Inhibitory effect of somatostatin Peptide analogues on DNA polymerase activity and human cancer cell proliferation.

BACKGROUND AND OBJECTIVES: It was previously reported that ten small peptides derived from TT-232, somatostatin structural analogue (compounds 1-10), synthesised by a solution-phase method, exhibited potent antitumour activity on human epithelial tumour (A431) cells. MATERIALS AND METHODS: The present study investigated the inhibitory activity of these peptide compounds against DNA polymerase (pol) and human cancer cell growth. RESULTS: Among the compounds tested, compounds 1-5, which contain a t-butyloxycarbonyl (Boc) group, inhibited the activity of mammalian pols. Compounds 2 (Boc-Tyr-D-Trp-1-adamantylamide) and 3 (Boc-Tyr-D-Trp-2-adamantylamide) strongly suppressed the growth of a human colon carcinoma (HCT116) cell line and also arrested HCT116 cells in S phase, suggesting that these phenomena observed in cancer cells may be due to the selective inhibition of mammalian pols, especially DNA replicative pol α, by these compounds. Compound 2 induced apoptosis of the cells, although compound 3 did not. CONCLUSION: Compounds 2 and 3 had an enhanced anticancer effect based on pol inhibition.

New cyclic somatostatin analogues containing a pyrazinone ring: importance of Tyr for antiproliferative activity.

Novel somatostatin analogues containing a pyrazinone ring, compounds 1 and 2, exhibited good antiproliferative activity on A431 tumor cells. To increase antitumor activity and binding affinity on somatostatin receptors (SSTRs), we substituted Tyr in the critical sequence, Tyr-D-Trp-Lys, with more hydrophobic aromatic residue. The substituted compounds dramatically lost antitumor activity, indicating that Tyr residue was an essential residue.

Synthesis of somatostatin analogues containing C-terminal adamantane and their antiproliferative properties.

On the basis of the structure of somatostatin analogue TT-232 (1), which exhibited a highly potent antitumor activity, we synthesized small linear peptide derivatives and evaluated their antitumor and apoptotic activity. Of them, Boc-Tyr-D-Trp-1-adamantylamide (5) had the most potent cell antiproliferative activity in SW480 and A431 cell lines, which was supported in A431 cell lines by FACS analysis that demonstrated a major increase in DNA fragmentation in the subG1 fraction.

Carnosic acid protects neuronal HT22 Cells through activation of the antioxidant-responsive element in free carboxylic acid- and catechol hydroxyl moieties-dependent manners.

In a previous study, we found that carnosic acid (CA) protected cortical neurons by activating the Keap1/Nrf2 pathway, which activation was initiated by S-alkylation of the critical cysteine thiol of the Keap1 protein by the "electrophilic"quinone-type of CA [T. Satoh, K. Kosaka, K. Itoh, A. Kobayashi, M. Yamamoto, Y. Shimojo, C. Kitajima, J. Cui, J. Kamins, S. Okamoto, T. Shirasawa, S.A. Lipton, Carnosic acid, a catechol-type electrophilic compound, protects neurons both in vitro and in vivo through activation of the Keap1/Nrf2 pathway via S-alkylation of targeted cysteines on Keap1. J Neurochem., in press]. In the present study, we used HT22 cells, a neuronal cell line, to test CA derivatives that might be more suitable for in vivo use, as an electrophile like CA might react with other molecules prior to reaching its intended target. CA and carnosol protected the HT22 cells against oxidative glutamate toxicity. CA activated the transcriptional antioxidant-responsive element of phase-2 genes including hemeoxygenase-1, NADPH-dependent quinone oxidoreductase, and gamma-glutamyl cysteine ligase, all of which provide neuroprotection by regulating cellular redox. This finding was confirmed by the result that CA significantly increased the level of glutathione. We synthesized a series of its analogues in which CA was esterified at its catechol hydroxyl moieties to prevent the oxidation from the catechol to quinone form or esterified at those moieties and its carbonic acid to stop the conversion from CA to carnosol. In both cases, the conversion and oxidation cannot occur until the alkyl groups are removed by an intracellular esterase. Thus, the most potent active form as the activator of the Keap1/Nrf2 pathway, the quinone-type CA, will be produced inside the cells. However, neither chemical modulation potentiated the neuroprotective effects, possibly because of increased lipophilicity. These results suggest that the neuroprotective effects of CA critically require both free carboxylic acid and catechol hydroxyl moieties. Thus, the hydrophilicity of CA might be a critical feature for its neuroprotective effects.

Synthesis of opioidmimetics, 3-[H-Dmt-NH(CH(2))(m)]-6-[H-Dmt-NH(CH(2))(n)]-2(1H)-pyrazinones, and studies on structure-activity relationships.

