Antifungal Activity of Tetra-Substituted Tetrahydrofuran Lignan, (−)-Virgatusin,and Its Structure-Activity Relationship

Antifungal activities of the optically pure (>99%ee) (?)- and (+)-virgatusin, a tetra-substituted tetrahydrofuran lignan, were tested. (?)-Virgatusin, which is a natural product, showed highest antifungal activity against Colletotrichum lagenarium. Research on its structure-activity relationship was also performed. It was shown that two methoxy groups on 9 and 9′ positions and a 3,4-methylenedioxyphenyl group on the 7 position of virgatusin were essential for high fungal growth inhibition. The part on 7′-phenyl group was not essential for activity. The 7′-(4-methoxyphenyl) derivative showed higher activity than that of (?)-virgatusin.     

Structure-antibacterial activity relationship for 9-O,9'-O-demethyl (+)-virgatusin

The relationship between the antibacterial activity and structure of 9-O,9'-O-demethyl (+)-virgatusin (Virg 3) was examined. The conversion of hydroxy groups on the 9 and 9' positions to amino groups increased the activity. It was found that the 3'-methoxy group was more important for higher activity than the 4'-methoxy group on the 7'-phenyl group, and that the 3,4-methylenedioxy group on the 7-phenyl group was necessary for activity.     

Structure-Antibacterial Activity Relationship for 9-O,9′-O-Demethyl (+)-virgatusin

The relationship between the antibacterial activity and structure of 9-O,9′-O-demethyl (+)-virgatusin (Virg 3) was examined. The conversion of hydroxy groups on the 9 and 9′ positions to amino groups increased the activity. It was found that the 3′-methoxy group was more important for higher activity than the 4′-methoxy group on the 7′-phenyl group, and that the 3,4-methylenedioxy group on the 7-phenyl group was necessary for activity.     

Mutagenic activity of 2-phenylbenzotriazole derivatives related to a mutagen, PBTA-1, in river water.

A mutagen, 2-[2-(acetylamino)-4-[bis(2-methoxyethyl)amino]-5-methoxyphenyl]5-ami no-7-bromo-4-chloro-2H-benzotriiazole (PBTA-1), isolated from water of the Nishitakase River in Kyoto exhibits potent mutagenic activity in Salmonella typhimurium TA98 with S9 mix and has characteristic moieties, including bromo, chloro, acetylamino, bis(2-methoxyethyl)amino and primary amino groups on a 2-phenylbenzotriazole skeleton. The mutagenicities of PBTA-1, its congeners and five related 2-phenylbenzotriazoles were examined in S. typhimurium TA98 with S9 mix in order to elucidate the structure-activity relationships. The data obtained suggest that a primary amino group plays an essential role in the mutagenic activity as do aromatic amines including heterocyclic amines in cooked foods. The effect of planarity of the 2-phenylbenzotriazole ring was significant, and in addition, halogen groups of PBTA-1 influenced the enhancement of the mutagenic activity.     

Disruption of ion homeostasis by verrucosin and a related compound

We have found that (-)-virgatusin and related compounds have antimicrobial and antifungal activity. To identify further biological activities of these compounds, we tested the activity of acridine orange efflux, which shows ionophore-like disruption of cellular ion homeostasis activity. After testing 31 compounds, we found that verrucosin and a related compound had disruption activity.     

Identification of a component that induces flowering of Lemna among the reaction products of alpha-ketol linolenic acid (FIF) and norepinephrine.

A stress-induced fatty acid [FIF; 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid] incubated with (-)-norepinephrine (NE) strongly induces flower formation in Lemna paucicostata [Yokoyama et al. (2000), Plant Cell Physiol. 41: 110). The increase of flower-inducing activity was well correlated with the decrease in FIF in the incubation mixture, and the reaction proceeded rapidly at higher pH. We detected small amounts of many active components in the mixture after incubation by HPLC analysis. In this study, two major components, named FN1 and FN2, of the reaction mixture were isolated, and their absolute stereostructures were determined. FN1 showed a strong flower-inducing activity and was identified as a tricyclic alpha-ketol fatty acid, 9(R)-11-[(2'R,8'R,10'S,11'S)-2',8'-dihydroxy-7'-oxo-11'-[(Z)-2-pentenyl]-9'-oxa-4'-azatricyclo[6.3.1.0(1.5)]dodec- 5'en-10'-yl]-9-hydroxy-10-oxoundecanoic acid [corrected]. FN2, the C-9 epimer of FN1, showed no flower-inducing activity. The absolute stereostructure of FIF was also determined by a modification of Mosher's method. The 9-hydroxyl group was found to be predominantly 9R, with an enantiomeric excess of 40% (70% 9R and 30% 9S). FN1 was derived from 9R-type FIF and FN2 from 9S-type FIF. Various catecholamines and related substances were investigated for the ability to develop flower-inducing activity upon incubation with FIF. The essential structures were catechol and ethylamine groups (dopamine).     

