Total synthesis and biological evaluation of novel C2–C6 region analogues of dictyostatin

By exploiting a Still–Gennari HWE coupling with a common C11–C26 aldehyde, a series of C2–C6 modified analogues of the microtubule-stabilising marine natural product dictyostatin were synthesised and evaluated in vitro for growth inhibition against a range of human cancer cell lines, including the (P-glycoprotein efflux-mediated) Taxol-resistant NCI/ADR cell line. Removal of the C6 methyl substituent in dictyostatin was found to be well tolerated and led to the retention of antiproliferative activity in the low nanomolar range (IC₅₀ = 43 nM in the NCI/ADR cell line), while partial and full saturation of the (2Z, 4E)-dienoate region led to a progressive reduction in biological potency. The lactone ring size was found to be critical, as C21 to C19 translactonisation to afford 20-membered isodictyostatin analogues led to a significant loss of cytotoxicity. In a series of incubatory experiments performed on the PANC-1 cell line, all three of the 22-membered macrolide analogues acted in an analogous fashion to dictyostatin, through a mechanism of microtubule stabilization, causing both an accumulation of cells at the G2/M phase and formation of characteristic dense intracellular microtubule bundles.     

Synthesis and biological evaluation of novel analogues of dictyostatin

Novel analogues of the microtubule-stabilising agent dictyostatin were designed using existing SAR information from the structurally related discodermolide, synthesised by a late-stage diversification strategy and evaluated in vitro for growth inhibition against a range of human cancer cell lines, including those known to exhibit Taxol-resistance (AsPC-1, DLD-1, PANC-1, NCI/ADR).     

In search of a cytostatic agent derived from the alkaloid lycorine: synthesis and growth inhibitory properties of lycorine derivatives

As a continuation of our studies aimed at the development of a new cytostatic agent derived from an Amaryllidaceae alkaloid lycorine, we synthesized 32 analogues of this natural product. This set of synthetic analogues included compounds incorporating selective derivatization of the C1 versus C2 hydroxyl groups, aromatized ring C, lactamized N6 nitrogen, dihydroxylated C3-C3a olefin functionality, transposed olefin from C3-C3a to C2-C3 or C3a-C4, and C1 long-chain fatty esters. All synthesized compounds were evaluated for antiproliferative activities in vitro in a panel of tumor cell lines including those exhibiting resistance to proapoptotic stimuli and representing solid cancers associated with dismal prognoses, such as melanoma, glioblastoma, and non-small-cell lung cancer. Most active analogues were not discriminatory between cancer cells displaying resistance or sensitivity to apoptosis, indicating that these compounds are thus able to overcome the intrinsic resistance of cancer cells to pro-apoptotic stimuli. 1,2-Di-O-allyllycorine was identified as a lycorine analogue, which is 100 times more potent against a U373 human glioblastoma model than the parent natural product. Furthermore, a number of synthetic analogues were identified as promising for the forthcoming in vivo studies.     

The intracellular mechanism of action on Escherichia coli of BF2-A/C, two analogues of the antimicrobial peptide Buforin 2

In the present study, the antimicrobial peptides BF2-A and BF2-C, two analogues of Buforin 2, were chemically synthesized and the activities were assayed. To elucidate the bactericidal mechanism of BF2-A/C and their different antimicrobial activities, the influence of peptides to E. coli cell membrane and targets of intracellular action were researched. Obviously, BF2-A and BF2-C did not induce the influx of PI into the E. coli cells, indicating nonmemebrane permeabilizing killing action. The FITC-labeled BF2-A/C could penetrate the E. coli cell membrane and BF2-C penetrated the cells more efficiently. Furthermore, BF2-A/C could bind to DNA and RNA respectively, and the affinity of BF2-C to DNA was powerful at least over 4 times than that of BF2-A. The present results implied that BF2-A and BF2-C inhibited the cellular functions by binding to DNA and RNA of cells after penetrating the cell membranes, resulting in the rapid cell death. The structure-activity relationship analysis of BF2-A/C revealed that the cell-penetrating efficiency and the affinity ability to DNA were critical factors for determining the antimicrobial potency of both peptides. The more efficient cell-penetrating and stronger affinity to DNA caused that BF2-C displayed more excellent antimicrobial activity and rapid killing kinetics than BF2-A.     

