Replacing the cyclohexene-linker of FR181157 leading to novel IP receptor agonists: orally active prostacyclin mimetics. Part 6

The synthesis and biological activity of novel derivatives of our previously reported IP receptor agonist FR181157 is described. SAR studies to replace the cyclohexene-linker of FR181157 led to the discovery of compound 1i (FR207845) as a potent non-prostanoid PGI2 mimetic with good oral bioavailability.     

FR207944, an antifungal antibiotic from Chaetomium sp. no. 217 I. Taxonomy, fermentation, and biological properties

An antifungal antibiotic, FR207944, was isolated from the culture broth of a fungal strain Chaetomium sp. no. 217. FR207944 is a triterpene glucoside with antifungal activity against Aspergillus fumigatus and Candida albicans. Specifically, FR207944 exhibits in vitro and in vivo antifungal activity against A. fumigatus. The effects of FR207944 on the morphology of A. fumigatus were shown to be similar to those of FR901379, a known 1,3-beta-glucan synthase inhibitor. The MECs of FR207944 against A. fumigatus FP1305 and C. albicans FP633 in micro-broth dilution test were 0.039 and 1.6 mug/ml respectively. FR207944 showed good potency by subcutaneous injection and oral administration against A. fumigatus in a murine systemic infection model, with ED(50)s of 5.7 and 17 mg/kg respectively.     

The spinal antinociceptive effect of FR140423 in mice involvement of the descending noradrenergic and serotonergic systems.

In this study, we investigated the role of descending monoaminergic systems in the antinociceptive activity of FR140423, 3-(difluoromethyl)-1-(4-methoxyphenyl)-5-[4-(methyl sulfinyl)phenyl]pyrazole, by using the tail-pinch test in mice and various monoamine depletors. The antinociceptive effects of FR140423 given p.o. and i.t. with ED50 values of 22 mg/kg and 3.5 microg/mouse, respectively, in the tail-pinch test were antagonized by reserpine, 6-hydroxydopamine plus nomifensine, and p-chlorophenylalanine treatment, whereas the antinociceptive action of morphine in this assay was attenuated by reserpine and 6-hydroxydopamine plus nomifensine but not by p-chlorophenylalanine treatment. These results suggest that both descending noradrenergic and serotonergic systems are involved in the antinociceptive activity of spinally and systemically administered FR140423 against mechanical noxious stimuli. The mechanisms underlying the suppressive action of FR140423 on the nociceptive response may differ from those of morphine in mice.     

Diaryl ester prodrugs of FR900098 with improved in vivo antimalarial activity

The fosmidomycin derivative FR900098 represents an inhibitor of the 1-deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase with potent antimalarial activity. Prodrugs of FR900098 with increased activity after oral administration were obtained by chemical modification of the phosphonate moiety to yield phosphodiaryl esters. One diaryl ester prodrug demonstrated efficacy in mice infected with the rodent malaria parasite Plasmodium vinckei comparable to i.p. drug administration.     

Effect of dietary restriction on estrous cyclicity and follicular reserves in aging C57BL/6J mice

Restricting the food intake of female mice by alternating days of feeding and fasting delayed the age-related loss of estrous cycling potential and retarded the rate of follicular depletion, as determined after reinstatement of ad libitum (AL) feeding. During the period of food restriction (FR; 3.5-10.5 mo), food intake and body weight were about 80% of AL values. Mice were acyclic and predominantly in a state of diestrus during FR, but after reinstatement of an AL diet at 10.5 mo all FR mice resumed cycling regularly. By contrast, 80% of AL controls had become acyclic by this age, and the cycles of the remaining mice were significantly longer than those of the reinstated FR mice. Follicular reserves of 12.5-mo-old FR mice were twice those of age-matched AL controls. Cycling performance of reinstated FR mice, measured by cycle length and the proportion of mice still cycling, was equivalent to that of AL mice when the latter were 2-5 mo younger. Ovarian age, measured by the size of the follicular reserve, was similarly retarded in FR mice. Based on these data and previous evidence that follicular depletion plays a major role in the cessation of cyclicity in this strain, we hypothesize that the delayed loss of estrous cyclicity in aging FR mice is mediated at least in part by the retarding effect of dietary restriction on the rate of follicular depletion.     

