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Structurally simplified macrocyclic ketone analogues of halichondrin B were prepared by total synthesis and found to retain the potent cell growth inhibitory activity in vitro, stability in mouse serum, and in vivo efficacy of the natural product.
Synthesis and HCV NS3 serine protease inhibitory activity of 4-hydroxyproline derived macrocyclic inhibitors and SAR around this macrocyclic core is described in this communication. X-ray structure of inhibitor 38 bound to the protease is discussed.
The design of a novel series of cyclin-dependent kinase (CDK) inhibitors containing a macrocyclic quinoxaline-2-one is reported. Structure-based drug design and optimization from the starting point of diarylurea 2, which we previously reported as a moderate CDK1,2,4,6 inhibitor [J. Biol.Chem.2001, 276, 27548], led to the discovery of potent CDK1,2,4,6 inhibitor that were suitable for iv administration for in vivo study.
Copper (I) promoted [3+2] Huisgen cycloaddition of azides with terminal alkynes was used to prepare triazole-containing macrocycles based on the Grb2 SH2 domain-binding motif, 'Pmp-Ac(6)c-Asn', where Pmp and Ac(6)c stand for 4-phosphonomethylphenylalanine and 1-aminocyclohexanecarboxylic acid, respectively. When cycloaddition reactions were conducted at 1mM substrate concentrations, cyclization of monomeric units occurred. At 2mM substrate concentrations the predominant products were macrocyclic dimers. In Grb2 SH2 domain-binding assays the monomeric (S)-Pmp-containing macrocycle exhibited a K(d) value of 0.23microM, while the corresponding dimeric macrocycle was found to have greater than 50-fold higher affinity. The open-chain dimer was also found to have affinity equal to the dimeric macrocycle. This work represents the first application of 'click chemistry' to the synthesis of SH2 domain-binding inhibitors and indicates its potential utility.
Synthesis of 10-membered bislactones by PCC oxidation of methyl 2,6-di-O-pivaloyl-alpha-D-glucopyranoside and methyl 4,6-O-benzylidene-alpha-D-glucopyranoside is described, with emphasis on their structure elucidation using the information gained by combination of NMR spectroscopic techniques with X-ray diffraction data. In alternative, the use of PCC and PCC adsorbed on silica gel or alumina for the regioselective oxidation of vicinal diols in sugars is also reported. Both bislactones showed antifungal activity against Candida albicans, and were slightly active against the bacteria Bacillus subtilis. The bislactone presenting pivaloyl protecting groups also promoted some growth inhibition of Staphylococcus aureus.
The crystal structure of an amphiphilic macrocycle [Velasco-Torrijos, T.; Murphy, P. V., Tetrahedron: Asymmetry2005, 16, 261-272] derived from saccharides shows extensive hydrogen-bonding networks, including participation of water, that may have relevance for the modelling of carbohydrate-carbohydrate recognition at cell-cell interfaces. The structure may provide a basis for understanding the binding of the macrocycle to hydrophobic probes.
A bacterial strain M6, isolated from soil and identified as Arthrobacter globiformis, produced a novel nonreducing oligosaccharide. The nonreducing oligosaccharide was produced from starch using a culture supernatant of the strain as enzyme preparation. The oligosaccharide was purified as a crystal preparation after alkaline treatment and deionization of the reaction mixture. The structure of the oligosaccharide was determined by methylation analysis, mass spectrometry, and (1)H and (13)C NMR spectroscopy, and it was demonstrated that the oligosaccharide had a cyclic structure consisting of four glucose residues joined by alternate alpha-(1-->4)- and alpha-(1-->6)-linkages. The cyclic tetrasaccharide, cyclo-{-->6)-alpha-D-Glcp(1-->4)-alpha-D-Glcp(1-->6)-alpha-D-Glcp(1-->4)-alpha-D-Glcp(1-->}, was found to be a novel oligosaccharide, and was tentatively called cyclic maltosyl-maltose (CMM). CMM was not hydrolyzed by various amylases, such as alpha-amylase, beta-amylase, glucoamylase, isoamylase, pullulanase, maltogenic alpha-amylase, and alpha-glucosidase, but hydrolyzed by isomalto-dextranase to give rise to isomaltose. This is the first report of the cyclic tetrasaccharide, which has alternate alpha-(1-->4)- and alpha-(1-->6)-glucosidic linkages.
