Cyclophilin inhibitors block arterivirus replication by interfering with viral RNA synthesis.

Virus replication strongly depends on cellular factors, in particular, on host proteins. Here we report that the replication of the arteriviruses equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) is strongly affected by low-micromolar concentrations of cyclosporine A (CsA), an inhibitor of members of the cyclophilin (Cyp) family. In infected cells, the expression of a green fluorescent protein (GFP) reporter gene inserted into the PRRSV genome was inhibited with a half-maximal inhibitory concentration (IC(50)) of 5.2 µM, whereas the GFP expression of an EAV-GFP reporter virus was inhibited with an IC(50) of 0.95 µM. Debio-064, a CsA analog that lacks its undesirable immunosuppressive properties, inhibited EAV replication with an IC(50) that was 3-fold lower than that of CsA, whereas PRRSV-GFP replication was inhibited with an IC(50) similar to that of CsA. The addition of 4 µM CsA after infection prevented viral RNA and protein synthesis in EAV-infected cells, and CsA treatment resulted in a 2.5- to 4-log-unit reduction of PRRSV or EAV infectious progeny. A complete block of EAV RNA synthesis was also observed in an in vitro assay using isolated viral replication structures. The small interfering RNA-mediated knockdown of Cyp family members revealed that EAV replication strongly depends on the expression of CypA but not CypB. Furthermore, upon fractionation of intracellular membranes in density gradients, CypA was found to cosediment with membranous EAV replication structures, which could be prevented by CsA treatment. This suggests that CypA is an essential component of the viral RNA-synthesizing machinery.

Structural insights into the specificity of Xyn10B from Paenibacillus barcinonensis and its improved stability by forced protein evolution.

Paenibacillus barcinonensis is a soil bacterium bearing a complex set of enzymes for xylan degradation, including several secreted enzymes and Xyn10B, one of the few intracellular xylanases reported to date. The crystal structure of Xyn10B has been determined by x-ray analysis. The enzyme folds into the typical (beta/alpha)(8) barrel of family 10 glycosyl hydrolases (GH10), with additional secondary structure elements within the beta/alpha motifs. One of these loops -L7- located at the beta7 C terminus, was essential for xylanase activity as its partial deletion yielded an inactive enzyme. The loop contains residues His(249)-Glu(250), which shape a pocket opened to solvent in close proximity to the +2 subsite, which has not been described in other GH10 enzymes. This wide cavity at the +2 subsite, where methyl-2,4-pentanediol from the crystallization medium was found, is a noteworthy feature of Xyn10B, as compared with the narrow crevice described for other GH10 xylanases. Docking analysis showed that this open cavity can accommodate glucuronic acid decorations of xylo-oligosaccharides. Co-crystallization experiments with conduramine derivative inhibitors supported the importance of this open cavity at the +2 subsite for Xyn10B activity. Several mutant derivatives of Xyn10B with improved thermal stability were obtained by forced evolution. Among them, mutant xylanases S15L and M93V showed increased half-life, whereas the double mutant S15L/M93V exhibited a further increase in stability, showing a 20-fold higher heat resistance than the wild type xylanase. All the mutations obtained were located on the surface of Xyn10B. Replacement of a Ser by a Leu residue in mutant xylanase S15L can increase hydrophobic packing efficiency and fill a superficial indentation of the protein, giving rise to a more compact structure of the enzyme.

New cationic polyprenyl derivative proposed as a lipofecting agent.

Cationic linear poly-cis-isoprenoid prepared from natural plant polyprenol in a mixture with dioleyl phosphatidylethanolamine was found to be an effective lipofection agent for eukaryotic cells. The transfecting activity is related to the poly-cis structure of the polyprenyl chain.

Search for alpha-glucosidase inhibitors: new N-substituted valienamine and conduramine F-1 derivatives.

A solid-phase synthesis of new N-substituted valienamines has been developed and new synthesis of (+/-)-conduramine F-1, (-)-conduramine F-1, and (+)-ent-conduramine F-1 is presented, together with the preparation of N-benzylated conduramines F-1. N-Benzylation of both valienamine and (+)-ent-conduramine F-1 improves their inhibitory activity toward alpha-glucosidases significantly. The additional hydroxymethyl group makes valienamine derivatives more active than their (+)-ent-conduramine F-1 analogues.