随机限食与重喂食小鼠的能量代谢和行为的可塑性

分别测定了随机限食和重喂食驯化的雌性KM小鼠的体重、摄食量、基础代谢率(BMR)、行为活动、身体脂肪和性腺重量.随机限食使摄食量增加、BMR和活动行为降低,生长发育迟缓,但对身体脂肪无显著影响.重喂食后上述指标均恢复到对照组水平,表现出显著的可塑性变化.结果表明,动物通过能量摄入和支出的权衡策略适应难以预测的食物资源变化,能量代谢和活动行为的可塑性调节在能量代谢的权衡策略中发挥重要作用.     

FR131535, a novel water-soluble echinocandin-like lipopeptide: synthesis and biological properties

The synthesis and biological properties of a novel water-soluble echinocandin-like lipopeptide, FR131535, are described. This compound displayed potent in vitro and in vivo antifungal activities. The hemolytic activity of FR901379 was reduced by replacing the acyl side chain. This compound showed good water-solubility, comparable to the natural product FR901379.     

Maintenance of NF-kappaB activation in T-lymphocytes and a naive T-cell population in autoimmune-prone (NZB/NZW)F(1) mice by feeding a food-restricted diet enriched with n-3 fatty acids

We have previously shown that feeding a fish oil (FO) supplemented diet in combination with 40% food restriction (FO/FR) has a greater impact on extending life span in lupus-prone (NZB x NZW)F1 mice than either FO ad libitum (FO/AL) or corn oil food restricted (CO/FR) alone. Lupus disease is associated with increased Th-2 (i.e., IL-6 and IL-10) cytokine production and reduced IL-2 production and NF-kappaB activation. We hypothesized that the mechanism of action by which FO/FR increases life span may involve alterations in T-lymphocyte signaling and subsequent cytokine production. To test this hypothesis, we isolated and then stimulated splenic T-lymphocytes ex vivo with anti-CD3 and -CD28 monoclonal antibodies. We report here that CO/FR and FO/FR and to a lesser extent FO/AL offset disease-associated losses in Th-1 cytokine production, CD69 expression, and NF-kappaB activation in splenic T-lymphocytes activated ex vivo. Similarly, CO/FR and FO/FR prevented the disease-dependent rise in Th-2 cytokine production ex vivo and CD69 expression in vivo. In essence, the T-lymphocyte phenotype in the old CO/FR and FO/FR groups was identical to that in the young disease-free mice. Taken together, the data suggest that both CO/FR and FO/FR increase life span, in part, by maintaining a youthful immune phenotype in autoimmune-prone mice. However, FO/FR appears to represent a more potent dietary strategy in delaying disease-associated immune dysregulation than CO/FR.     

Secreted FGFR3, but not FGFR1, inhibits lens fiber differentiation

The vertebrate lens has a distinct polarity with cuboidal epithelial cells on the anterior side and differentiated fiber cells on the posterior side. It has been proposed that the anterior-posterior polarity of the lens is imposed by factors present in the ocular media surrounding the lens (aqueous and vitreous humor). The differentiation factors have been hypothesized to be members of the fibroblast growth factor (FGF) family. Though FGFs have been shown to be sufficient for induction of lens differentiation both in vivo and in vitro, they have not been demonstrated to be necessary for endogenous initiation of fiber cell differentiation. To test this possibility, we have generated transgenic mice with ocular expression of secreted self-dimerizing versions of FGFR1 (FR1) and FGFR3 (FR3). Expression of FR3, but not FR1, leads to an expansion of proliferating epithelial cells from the anterior to the posterior side of the lens due to a delay in the initiation of fiber cell differentiation. This delay is most apparent postnatally and correlates with appropriate changes in expression of marker genes including p57(KIP2), Maf and Prox1. Phosphorylation of Erk1 and Erk2 was reduced in the lenses of FR3 mice compared with nontransgenic mice. Though differentiation was delayed in FR3 mice, the lens epithelial cells still retained their intrinsic ability to respond to FGF stimulation. Based on these results we propose that the initiation of lens fiber cell differentiation in mice requires FGF receptor signaling and that one of the lens differentiation signals in the vitreous humor is a ligand for FR3, and is therefore likely to be an FGF or FGF-like factor.     