Synthesis and antimicrobial activity of new 3-(1-R-3(5)-methyl-4-nitroso-1H-5(3)-pyrazolyl)-5-methylisoxazoles

A number of new 3-(1-R-3(5)-methyl-4-nitroso-1H-5(3)-pyrazolyl)-5-methylisoxazoles 6a–g (7b–f) were synthesized and tested for antibacterial and antifungal activity. Some of these compounds displayed antifungal activity at non-cytotoxic concentrations. Derivative 6c was 9 times more potent in vitro than miconazole and 20 times more selective against C. neoformans. 6c was also 8- and 125-fold more potent than amphotericin B and fluconazole, respectively. None of the compounds was active against bacteria. Preliminary structure–activity relationship (SAR) studies showed that the NO group at position 4 of the pyrazole ring is essential for the activity. Lipophilicity of the pyrazole moiety, N-alkyl chain length and planarity of the two heterocyclic rings appear to play a decisive role in modulating cytotoxicity and antifungal activity.     

Expression of matrix metalloproteinase -2, -9 and tissue inhibitors of metalloproteinase -1, -2, -3 mRNAs in rat uterus during early pregnancy

Zymography and in situ hybridizition were used to investigate matrix metalloproteinase-2, -9 (MMP-2, -9) activities, and expression of mRNAs for MMP-2, -9 and tissue inhibitors of matrix metalloproteinases (TIMP-1, -2, -3) in the rat uterus during early pregnancy (day 1-7). The zymography results showed two forms of MMP-2 (64 and 67 kDa) in the rat uteri during early pregnancy. The 64-kDa MMP-2 activity was the highest on day 2 (P < 0.01) and higher on day 5 and 6 (P < 0.05). The 67-kDa MMP-2 activity reached the highest on day 5 and 6 (P < 0.01). The 64-kDa MMP-2 activity at the implantation sites was higher than those at interimplantation sites (P < 0.05). Furthermore, the 67 kDa MMP-2 can be converted to 64 kDa forms by incubation with p-aminophenylmercuric acetate (APMA) and trypsin in vitro. The 92-kDa MMP-9 activity was only detected on day 5 and 6 of pregnancy (P < 0.01). In situ hybridization showed that on day 1-4 of pregnancy, both MMP-2 and TIMP-2 mRNAs were evidently localized in the basal stromal cells. On day 5, MMP-2 mRNA signals were decreased in the basal stromal cells and mRNA for TIMP-2 was expressed in the epithelial cells and subepithelial stromal cells. The mRNAs for MMP-9, TIMP-1, and -3 were mainly expressed in epithelial cells on day 1-5. At the implantation site on day 6, the mRNAs for MMP-2, -9, TIMP-1, -2, and -3 were highly expressed in the primary decidual zone surrounding the implanting embryo, and in the whole decidualized stromal cells (the primary and secondary decidual zones) at the implantation site on day 7. The intensities of mRNAs for the TIMPs in decidualized stromal cells at the implantation site on day 6 and 7 were stronger than those for the MMPs. The weak mRNAs for MMP-2, -9, TIMP-1, and -3 but not TIMP-2 were also observed in the ectoplacental cone/trophoblastic cells of the implanting embryos. However, at the interimplantation sites on day 6 and 7, MMP-2, -9, TIMP-1, -2, and -3 mRNAs were weakly expressed in the epithelial cells, subepithelial stromal cells, and myometrium. The results suggested that the implanting rat embryo strongly induced MMP-2 and -9 proteins and gene expression for decidulization and embryo invasion, which were strictly controlled and balanced by the simultaneous expression of TIMP-1, -2 and -3.     