Opioidmimetics containing 3-[H-Dmt-NH-(CH(2))(m)]-6-[H-Dmt-NH-(CH(2))(n)]-2(1H)-pyrazinone symmetric (m = n, 1-4) (1 - 4) and asymmetric (m, n = 1 - 4) aliphatic chains (5 - 16) were synthesized using dipeptidyl chloromethylketone intermediates. They had high mu-affinity (K(i)mu = 0.021 - 2.94 nM), delta-affinity (K(i)delta = 1.06 - 152.6 nM), and mu selectivity (K(i)delta/K(i)mu = 14 - 3,126). The opioidmimetics (1 - 16) exhibited mu agonism in proportion to their mu-receptor affinity. delta-Agonism was essentially lacking in the compounds except (4) and (16), and (1) and (2) indicated weak delta antagonism (pA(2) = 6.47 and 6.56, respectively). The data verify that a specific length of aliphatic linker is required between the Dmt pharmacophore and the pyrazinone ring to produce unique mu-opioid receptor ligands.

Design and synthesis of opioidmimetics containing 2',6'-dimethyl-L-tyrosine and a pyrazinone-ring platform.

Twelve 2',6'-dimethyl-L-tyrosine (Dmt) analogues linked to a pyrazinone platform were synthesized as 3- or 6-[H-Dmt-NH(CH(2))(n)],3- or 6-R-2(1H)-pyrazinone (n=1-4). 3-[H-Dmt-NH-(CH(2))(4)]-6-beta-phenethyl-5-methyl-2(1H)-pyrazinone 11 bound to mu-opioid receptors with high affinity (K(i)mu=0.13 nM; K(i)delta/K(i)mu=447) with mu-agonism (GPI IC(50)=15.9 nM) and weak delta-antagonism (MVD pA(2)=6.35). Key factors affecting opioid affinity and functional bioactivity are the length of the aminoalkyl chain linked to Dmt and the nature of the R residue. These data present a simplified method for the formation of pyrazinone opioidmimetics and new lead compounds.

Wogonin prevents glucocorticoid-induced thymocyte apoptosis without diminishing its anti-inflammatory action.

The effect of wogonin, a flavone highly purified from the roots of Scutellaria baicalensis, on apoptotic cell death was re-evaluated in rat thymocytes. This flavone inhibited glucocorticoid-induced apoptotic changes such as DNA fragmentation, phosphatidylserine translocation, and nuclear condensation in rat thymocytes. Similar inhibition was also observed in apoptosis induced by other inducers such as etoposide. No significant changes of these apoptotic features were observed in rat thymocytes treated with wogonin alone, suggesting that this flavone protects against glucocorticoid-mediated immunosuppression caused by thymocyte apoptosis. Wogonin was reported to possess anti-inflammatory action in some previous studies, but this flavone had no effect on carrageenan-induced paw edema in this study. The simultaneous treatment of wogonin and glucocorticoid neither enhanced nor reduced the anti-inflammatory effect of glucocorticoid. These results indicate that wogonin is likely to prevent the immunosuppression of glucocorticoid without diminishing its drug efficacy as an anti-inflammatory agent.

Wogonin prevents immunosuppressive action but not anti-inflammatory effect induced by glucocorticoid.

Glucocorticoid, such as dexamethasone, has anti-inflammatory and immunosuppressive action as major pharmacological effects. The latter action caused by lymphocyte apoptosis is not only a therapeutic effect but also an adverse reaction. Wogonin, a plant flavone found in Scutellaria baicalensis Georgi, inhibited dexamethasone-induced apoptotic changes, such as DNA fragmentation, nuclear condensation, phosphatidylserine translocation, and caspase activation in rat thymocytes. Since wogonin inhibited dexamethasone-induced DNA fragmentation in a noncompetitive manner, a target of this flavone is unlikely to be an antagonist of glucocorticoid receptor. Wogonin did not only act as an inhibitor of caspases, but also protected apoptosis induced by other glucocorticoids. Since wogonin reduced one of the major pharmacological effects of dexamethasone, we examined whether this flavone diminishes the anti-inflammatory action, another pharmacological effect. The anti-inflammatory action of dexamethasone was evaluated by carrageenan-induced paw edema model. Although dexamethasone significantly suppressed paw edema induced by carrageenan, wogonin had no effect on the anti-inflammatory action of dexamethasone. These results suggest that wogonin may be a useful compound to reduce the immunosuppressive side effect of glucocorticoid.

Wogonin, a plant flavone, potentiates etoposide-induced apoptosis in cancer cells.

