HUVEC cell affinity evaluation and integrin-mediated mechanism study on PHSRN-modified polymer

To investigate the role of the peptide Pro-His-Ser-Arg-Asn (PHSRN) in cell adhesion and growth, PHSRN- and Gly-Arg-Gly-Asp-Ser (GRGDS)-containing polymers (P-PN5 and P-GS5, respectively) were synthesized by modification of poly(D,L-lactide-co-beta-malic acid) (PLMA) with the corresponding peptides. The cell affinities of the modified polymers were evaluated by adhesion and proliferation experiments with human umbilical vein endothelial cells (HUVECs). The results showed that P-PN5 had comparable ability to that of P-GS5 in supporting HUVEC adhesion and growth. Furthermore, the integrin-mediated mechanism of cell-substrate interaction was investigated. The results showed that P-PN5 had similar binding affinity and binding strength towards α(5)β(1) compared to those of P-GS5. The findings suggest that PHSRN is capable of mediating the adhesion and growth of HUVECs independently and that PHSRN-modified polymers might be used as biologically compatible materials.     

Preparation and biological activities of a bivalent poly(ethylene glycol) hybrid containing an active site and its synergistic site of fibronectin

A bivalent poly(ethylene glycol) or PEG hybrid of fibronectin-related peptides was prepared. An active site peptide (RGD) and its synergistic site peptide (PHSRN) of fibronectin were conjugated with an amino acid-type PEG (aaPEG) to form PHSRN-aaPEG-RGD. A moderate spatial array between RGD and PHSRN in fibronectin may be required for synergic activity. The bivalent hybrid exhibited potent cell spreading activity and exhibited potent anti-metastatic activity in a model of experimental metastasis with B16-BL6 cells in mice. PEG may serve as a spacer for maintaining the desired spatial array.     

Synthesis of oligo(thiophene)-coated star-shaped ROMP polymers: unique emission properties by the precise integration of functionality

A facile synthesis of oligo(thiophene)-modified (coated) "soluble" star (ball)-shaped polymers has been achieved via sequential living ring-opening metathesis polymerization (ROMP) of norbornene and a cross-linking reagent using Mo(CHCMe(2)Ph)(N-2,6-(i)Pr(2)C(6)H(3))(O(t)Bu)(2) as the initiator and oligo(thiophene) carboxaldehydes for termination. The resultant star-shaped ROMP polymers containing ter- and tetrathiophene moieties exhibit unique emission properties due to an integration of the ROMP polymers (arranged functionalities): the blue emission was tuned to the white emission upon addition of 2-[2-[(E)-4-(dimethylamino)styryl]-6-methyl-4H-pyran-4-ylidene]malononitrile.     

Ring-opening metathesis polymerization of cis-5-norbornene-endo-2,3-dicarboxylic anhydride derivatives using the grubbs third generation catalyst

A series of cis-5-norbornene-endo-2,3-dicarboxylic anhydride(NDCA,M1) derivatives(M2-M4) with different types of nonpolar substituted groups were synthesized and characterized by ~1H/~(13)C-NMR and mass spectrometry(MS). Ringopening metathesis polymerization(ROMP) of these monomers using the Grubbs third generation catalyst(G3) generated high molecular weight polymers with much improved solubility compared with the NDCA's homopolymer. It was found that the solubility of these polymers increased with increased substituent's steric hindrance. The living polymerization of NDCA derivative containing the bulkiest substituent(M4) catalyzed by G3 in tetrahydrofuran was confirmed by the kinetic studies with low polydispersity indices(PDI)( 1.30). By using sequential ROMP, well-defined diblock copolymers containing anhydride groups were synthesized.     

Alkaloids from some amaryllidaceae species and their cholinesterase activity

Alkaloid extracts of four Amaryllidaceae species were studied with respect to their acetylcholinesterase and butyrylcholinesterase inhibitory activity and alkaloid patterns. Twenty-one alkaloids were determined by GC/MS, and seventeen of them identified from their mass spectra and retention times. The GC/MS analysis of the alkaloid extract of Nerine filamentosa is the first phytochemical investigation of this species. Promising erythrocytic acetylcholinesterase inhibitory activity was demonstrated by the alkaloid extracts of Narcissus poeticus var recurvus, Nerine filifolia and N. filamentosa (IC(50,HuAChE) = 6.0 +/- 0.1 microg/mL; IC(50,HuAChE) = 18.5 +/- 0.8 microg/mL, IC(50,HuAChE) = 21.6 +/- 1.1 microg/mL). The most potent inhibitory activity against serum butyrylcholinesterase was shown by extracts of Sternbergia lutea and Nerinefilamentosa (IC(50,HuBuChE) = 3.7 +/- 0.1 microg/mL; IC(50.HuBuChE) = 13.0 +/- 0.7 microg/mL).     

