(-)-Phenylahistin arrests cells in mitosis by inhibiting tubulin polymerization.
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(-)-Phenylahistin, a fungal diketopiperazine metabolite composed of phenylalanine and isoprenylated dehydrohistidine, arrested cells in mitosis and inhibited the proliferation of A549 cells. The microtubule network in A549 cells was disrupted by (-)-phenylahistin, which also inhibited the polymerization of both microtubule protein from bovine brain and phosphocellulose-purified tubulin in vitro. Competitive binding studies indicated that (-)-phenylahistin interacted with the colchicine binding site on tubulin but not with the vinblastine binding site. (+info)
Antitumor activity of phenylahistin in vitro and in vivo.
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Phenylahistin is a new cell cycle inhibitor produced by Aspergillus ustus. Since phenylahistin was produced as a scalemic mixture of (-)-phenylahistin and its enantiomer, we separated each enantiomer and evaluated their antitumor activity in vitro. (-)-Phenylahistin exhibited antitumor activity against 8 tumor cell lines with IC50 values ranging from 1.8 x 10(-7) to 3.7 x 10(-6), while (+)-phenylahistin exhibited 33-100-fold less potent activity than (-)-phenylahistin did. (-)-Phenylahistin also showed antitumor activity against P388 leukemia and Lewis lung carcinoma cells in vivo. (+info)
Reaction kinetics of solid-state cyclization of enalapril maleate investigated by isothermal FT-IR microscopic system.
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To investigate the reaction kinetics of the solid-state degradation process of enalapril maleate, a Fourier transform infrared microspectroscope equipped with thermal analyzer (thermal FT-IR microscopic system) was used. The isothermal stability study was conducted at 120-130 degrees C for 1-2 h and changes in the three-dimensional plots of the IR spectra of enalapril maleate with respect to heating time were observed. The study indicates that the bands at 1649, 1728, and 1751 cm(-1) assigned to intact enalapril maleate gradually reduced in peak intensity with heating time. However, the peak intensities at 1672 and 1738 cm(-1) (due to enalapril diketopiperazine (DKP) formation) and at 3250 cm(-1) (corresponding to water formation) gradually increased with heating time. The solid-state diketopiperazine formation and the degradation process of enalapril maleate via intramolecular cyclization were found to be simultaneous. The isothermal decomposition curves were sigmoidal and were characterized by induction and acceleration periods, indicating the presence of autocatalytic solid-state decompositions. Moreover, the power-law equation (n = 1/4) was found to provide the best fit to the kinetics of decomposition. This isothermal FT-IR microscopic system was easily used to investigate the degradation of enalapril maleate and the concomitant formation of DKP. The solid-state reaction of enalapril maleate required an activation energy of 195+/-12 kJ/mol to undergo the processes of decomposition and intramolecular cyclization. (+info)
Comparison of the rates of deamidation, diketopiperazine formation and oxidation in recombinant human vascular endothelial growth factor and model peptides.
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In this work, we examine the way in which stability information obtained from studies on small model peptides correlates with similar information acquired from a protein. The rates of deamidation, oxidation, and diketopiperazine reactions in model peptide systems were compared to those of recombinant human vascular endothelial growth factor (rhVEGF). The N-terminal residues of rhVEGF, a potent mitogen in angiogenesis, are susceptible to the aforementioned reactions. The degradation of the peptides L-Ala-L-Pro-L-Met (APM) and Gly-L-Gln-L-Asn-L-His-L-His (GQNHH), residues 1-3 and 8-12 of rhVEGF, respectively, and rhVEGF were examined at pH 5 and 8 at 37 degrees C. Capillary electrophoresis and high-performance liquid chromatography (HPLC) stability-indicating assays were developed to monitor the degradation of the penta- and tripeptides, respectively. The degradation of rhVEGF was determined by tryptic mapping and quantified by RP-HPLC. The rates of degradation of both peptides and the protein followed apparent first-order kinetics and increased with increasing pH. The tripeptide APM underwent diketopiperazine formation (Ala-Pro-diketopiperazine) and oxidation of the Met residue, whereas the pentapeptide GQNHH degraded via the deamidation pathway. The results indicate that the rates of deamidation and oxidation of the protein are comparable to those observed in the model peptides at both pH values. However, the rate of the diketo-piperazine reaction was slower in the protein than in the model peptide, which may be the result of differences in the cis-trans equilibrium of the X-Pro peptide bonds in the 2 molecules. (+info)
The albonoursin gene Cluster of S noursei biosynthesis of diketopiperazine metabolites independent of nonribosomal peptide synthetases.