Etoposide, a podophylotoxin anticancer agent, induces apoptotic cell death in normal and cancer cells. Etoposide-induced apoptosis plays a role in not only anticancer effect but also adverse reaction, such as myelosuppression. Since we have found that wogonin, a flavone found in Scutellaria baicalensis Georgi, prevents thymocyte apoptosis induced by various compounds including etoposide, we examined the effect of this flavone on etoposide-induced apoptosis in cancer cells. Although 100 muM wogonin itself significantly increased DNA fragmentation in HL-60 cells, this change was not observed in Jurkat cells. On the other hand, this flavone significantly potentiated etoposide-induced apoptosis in Jurkat and HL-60 cells. Similarly, wogonin accelerated etoposide-induced cell death in lung cancer cells. Since wogonin had no effect on the action of other anticancer agents, such as 5-FU and cisplatin, this flavone seems to accelerate only etoposide-induced apoptotic cell death in cancer cells. These results suggest that the modification of etoposide-induced apoptosis by wogonin may be available to reduce the adverse reaction of this agent.

Studies on the mechanism of 1,2-dihydropyrazin-2-one ring formation from dipeptidyl chloromethyl ketone and its chemical properties: immediate deamination during catalytic hydrogenation.

1,2-Dihydropyrazin-2-one derivatives, which have two aminoalkyl groups at the positions 3 and 6, were found to be efficient tools for the construction of potent, selective and long-acting opioid mimetics. During the course of preparation, we found that the catalytic hydrogenation of 3,6-bis(benzyloxycarbonylaminomethyl)-5-methyl-1,2-dihydropyrazin-2-one to remove the benzyloxycarbonyl groups resulted in a side reaction. By MS and NMR studies and by preparation of additional 1,2-dihydropyrazin-2-one derivatives, the structure of the by-product was identified as 3-aminomethyl-5,6-dimethyl-1,2-dihydropyrazin-2-one. Preparation of additional compounds substituted with deuterium provided us with sufficient information to confirm the structure of the product and to support a cyclization mechanism in its formation.

Studies on the structure-activity relationship of 2',6'-dimethyl-l-tyrosine (Dmt) derivatives: bioactivity profile of H-Dmt-NH-CH(3).

The 2',6'-dimethyl-l-tyrosine (Dmt) enhances receptor affinity, functional bioactivity and in vivo analgesia of opioid peptides. To further investigate its direct influence on these opioid parameters, we developed a series of compounds (H-Dmt-NH-X). Among them, H-Dmt-NH-CH(3) showed the highest affinity (K(i)mu=7.45 nM) equal to that of morphine, partial mu-opioid agonism (E(max)=66.6%) in vitro and a moderate antinociception in mice.

Development of potent bifunctional endomorphin-2 analogues with mixed mu-/delta-opioid agonist and delta-opioid antagonist properties.

The C terminus of endomorphin-2 (EM-2) analogues (Tyr-Pro-Phe-NH-X) was modified with aromatic, heteroaromatic, or aliphatic groups (X = phenethyl,benzyl, phenyl, naphthyl, pyridyl, quinolyl, isoquinolyl, tert-butyl, cyclohexyl, or adamantyl; 3-18) to study their effect on opioid activity. Only 9 (1-naphthyl), 11 (5-quinolyl), 16 (cyclohexyl), and 18 (2-adamantyl) exhibited mu-opioid receptor affinity in the nanomolar range (K(i) = 2.41-6.59 nM), which, however, was 3- to 10-fold less than the parent peptide. Replacement of Tyr(1) by Dmt (2',6'-dimethyl-l-tyrosine) (19-32) exerted profound effects: (i) acquisition of high mu-opioid receptor affinity (K(i) = 0.11-0.52 nM) except 23 (Ph); (ii) presence of potent functional mu-opioid receptor agonism (IC(50) < 1 nM) for 19 ([Dmt(1)]EM-2), 27 (1-naphthyl), 29 (5-quinolyl), and 32 (5-isolquinolyl); (iii) association of weak delta-opioid antagonist activity (pA(2) = 5.41-7.18) except 19 ([Dmt(1)]EM-2), 20 (H), 27 (1-naphthyl), and in particular 29 (5-quinolyl) with its potent delta-agonism (IC(50) = 0.62 nM, pA(2) = 5.88); (iv) production of antinociception after ic administration of 32 (5-isoquinolyl) in mice, a bioactivity absent in the corresponding Tyr(1) analogue (14); and (v) preferential cis orientation (cis/trans = 3:2 to 7:3) at the Dmt-Pro amide bond, in contrast to the Tyr-Pro amide trans orientation (cis/trans = 1:2 to 1:3). Thus, [Dmt(1)]EM-2 analogues with hydrophobic C-terminal extensions provide model compounds with potent mu-opioid receptor bioactivity and dual functional agonism.

Oral bioavailability of a new class of micro-opioid receptor agonists containing 3,6-bis[Dmt-NH(CH(2))(n)]-2(1H)-pyrazinone with central-mediated analgesia.

