Novel, potent, and selective phosphodiesterase 5 inhibitors: synthesis and biological activities of a series of 4-aryl-1-isoquinolinone derivatives.

A novel class of potent and selective phosphodiesterase 5 (PDE5) inhibitors, 4-aryl-1-isoquinolinone derivatives, which have been designed by the comparison of the structure of cGMP and a previously reported 1-arylnaphthalene lignan, was disclosed. Among these compounds, methyl 2-(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-3-isoquinoline carboxylate dihydrochloride (36a) exhibited potent PDE5 inhibitory activity (IC(50) = 1.0 nM) with high isozyme selectivities (IC(50) ratio: PDE1/PDE5 = 1300, PDE2/PDE5 > 10 000, PDE3/PDE5 > 10 000, PDE4/PDE5 = 4700, PDE6/PDE5 = 28). Compound 36a also showed the most potent relaxant effect on isolated rabbit corpus cavernosum (EC(30) = 7.9 nM). Compound 63 (T-1032), the sulfate form of 36a, was selected for further biological and pharmacological evaluation of erectile dysfunction.

Alpha helix content of G protein alpha subunit is decreased upon activation by receptor mimetics.

To elucidate the mechanism whereby liganded receptor molecules enhance nucleotide exchange of GTP-binding regulatory proteins (G proteins), changes in the secondary structure of the recombinant Gi1 alpha subunit (Gi1alpha) upon binding with receptor mimetics, compound 48/80 and mastoparan, were analyzed by circular dichroism spectroscopy. Compound 48/80 enhanced the initial rate of GTPgammaS binding to soluble Gi1alpha 2.6-fold with an EC50 of 30 microg/ml. With the same EC50, the mimetic decreased the magnitude of ellipticity, which is ascribed to a reduction in alpha helix content of the Gi1alpha by 7%. Likewise, mastoparan also enhanced the rate of GTPgammaS binding by 3.0-fold and decreased the magnitude of ellipticity of Gi1alpha similar to compound 48/80. In corresponding experiments using a K349P-Gi1alpha, a Gi1alpha counterpart of the unc mutant in Gsalpha in which Pro was substituted for Lys349, enhancement of the GTPgammaS binding rate by both activators was quite small. In addition, compound 48/80 showed a negligible effect on the circular dichroism spectrum of the mutant. On the other hand, a proteolytic fragment of Gi1alpha lacking the N-terminal 29 residues was activated and showed decreased ellipticity upon interaction with the compound, as did the wild-type Gi1alpha. Taken together, our results strongly suggest that the activator-induced unwinding of the alpha helix of the G protein alpha subunit is mechanically coupled to the enhanced release of bound GDP from the alpha subunit.

A potent dipeptide inhibitor of dipeptidyl peptidase IV.

A series of novel potent inhibitors of dipeptidyl peptidase IV (DPP-IV) has been developed. A brief structure-activity relationship of the inhibitors was investigated. The dipeptide TSL-225, tryptophyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, was identified with the critical structure for the inhibitory activity.

A Gs-selective analog of the receptor-mimetic peptide mastoparan binds to Gs alpha in a kinked helical conformation.

Mastoparan, a 14-residue peptide, stimulates GDP/GTP exchange on G proteins in a manner strikingly analogous to that of agonist-bound receptors. Presumably, the peptide structurally mimics a receptor's G protein-binding domain. We previously reported that mastoparan-X binds to alpha-subunits of Gi and Go in a predominantly alpha-helical conformation [Sukumar, M., & Higashijima, T. (1992) J. Biol. Chem. 267, 21421-21424]. We have now developed an analogous peptide, INWKGIASM-alpha-aminoisobutyryl (Aib)-RQVL-NH2 (MP-S), which is a selective activator of Gs. We report the conformation of MP-S when it is bound to Gs alpha, determined from distance geometry calculations based on transferred nuclear Overhauser effects (TRNOEs). The Gs-bound conformation of MP-S is an alpha-helix that is kinked at residue 9. The conformations of MP-S when bound to Gi alpha or Go alpha are similar to the Gs alpha-bound conformation. In contrast, the lipid-bound conformation of MP-S is a straight helix. On the basis of the Gs-bound conformation of MP-S, directions for the design of Gs-selective peptidergic mimics of receptors are suggested.

Gonadotropin-releasing hormone agonist therapy induces apoptosis in uterine leiomyoma.