A novel cyclic pentasaccharide (CPS) and a branched cyclic pentasaccharide (6G-CPS) consisting of d-glucopyranose were synthesized with 6-alpha-glucosyltransferase (6GT) and 3-alpha-isomaltosyltransferase (IMT) from Bacillus globisporus N75. The structure of CPS was cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->]. The other, 6G-CPS, had the structure cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-[alpha-D-Glcp-(1-->6)]-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->]. The formation of CPS was presumed to occur after the following four successive reactions: a 6-glucosyltransfer reaction with 6GT, a 4-glucosyltransfer reaction with 6GT, a 3-isomaltosyltransfer reaction with IMT, and a cyclization reaction with IMT.
Changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) control the setting up of the neuro-muscular synapse in vitro and probably in vivo. Dissociated cultures of purified embryonic (E15) rat motoneurons were used to explore the molecular mechanisms by which endoplasmic reticulum Ca(2+) stores, via both ryanodine-sensitive and IP(3)-sensitive intracellular Ca(2+) channels control [Ca(2+)](i) homeostasis in these neurons during ontogenesis. Fura-2 microspectrofluorimetry monitorings in single neurons showed that caffeine-induced responses of [Ca(2+)](i) increased progressively from days 1-7 in culture. These responses were blocked by ryanodine and nicardipine but not by omega-conotoxin-GVIA or omega-conotoxin-MVIIC suggesting a close functional relationship between ryanodine-sensitive and L-type Ca(v)1 Ca(2+) channels. Moreover, after 6 days in vitro, neurons exhibited spontaneous or caffeine-induced Ca(2+) oscillations that were attenuated by nicardipine. In 1-day-old neurons, both thapsigargin or CPA, which deplete Ca(2+) stores from the endoplasmic reticulum, induced an increase in [Ca(2+)](i) in 75% of the neurons tested. The number of responding motoneurons declined to 25% at 5-6 days in vitro. Xestospongin-C, a membrane-permeable IP(3) receptor inhibitor blocked the CPA-induced [Ca(2+)](i) response in all stages. RT-PCR studies investigating the expression pattern of RYR and IP(3) Ca(2+) channels isoforms confirmed the presence of their different isoforms and provided evidence for a specific pattern of development for RYR channels during the first week in vitro. Taken together, present results show that the control of motoneuronal [Ca(2+)](i) homeostasis is developmentally regulated and suggest the presence of an intracellular ryanodine-sensitive Ca(2+) channel responsible for a Ca(2+)-induced Ca(2+) release in embryonic motoneurons following voltage-dependent Ca(2+) entry via L-type Ca(2+) channels.
Human umbilical vein endothelial cells were exposed to sinusoidal electric fields of 0.3 or 30 kV/m, 50 Hz, for 24 h. Changes in intracellular calcium concentration ([Ca(2+)](i)) induced by ATP-stimulation in the absence of extracellular Ca(2+) were observed in individual cells. No differences were observed between the exposure and sham-exposure groups in [Ca(2+)](i) resting level before ATP-stimulation, or in the [Ca(2+)](i) peak levels induced by stimulation. However, the duration of the initial transients in [Ca(2+)](i) following an ATP stimulus was significantly prolonged by exposure to a 30 kV/m field. The inositol trisphosphate receptor inhibitor, xestospongin C, inhibited the ATP-induced elevation in [Ca(2+)](i) in both the exposure and sham-exposure groups. The ATP-receptor P2Y appeared to play an important role in the increase of [Ca(2+)](i). The present results suggest that an extremely low-frequency electric field affects the function of vascular endothelial cells by a mechanism involving activation of P2Y.
Macrocyclization carried out by thioesterase domains of multimodular nonribosomal peptide synthetases (NRPSs) is a key step in the biosynthesis of many biologically active peptides. The thioesterase excised from tyrocidine synthetase is a versatile macrocyclization catalyst and a useful tool for chemoenzymatic synthesis of diverse cyclic peptides. However, its utility is limited by its short lifetime of catalytic activity as well as significant flux of the acyl-enzyme intermediate to hydrolysis. The addition of Brij 58, a nonionic detergent, above the critical micelle concentration, has dramatic effects on enzyme activity: catalytic activity is extended to >60 min and the rate of cyclization (but not hydrolysis) increases 6-fold, resulting in a net 150- to 300-fold increase in cyclic product yields. This enhanced activity allowed enzymatic macrocyclization of a solid phase library of tyrocidine decapeptides to identify acceptable substitutions at the Orn9 position which had previously been inaccessible for diversification.