蚜虫报警信息素类似物的结构与生物活性关系

利用AccuModel和PowerFit软件比较了蚜虫报警信息素［反］-β-法尼烯（EBF）的氟取代类似物与EBF的结构相似性,并对非EBF系列物结构活性关系进行了研究。结果发现对于一氟取代化合物来说,在分子骨架两端进行取代修饰,所得化合物EBF04, EBF05, EBF12与EBF结构最相似;非EBF系列化合物的扭转角(H25-C4-C5-C1和C5-C6-C7-C8)数值的正负取向对报警活性有明显影响。     

Tubulin assembly, taxoid site binding, and cellular effects of the microtubule-stabilizing agent dictyostatin

(-)-Dictyostatin is a sponge-derived, 22-member macrolactone natural product shown to cause cells to accumulate in the G2/M phase of the cell cycle, with changes in intracellular microtubules analogous to those observed with paclitaxel treatment. Dictyostatin also induces assembly of purified tubulin more rapidly than does paclitaxel, and nearly as vigorously as does dictyostatin's close structural congener, (+)-discodermolide (Isbrucker et al. (2003), Biochem. Pharmacol. 65, 75-82). We used synthetic (-)-dictyostatin to study its biochemical and cytological activities in greater detail. The antiproliferative activity of dictyostatin did not differ greatly from that of paclitaxel or discodermolide. Like discodermolide, dictyostatin retained antiproliferative activity against human ovarian carcinoma cells resistant to paclitaxel due to beta-tubulin mutations and caused conversion of cellular soluble tubulin pools to microtubules. Detailed comparison of the abilities of dictyostatin and discodermolide to induce tubulin assembly demonstrated that the compounds had similar potencies. Dictyostatin inhibited the binding of radiolabeled discodermolide to microtubules more potently than any other compound examined, and dictyostatin and discodermolide had equivalent activity as inhibitors of the binding of both radiolabeled epothilone B and paclitaxel to microtubules. These results are consistent with the idea that the macrocyclic structure of dictyostatin represents the template for the bioactive conformation of discodermolide.     

Chemical structures and biological activities of rhamnolipids produced by Pseudomonas aeruginosa B189 isolated from milk factory waste

The aim of this work was to study chemical structures and biological activities of rhamnolipids produced by Pseudomonas aeruginosa B189 isolated from milk factory waste. The culture produced two biosurfactants, a and b, which showed strong activity and were identified as L-rhamnopyranosyl-L-rhamnopyranosyl-beta-hydroxydecanoyl-beta-hydroxydecanoate or Rha-Rha C10-C10 and L-rhamnopyranosyl-L-rhamnopyranosyl-beta-hydroxydecanoyl-beta-hydroxydodecanoate or Rha-Rha C(10)-C(12), respectively. Both compounds exhibited higher surfactant activities tested by the drop collapse test than several artificial surfactants such as SDS and Tween 80. Rhamnolipid a showed significant antiproliferative activity against human breast cancer cell line (MCF-7) at minimum inhibitory concentration (MIC) at 6.25 microg/mL while rhamnolipid b showed MIC against insect cell line C6/36 at 50 microg/mL.     

Chemical structures and biological activities of rhamnolipids produced by Pseudomonas aeruginosa B189 isolated from milk factory waste

The aim of this work was to study chemical structures and biological activities of rhamnolipids produced by Pseudomonas aeruginosa B189 isolated from milk factory waste. The culture produced two biosurfactants, a and b, which showed strong activity and were identified as L-rhamnopyranosyl-L-rhamnopyranosyl-beta-hydroxydecanoyl-beta-hydroxydecanoate or Rha-Rha-C10-C10 and L-rhamnopyranosyl-L-rhamnopyranosyl-beta-hydroxydecanoyl-beta-hydroxydodecanoate or Rha-Rha-C10-C12, respectively. Both compounds exhibited higher surfactant activities tested by the drop collapse test than several artificial surfactants such as SDS and Tween 80. Rhamnolipid a showed significant antiproliferative activity against human breast cancer cell line (MCF-7) at minimum inhibitory concentration (MIC) at 6.25 microg/mL while rhamnolipid b showed MIC against insect cell line C6/36 at 50 microg/mL.     