OBJECTIVE: We examined uterine tissue samples obtained from premenopausal women with uterine leiomyoma treated with gonadotropin-releasing hormone agonist (GnRHa) to investigate the mechanism of the effects of GnRHa. STUDY DESIGN: Surgically resected myoma tissue obtained from 26 premenopausal patients with uterine leiomyoma treated with GnRHa, 20 premenopausal patients with uterine leiomyoma who did not receive GnRHa treatment, and 15 postmenopausal women with uterine leiomyoma were examined histologically. RESULTS: GnRHa treatment reduced the size of uterine leiomyomata and induced significant hyaline degeneration in tumor tissue. Le(Y)-antigen expression was detected in 18 (69.3%) of 26 GnRHa-treated patients (P < 0.02) and in 12 (80.0%) of 15 postmenopausal women (P < 0.05), but in only eight (40.0%) of the 20 premenopausal patients who did not receive GnRHa. Apoptotic cells, detected by the nick-end labeling method were observed in 14 patients (53.8%) in the GnRHa-treated group, 10 patients (50.0%) in the non-treated group, and 12 postmenopausal women (80.0%). CONCLUSION: Our findings suggest that induction of apoptosis may be a mechanism of the effect of GnRHa in leiomyoma.

Scanning of polypoid gallbladder lesions by ultrasonic microprobes using transpapillary catheterization.

BACKGROUND AND STUDY AIMS: Polypoid lesions of the gallbladder can be diagnosed by transabdominal ultrasonography and endoscopic ultrasonography. However, the image resolution is limited, and the histopathology of these lesions is difficult to determine. We report here on our preliminary results in four patients using a high-frequency (20 MHz) ultrasonic microprobe introduced into the gallbladder via the transpapillary approach. PATIENTS AND METHODS: Insertion of the microprobe into the gallbladder through the papilla of Vater was attempted in four patients with gallbladder diseases without preceding endoscopic sphincterotomy. RESULTS: The lesions in the gallbladder were clearly visualized in three of the four patients. We tried to correlate the characteristic ultrasound findings with the histopathology of the lesions, which were found to be early cancer, adenoma, and adenomyomatosis on histopathology of the specimens after cholecystectomy. No complications occurred. CONCLUSIONS: High-resolution endoscopic transpapillary ultrasonography of the gallbladder is feasible and safe, and may contribute to the diagnosis of gallbladder diseases, particularly of small polypoid lesions.

The upstream region of the isocitrate lyase gene (UPR-ICL) of Candida tropicalis induces gene expression in both Saccharomyces cerevisiae and Escherichia coli by acetate via two distinct promoters.

The upstream region of the isocitrate lyase gene (UPR-ICL, 1530bp) of an n-alkane-utilizable yeast, Candida tropicalis, induced gene expression in another yeast, Saccharomyces cerevisiae, when the yeasts were grown on acetate. Surprisingly, UPR-ICL displayed the same regulatory function in the bacterium Escherichia coli when grown on acetate. We determined the interesting nucleotide sequence of UPR-ICL. The deletion analysis of UPR-ICL in both cells revealed the presence of two distinct promoters: one was localized at -394 to -379 and regulated gene expression in S. cerevisiae; the other was located near the initiation codon and regulated gene expression in E. coli. The two promoter sequences were similar, but not identical to regulatory elements that have been previously reported in S. cerevisiae and E. coli, respectively. Accordingly, the possibility of novel regulatory mechanisms could not be excluded. This is an interesting example of the presence of distinct cis-acting regulatory elements responsible for the induction of gene expression in one gene by acetate in both S. cerevisiae and E. coli. Preservation of such promoters through evolution is also discussed.

[The effects of sex steroid hormones, gonadotropins, and gonadotropin releasing hormone agonist on the proliferation of ovarian cancer cell line (KOC-2S)].

The present study was designed to determine the effects of sex steroid hormones (estradiol, progesterone), gonadotropins (FSH, hCG), and gonadotropin releasing hormone agonist (GnRHa: Buserelin) on the proliferation of the ovarian serous adenocarcinoma cell line KOC-2S in vitro. The results showed: 1. The colony formation rate and fraction growth in the colony stem cell assay were suppressed by estradiol (5,000 pg/ml) and progesterone (50 ng/ml). The colony formation rates were 2.96% (control); 2.49%, 0.99% (estradiol: 500 pg/ml, 5,000 pg/ml); 2.58%, 0.53% (progesterone: 5.0 ng/ml, 50.0 ng/ml); 4.80%, 3.34% (hCG: 10 mIU/ml, 100 mIU/ml); and 1.79%, 2.96% (GnRHa: 1.0 ng/ml, 10 ng/ml). 2. The doubling time (DT) in the growth inhibition test was shortened by FSH and the saturation density (SD) became greater. The SD was suppressed by hCG. 3. The cytogenic features of the cells treated with FSH, estradiol and GnRHa did not show obvious morphologic change. The spontaneous floating cells were observed following treatment with progesterone. Cytoplasmic enlargement was observed following treatment with hCG. 4. Neither erb B nor erb B-2 was expressed in KOC-2S cells, and neither was induced by sex steroid hormones, gonadotropins or GnRHa.