Synthesis and Insecticidal Activity of Lignan Analogs (III)

Ten haedoxan analogs with the bond split between 2C and 3C of the 6-methoxy-2-methoxymethyl-3-(3,4-methylenedioxy)phenyl-1,4-benzodioxan-7-yl group of haedoxans were synthesized, and their insecticidal activity was assessed on the housefly. The inactivity of an analog having a 2-methoxy-5-(2-methoxyethoxy)-4-(3,4-methylenedioxybenzyloxy)phenyI instead of the 1,4-benzodioxan-7-yl group made it evident that the benzodioxane framework is essential for the activity of haedoxans.     

Effect of alterations in the cyclopentane ring on bone resorptive activity of prostaglandin

Previous studies have shown that the natural prostanoids, PGE2, PGE1 and PGF2 alpha are potent stimulators of bone resorption. In this study, we have examined the effects of alterations in the cyclopentane ring of these prostanoids for their effect on the resorptive response of cultured long bones from 19-day fetal rats as measured by the release of previously incorporated 45Ca. Indomethacin (10(-6)M) was added to minimize endogenous prostaglandin production. In this system PGE2 and PGE1, the 9 keto, 11 alpha hydroxy compounds, were approximately equally effective at concentrations of 10(-8) to 10(-6) M. The 9 alpha hydroxy, 11 alpha hydroxy compound, PGF2 alpha, was active at 10(-7) to 10(-5) M. In contrast, the 9 alpha hydroxy, 11-keto compound, PGD2, showed only a minimal stimulation of bone resorption at 10(-5) M. While these data suggested that the 11 alpha hydroxy group was important for bone resorbing activity, 11 beta PGE2 and 11-deoxy PGE1 were only slightly less potent than their physiologic counterparts. Both 9 beta, 11 alpha PGF2 and 9 alpha, 11 beta PGF2 were less potent than PGF2 alpha but did cause substantial stimulation of bone resorption and were equally effective at 10(-6) to 10(-5) M. 9 alpha, 11 beta PGF2 alpha is of particular interest since it is major metabolite of PGD2. These results suggest that the binding of prostanoids to the receptor which mediates bone resorption is affected by changes at the 9 and 11 positions of the pentane ring but do not support the hypothesis that the 11 alpha OH function is essential for this biological activity.     

Effects of E-cadherin on mouse embryo implantation and expression of matrix metalloproteinase-2 and -9

E-cadherin is a cell surface glycoprotein, which is responsible for adhesion between epithelial cells. Whether it is involved in embryo implantation is still unknown. In a mouse intrauterine horn injection model, one uterine horn in each mouse was injected with different doses of E-cadherin antibody on day 3 of pregnancy. The results showed that embryo implantation was significantly inhibited in the mice injected with 3 microg E-cadherin antibody. The mouse uteri in this group were collected on days 5, 6, and 7 of pregnancy and expressions of MMP-2 and -9 were studied. In situ hybridization and RT-PCR results showed that the expression of MMP-2 and -9 mRNAs in uteri of E-cadherin antibody treated group was increased on days 5-7. The results of gelatin zymography of MMPs showed that the activities of pro-MMP-2, MMP-2, and pro-MMP-9 were increased significantly on days 5 and 6, and pro-MMP-9 activity was increased on day 7. The present study suggested that E-cadherin was involved in embryo implantation through decreasing the expressions and activities of MMP-2 and -9.     

Identification and egg hatching activity of monohydroxy fatty acid eicosanoids in the barnacle Balanus balanoides.

Monohydroxy fatty acids (MHFAs) were isolated from homogenates of the barnacle Balanus balanoides and identified by gas chromatography-mass spectrometry (GC-MS) as 14- and 17-hydroxy docosahexaenoic acids, 8-, 11-, 12-, 15- and 18-hydroxy eicosapentaenoic acids, 13- and 16-hydroxyoctadecatrienoic acids and 9-, 13- and 15-hydroxyoctadecadienoic acids. Each monohydroxy fatty acid was tested for egg hatching activity in a bioassay using Elminius modestus egg masses, but 8-hydroxy-5, 9, 11, 14, 17-eicosapentaenoic acid (8-HEPE) was the only MHFA with barnacle egg hatching activity. Studies on the egg hatching activity of MHFAs prepared from the oxidation of polyunsaturated fatty acids showed that activity was confined to the 8-hydroxy isomer of eicosapentaenoic acid and arachidonic acid, and that unsaturation at C5 and C14, but not C17, was essential for activity. In addition, the 8(R) conformation is necessary for activity, as 8(R)-HEPE caused egg hatching at 10(-7) M whereas the enantiomer 8(S)-HEPE was inactive.     