The 35 kDa thioesterase (TE) domain excised from the megadalton tyrocidine synthetase (Tyc Syn) retains autonomous capacity to macrocyclize peptidyl thioesters to D-Phe1-L-Leu10-macrolactams. Since a number of nonribosomal peptides undergo O-glycosylation events during tailoring to gain biological activity, the Tyc Syn TE domain was evaluated for cyclization capacity with glycosylated peptidyl-S-NAC substrates. First, Tyr7 was replaced with Tyr(beta-D-Gal) and Tyr(beta-D-Glc) as well as with Ser-containing beta-linked D-Gal, D-Glc, D-GlcNAc, and D-GlcNH2, and these new analogs were shown to be cyclized with comparable kcat/Km catalytic efficiency. Similarly, Gal- or tetra-O-acetyl-Gal-Ser could also be substituted at residues 5, 6, and 8 in the linear decapeptidyl-S-NAC sequences and cyclized without substantial loss in catalytic efficiency by Tyc Syn TE. The cyclic glycopeptides retained antibiotic activity as membrane perturbants in MIC assays, opening the possibility for library construction of cyclic glycopeptides by enzymatic macrocyclization.
Polarized migration and spreading of epithelial sheets is important during many processes in vivo, including embryogenesis and wound healing. However, the signaling pathways that regulate epithelial migrations are poorly understood. To identify molecular components that regulate the spreading of epithelial sheets, we performed a screen for mutations that perturb epidermal cell migration during embryogenesis in Caenorhabditis elegans. We identified one mutant (jc5) as a weak mutation in itr-1, which encodes the single inositol 1,4,5-trisphosphate receptor (ITR) in C. elegans. During the migration of the embryonic epidermis, jc5 embryos display defects including misdirected migration or premature cessation of migration. Cells that halt their migration have disorganized F-actin and display reduced filopodial protrusive activity at their leading edge. Furthermore, some filopodia formed by epidermal cells in itr-1(jc5) embryos exhibit abnormally long lifetimes. Pharmacological studies with the inositol 1,4,5-trisphosphate antagonist xestospongin C phenocopy these defects, confirming that ITR function is important for proper epidermal migration. Our results provide the first molecular evidence that movements of embryonic epithelial cell sheets can be controlled by ITRs and suggest that such regulation may be a widespread mechanism for coordinating epithelial cell movements during embryogenesis.
The reactions of 1,3-bis(alpha,omega-bromoalkyl)-6-methyluracils with 1,3-bis(alpha,omega-ethylaminoalkyl)-6-methyluracils or 1,3-bis(bromopentyl)thymine with butylamine afforded pyrimidinophanes containing one or two uracil units and nitrogen atoms in bridging polymethylene chains. In some cases individual geometric isomers of pyrimidinophanes differing in the mutual arrangement of the carbonyl and methyl groups at different pyrimidine rings were isolated. Quaternization of the bridging nitrogen atom with o-nitrobenzyl bromide, benzyl bromide, n-decyl bromide gave rise to water-soluble pyrimidinophanes which were evaluated for their antibacterial and antifungal activity. The arrangement of the carbonyl groups in macrocycles doesn't affect the activity. Antibacterial and antifungal activity of pyrimidinophanes increases with the increase of polymethylene N(pyr)-N-chain length and dramatically increases upon the introduction of n-decyl substituent at nitrogen atoms in spacers. Pyrimidinophanes with 5 and 6 methylene groups in N(pyr)-N-chain and n-decyl substituent showed significant bacteriostatic, fungistatic, bactericidal, fungicidal activity which comparable with standard antibacterial and antifungal drugs. Acyclic counterpart demonstrated the highest activity against fungi. Toxicity of more effective pyrimidinophanes was determined for mice and Daphnia magna Straus.
Interleukin-1beta (IL-1beta) plays an important role in neuroprotective and neurodegenerative events in the central nervous system. To clarify the mechanism of controversial actions of IL-1beta, we determined the effect of IL-1beta, as well as the interaction between IL-1beta and Ca(2+)-induced Ca2+ releasing system (CICR), on adenosine releases in mice hippocampus using mini-slices method. Basal and K(+)-stimulated adenosine releases were regulated by two types of CICRs, including inositol-1,4,5-trisphosphate (IP3) receptor and ryanodine receptor. Lower concentration of IL-1beta increased both adenosine releases, whereas higher concentration did not affect their releases. The stimulatory effect of IL-1beta on basal adenosine release was reduced by removal of extracellular Ca2+ and IP3 receptor inhibitor, while the stimulatory effect of IL-1beta on K(+)-stimulated adenosine release was reduced by ryanodine receptor inhibitor. These results suggest that the potent effect of IL-1beta upon adenosine release might contribute to the neuroprotective action of IL-1beta, whereas IL-1beta-induced neurodegeneration might be due to the overload response of Ca2+ mobilization and the inactivation of adenosine exocytosis.
