Scutellarin-graft cationic ß-cyclodextrin-polyrotaxane: Synthesis, characterization and DNA condensation.

As a prerequisite of gene delivery in living cells, DNA condensation has attracted more and more attention. In order to improve the efficiencies of polyamine-ß-cyclodextrin-based cationic polyrotaxanes (PR-EDA and PR-DETA) as DNA condensation materials, we have designed and prepared two novel scutellarin-grafted cationic polyrotaxanes (PR-EDA-SCU and PR-DETA-SCU), in which scutellarins (SCU), the planar molecules, were conjugated on the cyclodextrin molecules of PR-EDA and PR-DETA. These materials were characterized by 1D and 2D NMR, XRD, TG and DSC. The electrophoresis assays showed that pDNA condensation efficiencies of PR-EDA and PR-DETA were better than that of PR-EDA and PR-DETA. The complexes of PR-EDA, PR-DETA, PR-EDA-SCU and PR-DETA-SCU with pDNA were further investigated by zeta potential and atomic force microscopy analysis. The results indicated that the planar structure of SCU played an important role in improvement of pDNA condensation efficiencies of PR-EDA-SCU and PR-DETA-SCU. The satisfactory pDNA condensation abilities of PR-EDA-SCU and PR-DETA-SCU could be helpful in designing non-viral gene delivery vectors to control gene expression and delivery.

Production and purification of recombinants of mouse MASP-2 and sMAP.

Small mannose-binding lectin (MBL)-associated protein (sMAP) is a component of the complex consisting of MBL and MBL-associated serine proteases (MASPs) in the lectin complement pathway. sMAP is a truncated form of MASP-2, which is generated by an alternative splicing from a single structural MASP-2 gene. Upon activation of the MBL-MASPs complex, MASP-2 cleaves the complement C4, but the role of sMAP which lacks the serine protease domain is not clear. To clarify the role of sMAP in activation of the lectin pathway, we have generated sMAP-gene deficient mice which are also deficient for MASP-2. In this study, we generated and purified mouse recombinant sMAP (rsMAP) and rMASP-2 using the Drosophila expression system for the reconstitution assay of the deficient mice. In preliminary experiments, these purified recombinants were able to reconstitute the MBL-MASPs-sMAP complexes and the addition of rMASP-2 to deficient serum restored the C4 cleavage activity of the MBL-MASPs complex. From these data, rsMAP and rMASP-2 generated in this study seem to be useful in analysis of the deficient mice.

Peptide-based inhibitors of hepatitis C virus full-length NS3 (protease-helicase/NTPase): model compounds towards small molecule inhibitors.

From L-alpha-aminobutyric acid (Abu) a set of electrophilic and non-electrophilic replacements for the P1 cysteine of substrate and product inhibitors of hepatitis C virus full-length NS3 (protease-helicase/NTPase) serine protease have been synthesised and coupled to a model pentapeptide furnishing a set of hexapeptide inhibitors. Promising inhibitory activities with K(i) values of 0.18 microM (11b, P1 electrophilic alpha,beta-unsaturated ketone), 0.46 microM (12e, P1 electrophilic alkyl ketone) and 0.98 microM (10e, P1 non-electrophilic alkenyl alcohol as diastereomeric mixture). The reference hexapeptide product inhibitor had a K(i) value of 1.54 microM (14, P1 Abu-OH). The electrophilic inhibitors exhibit increased potency as compared with the corresponding product inhibitor, and notably also the non-electrophilic P1 alkenyl alcohol 10e. This represents the first example of non-electrophilic inhibitors that are not P1 amides or product inhibitors.

A convergent solution-phase synthesis of the macrocycle Ac-Phe-[Orn-Pro-D-Cha-Trp-Arg], a potent new antiinflammatory drug.

Relatively few cyclic peptides have reached the pharmaceutical marketplace during the past decade, most produced through fermentation rather than made synthetically. Generally, this class of compounds is synthesized for research purposes on milligram scales by solid-phase methods, but if the potential of macrocyclic peptidomimetics is to be realized, low-cost larger scale solution-phase syntheses need to be devised and optimized to provide sufficient quantities for preclinical, clinical, and commercial uses. Here, we describe a cheap, medium-scale, solution-phase synthesis of the first reported highly potent, selective, and orally active antagonist of the human C5a receptor. This compound, Ac-Phe[Orn-Pro-d-Cha-Trp-Arg], known as 3D53, is a macrocyclic peptidomimetic of the human plasma protein C5a and displays excellent antiinflammatory activity in numerous animal models of human disease. In a convergent approach, two tripeptide fragments Ac-Phe-Orn(Boc)-Pro-OH and H-d-Cha-Trp(For)-Arg-OEt were first prepared by high-yielding solution-phase couplings using a mixed anhydride method before coupling them to give a linear hexapeptide which, after deprotection, was obtained in 38% overall yield from the commercially available amino acids. Cyclization in solution using BOP reagent gave the antagonist in 33% yield (13% overall) after HPLC purification. Significant features of the synthesis were that the Arg side chain was left unprotected throughout, the component Boc-d-Cha-OH was obtained very efficiently via hydrogenation of d-Phe with PtO(2) in TFA/water, the tripeptides were coupled at the Pro-Cha junction to minimize racemization via the oxazolone pathway, and the entire synthesis was carried out without purification of any intermediates. The target cyclic product was purified (>97%) by reversed-phase HPLC. This convergent synthesis with minimal use of protecting groups allowed batches of 50-100 g to be prepared efficiently in high yield using standard laboratory equipment. This type of procedure should be useful for making even larger quantities of this and other macrocyclic peptidomimetic drugs.