Relationships between structure and molting hormonal activity of tebufenozide, methoxyfenozide, and their analogs in cultured integument system of Chilo suppressalis Walker

The molting hormonal activity of methoxyfenozide (RH-2485), tebufenozide (RH-5992), five analogs with various alkyl groups, and 18 acyl analogs was measured by using cultured integument of rice stem borers, Chilo suppressalis Walker. The hormonal activity of methoxyfenozide was remarkably high (EC(50) = 1.1 x 10(-9) M), being equivalent to that of tebufenozide (RH-5992). The hormonal activity of several tebufenozide analogs with varying alkyl groups such as CH(3), n-C(3)H(7), i-C(3)H(7), n-C(4)H(9) and n-C(5)H(11) at the para-position of the benzene ring furthest from the tert-butyl group was lower than that of tebufenozide (alkyl group is C(2)H(5)). The activity decreased to varying degrees as a result of replacement of the 3,5-dimethylphenyl moiety of tebufenozide with either a phenyl, naphthyl, or cyclohexyl group. Both 1- and 2-naphthyl derivatives were very active (EC(50) = 4.3 x 10(-8) M and 3.2 x 10(-8) M, respectively) without any significant difference between them. The activity of the 1-cyclohexenyl analog (EC(50) = 1.0 x 10(-7) M) was about 40x that of the corresponding 3-cyclohexenyl analog (EC(50) = 4.4 x 10(-6) M), but 1/100 that of tebufenozide. The activity varied parabolically with respect to the molecular hydrophobicity, and decreased with longer acyl moieties.     

Antioxidant function of phenethyl-5-bromo-pyridyl thiourea compounds with potent anti-HIV activity

In a systematic search for novel dual function antioxidants with potent anti-HIV activity, we evaluated 9 rationally designed non-nucleoside inhibitors (NNI) of HIV-1 RT for antioxidant and anti-HIV activities. Our lead phenethyl-5-bromopyridyl thiourea (PEPT) compounds, N-[2-(2-methoxyphenylethyl)]-N'-[2-(5-bromopyridyl)]-thioure a (2) and N-[2-(2-chlorophenylethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (9), inhibited the oxidation of ABTS to ABTS*+ by metmyoglobin in the presence of hydrogen peroxide with EC50 values of 79 and 75 microM, respectively. Both compounds effectively inhibited the oxidation-induced green fluorescence emission from the free radical-sensitive indicator dye 2',7'-dichlorodihydrofluorescein diacetate in CEM human T-cells and Nalm-6 human B-cells exposed to hydrogen peroxide. To our knowledge, compounds 2 and 9 are the first NNI of HIV-1 RT with potent anti-oxidant activity. Furthermore, the activity center was defined as the sulfhydryl group since alkylated PEPT derivatives were inactive. The presence of a free thiourea group was also essential for the anti-HIV activity of the PEPT compounds.     

Mutational analyses of Aquifex pyrophilus DNA ligase define essential domains for self-adenylation and DNA binding activity

We constructed nine deletion mutants of NAD+-dependent DNA ligase from Aquifex pyrophilus to characterize the functional domains. All of DNA ligase deletion mutants were analyzed in biochemical assays for NAD+-dependent self-adenylation, DNA binding, and nick-closing activity. Although the mutant lsub1 (91-362) included the active site lysine (KxDG), self-adenylation was not shown. However, the mutants lsub6 (1-362), lsub7 (1-516), and lsub9 (1-635) showed the same adenylation activity as that of wild type. The lsub5 (91-719), which has the C-terminal domain (487-719) as to lsub4 (91-486), showed minimal adenylation activity. These results suggest that the presence of N-terminal 90 residues is essential for the formation of an enzyme-AMP complex, while C-terminal domain (487-719) appears to play a minimal role in adenylation. It was found that the presence of C-terminal domain (487-719) is indispensable for DNA binding activity of lsub5 (91-719). The mutant lsub9 (1-635) showed reduced DNA binding activity compared to that of wild type, suggesting the contribution of the domain (636-719) for the DNA binding activity. Thus, we concluded that the N-terminal 90 residues and C-terminal domain (487-719) of NAD+-dependent DNA ligase from A. pyrophilus are mutually indispensable for binding of DNA substrate.     

