Anti-Helicobacter pylori agents. 4. 2-(Substituted guanidino)-4-phenylthiazoles and some structurally rigid derivatives.

In order to find a new class of anti-Helicobacter pylori (H. pylori) agents, a series of 4-[(3-acetamido)phenyl]-2-(substituted guanidino)thiazoles and some structurally rigid analoges were synthesized and evaluated for antimicrobial activity against H. pylori. Among the compounds obtained, high anti-H. pyrori activities were observed in benzyl derivative 34 (MIC = 0.025 microg/mL) and phenethyl derivatives 35 and 36 (MIC = 0.037 microg/mL and 0.017 microg/mL). Though alkyl derivatives generally showed lower activity, the 2-methoxyethyl derivative 28 preserved significant activity (MIC = 0.32 microg/mL) and also exhibited more potent gastric antisecretory activity than ranitidine. Structural restriction by bridging between the thiazole and the phenyl rings with an alkyl chain did not improve the activity in this series.

Activation of maturation promoting factor and 26S proteasome assembly accelerated by a high concentration of 1-methyladenine in starfish oocytes.

In the oocyte maturation process of the starfish Asterina pectinifera, the extent of inhibition of germinal vesicle breakdown (GVBD) by the proteasome inhibitor MG115 (benzyloxycarbonyl-leucyl-leucyl-norvalinal), as well as the timing of activation of pre-MPF (inactive maturation promoting factor) and 26S proteasome assembly, were found to be dependent on the concentration of the maturation-inducing hormone 1-methyladenine (1-MeAde). Activation of pre-MPF was accelerated by increasing the concentration of 1-MeAde, while there was little effect on the time required for GVBD. Assembly of the 26S proteasome was also accelerated by increasing the concentration of 1-MeAde. These results indicate that a higher concentration of 1-MeAde triggers acceleration of the assembly and increase in the activity of the 26S proteasome, which results in activation of pre-MPF, although there is little effect on the timing of GVBD. It was also clarified that the timing of GVBD is controlled by a rate-liming step after MPF-activation.

Isolation and characterization of a novel 530-kDa protein complex (PC530) capable of associating with the 20S proteasome from starfish oocytes.

A novel protein complex called PC530 was purified concomitantly with proteasomes from oocytes of the starfish, Asterina pectinifera, by chromatography with DEAE-cellulose, phosphocellulose, Mono Q, and Superose 6 columns. The molecular mass of this complex was estimated to be 530 kDa by Ferguson plot analysis and about 500 kDa by Superose 6 gel filtration. Since the 1500-kDa proteasome fractions contain the PC530 subunits as well as the 20S proteasomal subunits, and also since the purified PC530 and the 20S proteasome were cross-linked with a bifunctional cross-linking reagent, it is thought that PC530 is able to associate with the 20S proteasome. The PC530 comprises six main subunits with molecular masses of 105, 70, 50, 34, 30, and 23 kDa. The 70-kDa subunit showed a sequence similarity to the S3/p58/Sun2/Rpn3p subunit of the 26S proteasome, whereas the other subunits showed little or no appreciable similarity to the mammalian and yeast regulatory subunits. These results indicate that starfish oocytes contain a novel 530-kDa protein complex capable of associating with the 20S proteasome, which is distinctly different from PA700 or the 19S regulatory complex in molecular size and subunit composition.

Anti-Helicobacter pylori agents. 3. 2-[(Arylalkyl)guanidino]-4-furylthiazoles.

A series of 2-[(arylalkyl)guanidino]-4-[(5-acetamidomethyl)furan-2-yl]thiazole s and some 4-acetamidomethyl positional isomers were synthesized and evaluated for antimicrobial activity against Helicobacter pylori. Among the compounds that had potent antimicrobial activity (MIC < 0. 1 microgram/mL), compounds 31 and 36 additionally possessed H2 antagonist and gastric antisecretory activities. Though compound 51, an analogue incorporating a methyl group onto the furan nucleus of 36, and compound 54, a positional isomer of 51, also showed potent anti-H. pylori activity, the H2 antagonism profile was eliminated from these compounds. Thus, two types of potent anti-H. pylori agents could be derived from the same scaffold.

The 26S proteasome assembly is regulated by a maturation-inducing hormone in starfish oocytes.