Apoptosis induced by the histone deacetylase inhibitor FR901228 in human T-cell leukemia virus type 1-infected T-cell lines and primary adult T-cell leukemia cells

Inhibition of histone deacetylase (HDAC) activity induces growth arrest, differentiation, and, in certain cell types, apoptosis. FR901228, FK228, or depsipeptide, is an HDAC inhibitor effective in T-cell lymphomas. Adult T-cell leukemia (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1) and remains incurable. We examined whether FR901228 is effective for treatment of ATL by assessing its ability to induce apoptosis of HTLV-1-infected T-cell lines and primary leukemic cells from ATL patients. FR901228 induced apoptosis of Tax-expressing and -unexpressing HTLV-1-infected T-cell lines and selective apoptosis of primary ATL cells, especially those of patients with acute ATL. FR901228 also efficiently reduced the DNA binding of NF-kappaB and AP-1 in HTLV-1-infected T-cell lines and primary ATL cells and down-regulated the expression of Bcl-x(L) and cyclin D2, regulated by NF-kappaB. Although the viral protein Tax is an activator of NF-kappaB and AP-1, FR901228-induced apoptosis was not associated with reduced expression of Tax. In vivo use of FR901228 partly inhibited the growth of tumors of HTLV-1-infected T cells transplanted subcutaneously in SCID mice. Our results indicated that FR901228 could induce apoptosis of these cells and suppress the expression of NF-kappaB and AP-1 and suggest that FR901228 could be therapeutically effective in ATL.     

Body mass and behavior in Swiss mice subjected to continuous or discontinuous food restriction and refeeding

Food restriction (FR) is hypothesized to decrease body fat content of an animal and thus prevent obesity. However, the response of energy budget to a continuous (CFR) or discontinuous FR (DFR) remains inconsistent. In the present study, effects of CFR or DFR and refeeding on energy budget and behavior were examined in male Swiss mice. CFR significantly decreased the energy expenditure associated with basal metabolic rate (BMR) and activity behavior, but not sufficiently to compensate for energy deficit and thus resulted in lower body mass and fat content. DFR mice had a significantly higher food intake on ad libitum days and showed increases in BMR and activity after 4 weeks’ DFR, which might resulted in lower body mass and less body fat than controls. After being refed ad libitum, both CFR and DFR mice had similar body mass, BMR, and behavioral patterns to controls but had 95% and 75% higher fat content. This suggested that not only CFR but also DFR would be a significant factor in the process of obesity for animals that were refed ad libitum. It also indicated that food restriction interrupted many times by periods of ad libitum feeding had the same long-term effects like continuous underfeeding.     

FR290581, a novel sordarin derivative: Synthesis and antifungal activity

Sordarin is a unique natural product antifungal agent that is an inhibitor of elongation factor 2. To improve biological activity, we synthesized various compounds by novel modification of the aglycone, sordaricin. As a result, we have discovered the novel sordarin derivative FR290581. This compound exhibited superior activity and a good pharmacokinetic profile, and also displayed good in vivo activity against Candida albicans.     