Role of Asn(2) and Glu(7) residues in the oxidative folding and on the conformation of the N-terminal loop of apamin

The X-ray structure of [N-acetyl]-apamin has been solved at 0.95 A resolution. It consists of an 1-7 N-terminal loop stabilized by an Asn-beta-turn motif (2-5 residues) and a helical structure spanning the 9-18 residues tightly linked together by two disulfide bonds. However, neither this accurate X-ray nor the available solution structures allowed us to rationally explain the unusual downfield shifts observed for the Asn(2) and Glu(7) amide signals upon Glu(7) carboxylic group ionization. Thus, apamin and its [N-acetyl], [Glu(7)Gln], [Glu(7)Asp], and [Asn(2)Abu] analogues and submitted to NMR structural studies as a function of pH. We first demonstrated that the Glu(7) carboxylate group is responsible for the large downfield shifts of the Asn(2) and Glu(7) amide signals. Then, molecular dynamics (MD) simulations suggested unexpected interactions between the carboxylate group and the Asn(2) and Glu(7) amide protons as well as the N-terminal alpha-amino group, through subtle conformational changes that do not alter the global fold of apamin. In addition, a structural study of the [Asn(2)Abu] analogue, revealed an essential role of Asn(2) in the beta-turn stability and the cis/trans isomerization of the Ala(5)-Pro(6) amide bond. Interestingly, this proline isomerization was shown to also depend on the ionization state of the Glu(7) carboxyl group. However, neither destabilization of the beta-turn nor proline isomerization drastically altered the helical structure that contains the residues essential for binding. Altogether, the Asn(2) and Glu(7) residues appeared essential for the N-terminal loop conformation and thus for the selective formation of the native disulfide bonds but not for the activity.     

Synthesis and Insecticidal Activity of Lignan Analogs (II)

The influence of the 6-methoxy-2-methoxymethyl-3-(3,4-methylenedioxyphenyl)-1,4-benzodioxan-7-yl group on the insecticidal activity of haedoxans was studied by synthesizing an analog without the (methoxymethyl)methinoxy moiety of the benzodioxanyl group to test for its activity on the housefly. The inactivity of the analog and its satellite compounds implies that the (methoxymethyl)methinoxy moiety is essential for the biological activity of haedoxans.     

Macromolecular Composition Dictates Receptor and G Protein Selectivity of Regulator of G Protein Signaling (RGS) 7 and 9-2 Protein Complexes in Living Cells

Regulator of G protein signaling (RGS) proteins play essential roles in the regulation of signaling via G protein-coupled receptors (GPCRs). With hundreds of GPCRs and dozens of G proteins, it is important to understand how RGS regulates selective GPCR-G protein signaling. In neurons of the striatum, two RGS proteins, RGS7 and RGS9-2, regulate signaling by μ-opioid receptor (MOR) and dopamine D2 receptor (D2R) and are implicated in drug addiction, movement disorders, and nociception. Both proteins form trimeric complexes with the atypical G protein β subunit Gβ5 and a membrane anchor, R7BP. In this study, we examined GTPase-accelerating protein (GAP) activity as well as Gα and GPCR selectivity of RGS7 and RGS9-2 complexes in live cells using a bioluminescence resonance energy transfer-based assay that monitors dissociation of G protein subunits. We showed that RGS9-2/Gβ5 regulated both Gi and Go with a bias toward Go, but RGS7/Gβ5 could serve as a GAP only for Go. Interestingly, R7BP enhanced GAP activity of RGS7 and RGS9-2 toward Go and Gi and enabled RGS7 to regulate Gi signaling. Neither RGS7 nor RGS9-2 had any activity toward Gz, Gs, or Gq in the absence or presence of R7BP. We also observed no effect of GPCRs (MOR and D2R) on the G protein bias of R7 RGS proteins. However, the GAP activity of RGS9-2 showed a strong receptor preference for D2R over MOR. Finally, RGS7 displayed an four times greater GAP activity relative to RGS9-2. These findings illustrate the principles involved in establishing G protein and GPCR selectivity of striatal RGS proteins.     

Synthesis and antiviral activity of carbocyclic nucleosides incorporating a modified cyclopentane ring. Part 3: Adenosine and uridine analogues

Six new carbocyclic nucleosides were prepared by mounting a purine (compounds 4-6), 8-azapurine (7 and 8) or uridine (9) base on the amino group of (1S,3R)-3-amino-2,2,3-trimethylcyclopentylmethanol (10). At subtoxic concentrations, compounds 5-9 showed at best marginal antiviral activity.