Reactive oxygen species are believed to be the central mediators of beta-cell destruction that leads to type 1 and 2 diabetes, and calcium has been reported to be an important mediator of beta cell death. In the present study, the authors investigated whether Ca(2+) plays a role in hydrogen peroxide (H(2)O(2))-induced MIN6N8a mouse beta cell death. Treatment with low concentration H(2)O(2) (50 microM) was found to be sufficient to reduce MIN6N8a cell viability by 55%, largely via apoptosis. However, this H(2)O(2)-induced cell death was near completely blocked by pretreatment with BAPTA/AM (5 microM), a chelator of intracellular Ca(2+). Moreover, the intracellular calcium store channel blockers, such as, xestospongin c and ryanodine, significant protected cells from 50 microM H(2)O(2)-induced cell death and under extracellular Ca(2+)-free conditions, 50 microM H(2)O(2) elicited transient [Ca(2+)](i) increases. In addition, pharmacologic inhibitors of calpain, calcineurin, and calcium/calmodulin-dependent protein kinase II were found to have a protective effect on H(2)O(2)-induced death. Moreover, H(2)O(2)-induced apoptotic signals, such as c-JUN N-terminal kinase activation, cytochrome c release, caspase 3 activation, and poly (ADP-ribose) polymerase cleavage were all down-regulated by the intracellular Ca(2+) chelation. These findings show that [Ca(2+)](i) elevation, possibly due to release from intracellular calcium stores and the subsequent activation of Ca(2+)-mediated apoptotic signals, critically mediates low concentration H(2)O(2)-induced MIN6N8a cell death. These findings suggest that a breakdown of calcium homeostasis by low level of reactive oxygen species may be involved in beta cell destruction during diabetes development.
Xestospongin B, a macrocyclic bis-1-oxaquinolizidine alkaloid extracted from the marine sponge Xestospongia exigua, was highly purified and tested for its ability to block inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release. In a concentration-dependent manner xestospongin B displaced [(3)H]IP(3) from both rat cerebellar membranes and rat skeletal myotube homogenates with an EC(50) of 44.6 +/- 1.1 microM and 27.4 +/- 1.1 microM, respectively. Xestospongin B, depending on the dose, suppressed bradykinin-induced Ca(2+) signals in neuroblastoma (NG108-15) cells, and also selectively blocked the slow intracellular Ca(2+) signal induced by membrane depolarization with high external K(+) (47 mM) in rat skeletal myotubes. This slow Ca(2+) signal is unrelated to muscle contraction, and involves IP(3) receptors. In highly purified isolated nuclei from rat skeletal myotubes, Xestospongin B reduced, or suppressed IP(3)-induced Ca(2+) oscillations with an EC(50) = 18.9 +/- 1.35 microM. In rat myotubes exposed to a Ca(2+)-free medium, Xestospongin B neither depleted sarcoplasmic reticulum Ca(2+) stores, nor modified thapsigargin action and did not affect capacitative Ca(2+) entry after thapsigargin-induced depletion of Ca(2+) stores. Ca(2+)-ATPase activity measured in skeletal myotube homogenates remained unaffected by Xestospongin B. It is concluded that xestospongin B is an effective cell-permeant, competitive inhibitor of IP(3) receptors in cultured rat myotubes, isolated myonuclei, and neuroblastoma (NG108-15) cells.
The stability constants of La(3+), Sm(3+) and Ho(3+) complexes with 13- and 14-membered macrocycles having methylcarboxylate (trita and teta) or methylphosphonate (tritp and tetp) arms were determined. All the ligands were labelled with (153)Sm and (166)Ho in order to evaluate the effect of the macrocyclic cavity size and type of appended arms on their in vitro and in vivo behaviour. The radiolabelling efficiency was found to be higher than 98% for all the complexes, except for those of tetp. All radiocomplexes studied are hydrophilic with an overall negative charge and low plasmatic protein binding. Good in vitro stability in physiological media and human serum was found for all complexes, except the (153)Sm/(166)Ho-teta, which are unstable in phosphate buffer (pH 7.4). In vitro hydroxyapatite (HA) adsorption studies indicated that (153)Sm/(166)Ho-tritp complexes bind to HA having the (166)Ho complex the highest degree of adsorption (>80%, 10 mg). Biodistribution studies in mice demonstrated that (153)Sm/(166)Ho-trita complexes have a fast tissue clearance with more than 95% of the injected activity excreted after 2 h, value that is comparable to the corresponding dota complexes. In contrast, the (153)Sm-teta complex has a significantly lower total excretion. (153)Sm/(166)Ho-tritp complexes are retained by the bone, particularly (166)Ho-tritp that has 5-6% (% I.D./g) bone uptake and also a high rate of total excretion. Thus, these studies support the potential interest of (153)Sm/(166)Ho-trita complexes for therapy when conjugated to a biomolecule and the potential usefulness of the (166)Ho-tritp complex in bone pain palliation.