Increasing maternal age of blastocyst affects on efficient derivation and behavior of mouse embryonic stem cells

Mouse embryonic stem cells (mESCs) have the capability to undergo unlimited cell division and differentiate into derivatives of all three embryonic germ layers. These fundamental features enable mESCs to potentially be appropriate, efficient models for biological and medical research. Therefore, it is essential to produce high-performance mESCs. In the current study, we have produced mESCs from blastocysts that developed from fertilized oocytes of 2 (2-C57)-, 4 (4-C57)-, and 6 (6-C57)-month-old C57BL/6 mice. A comparison of isolated stem cells was done from the viewpoint of the efficiency of mESC derivation, self-renewal, and their differentiation capacity. All generated mESCs showed a similar expression of the molecular markers protein of pluripotency and AP activity. In the 3i medium, there was a significant decrease in undifferentiated marker genes expression in the 2-C57 cells compared with the other two groups ( P < 0.05) but developmental genes significantly increased in the 4-C57 and 6-C57 cells compared with the 2-C57 cells ( P < 0.05). The differentiation capacity into three germ layers through the embryoid body formation and percentage of cell lines with normal numbers of chromosomes reduced with increased maternal age. The highest DT and highest percentage of cells in the S phase belonged to 2-C57 cells. These data demonstrated that blastocysts which developed from fertilized oocytes of 2-, 4-, and 6-month-old C57BL/6 mice can generate pluripotent stem cells, and suggested that both the efficiency of mESC isolation and the behavior of these isolated mESCs including pluripotency, self-renewal, cell cycle, and DT changed with increasing maternal age.     

Susceptibility of<Emphasis Type="Italic">Escherichia coli</Emphasis> to C<Subscript>2</Subscript>-C<Subscript>18</Subscript> fatty acids

The antimicrobial activity of C2-C18 fatty acids was determined in vitro in cultures of two strains of Escherichia coli grown on glucose. Antimicrobial activity was expressed as IC50 (a concentration at which only 50% of the initial glucose in the cultures was utilized). Utilization of glucose was inhibited by caprylic acid (IC50 0.30-0.85 g/L) and capric acid (IC50 1.25-2.03 g/L). Neither short-chain fatty acids (C2-C6) nor fatty acids with longer chain (C12-C18) influenced substrate utilization. Caproic acid, however, decreased cell yield in cultures of E. coli in a dose-dependent manner. No inhibition of glucose utilization was produced with unsaturated fatty acids, oleic and linoleic. Calcium ions added in excess reversed the antimicrobial effect of capric acid, but not that of caprylic acid. Antimicrobial activity of caprylic and capric acid decreased when the bacteria were grown in the presence of straw particles, or repeatedly subcultured in a medium containing these compounds at low concentrations. Counts of viable bacteria determined by plating decreased after incubation with caprylic and capric acid (30 min; 1 g/L) at pH 5.2 from > 10(9) to approximately 10(2)/mL. A reduction of a mere 0.94-1.96 log10 CFU was observed at pH 6.5-6.6. It can be concluded that caprylic acid, and to a lesser extent also capric acid, has a significant antimicrobial activity toward E. coli. Effects of other fatty acids were not significant or absent.     