The inability of opioid peptides to be transported through epithelial membranes in the gastrointestinal tract and pass the blood-brain barrier limits their effectiveness for oral application in an antinociceptive treatment regime. To overcome this limitation, we enhanced the hydrophobicity while maintaining the aqueous solubility properties in a class of opioid-mimetic substances by inclusion of two identical N-termini consisting of Dmt (2',6'-dimethyl-l-tyrosine) coupled to a pyrazinone ring platform by means of alkyl chains to yield the class of 3,6-bis[Dmt-NH-(CH(2))(n)]-2(1H)-pyrazinones. These compounds displayed high micro-opioid receptor affinity (K(i)micro = 0.042-0.115 nM) and selectivity (K(i)delta/K(i)micro = 204-307) and functional micro-opioid receptor agonism (guinea-pig ileum, IC(50) = 1.3-1.9 nM) with little or undetectable bioactivity toward delta-opioid receptors (mouse vas deferens) and produced analgesia in mice in a naloxone reversible manner when administered centrally (intracerebroventricular, i.c.v.) or systemically (subcutaneously and orally). Furthermore, the most potent compound, 3,6-bis(3'-Dmt-aminopropyl)-5-methyl-2(1H)-pyrazinone (7'), lacked functional delta-opioid receptor bioactivity and was 50-63-fold and 18-21-fold more active than morphine by icv administration as measured analgesia using tail-flick (spinal involvement) and hot-plate (supraspinal effect) tests, respectively; the compound ranged from 16 to 63% as potent upon systemic injection. These analgesic effects are many times greater than unmodified opioid peptides. The data open new possibilities for the rational design of potential opioid-mimetic drugs that pass through the epithelium of the gastrointestinal tract and the blood-brain barrier to target brain receptors.

Carnosic acid, a component of rosemary (Rosmarinus officinalis L.), promotes synthesis of nerve growth factor in T98G human glioblastoma cells.

Nerve growth factor (NGF) is a factor vital for the growth and functional maintenance of nerve tissue. The authors found that a rosemary (Rosmarinus officinalis L.) extract enhanced the production of NGF in T98G human glioblastoma cells. Furthermore, the results indicated that carnosic acid and carnosol, which are major components of the rosemary extract, were able to promote markedly enhanced synthesis of NGF.

Unique high-affinity synthetic mu-opioid receptor agonists with central- and systemic-mediated analgesia.

Unique opioid mimetic substances containing identical N-terminal aromatic residues separated by an unbranched alkyl chain containing two to eight methylene groups were developed. Regardless of the length of interposing alkyl chain, the bis-Tyr and bis-Phe compounds were inactive; however, replacement by a single Dmt (2',6'-dimethyl-L-tyrosine) residue enhanced activity by orders of magnitude. Moreover, the bis-Dmt compounds were another 10-fold more potent with an optimum intra-aromatic ring distance of about four to six methylene units. 1,4-Bis(Dmt-NH)butane (7) had high mu-opioid receptor affinity (K(i) = 0.041 nM) and functional mu-opioid agonist bioactivity (IC(50) = 5.3 nM) with in vivo central (intracerebroventricular) and systemic (subcutaneous) analgesia in mice (1.5- to 2.5-fold greater than and 10-12% relative to morphine, respectively); these activities were reversed by naloxone to the same degree. It appears that the bis-Dmt compounds indiscriminately act as both message and address domains.

Structural studies of [2',6'-dimethyl-L-tyrosine1]endomorphin-2 analogues: enhanced activity and cis orientation of the Dmt-Pro amide bond.

Analogues of endomorphin-2 (EM-2: Tyr-Pro-Phe-Phe-NH(2)) (1) were designed to examine the importance of each residue on mu-opioid receptor interaction. Replacement of Tyr(1) by 2',6'-dimethyl-L-tyrosine (Dmt) (9-12) exerted profound effects: [Dmt(1)]EM-2 (9) elevated mu-opioid affinity 4.6-fold (K(i mu=0.15 nM) yet selectivity fell 330-fold as delta-affinity rose (K(i)delta=28.2 nM). This simultaneous increased mu- and delta-receptor bioactivities resulted in dual agonism (IC(50)=0.07 and 1.87 nM, respectively). While substitution of Phe(4) by a phenethyl group (4) decreased mu affinity (K(i)mu=13.3 nM), the same derivative containing Dmt (12) was comparable to EM-2 but also acquired weak delta antagonism (pA(2)=7.05). 1H NMR spectroscopy revealed a trans configuration (1:2 to 1:3, cis/trans) in the Tyr-Pro amide bond, but a cis configuration (5:3 to 13:7, cis/trans) with Dmt-Pro analogues.