resulted in a great loss in receptor selectivity in each of the peptides. The specific receptor interactions of 1 and 3 were confirmed by in vitro bioassays. Analogues 1 and 3 seem to be useful as pharmacological tools for the study of opioid systems.

ANTINOCICEPTIVE ACTIVITY OF DELTORPHIN ANALOGS IN THE FORMALIN TEST

Two deltorphin (DLT: Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2) analogs, [N alpha-n-butyl-Gly6]DLT ([nBuG6]DLT) and [N alpha-iso-butyl-Gly6]DLT ([isoBuG6]DLT), were examined for their antinociceptive activities by a formalin test in mice after subcutaneous (s.c.) injection. [nBuG6]DLT exhibited potent dose-dependent antinociceptive activities at doses of more than 0.02 mumol/kg at the first and second phases, while morphine similarly inhibited of the pain responses at doses more than 0.01 mumol/kg in the formalin test. [isoBuG6]DLT showed potent antinociceptive activity at the second phase, but did not inhibit the pain response at the first phase. This phenomenon may be caused by a mu-antagonist/delta-agonist property of this compound. The antinociceptive effects of these analogs were antagonized by delta-antagonist naltrindole, but not by the mu-antagonist naloxone. These findings suggest that the antinociceptive effects were mediated via delta-receptors. These compounds may be useful as delta-agonists for clarifying the mechanism of analgesia mediated by delta-opioid receptors.

Role of benzimidazole (Bid) in the δ-opioid agonist pseudopeptide H-Dmt-Tic-NH-CH2-Bid (UFP-502)

H-Dmt-Tic-NH-CH(2)-Bid (UFP-502) was the first delta-opioid agonist prepared from the Dmt-Tic pharmacophore. It showed interesting pharmacological properties, such as stimulation of mRNA BDNF expression and antidepression. To evaluate the importance of 1H-benzimidazol-2-yl (Bid) in the induction of delta-agonism, it was substituted by similar heterocycles: The substitution of NH(1) by O or S transforms the reference delta-agonist into delta-antagonists. Phenyl ring of benzimidazole is not important for delta-agonism; in fact 1H-imidazole-2-yl retains delta-agonist activity.

Influence of the side chain next to C-terminal benzimidazole in opioid pseudopeptides containing the Dmt-Tic pharmacophore

To improve the structure-activity studies of the lead delta opioid agonist H-Dmt-Tic-Asp*-Bid, we synthesized and pharmacologically characterized a series of analogues in which the side chain next to 1H-benzimidazole-2-yl (Bid) was substituted by those endowed with different chemical properties. Interesting results were obtained: (1) only Gly, Ala, and Asp resulted in delta agonism, (2) Phe yielded delta antagonism, (3) and all other residues except Glu (devoid of any activity) gave mu agonism.