Two novel antifungal peptides distinct with a five-disulfide motif from the bark of Eucommia ulmoides Oliv

Two antifungal peptides, named EAFP1 and EAFP2, have been purified from the bark of Eucommia ulmoides Oliv. Each of the sequences consists of 41 residues with a N-terminal blockage by pyroglutamic acid determined by automated Edman degradation in combination with the tandem mass spectroscopy and the C-terminal ladder sequencing analysis. The primary structurs all contain 10 cysteines, which are cross-linked to form five disulfide bridges with a pairing pattern (C1-C5, C2-C9, C3-C6, C4-C7, C8-C10). This is the first finding of a plant antifungal peptide with a five-disulfide motif. EAFP1 and EAFP2 show characteristics of hevein domain and exhibit chitin-binding properties similar to the previously identified hevein-like peptides. They exhibit relatively broad spectra of antifungal activities against eight pathogenic fungi from cotton, wheat, potato, tomato and tobacco. The inhibition activity of EAFP1 and EAFP2 can be effective on both chitin-containing and chitin-free fungi. The values of IC(50) range from 35 to 155 microg/ml for EAFP1 and 18 to 109 microg/ml for EAFP2. Their antifungal effects are strongly antagonized by calcium ions.     

Structural characterization of rhamnolipid produced by Pseudonomas aeruginosa strain FIN2 isolated from oil reservoir water

Biosurfactant-producing microorganisms inhabiting oil reservoirs are of great potential in industrial applications. Yet, till now, the knowledge about the structure and physicochemical property of their metabolites are still limited. The aim of this study was to purify and structurally characterize the biosurfactant from Pseudomonas aeruginosa strain FIN2, a newly isolated strain from an oil reservoir. The purification was conducted by silica gel column chromatography followed by pre-RP HPLC and the structural characterization was carried out by GC–MS combined with MS/MS. The results show that the biosurfactant produced by FIN2 is rhamnolipid in nature and its four main fractions were identified to be Rha-C10-C10(46.1 %), Rha–Rha-C10-C10(20.1 %), Rha-C8-C10 (7.5 %) and Rha-C10-C12:1(5.5 %), respectively. Meanwhile, the rarely reported rhamnolipid congeners containing β-hydroxy fatty acids of C6, C9, C10:1 and C11 were also proved to be present in the rhamnolipid mixture produced. The rhamnolipid mixture exhibited a strong surface activity by lowering the surface tension of distilled water to 28.6 mN/m with a CMC value of 195 mg/l.     

Impact of N-terminal domains for corticotropin-releasing factor (CRF) receptor-ligand interactions

The large extracellular N-terminal domains (NTs) of class B G protein-coupled receptors serve as major ligand binding sites. However, little is known about the ligand requirements for interactions with these receptor domains. Recently, we have shown that the most potent CRF receptor agonist urocortin 1 (Ucn1) has two segregated receptor binding sites Ucn1(1-21) and Ucn1(32-40). For locating the receptor domains interacting with these two sites, we have investigated the binding of appropriate Ucn1 analogues to the receptor N-termini compared to the corresponding full-length receptors. For this purpose receptor NTs of CRF(rat) subtypes 1 and 2(alpha) without their signal sequences were overexpressed in Escherichia coli and folded in vitro. For CRF2(a)-rNT, which bears five cysteine residues (C2-C6), the disulfide arrangement C2-C5 and C4-C6 was found, leaving C3 free. This is consistent with the disulfide pattern of CRF1-rNT, which has six cysteines and in which C1 is paired with C3. Binding studies of N-terminally truncated or C-terminally modified Ucn1 analogues demonstrate that it is the C-terminal part, Ucn1(11-40), that binds to receptor NT, indicating a two-domain binding mechanism for Ucn binding to receptor NT. Since the binding of Ucn1 to the juxtamembrane domain has been shown to be segregated from binding to the receptor N-terminus [Hoare et al. (2004) Biochemistry 43, 3996-4011], a third binding domain should exist, probably comprising residues 8-10 of Ucn, which particularly contribute to a high-affinity binding to full-length receptors but not to receptor NT.     