Total asymmetric synthesis of (-)-conduramine B-1 and of its enantiomer. N-Benzyl derivatives of conduramine B-1 are beta-glucosidase inhibitors.

The 'naked sugars' (+)- and (-)-7-oxabicyclo[2.2.1]hept-5-en-2-one have been converted into (-)-conduramine B-1 ((-)-3) and its enantiomer (+)-3, respectively. They have been condensed with a variety of aldehydes in the presence of NaBH(OAc)(3). The N-substituted derivatives 4 and ent-4 so-obtained have been tested against two alpha-glucosidases, two amyloglucosidases, two beta-glucosidases and one beta-xylosidase for their inhibitory activities. Although (-)-3 and (+)-3 do not inhibit any of these enzymes at 1mM concentration, N-benzylated derivatives of (-)-conduramine B-1 are selective and competitive inhibitors of beta-glucosidases with K(i) in low micromolecular range.

Strategies for the stereocontrolled formation of oxygen analogues of penicillins and cephalosporins.

The synthesis of oxacephalotin and oxacephamandol, which are more active than natural, sulfur-containing congeners, and the isolation of clavulanic acid, a potent inhibitor of beta-lactamase enzymes, directed attention of many academic and industrial laboratories the synthesis of oxygen analogues of penicillins and cephalosporins. The present review focuses attention on the problem of stereocontrol in the formation of a desired configuration of the bridgehead carbon atom in the title compounds. Five feasible synthetic methods leading to the basic skeletons of clavams and 5-oxacephams are discussed. Three of them involve the nucleophilic substitution at C-4 of the azetidin- 2-ones performed as inter- or intramolecular process and the remaining two involve cycloaddition reactions between ketenes and iminoethers, or between vinyl ethers and isocyanates. Owing to the general application, stereospecificity and high asymmetric induction, the last method seems to be most advantageous. The weak point of the nucleophilic substitution methodology is that a nucleophile approaches the 3-substituted azetidin-2-one ring preferentially anti to the existing substituent and in the case where there is no substituent at C-3, that the stereoselectivity of formation of the new chirality center at C-4 is low. All discussed methods are illustrated by the examples taken from the literature.

Practical use of DD-peptidase 64-575 for the assay of inhibition activity of natural and synthetic beta-lactam compounds.

DD-peptidase 64-575 is produced by Saccharopolyspora erythrea PZH TZ 64-575. This enzyme is very sensitive to beta-lactam antibiotics. It could be used to assay the affinity of natural and synthetic beta-lactam compounds. In this paper an enzymatic method is described for investigation of synthetic, insoluble in water beta-lactams.

Structure-chiroptical properties relationship in oxabicyclic beta-lactam derivatives.

The relationship between molecular structure of 5-dethia-5-oxacephams and clavams and their chiroptical properties was investigated by means of X-ray diffraction analysis, molecular modeling calculations and circular dichroism spectroscopy. It was found that the amide chromophore of the beta-lactam unit in these compounds is nonplanar with nitrogen atom having a pyramidal configuration. It was also found that the helicity of the lactam moiety in investigated oxacephams and clavams is controlled by the absolute configuration at the C-6 and C-5 carbon atom, respectively. Thus, the applicability of helicity rule correlating a positive (negative) torsional angle of the beta-lactam subunit O=C-N-C with a negative (positive) sign of the n-->pi* CE, previously applied to oxacephams, is now extended to clavams.

5-Dethia-5-oxacephams: toward correlation of absolute configuration and chiroptical properties.

The relationship between chiroptical properties of differently substituted 5-dethia-5-oxacephams and their respective molecular structures was investigated. The amide chromophore of the beta-lactam unit in these compounds was found to be nonplanar with a shallow pyramidal configuration at the nitrogen atom. Due to the nonplanarity, the beta-lactam system becomes inherently dissymmetric, which is supported by a high magnitude of the n --> pi* CD band. It was also found that the helicity of the lactam moiety in investigated oxacephams is controlled by the absolute configuration at the C(6) carbon atom. On this basis, a helicity rule correlating a positive (negative) sign of the n right arrow pi Cotton effect with a negative (positive) O [double bond] C [bond] N [bond] C(6) torsional angle for policyclic beta-lactam derivatives possessing a nonplanar amide chromophore was formulated.