Modulation of gene expression in autoimmune disease and aging by food restriction and dietary lipids

Several recent observations carried out by many investigators have offered some clues in understanding the mechanism of how food restriction (FR) acts in the prolongation of life-span, but the precise mechanisms involved in modulating the immune system have not been clearly understood. Our own ongoing studies indicate that FR may act at the molecular level and may extend the life-span by modulating functional activities of several genes in various target tissues. For instance, while cytochrome P-450 IIB1 and IIB2 expression is known to decline with age in ad libitum-fed rats, FR prevented the loss of (drug-inducible) P-450 enzymes in liver tissues. In addition, both alpha 2u-globulin and senescence marker protein 2 expressions, which are regulated by hormones, were also modulated during aging by FR in Fischer 344 male rats. In short-lived autoimmune-prone mice, both FR and omega-3 (n-3) fatty acids diet lowered the severity of autoimmune disease both in lupus-prone (NZB x NZW)F1 mice and in mice prone to develop lymphoproliferative and renal diseases, whereas saturated (n-9) and polyunsaturated (n-6) dietary lipids not only exacerbated autoimmune disease, but also significantly enhanced expression of several oncogenes in lymphoid tissues. FR and omega-3 fatty acids decreased the expression of certain oncogenes. Both FR and omega-3 fatty acids may modulate the aging and autoimmune disease processes by not only altering the fatty acid composition, membrane fluidity, and signal transduction, but also by modulating the lymphokine hormone receptors and their functions and thereby modulating expression of several genes in various tissues during the aging process.     

Acyloxyalkyl ester prodrugs of FR900098 with improved in vivo anti-malarial activity

FR900098 represents an improved derivative of the new antimalarial drug fosmidomycin and acts through inhibition of the 1-deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase, an essential enzyme of the mevalonate independent pathway of isoprenoid biosynthesis. Prodrugs with increased activity after oral administration were obtained by chemical modification of the phosphonate moiety to yield acyloxyalkyl esters. The most successful compound demonstrated 2-fold increased activity in mice infected with the rodent malaria parasite Plasmodium vinckei.     

90Y Labeling of monoclonal antibody MOv18 and preclinical validation for radioimmunotherapy of human ovarian carcinomas

The monoclonal antibody (mAb) MOv18 binds the membrane alpha isoform of the folate receptor (FR) which is overexpressed in human ovarian carcinoma cells. Exploiting the targeting capacity of this mAb, we developed and preclinically validated a protocol for the stable labeling of the mAb with ⁹⁰Y, an isotope which has shown promise in cancer radioimmunotherapy. MOv18 was derivatized with the stable macrocyclic ligand p-isothiocyanatobenzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10- tetraacetic acid (Bz-DOTA). MOv18-Bz-DOTA conjugates were labeled with ⁹⁰Y or ¹¹¹In under metal-free and good laboratory practice conditions. At the optimal Bz-DOTA/mAb derivatization ratio of 4–5, conjugates maintained binding activity up to 6 months, were efficiently labeled with ⁹⁰Y or ¹¹¹In (mean labeling yield 85 and 64%, associated to a final mean specific activity of 74 and 37 MBq/mg) and displayed a mean immunoreactivity of 60 and 58%, respectively. The radiolabeled preparations were stable in human serum, with >97% radioactivity associated to mAb at 48 h after labeling. The ability of ⁹⁰Y- and ¹¹¹In-MOv18 to localize FR on tumors in vivo was analyzed in nude mice bearing tumors induced by isogenic cell lines differing only in the presence or absence of the relevant antigen [A431FR (FR-positive) and A431tMock (FR-negative)]. In vivo biodistribution in organs other than tumor was comparable in non-tumor-, A431tMock- and A431FR-bearing mice, whereas the median tumor uptake of the radiolabeled reagents, expressed as area under the curve (AUC) and maximum uptake (U_max), was significantly higher (sixfold to sevenfold) in A431FR than in A431tMock tumors (P=0.0465 and P=0.0332, respectively). Mean maximum uptake (% ID/g) for ⁹⁰Y-MOv18 was 53.7 and 7.4 in A431FR and A431tMock respectively; corresponding values for ¹¹¹In-Mov18 were 45.0 and 11.3. These data demonstrate the feasibility of ⁹⁰Y-labeling of MOv18 without compromising antibody binding ability and the immunoreagent-specific localization in vivo on FR-expressing tumors, suggesting the suitability of ⁹⁰Y-MOv18 for clinical studies.Angela Coliva and Alberto Zacchetti contributed equally to this work.     