Design and synthesis of peptides that bind alpha-bungarotoxin with high affinity

BACKGROUND: Alpha-bungarotoxin (alpha-BTX) is a highly toxic snake venom alpha-neurotoxin that binds to acetylcholine receptor (AChR) at the neuromuscular junction, and is a potent inhibitor of this receptor. We describe the design and synthesis of peptides that bind alpha-BTX with high affinity, and inhibit its interaction with AChR with an IC(50) of 2 nM. The design of these peptides was based on a lead peptide with an IC(50) of 3x10(-7) M, previously identified by us [M. Balass et al., Proc. Natl. Acad. Sci. USA 94 (1997) 6054] using a phage-display peptide library. RESULTS: Employing nuclear magnetic resonance-derived structural information [T. Scherf et al., Proc. Natl. Acad. Sci. USA 94 (1997) 6059] of the complex of alpha-BTX with the lead peptide, as well as structure-function analysis of the ligand-binding site of AChR, a systematic residue replacement of the lead peptide, one position at a time, yielded 45 different 13-mer peptides. Of these, two peptides exhibited a one order of magnitude increase in inhibitory potency in comparison to the lead peptide. The design of additional peptides, with two or three replacements, resulted in peptides that exhibited a further increase in inhibitory potency (IC(50) values of 2 nM), that is more than two orders of magnitude better than that of the original lead peptide, and better than that of any known peptide derived from AChR sequence. The high affinity peptides had a protective effect on mice against alpha-BTX lethality. CONCLUSIONS: Synthetic peptides with high affinity to alpha-BTX may be used as potential lead compounds for developing effective antidotes against alpha-BTX poisoning. Moreover, the procedure employed in this study may serve as a general approach for the design and synthesis of peptides that interact with high affinity with any desired biological target.     

Structure determination of bioactive galloyl derivatives by NMR spectroscopy

The investigation of the chemical constituents of Symplocos racemosa Roxb led to the isolation of two new glycosides, symcomoside A (1) and symcomoside B (2), together with one known glycoside, tortoside C (3), which is reported for the first time from this plant. The structures of the new compounds were determined by 1D and 2D homonuclear and heteronuclear NMR spectroscopy, from chemical evidence and by comparison with published data for closely related compounds. Symcomoside B (2) showed potent inhibitory activity against alpha-glucosidase in a concentration-dependent fashion with an IC50 value of 0.733 +/- 0.033 mM whereas symcomoside A (1) showed very weak inhibitory activity against alpha-glucosidase (9.90% in 0.70 mM).     

Topochemical exploration of potent compounds using retro-enantiomer libraries of cyclic pentapeptides

Cyclic pentapeptides have been adopted as conformationally restricted peptide templates to dispose pharmacophores of bioactive peptides. In our recent study, use of two orthogonal cyclic pentapeptide libraries involving conformation-based and sequence-based libraries containing critical residues of a bioactive peptide led to the discovery of potent downsized peptides that possess activity comparable to that of the parent peptide. The present study demonstrates that a third library consisting of retro-enantiomers (retro-inverso peptides) that possess not only all residues with the opposite configuration to those in the corresponding original peptide but also amino acid sequences with reversed arrangement, is important as an alternative library for rationally finding active compounds.     

Increased polymer length of oligopeptide-substituted polynorbornenes with LiCl

The ring-opening metathesis polymerization (ROMP) reaction is extraordinarily useful for the preparation of a large variety of polymers. We report that the length (n = 25-50) of high-substituent-density oligopeptide polymers synthesized by ROMP is dramatically improved upon addition of LiCl to reduce polymer and oligopeptide aggregation. This methodology should significantly expand the variety of polymers that may be prepared by ROMP and be of general use with norbornyl oligopeptides of any sequence.     