Vesicle-bound conformation of melittin: transferred nuclear Overhauser enhancement analysis in the presence of perdeuterated phosphatidylcholine vesicles.

We determined a detailed conformation of the honeybee venom peptide melittin when bound to phosphatidylcholine vesicles using proton NMR. In the presence of vesicles of perdeuterated dipalmitoylglycerophosphocholine, two-dimensional transferred nuclear Overhauser enhancement (TRNOE) experiments were carried out. By a distance geometry calculation using NOE-derived distance constraints followed by a simulated annealing refinement, the N-terminal (Leu6-Leu10) and C-terminal (Leu13-Lys21) parts were found to have an alpha-helical conformation, whereas five C-terminal residues (Arg22-Gln26) did not show a unique conformation in the vesicle-bound state. The two alpha-helices were connected via a less structured segment (Thr11-Gly12) with a helix bend angle of 86 degrees +/- 34 degrees. Model distance geometry calculations using distance constraints extracted from a tetrameric melittin molecule in crystal assured us that the NOE constraints can accurately reproduce melittin's structure, as well as helping to interpret the NMR structures. Although the vesicle-bound conformation of melittin is similar to that occurring in a methanol solution and in dodecylphosphocholine micelles, significant differences were found in the conformation of C-terminal basic residues and the helix bend angle. This is the first study to clearly demonstrate conformation differences in micelle- and vesicle-bound peptides. In addition, lytic activity of melittin and its analogs showed better correlation with a peptide conformation in vesicles than in either methanol or micelles.

Circular dichroism studies of the interaction between synthetic peptides corresponding to intracellular loops of beta-adrenergic receptors and phospholipid vesicles.

We previously showed that peptides corresponding to the N-terminal parts of the third intracellular loops of turkey and hamster beta-adrenergic receptors (tu beta I3N and ha beta I3N, respectively) can activate the GS protein (one of the GTP-binding regulatory proteins which couples to the beta-adrenergic receptor) reconstituted in phospholipid vesicles, and also that such activation can be greatly enhanced by a modification which increases the hydrophobicity of the peptides. These observed phenomena suggest that the interaction with phospholipid membranes is important for the activity of these peptides; hence, in the present study we employed circular dichroism to analyze the interaction of the synthetic peptides corresponding to the intracellular loops of G protein-coupled receptors with phosphatidylserine/phosphatidylcholine mixed vesicles. The tu beta I3N and ha beta I3N peptides were subsequently found to take on an alpha-helical conformation upon binding with the vesicles, whereas those corresponding to the intracellular loops of m1 and m2 muscarinic acetylcholine receptors in contrast did not interact with the vesicles. The positions of several side chains of the membrane-bound loop peptides were also determined. Our results show for the first time the interaction occurring between the intracellular loops of beta-adrenergic receptors and a phospholipid membrane.

GAP-43 augments G protein-coupled receptor transduction in Xenopus laevis oocytes.

The neuronal protein GAP-43 is thought to play a role in determining growth-cone motility, perhaps as an intracellular regulator of signal transduction, but its molecular mechanism of action has remained unclear. We find that GAP-43, when microinjected into Xenopus laevis oocytes, increases the oocyte response to G protein-coupled receptor agonists by 10- to 100-fold. Higher levels of GAP-43 cause a transient current flow, even without receptor stimulation. The GAP-43-induced current, like receptor-stimulated currents, is mediated by a calcium-activated chloride channel and can be desensitized by injection of inositol 1,4,5-trisphosphate. This suggests that neuronal GAP-43 may serve as an intracellular signal to greatly enhance the sensitivity of G protein-coupled receptor transduction.

The mechanism of aluminum-independent G-protein activation by fluoride and magnesium. 31P NMR spectroscopy and fluorescence kinetic studies.