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Albonoursin [cyclo(deltaPhe-DeltaLeu)], an antibacterial peptide produced by Streptomyces noursei, is one of the simplest representatives of the large diketopiperazine (DKP) family. Formation of alpha,beta unsaturations was previously shown to occur on cyclo(L-Phe-L-Leu), catalyzed by the cyclic dipeptide oxidase (CDO). We used CDO peptide sequence information to isolate a 3.8 kb S. noursei DNA fragment that directs albonoursin biosynthesis in Streptomyces lividans. This fragment encompasses four complete genes: albA and albB, necessary for CDO activity; albC, sufficient for cyclic dipeptide precursor formation, although displaying no similarity to non ribosomal peptide synthetase (NRPS) genes; and albD, encoding a putative membrane protein. This first isolated DKP biosynthetic gene cluster should help to elucidate the mechanism of DKP formation, totally independent of NRPS, and to characterize novel DKP biosynthetic pathways that could be engineered to increase the molecular diversity of DKP derivatives. (+info)
A novel potent cell cycle inhibitor dehydrophenylahistin--enzymatic synthesis and inhibitory activity toward sea urchin embryo.
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A novel dehydrogenated cyclic dipeptide named as dehydrophenylahistin (deltaPLH) was effectively prepared from a fungal metabolite (-)-phenylahistin by the enzymatic conversion catalyzed by the cell-free extract of Streptomyces albulus KO-23, an albonoursin-producing actinomycete. deltaPLH exhibited more than 1,000 times as high potent inhibitory activity toward the first cleavage of sea urchin embryos as (-)-phenylahisitn which has been reported to be a cell cycle inhibitor and more than 10,000 as high as albonoursin, indicating that deltaPLH is a promising leading compound for anticancer drugs. (+info)
Elimination of water from the carboxyl group of GlyGlyH+.
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The elimination of water from the carboxyl group of protonated diglycine has been investigated by density functional theory calculations. The resulting structure is identical to the b(2) ion formed in the mass spectrometric fragmentation of protonated peptides (therefore named "b2" in this study). The most stable geometry of the fragment ion ("b2") is an O-protonated diketopiperazine. However, its formation is kinetically disfavored as it requires a free energy of 58.2 kcal/mol. The experimentally observed N-protonated oxazolone is 3.0 kcal/mol less stable. The lowest energy pathway for the formation of the "b2" ion requires a free energy of 37.5 kcal/mol and involves the proton transfer from the amide oxygen of protonated diglycine to the hydroxyl oxygen. Fragmentation initiated by proton transfer from the terminal nitrogen has also a comparable free energy of activation (39.4 kcal/mol). Proton transfer initiating the fragmentation, from the highly basic terminal nitrogen or amide oxygen to the less basic hydroxyl oxygen is feasible at energies reached in usual mass spectrometric experiments. Amide N-protonated diglycine structures are precursors of mainly y(1) ions rather than "b2" ions. In the lowest energy fragmentation channels, proton transfer to the hydroxylic oxygen, bond breaking and formation of an oxazolone ring occur concertedly but asynchronously. Proton transfer to hydroxyl oxygen and cleavage of the corresponding C-O bond take place at the early stages of the fragmentation step, while ring closure to form an oxazolone geometry occurs at the later stages of the transition. The experimentally observed low kinetic energy release is expected to be due to the existence of a strongly hydrogen bonded protonated oxazolone-water complex in the exit channel. Whereas the threshold energy for "b2" ion formation (37.1 kcal/mol) is lower than for the y(1) ion (38.4 kcal/mol), the former requires a tight transition state with an activation entropy, DeltaS++ = -1.2 cal/mol.K and the latter has a loose transition state with DeltaS++ = +8.8 cal/mol.K. This leads to y(1) being the major fragment ion over a wide energy range. (+info)
Experimental and theoretical investigation of the main fragmentation pathways of protonated H-Gly-Gly-Sar-OH and H-Gly-Sar-Sar-OH.
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The fragmentation pathways of protonated H-Gly-Gly-Sar-OH and H-Gly-Sar-Sar-OH are investigated by using both computational and experimental techniques. The main goal of these studies is to further investigate which factors determine the branching ratio of the b2-y1 (Paizs, B.; Suhai, S. Rapid Commun. Mass Spectrom. 2002, 16, 375.) and "diketopiperazine" (Cordero, M. M.; Houser, J. J.; Wesdemiotis, C. Anal. Chem. 1993, 65, 1594.) pathways of protonated tripeptides. Protonated H-Gly-Sar-Sar-OH represents a sensitive test for the branching ratio of the b2-y1 and "diketopiperazine" pathways since this ion cannot produce y1 ions on the b(-y1 channel but only b2 ions. Protonated H-Gly-Gly-Sar-OH and H-Gly-Sar-Sar-OH exhibit very different fragmentation behavior under the investigated experimental conditions. The former fragments forming mainly y1 ions (maximum abundance of the b2 and y2 ions is approximately 15%), while the latter produces mainly b2 ions while at larger internal energies the a2, y2, and y1 ions become also moderately abundant. Theoretical modeling and analysis of the main fragmentation pathways indicate that the majority of the b2 and y1 ions of protonated H-Gly-Gly-Sar-OH and the b2 ions of H-Gly-Sar-Sar-OH are formed on the b2-y1 pathway. (+info)