Acetylcholinesterase and butyrylcholinesterase inhibitory compounds from Corydalis cava (Fumariaceae)

Tubers of Corydalis cava were extracted with ethanol and fractionated using n-hexane, chloroform and ethanol. Repeated column chromatography, preparative TLC and crystallization led to the isolation of fifteen isoquinoline alkaloids. The chemical structures of the isolated compounds were determined on the basis of spectroscopic techniques and by comparison with literature data. All isolated compounds were tested for human blood acetylcholinesterase (HuAChE) and human plasma butyrylcholinesterase (HuBuChE) inhibitory activity. (+)-Canadaline inhibited acetylcholinesterase as well as butyrylcholinesterase in a dose-dependent manner with IC50 values of 20.1 +/- 1.1 microM and 85.2 +/- 3.2 microM, respectively. (+)-Canadine, with an IC50 value of 12.4 +/- 0.9 microM, was the most potent inhibitor of acetylcholinesterase, whilst (+/-)-corycavidine and (+)-bulbocapnine were effective inhibitors of butyrylcholinesterase with IC50 values of 46.2 +/- 2.4 microM and 67.0 +/- 2.1 microM, respectively. The other isolated alkaloids were considered inactive (IC50 > 100 microM).     

Design and synthesis of sulfur based inhibitors of matrix metalloproteinase-1

Fibroblast collagenase (MMP-1), a member of the matrix metalloproteinases family, is believed to be a pathogenesis of arthritis, by cleaving triple-helical type II collagen in cartilage. From the similarity of the active site zinc binding mode with hydroxamate, we designed and synthesized alpha-mercaptocarbonyl possessing compounds (3-5), which incorporated various peptide sequences as enzyme recognition sites. The P4-P1 peptide incorporating compound (3) exhibited as potent inhibition as the hydroxamate (1) and the carboxylate (2) type inhibitors, with an IC50 of 10(-6) M order against MMP-1. But the inhibitor (3) related compounds (6-8) displayed decreased or no inhibitory potencies. These results suggest that the existence of both the carbonyl and thiol groups might be critical for the inhibition, and the distance between the two functional groups is important for inhibitory potency. For Pn' peptide incorporating compounds (4a-k), except for 4h and 4k, all compounds showed IC50 values under sub-nanomolar. Among them, for potent inhibition, Leu was better than Phe and Val as the P1' amino acid, and the P2' position amino acid was necessary, and preferentially Phe. Insertion of the Pn peptide into 4d or 4k, giving compounds 5a-c, did not increase the activities of 4d and 4k. Substitution of the mercapto group with other functional groups lost the activity of compound 4a. The stereochemical preference at the thiol-attached position was also determined by preparation of both isomers of 4a. It was found that the S configuration compound (36b) is approximately 100 times more potent than the corresponding R-isomer (36a).     

血红蛋白片段的合成及生物活性

采用多肽固相合成方法, 以Wang 树脂为载体, Fmoc为N-端氨基酸保护基, HOBt-HBTU为缩合试剂, 合成了一系列血红蛋白α链的片段, 产物经RP-HPLC和质谱进行了确定. 生物活性研究结果表明, 该系列多肽具有较高的血管紧张素Ⅰ转换酶抑制活性, 但不具有α-葡萄糖苷酶抑制活性.     

Synthesis of bisubstrate and donor analogues of sialyltransferase and their inhibitory activities

[reaction: see text] Sialyltransferases (STs) are involved in the biosynthesis of glycoconjugates with important biological activities. Most STs utilize cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) as a common donor substrate. A bisubstrate analogue containing the donor substrate (CMP-Neu5Ac mimic) and the acceptor substrate (galactose) was synthesized. Four donor analogues having the partial structure of the bisubstrate analogue were also synthesized to support study of the structure-activity relationship. Each analogue contains an ethylene group in place of the exocyclic anomeric oxygen of CMP-Neu5Ac. The bisubstrate analogue exhibited only weak inhibitory activity to rat recombinant alpha-2,3- and alpha-2,6-ST (IC(50) = 1.3, 2.4 mM). Conversion of the C-1 carboxylate of the Neu5Ac moiety to carboxyamide, hydroxymethyl, or methylene phosphate each resulted in a reduction in inhibitory activity. Among the synthesized analogues, cytidin-5'-yl sialylethylphosphonate (4) was the most potent inhibitor against rat recombinant alpha-2,3- and alpha-2,6-ST (IC(50) = 0.047, 0.34 mM).     