Effects of acyclic analogs of atrial natriuretic factor on proliferative processes of the epithelial tissue in white rats]

The effect of atrial natriuretic factor (ANF) synthetic linear truncated analogues AP-H-6-OH and AP-FOR-6-OH on corneal, skin, duodenum and colon epithelium proliferation has been studied on male rats. The epithelium mitotic activity and DNA synthesis were evaluated 4 and 24 h after intraperitoneal injection of 10 or 100 micrograms/kg peptides. In a dose of 10 micrograms/kg both ANF synthetic analogues inhibited proliferation processes in corneal epithelium, but activated the DNA synthesis in duodenum and colon epithelium. AP-FOR-6-OH (10 micrograms/kg) decreased the mitotic activity of skin epithelium and increased the silver grain density over the cell nuclei at the same time. 100 micrograms/kg ANF analogues stimulated cell mitogenesis in all organs studied. According to the data obtained ANF linear truncated analogues influence on epithelium proliferation is similar to effector of previously studied cyclic atriopeptin AP II.     

Synthesis of the first example of a C2-C3/C2'-C3'-endo unsaturated pyrrolo[2,1-c][1,4]benzodiazepine dimer

We report the first example of a C2-C3/C2'-C3'-endo unsaturated pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimer 16 synthesised through a new and efficient route, thus establishing that C2-C3-endo unsaturation enhances both cytotoxicity and DNA-binding affinity in A-Ring-linked PBD dimers but to a lesser extent than C2/C2'-exo-unsaturation. This new route has allowed the preparation of multi-gram quantities of the related clinical candidate 1 and should lead to more structurally diverse PBD dimer analogues.     

Structure-activity studies of melatonin analogues in prepubertal male rats

Comparison has been made between the activity of the pineal hormone melatonin, and several analogues and metabolites in inhibiting sexual development in a protein-restricted prepubertal rat model. Eleven melatonin analogues or metabolites were tested with the aim of evaluating the model as a test of the hypothesis that melatonin acts as a prohormone and that the ring schism metabolites (kynurenamines) mediate many of the effects attributable to melatonin. Although the hypothesis could not be confirmed, modification of the melatonin structure by lengthening the acrylamide side chain or by replacing the 5 methoxy function with fluorine resulted in loss of biological potency. Modification of the melatonin structure to block the two known points of metabolism resulted in no significant alteration in biological activity. Thus 6-chloromelatonin (blocking 6-hydroxylation) and 2,3-dihydromelatonin (blocking oxidative cleavage of the C2-C3 bond) and 6-chloro-2,3-dihydromelatonin remained biologically active. The metabolic products of brain indoleamine-2,3-dioxygenase, N-acetyl-N2-formyl-5-methoxy kynurenamine (aFoMK) and N-acetyl-5-methoxy kynurenamine (aMK), paradoxically were also biologically active.     

A comparative molecular modeling study of dydrogesterone with other progestational agents through theoretical calculations and nuclear magnetic resonance spectroscopy

6-Dehydroretroprogesterone (dydrogesterone) and three other natural or synthetic progestins (progesterone, retroprogesterone, and 6-dehydroprogesterone) were submitted to a conformational study through theoretical calculations at the B3LYP/6-31G(*) level and high field NMR spectroscopy. The study allows to define the role of the two structural features which differentiate these steroids, i.e., the C9 and C10 configuration and the C6-C7 unsaturation. The combined effects of the conformational preference of A ring, determined by the configuration at C9 and C10, and the enhanced rigidity due to the C6-C7 double bond, could account both for the higher activity and selectivity of dydrogesterone with respect to the other three steroids.     

Structural basis for the decreased procoagulant activity of human factor VIII compared to the porcine homolog

