Design, synthesis, and biological evaluation of novel 2H-pyran-2-one derivatives as potential HIV-1 reverse transcriptase inhibitors.

In search for more effective drugs against HIV infection acting as non-nucleoside reverse transcriptase inhibitors (NNRTIs), a series of new molecules with hybrid structures based on the natural product (+)-calanolide A and the synthetic molecule α-APA, known as potent and selective inhibitors of this enzyme, were selected by docking calculations. A convergent synthetic strategy gave 21 compounds with a 2H-pyran-2-one structural unit and bearing isosteric modifications, which were tested against HIV-infected CEM cell cultures. Only compound 6 (4-((2-(1H-indol-3-yl)ethyl)amino)-6-methyl-2H-pyran-2-one) displayed inhibitory activity (EC50 : 25-50 µM). However, it was associated with a relatively high cytostatic effect on human T lymphocyte (CEM) cell cultures, not easily predictable, neither by the chemical structure nor by the computational approach. Although this drug design has failed in selecting a novel scaffold for NNRTIs, the results have driven the interest towards new potential antitumor molecules showing activity against L1210 murine leukemia and HeLa cervix carcinoma cells, among which compound 21 (6-methyl-4-((2-(naphthalen-1-yl)ethyl)sulfonyl)-2H-pyran-2-one) was the most effective (IC50 : 0.95 and 2.9 µM, respectively).

EPR and FTIR studies reveal the importance of highly ordered sterol-enriched membrane domains for ostreolysin activity.

Ostreolysin is a cytolytic protein from the edible oyster mushroom (Pleurotus ostreatus), which recognizes specifically and binds to raft-like sterol-enriched membrane domains that exist in the liquid-ordered phase. Its binding can be abolished by micromolar concentrations of lysophospholipids and fatty acids. The membrane activity of ostreolysin, however, does not completely correlate with the ability of a certain sterol to induce the formation of a liquid-ordered phase, suggesting that the protein requires an additional structural organization of the membrane to exert its activity. The aim of this study was to further characterize the lipid membranes that facilitate ostreolysin binding by analyzing their lipid phase domain structure. Fourier-transformed infrared spectroscopy (FTIR) and electron paramagnetic resonance (EPR) were used to analyze the ordering and dynamics of membrane lipids and the membrane domain structure of a series of unilamellar liposomes prepared by systematically changing the lipid components and their ratios. Our results corroborate the earlier conclusion that the average membrane fluidity of ostreolysin-susceptible liposomes alone cannot account for the membrane activity of the protein. Combined with previous data computer-aided interpretation of EPR spectra strongly suggests that chemical properties of membrane constituents, their specific distribution, and physical characteristics of membrane nanodomains, resulting from the presence of sterol and sphingomyelin (or a highly ordered phospholipid, dipalmitoylphosphatidylcholine), are essential prerequisites for ostreolysin membrane binding and pore-formation.

Reductive activation of ricin and ricin A-chain immunotoxins by protein disulfide isomerase and thioredoxin reductase.

Intracellular activation of ricin and of the ricin A-chain (RTA) immunotoxins requires reduction of their intersubunit disulfide(s). This crucial event is likely to be catalyzed by disulfide oxidoreductases and precedes dislocation of the toxic subunit to the cytosol. We investigated the role of protein disulfide isomerase (EC 5.3.4.1, PDI), thioredoxin (Trx), and thioredoxin reductase (EC 1.8.1.9, TrxR) in the reduction of ricin and of a ricin A-chain immunotoxin by combining enzymatic assays, SDS-PAGE separation and immunoblotting. We found that, whereas PDI, Trx, and TrxR used separately were unable to directly reduce ricin and the immunotoxin, PDI and Trx in the presence of TrxR and NADPH could reduce both ricin and immunotoxin in vitro. PDI functioned only after pre-incubation with TrxR and the reductive activation of ricin was more efficient in the presence of glutathione. Similar results were obtained with microsomal membranes or crude cell extracts. Pre-incubation with the gold(I) compound auranofin, which irreversibly inactivates TrxR, resulted in a dose-dependent inhibition of ricin and immunotoxin reduction. Reductive activation of ricin and immunotoxin decreased or was abolished in microsomes depleted of TrxR and in cell extracts depleted of both PDI and Trx. Pre-incubation of U-937, Molt-3, Jurkat, and DU145 cells with auranofin significantly decreased ricin cytotoxicity with respect to mock-treated controls (P<0.05). Conversely, auranofin failed to protect cells from the toxicity of pre-reduced ricin which does not require intracellular reduction of disulfide between the two ricin subunits. We conclude that TrxR, by activating disulfide reductase activity of PDI, can ultimately lead to reduction/activation of ricin and immunotoxin in the cell.

Metal binding in amyloid beta-peptides shows intra- and inter-peptide coordination modes.

X-ray absorption spectroscopy data show different metal binding site structures in beta-amyloid peptides according to whether they are complexed with Cu(2+) or Zn(2+) ions. While the geometry around copper is stably consistent with an intra-peptide binding with three metal-coordinated Histidine residues, the zinc coordination mode depends on specific solution conditions. In particular, different sample preparations are seen to lead to different geometries around the absorber that are compatible with either an intra- or an inter-peptide coordination mode. This result reinforces the hypothesis that assigns different physiological roles to the two metals, with zinc favoring peptide aggregation and, as a consequence, plaque formation.

A fluorescence-based assay for the reductase activity of protein disulfide isomerase.

We report on a new spectrofluorimetric assay for the measurement of reductase activity of proteins belonging to the superfamily of thioredoxins such as protein disulfide isomerase (PDI). The assay relies on the preparation of a fluorescence-quenched substrate easily accessible in two steps through functional group transformations of the peptide Gly-Cys-Asp. In the first step fluorescein isothiocyanate is linked to the Gly-NH(2) terminus and in the second step the Cys-SH groups are converted into a disulfide bond. Both intermediate and final substrate have been fully characterized by mass spectrometric and nuclear magnetic resonance measurements. Dimethyl sulfoxide is here reported to be a mild oxidizing agent allowing us to obtain in good overall yield the assay substrate in a single synthetic step. A reliable estimation of PDI reductase activity is obtained via the detection of a strong fluorescence enhancement after enzymatic reduction. Moreover, our assay provides further support for the key role played by thioredoxin reductase in enabling disulfide reductase activity of PDI.

N-terminal deletion affects catalytic activity of saporin toxin.

Single-chain ribosome inactivating proteins (RIPs) are cytotoxic components of macromolecular pharmaceutics for immunotherapy of cancer and other human diseases. Saporin belongs to a family of single-chain RIPs sharing sequence and structure homology. In a preliminary attempt to define an active saporin polypeptide of minimum size we have generated proteins with deletions at the N-terminus and at the C-terminus. An N-terminal (sapDelta1-20) deletion mutant of saporin displayed defective catalytic activity, drastically reduced cytotoxicity but increased ability to interact with liposomes inducing their permeabilization at low pH. A C-terminal (sapDelta239-253) deletion mutant showed instead a moderate reduction in cytotoxic activity. A substantial alteration of secondary structure was evidenced by Fourier transformed infrared spectroscopy (FTIR) in the sapDelta1-20 mutant. It can be hypothesized that the defective functions of sapDelta1-20 are due to alterations of its spatial configuration.