DNA binding and biological activity of some platinum(II) intercalating compounds containing methyl-substituted 1,10-phenanthrolines

This study documents the first detailed investigation into the relationship between molecular structure and biological activity of platinum(II) complexes containing methylated derivatives of 1,10-phenanthroline (phen). A series of square planar platinum(II) compounds incorporating methylated derivatives of phen, 4-methyl-1,10-phenanthroline (4-Mephen), 5-methyl-1,10-phenanthroline (5-Mephen), 4,7-dimethyl-1,10-phenanthroline (4,7-Me2phen), 5,6-dimethyl-1,10-phenanthroline (5,6-Me2phen) and 3,4,7,8-tetramethyl-1,10-phenanthroline (3,4,7,8-Me4phen) were synthesised and the relationship between their structure and biological activity investigated. The biological activity of these compounds was quantified using the in vitro cytotoxicity assay against the L1210 Murine leukaemia cell line. Large variation in cytotoxicities with different methylation was observed. The 5- and 5,6-methylated derivatives of phen displayed a greater biological activity, with IC50 values of 2.8 +/- 0.8 microM and 1.5 +/- 0.3 microM respectively, compared with the phen compound, with an IC50 value of 9.7 +/- 0.3 microM, while all the others were inactive with IC50 values over 50 microM. Binding constants were determined using circular dichroism spectroscopy (CD) and induced circular dichroism (ICD). ICD was used to highlight any differences in the spectra. Viscometry studies and linear dichroism (LD) experiments indicate that the platinum(II) complexes intercalate although for [Pt(en)(4-Mephen)]Cl2 and [Pt(en)(4,7-Me2phen)]Cl2 this mode of binding appears to be concentration dependent. The binding of the platinum(II) complexes to the oligonucleotide d(GTCGAC)2 was studied using two-dimensional 1H NMR spectroscopy. The addition of each metal complex to the hexamer d(GTCGAC)2 produced upfield shifts of the metal complex resonances, characteristic of intercalation. Through the observation of NOE cross-peaks, two-dimensional NMR studies provided insight into the site and groove preferences of these compounds when binding to DNA.     

Characterization of polyethylene with partially random chlorine substitution

A series of chlorine‐containing polymers were prepared by ring‐opening metathesis polymerization (ROMP) followed by hydrogenation. This synthesis route was chosen specifically so that chain microstructures would be obtained that resembled copolymers of ethylene and vinyl chloride. The chlorine content was varied by the copolymerization of 5‐chlorocyclooctene and cyclooctene. Differential scanning calorimetry, light microscopy, tapping‐mode atomic force microscopy, wide‐angle X‐ray diffraction (WAXD), and density were employed to characterize the polymers. The copolymers had certain restrictions on the length of the methylene sequence between substituted carbons, however, ROMP copolymerization introduced enough variation in the methylene sequence length that model copolymers with the equivalent of 14 mol % vinyl chloride or less closely resembled random copolymers of ethylene and vinyl chloride. These materials organized as spherulites and exhibited the orthorhombic crystal form. Constraints on the placement of chlorine atoms strongly affected the crystallization of polymers with more than the equivalent of 14 mol % vinyl chloride. More regular chlorine substitution along the polyethylene chain translated into better ordered crystal structures with sharp melting peaks. The granular morphology of these materials at ambient temperature was interpreted as fringed micellar crystals. The WAXD patterns provided definitive evidence that chains in the fringed micelle took the hexagonal crystal form. The lower density hexagonal form facilitated the crystallization of short ethylene sequences and accommodated chlorine atoms more easily than the orthorhombic form. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 2062–2070, 2003     

Phosphorus-based SAHA analogues as histone deacetylase inhibitors

[structure: see text] Three analogues of suberoyl anilide hydroxamic acid (SAHA) with phosphorus metal-chelating functionalities were synthesized as inhibitors of histone deacetylases (HDACs). The compounds showed weak activity for HeLa nuclear extracts (IC(50) = 0.57-6.1 mM), HDAC8 (IC(50) = 0.28-0.41 mM), and histone-deacetylase-like protein (HDLP, IC(50) = 0.33-1.9 mM), suggesting that the transition state of HDAC is not analogous to zinc proteases. Antiproliferative activity against A2780 cancer cells (IC(50) = 0.11-0.12 mM), comparable to SAHA (0.15 mM), was observed.     

Biomolecular engineering by combinatorial design and high-throughput screening: small, soluble peptides that permeabilize membranes

Rational design and engineering of membrane-active peptides remains a largely unsatisfied goal. We have hypothesized that this is due, in part, to the fact that some membrane activities, such as permeabilization, are not dependent on specific amino acid sequences or specific three-dimensional peptide structures. Instead they depend on interfacial activity: the ability of a molecule to partition into the membrane-water interface and to alter the packing and organization of lipids. Here we test that idea by taking a nonclassical approach to biomolecular engineering and design of membrane-active peptides. A 16,384-member rational combinatorial peptide library, containing peptides of 9-15 amino acids in length, was screened for soluble members that permeabilize phospholipid membranes. A stringent, two-phase, high-throughput screen was used to identify 10 unique peptides that had potent membrane-permeabilizing activity but were also water soluble. These rare and uniquely active peptides do not share any particular sequence motif, peptide length, or net charge, but instead they share common compositional features, secondary structure, and core hydrophobicity. We show that they function by a common mechanism that depends mostly on interfacial activity and leads to transient pore formation. We demonstrate here that composition-space peptide libraries coupled with function-based high-throughput screens can lead to the discovery of diverse, soluble, and highly potent membrane-permeabilizing peptides.     