Evaluation of a two-stage screening procedure in the combinatorial search for serine protease-like activity.

A series of peptidosteroid derivatives containing two independent peptide chains in which Ser and His are incorporated were synthesized by solid-phase peptide synthesis. The activity of the different compounds in the hydrolysis of the activated substrate NF31 was assessed in a stepwise fashion. First, the different resin-bound derivatives 6a-l and 6x-z were individually assayed for serine esterification in the absence of water. The use of a colored substrate allowed for a visual identification of the most active compounds. Through the inclusion of control substances, the involvement of histidine in the mechanism for serine acylation was shown. Second, the hydrolysis and methanolysis of the different acylated derivatives 8a-l and 8x were evaluated using UV spectroscopy, again indicating the involvement of histidine. The feasibility of applying the above procedures in a combinatorial context was proven via the screening of artificial libraries, created by mixing the different resin-bound peptidosteroid compounds. In this respect, the use of a photocleavable linker allowed for the unambiguous structural characterization of the selected members via application of single-bead electrospray tandem mass spectrometry.

Peptide design aided by neural networks: biological activity of artificial signal peptidase I cleavage sites.

De novo designed signal peptidase I cleavage sites were tested for their biological activity in vivo in an Escherichia coli expression and secretion system. The artificial cleavage site sequences were generated by two different computer-based design techniques, a simple statistical method, and a neural network approach. In previous experiments, a neural network was used for feature extraction from a set of known signal peptidase I cleavage sites and served as the fitness function in an evolutionary design cycle leading to idealized cleavage site sequences. The cleavage sites proposed by the two algorithms were active in vivo as predicted. There seems to be an interdependence between several cleavage site features for the constitution of sequences recognized by signal peptidase. It is concluded that neural networks are useful tools for sequence-oriented peptide design.

Solution structure of the epidermal growth factor (EGF)-like module of human complement protease C1r, an atypical member of the EGF family.

The calcium-dependent interaction between C1r and C1s, the two homologous serine proteases of the first component of human complement C1, is mediated by their N-terminal regions. The latter comprise an epidermal growth factor (EGF)-like module exhibiting the consensus sequence characteristic of Ca(2+)-binding EGF modules, surrounded by two CUB modules. Due to its Ca2+ binding ability, the C1r EGF-like module (C1r-EGF) is supposed to participate in the C1r-C1s interaction. An additional interesting feature of C1r-EGF is the unusually large loop connecting the first two conserved cysteine residues. The solution structure of synthetic C1r-EGF (residues 123-175) has been determined using nuclear magnetic resonance and combined simulated annealing-restrained molecular dynamics calculations. The resulting family of 19 structures is characterized by a well-ordered C-terminal part (residues Cys 144-Ala174) with a backbone rmsd of 0.7 A and a disordered N-terminal, including the large loop between the first two cysteines (Cys129 and Cys144). This loop is known to be surface exposed and may be expected to participate in domain-domain or protein-protein interactions. In its C-terminal part, C1r-EGF possesses the characteristic EGF fold with a major and a minor beta-sheet. The latter comprises a beta-bulge, and comparison with other EGF-like modules reveals the existence of two distinct structural and sequential motifs in the bulged part. Additional experiments in the presence of 80 mM Ca2+ did not show significant structural variation of C1r-EGF, in keeping with previous observations on blood-clotting factors IX and X.

Liquid-phase combinatorial synthesis: in search of small-molecule enzyme mimics.

The applications, advantages and recent advances in liquid-phase combinatorial chemistry using poly-(ethyleneglycol) as a soluble polymer support are reviewed. Our recent efforts towards the synthesis of peptide-based catalysts on polyethyleneglycol are reported. The screening of libraries of peptides for catalysis is discussed.

Characterization and inhibition of a cholecystokinin-inactivating serine peptidase.

A cholecystokinin (CCK)-inactivating peptidase was purified and identified as a membrane-bound isoform of tripeptidyl peptidase II (EC 3.4.14.10), a cytosolic subtilisin-like peptidase of previously unknown functions. The peptidase was found in neurons responding to cholecystokinin, as well as in non-neuronal cells. Butabindide, a potent and specific inhibitor, was designed and shown to protect endogenous cholecystokinin from inactivation and to display pro-satiating effects mediated by the CCKA receptor.