The stability of activated human and porcine factor VIII (fVIII) differ, but a direct comparison of their structural and functional properties has not been made. Highly purified, heterodimeric human recombinant and porcine plasma-derived fVIII were exchanged into a common buffer and some minor contaminants were removed by anion-exchange chromatography. The activations of human and porcine fVIII by thrombin were studied by a two-stage coagulation assay using human citrated plasma as the standard. The peak activation of porcine fVIII was 10-fold greater than human fVIII (1.1 x 10(6) unit/mg versus 1.1 x 10(5) unit/mg). The proteolytic fragmentation of fVIII by thrombin was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was not different between human and porcine fVIII, yielding previously identified bands corresponding to fragments A1, A2, A3-C1-C2, and the B domain. Following activation by thrombin, human fVIII was subjected to cation-exchange (Mono S) high performance liquid chromatography at pH 6.0 under conditions that yields stable, heterotrimeric (A1/A2/A3-C1-C2) porcine fVIIIaIIa (Lollar, P., and Parker, C.G. (1990) Biochemistry 28, 666-674). Coagulant activity was recovered in a single peak that was less than 0.5% that of porcine fVIIIaIIa (1.2 x 10(4) unit/mg versus 2.6 x 10(6) unit/mg). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the peak fraction revealed bands corresponding to the A3-C1-C2 and A1 fragments but only trace levels of the A2 fragment. In contrast, activation of human fVIII by thrombin followed by Mono S HPLC at pH 5.0 produced a peak with 10-fold greater activity (1.2 x 10(5) unit/mg) than at pH 6.0 and which contained significant amounts of the A2 fragment. We conclude that human fVIIIIIa, like porcine fVIIIIIa, is a heterotrimer and propose that its apparent decreased coagulant activity is due to weaker association of the A2 subunit.     

Structurally and functionally conserved domains in the diverse hydrophilic carboxy-terminal halves of various yeast and fungal Na+/H+ antiporters (Nha1p)

Genes encoding the Na(+)/H(+) antiporter (Nha1p) from Candida tropicalis (C.t.), Hansenula anomala (H.a.) (also named Pichia anomala), and Aspergillus nidulans (A.n.) were cloned, and the nucleotide sequences were determined. The deduced primary sequences revealed highly conserved hydrophobic regions and rather diverse hydrophilic regions. Among the seven known Nha1p sequences, Schizosaccharomyces pombe (S.p.) Nha1p is exceptional in lacking the hydrophilic region. Within the diverse hydrophilic regions, we found six conserved regions (C1-C6). Expression of C.t. Nha1p in Saccharomyces cerevisiae (S.c.) cells lacking NHA1 and ENA1 (Na(+)-ATPase) complemented the salinity-sensitive phenotype, suggesting that C.t. Nha1p is functionally related to S.c. Nha1p. Expression of various truncated forms of the C-terminal half of S.c. and C.t. Nha1p showed essentially the same phenotype for both species: deletion of the C4-C6 region caused cell growth to be more resistant to high salinity than the wild type, suggesting an inhibitory function of these domains on the antiporter activity. However, complete loss of C1-C6 caused a severe growth defect under conditions of high salinity, suggesting a defect in antiporter activity. The DeltaC2-C6 form of C.t. Nha1p, containing only C1, restored the retarded cell growth at high salinity more than the control vector alone, but to a value lower than the wild type. These results suggest an essential role for C1 and an activating role of the C2-C3 region in the functional expression of Nha1. High expression of the DeltaC2-C6 form of S.c. Nha1p was toxic for yeast cells, although low expression was not, suggesting that the overexpression of C1 is toxic. The results in this study suggest that the diverse hydrophilic region of yeast and fungal Nha1p has six conserved domains with conserved functions in terms of expression of Nha1p activity.     

Vasopressin-dependent control of basolateral Na/H-exchange in highly differentiated A6-cell monolayers

We have used a well-differentiated A6-cell preparation (A6-C1) to study cellular location and vasopressin control of Na/H-exchange activity. After cell acidification, cell pH_i (measured by BCECF-fluorescence) only recovered by the addition of Na medium to the basolateral cell surface; this pH_i recovery was inhibited by dimethylamiloride (2 m) consistent with basolateral location of Na/H-exchange activity. Addition of vasopressin produced stimulation of Na/H-exchange activity and increased the affinity of the exchanger for Na⁺. Stimulation of Na/H exchange was mimicked by pharmacological activation of protein kinase A (forskolin, 8-Br-cAMP) and not by pharmacological activation of protein kinase C (TPA). It is concluded that basolaterally located Na/H-exchange in A6-C1 cells is activated by vasopressin.