Contractile activity of porcine neuromedin U-25 and various neuromedin U-related peptide fragments on isolated chicken crop smooth muscle.

Contractile activity of porcine neuromedin U-25 (p-NMU-25) and various neuromedin U (NMU) peptide fragment amides was examined on chicken crop smooth muscle preparation. The relative activity (expressed as RA value) of p-NMU-25 to porcine neuromedin U-8 (p-NMU-8) was 5.51 +/- 0.09, and p-NMU-25 (15-25) was the most potent fragment with an RA value of 7.78 +/- 0.05. All C-terminal 11-peptide amides of rat, rabbit, and frog NMU peptides retained activity about three-fold higher than the corresponding C-terminal 8-peptide amides. The peptide segment Asn15-Arg-Arg17 of p-NMU-25, as well as the corresponding positions of various NMU peptides: Ser13-Gly-Gly15 of rat NMU and Ser15-Arg-Gly17 of rabbit and frog NMUs, appeared to be involved in the structural requirements for increased contractile activity in the assay system.     

Design, synthesis and antifibrotic activities of carbohydrate-modified 1-(substituted aryl)-5-trifluoromethyl-2(1H) pyridones

Pirfenidone, a pyridone compound, is an effective and novel antifibrotic agent. In this article, we describe the design, synthesis and activity evaluation of novel antifibrotic agents, 1-(substituted aryl)-5-trifluoromethyl-2(1H) pyridones modified with carbohydrate. Most of the title compounds exhibited comparable or better inhibitory activity than fluorofenidone. Notably, compound 19a demonstrated the highest cell-based inhibitory activity against NIH 3T3 (IC(50) = 0.17 mM).     

Sequence-selective DNA cleavage by a chimeric metallopeptide

A chimeric metallopeptide derived from the sequences of two structurally superimposable motifs was designed as an artificial nuclease. Both DNA recognition and nuclease activity have been incorporated into a small peptide sequence. P3W, a 33-mer peptide comprising helices alpha2 and alpha3 from the engrailed homeodomain and the consensus EF-hand Ca-binding loop binds one equivalent of lanthanides or calcium and folds upon metal binding. The conditional formation constants (in the presence of 50 mM Tris) of P3W for Eu(III) (K(a) = (2.1 +/- 0.1) x 10(5) M(-1)) and Ce(IV) (K(a) = (2.6 +/- 0.1) x 10(5) M(-1)) are typical of isolated EF-hand peptides. Circular dichroism studies show that 1:1 CeP3W is 26% alpha-helical and EuP3W is up to 40% alpha-helical in the presence of excess metal. The predicted helicity of the folded peptide based on helix length and end effects is about 50%, showing the metallopeptides are significantly folded. EuP3W has considerably more secondary structure than our previously reported chimeras (Welch, J. T.; Sirish, M.; Lindstrom, K. M.; Franklin, S. J. Inorg. Chem. 2001, 40, 1982-1984). Eu(III)P3W and Ce(IV)P3W nick supercoiled DNA at pH 6.9, although EuP3W is more active at pH 8. CeP3W cleaves linearized, duplex DNA as well as supercoiled plasmid. The cleavage of a 5'-(32)P-labeled 121-mer DNA fragment was followed by polyacrylamide gel electrophoresis. The cleavage products are 3'-OPO(3) termini exclusively, suggesting a regioselective or multistep mechanism. In contrast, uncomplexed Ce(IV) and Eu(III) ions produce both 3'-OPO(3) and 3'-OH, and no evidence of 4'-oxidative cleavage termini with either metal. The complementary 3'-(32)P-labeled oligonucleotide experiment also showed both 5'-OPO(3) and 5'-OH termini were produced by the free ions, whereas CeP3W produces only 5'-OPO(3) termini. In addition to apparent regioselectivity, the metallopeptides cut DNA with modest sequence discrimination, which suggests that the HTH motif binds DNA as a folded domain and thus cleaves selected sequences. The de novo artificial nuclease LnP3W represents the first small, underivatized peptide that is both active as a nuclease and sequence selective.