Influence on adipocyte plasma membrane bound protein kinase by feedback regulator.

Protein kinase, phosphodiesterase and adenylate cyclase of plasma membrane of adipocytes and the effect of the feedback regulator (FR) on these three enzymes was measured and compared. The basal level ratio of adenylate cyclase to phosphodiesterase to protein kinase was 1:1.9:3.0. Epinephrine and/or FR alters this ratio. FR stimulated protein kinase activity up to 3 fold in the presence of a wide range of enzyme concentrations, 5-50 mug membrane protein/tube. The concentration of FR effective for stimulation of membrane protein kinase was much greater than that needed for inhibition of adenylate cyclase and phosphodiesterases. The inhibition by FR on adenylate cyclase was the most potent effect among the 3 enzymes. 1 U (or 2 U/ml) of FR inhibited 50% of the adenylate cyclase activity in a defined system. The maximum effective concentration of FR for stimulation of membrane protein kinase was greater than 10 U/ml. Histone type 11A was the best substrate for protein phosphorylation so far observed. The FR stimulatory effect was observed at all substrate concentrations used ranging from 1-5 mg/ml. A NaF concentration curve shows that 15 mM NaF gave maximum phosphorylation. The stimulatory effect of FR was observed both in the presence and absence of NaF. Protein kinase of adipocyte plasma membrane was mainly cAMP-independent. The effect of FR (20 U/ml) in stimulation of protein phosphorylation was much greater than that of cAMP (1 X 10(-6) M). The cAMP and FR effects seemed to be additive. Preincubation of plasma membrane with FR in the absence of ATP resulted in no decrease but slight increase in protein kinase activity. A shift in protein kinase, phosphodiesterase and adenylate cyclase ratios by FR suggests the regulatory role of FR in cAMP metabolism in adipocytes.     

Phytochrome Control of Cellulase Activity in Datura ferox L. Seeds and its Relationship with Germination

The influence of phytochrome on endosperm softening and cellulaseactivity was studied on light-stimulated Datura ferox seeds.Endosperm softening preceded the earliest signs of radicle protrusion,and there was good correlation between the % of seeds with softendosperm at 48 h after R and germination at 96 h after R. Cellulaseactivity was stimulated by R and the increase in activity preceded,by more than 24h, radicle protrusion and endosperm softening.The effect of R was reversed by FR, but, by delaying the irradiationwith FR until cellulase activity had increased significantly,it was observed that removing Pfr did more than just stop anyfurther increase, the level of cellulase activity decreasedin about 24 h close to the dark controls. Cellulase activitywas decreased by a FR irradiation even when more than 60% germinationhad escaped from reversion. These results indicate that phytochromeinfluence on cellulase is not an indirect consequence of thestimulus of germination and that the continuous presence ofPfr is required for the cellulase activity to remain high. Thepossibility that cellulase and other degrading enzymes may bepart of the mechanism of light-induced germination is discussed. Key words: Phytochrome, germination, cellulase     

Structure-based de novo design of non-nucleoside adenosine deaminase inhibitors

We searched for non-nucleoside inhibitors of adenosine deaminase by rational structure-based de novo design and succeeded in the discovery of 1-(1-hydroxy-4-phenyl-2-butyl)imidazole-4-carboxamide (FR221647: K(i)=5.9 microM to human ADA) as a novel inhibitor with moderate activity and good pharmacokinetics compared with the known inhibitors pentostatin and EHNA.