Changes in proteasome activities were observed during starfish oocyte maturation induced by a maturation-inducing hormone, 1-methyladenine. Succinyl-Leu-Leu-Val-Tyr-MCA-hydrolyzing proteasome activity in immature oocytes showed a main peak of a 1500-kDa fraction and a shoulder centered at a 650-kDa fraction on Superose 6 gel-filtration chromatography in the presence of ATP and glycerole. The 1500-kDa activity transiently decreased and then increased at about a half the time required for germinal vesicle breakdown (GVBD). In contrast, the 650-kDa activity showed only a slight change during the maturation process. The activity of the 1500-kDa complex, unlike that of the 650-kDa complex, was immunoprecipitated with an antibody raised against regulatory subunits of mammalian 26S proteasomes, whereas both 1500- and 650-kDa activities were immunoprecipitated with anti-20S proteasome antibody. In addition, the 1500-kDa complex showed an ATP/ubiquitin-dependent proteolytic activity. These results indicate that the 1500- and 650-kDa complexes correspond to the mammalian 26S and 20S proteasomes, respectively. Immunoblot analysis revealed that the change in the 26S proteasomal activity is due to the change in the amount of the 20S proteasome subcomplex. Taken together, the proteasome undergoes changes in molecular assembly and activities during hormone-induced oocyte maturation.

Studies on anti-Helicobacter pylori agents. Part 1: Benzyloxyisoquinoline derivatives.

The synthesis and optimization of the anti-Helicobacter pylori activity of a novel series of benzyloxyisoquinoline derivatives that was discovered by a random screening process, are described. In the in vitro assay, compound 10c containing a 3-acetamido-2,6-dichlorobenzyl substituent was found to have extremely potent activity against H. pylori and no activity against other common bacteria. The anti-H. pylori activity of 10c was superior to that of amoxicillin (AMPC) (1) and clarithromycin (CAM) (2). However, 10c did not show in vivo efficacy in a mouse infection model; a feature attributed to the lack of strong bactericidal activity at short contact times.

Discovery of a novel benzyloxyisoquinoline derivative with potent anti-Helicobacter pylori activity.

The synthesis and in vitro optimization of the anti-Helicobacter pylori activity of a novel series of benzyloxyisoquinoline derivatives discovered by a random screening process, are described. FR180102 (7f), having a 3-acetamido-2,6-dichlorobenzyl moiety, was found to have extremely potent activity against H. pylori and no effect against a series of common Gram-positive and Gram-negative bacteria.

The proteasome is an essential mediator of the activation of pre-MPF during starfish oocyte maturation.

Starfish oocyte maturation was blocked by the addition of 100 microM MG115, a potent proteasome inhibitor, whereas no inhibition was observed by membrane permeable cysteine protease inhibitor, E-64-d. The inhibition by MG115 was diminished by adding at a time corresponding to the half time required for germinal vesicle breakdown. Potent inhibition of germinal vesicle breakdown was also observed by microinjection of anti-proteasome-a-subunit antibodies. The antibody-injected oocytes failed to activate pre-maturation promoting factor (pre-MPF), since the dephosphorylation of phospho-Tyr15 in cdc2 kinase was not observed even in the presence of 1-methyadenine, a maturation-inducing hormone. These results indicate that the proteasome triggers the activation of pre-MPF via the dephosphorylation of cdc2 kinase in the signal transduction pathway in response to the hormonal stimulus during starfish oocyte maturation.

Protease triggers dephosphorylation of cdc2 kinase during starfish oocyte maturation.

We previously presented evidence that a Z-Phe-Ser-argininal-susceptible protease which is involved in oocyte maturation of the starfish, Asterina pectinifera is the proteasome (Takagi Sawada et al, Dev. Biol. 150, 414-418 (1992)). In the present study, we investigated the timing of the function of and the role of the protease in oocyte maturation using Z-Phe-Ser-argininal. By adding the inhibitor in maturing oocytes at various times after 1-methyladenine treatment, the inhibitory ability was markedly reduced in half the time required for germinal vesicle breakdown. Furthermore, the inhibitor potently blocked the activation of histone H1 kinase and the dephosphorylation of cdc2 kinase during oocyte maturation. These results indicate that the Z-Phe-Ser-argininal-susceptible protease, probably the proteasome, plays a key role in the step of the signal transduction pathway that triggers the dephosphorylation of cdc2 kinase in response to the maturation-inducing hormone.