With magnesium present, fluoride and aluminum ions activate heterotrimeric G-proteins by forming AlFx complexes that mimic the gamma phosphate of a GTP. We report compelling evidence for a newly proposed process of G-protein activation by fluoride and magnesium, without Al3+. With millimolar Mg2+ and F-, Gs and Gt activate adenylylcyclase and cGMP-phosphodiesterase, respectively. In 31P NMR, addition of magnesium to Gi1 alpha GDP or Gt alpha GDP solutions containing fluoride, but no Al3+, modifies the chemical shift of the GDP beta phosphorus, suggesting that magnesium interacts with the beta phosphate. Titration of this effect indicates that two Mg2+ are bound per G alpha. Biphasic activation kinetics, monitored by G alpha tryptophan fluorescence, suggests the rapid binding of one Mg2+ to G alpha GDP and the slow association of another Mg2+, in correlation with fluoride binding and G alpha activation. The deactivation rate upon fluoride dilution shows a second order dependence with respect to the residual F- concentration, suggesting the sequential release of at least three F-/G alpha. Thus, in millimolar Mg2+ and F-, and without Al3+, two Mg2+ and three F- bind sequentially to G alpha GDP and induce the switch to an active G alpha (GDP-MgF3)Mg state, which is structurally analogous to G alpha (GDP-AlFx)Mg and to G alpha (GTP)Mg.

G protein-bound conformation of mastoparan-X, a receptor-mimetic peptide.

Mastoparans are a family of 14-residue peptide toxins from wasp venom which have been proposed to stimulate secretion from a variety of cells, by directly activating GTP-binding regulatory proteins (G proteins). In vitro studies have shown that mastoparans activate G proteins by a mechanism remarkably similar to that used by agonist-bound receptors (Higashijima, T., Uzu, S., Nakajima, T., and Ross, E. M. (1988) J. Biol. Chem. 263, 6491-6494). Here, we report the conformation of mastoparan-X (INWKGIAAMAKKLL-NH2) when it is bound to the alpha subunits of recombinant G(i) and G(o), derived from an analysis of transferred nuclear Overhauser effects in a two-dimensional 1H NMR spectrum of mastoparan-X obtained in the presence of these G proteins. Restrained molecular dynamic simulations with NMR-derived distance constraints were used to determine conformations consistent with NMR data. The G(i)- and G(o)-bound conformations of mastoparan-X are very similar, and in both cases, a major part of the molecule adopts an amphiphilic alpha-helical conformation. The lysine residues are known to be crucial for activity, and it is thus likely that at least the polar face of the amphiphilic helix is in contact with the G proteins. These conclusions should be useful in the design of potent and selective analogs of mastoparan and in the development of models for receptor-G protein interaction.

G protein antagonists. A novel hydrophobic peptide competes with receptor for G protein binding.

A substance P (SP) analog, [D-Pro4,D-Trp7,9,10] SP4-11, is known to inhibit the actions of various structurally unrelated messenger molecules as well as SP. Our studies on the effects of this peptide on the regulation of purified G proteins by receptor showed that at least some of the biological effects of the peptide can be explained by the ability of the peptide to block the activation of G proteins by receptors. Here we report that a novel truncated SP-related peptide, pGlu-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH2, inhibited the activation of G(i) or G(o) by M2 muscarinic cholinergic receptor (M2 mAChR) or of Gs by beta-adrenergic receptor in the reconstituted phospholipid vesicles, assayed by receptor-promoted GTP hydrolysis. The inhibition by the peptide was apparently reversible and competitive with respect to receptor binding to G proteins; the inhibition could be overcome by increasing the concentration of receptor in the vesicles and was not altered by changes in the concentration of G protein. The competing effects of the peptide were used to analyze the effect of agonist on receptor-G protein interaction. The concentration change of muscarinic agonist did not alter the inhibitory effects of the peptide on M2 mAChR-promoted GTPase by G(o), which is consistent with the idea that agonist increases the regulatory efficiency of the receptor but does not alter its affinity for G proteins. This new group of compounds (G protein antagonists) is a promising tool to study receptor-G protein interaction quantitatively.

Membrane-bound conformation of mastoparan-X, a G-protein-activating peptide.

Mastoparan-X, a tetradecapeptide from wasp venom, has been proposed to cause secretion from various kinds of cells by the direct activation of GTP-binding regulatory proteins (G proteins) that couple to phospholipase C. The mechanism of the activation has been shown to be very similar to that of G-protein-coupled receptors in vitro, and the interaction with membranes seems to be very important for the activation of G proteins that are membrane-bound [Higashijima, T., Uzu, S., Nakajima, T., & Ross, E. M. (1988) J. Biol. Chem. 263, 6491-6494]. We report here the precise vesicle-bound conformation of mastoparan-X in the presence of perdeuterated phospholipid vesicles, determined by two-dimensional 1H-NMR analyses of transferred nuclear Overhauser effects, combined with distance geometry and molecular dynamics calculations. Of 14 amino acid residues, the C-terminal 12 residues take an alpha-helical conformation upon binding to the phospholipid bilayer. The overall structure of the alpha-helix is amphiphilic, with three lysine side chains located on one side and with hydrophobic side chains on the other side. This conformation of mastoparan-X was maintained both in the gel and in the liquid-crystalline phases of the membranes. The conformation described herein will provide a useful basis for understanding conformation-activity relationships of mastoparan analogs as activators of G proteins. These studies will help to design novel potent analogs for the regulation of G proteins and to analyze receptor-G-protein interactions.

[Effects of chloride ion on the activation of G proteins].

G proteins act as signal transducers that couple receptors to effectors through membranes. G proteins are in a GDP-bound form in the basal state, which can interact with receptors. The hormone-bound receptor promotes GDP-GTP exchange of G proteins. The GTP, Mg(2+)-bound G proteins can activate effector molecules, which in turn produce second messengers. The GTP, Mg(2+)-form is converted to the GDP-form by the GTP-hydrolysis activity of G protein alpha-subunits. Cl- ions modulate the GDP-GTP cycle of G proteins. Cl- ions, in the presence of Mg2+, decelerate the receptor-independent spontaneous release of GDP (ca. 6-fold by 100 mM NaCl), which will result in a lowering of the basal level of second messengers. On the other hand, the GTP-hydrolysis activity of G proteins was suppressed by 3-fold with 100 mM NaCl, which will result in keeping the G proteins in an active form for a longer time. In the absence of Mg2+, Cl- ion mimics Mg2+ to convert GTP-bound G proteins to an active form (dissociated form), although the effects are weaker than those of Mg2+. These effects are very different from F-, which is another halogen ion that interacts with G proteins.

Reconstitution of agonist-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis using purified m1 muscarinic receptor, Gq/11, and phospholipase C-beta 1.

We describe the reconstitution using purified proteins of the m1 muscarinic cholinergic pathway that activates phosphatidylinositol 4,5-bisphosphate-specific phospholipase C via the G protein Gq/11. Recombinant m1 muscarinic receptor was co-reconstituted in lipid vesicles with either hepatic Gq/11 or with cerebral alpha q/11 and beta gamma subunits. The rate of [35S]GTP gamma S binding to the reconstituted vesicles was stimulated 20-50-fold by agonist. Maximal receptor-catalyzed binding was 7 mol of GTP gamma S bound per mol of receptor. The m2 muscarinic receptor was a poor activator of Gq/11. The binding of [alpha-32P]GTP to [gamma-32P]GTP to m1/Gq/11 vesicles indicated that the receptor could maintain up to 40% of the total coupled Gq/11 in the GTP bound state. The rate of hydrolysis of bound GTP, 0.8 min-1, is consistent with the rate predicted from the GTP binding data but is 3-5-fold lower than rates reported for other trimeric G proteins. Agonist-stimulated photo-affinity labeling with gamma-(4-azidoanilido)-[alpha-32P]GTP indicated that the receptor catalyzed binding to both alpha q and alpha 11 with about equal efficiency. Receptor-catalyzed activation of Gq/11 by GTP gamma S, measured as the ability to activate purified phospholipase C-beta 1, paralleled receptor-catalyzed [35S]GTP gamma S binding. Co-reconstitution of receptor, Gq/11, and phospholipase C-beta 1 restored GTP gamma S-dependent carbachol-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate. The m1 receptor, Gq/11, and phospholipase C-beta 1 are thus sufficient to initiate the hormonal inositol trisphosphate/diacylglycerol signaling pathway without additional proteins.

[Immunohistochemical studies of ras oncogene product p21 in 7,12 dimethylbenz (a) anthracene-induced rat ovarian tumors].

In the present study, ras oncogene product p21 was analyzed immunohistochemically in rat ovarian tumors induced by 7,12 dimethylbenz (a) anthracene (DMBA). 1) After 28-38 (average 35.6) weeks, the tumors -6 adenomas, 30 adenocarcinomas, 3 sarcomas, one mixed müllerian tumor and 2 epidermal cysts--were produced in the rat ovaries. 2) The p21 positive reactions could be seen in the cytoplasm of the tumor cells. The positive rates were as follows: adenomas 83%, adenocarcinomas 57%, sarcomas 67%, mixed müllerian tumors 0%, epidermal cysts 100%, in which the positive reactions were obtained in squamous cells. Both of the serial-allografted tumor and DMBA-OC-1 were positive. 3) The present study showed that the ras oncogene is not a specific gene in ovarian sarcoma or in other tumors. 4) The present study has suggested that the ras oncogene plays a role in tumor genesis including benign tumors, by showing p21 positive in adenomas and epidermal cysts.