Total synthesis of (+)-A83586C, (+)-kettapeptin and (+)-azinothricin: powerful new inhibitors of beta-catenin/TCF4-and E2F-mediated gene transcription

Herein we describe our asymmetric total syntheses of (+)-A83586C, (+)-kettapeptin and (+)-azinothricin. We also demonstrate that molecules of this class powerfully inhibit beta-catenin/TCF4- and E2F-mediated gene transcription within malignant human colon cancer cells at low drug concentrations.

Characterization of bafinivirus main protease autoprocessing activities

The production of functional nidovirus replication-transcription complexes involves extensive proteolytic processing by virus-encoded proteases. In this study, we characterized the viral main protease (Mpro) of the type species, White bream virus (WBV), of the newly established genus Bafinivirus (order Nidovirales, family Coronaviridae, subfamily Torovirinae). Comparative sequence analysis and mutagenesis data confirmed that the WBV Mpro is a picornavirus 3C-like serine protease that uses a Ser-His-Asp catalytic triad embedded in a predicted two-ß-barrel fold, which is extended by a third domain at its C terminus. Bacterially expressed WBV Mpro autocatalytically released itself from flanking sequences and was able to mediate proteolytic processing in trans. Using N-terminal sequencing of autoproteolytic processing products we tentatively identified Gln?(Ala, Thr) as a substrate consensus sequence. Mutagenesis data provided evidence to suggest that two conserved His and Thr residues are part of the S1 subsite of the enzyme's substrate-binding pocket. Interestingly, we observed two N-proximal and two C-proximal autoprocessing sites in the bacterial expression system. The detection of two major forms of Mpro, resulting from processing at two different N-proximal and one C-proximal site, in WBV-infected epithelioma papulosum cyprini cells confirmed the biological relevance of the biochemical data obtained in heterologous expression systems. To our knowledge, the use of alternative Mpro autoprocessing sites has not been described previously for other nidovirus Mpro domains. The data presented in this study lend further support to our previous conclusion that bafiniviruses represent a distinct group of viruses that significantly diverged from other phylogenetic clusters of the order Nidovirales.

Increased endocytosis in retinal vascular endothelial cells grown in high glucose medium is modulated by inhibitors of nonenzymatic glycosylation

We sought to determine if hyperglycaemia is responsible for increased retinal vascular endothelial-cell (RVEC) endocytosis in diabetes and to assess the role of nonenzymatic glycosylation in mediation of this novel endothelial-cell pathology. RVECs were propagated in media containing either 5 or 25 mmol/l glucose for up to 10 days after which they were exposed to the protein tracer horseradish peroxidase for 30 min. The level of RVEC endocytosis was quantified in intact cell monolayers by electron microscopic stereology, and in cell lysates by a simple spectrophotometric method. The effect of the nonenzymatic glycosylation inhibitors, aminoguanidine and D-lysine, on high-glucose medium induced changes in RVEC endocytosis was tested by inclusion of these agents in the culture medium. RVECs exposed to 25 mmol/l glucose showed a stepwise increase in endocytosis of horseradish peroxidase culminating in a two- to threefold increase after 10 days. Endocytosis returned to normal levels after a further 10 days in 5 mmol/l glucose medium. The increase in RVEC endocytosis was markedly reduced, but not completely normalised, by aminoguanidine and D-lysine. Exposure of cultured RVECs to 25 mmol/l glucose causes an increase in endocytosis of similar magnitude to that experienced by RVEC in early diabetes, and implicates hyperglycaemia in the latter situation. A significant component of the increase in RVEC endocytosis appears to be mediated by nonenzymatic glycosylation.

Probing the mid-gorge of cholinesterases with spacer-modified bivalent quinazolinimines leads to highly potent and selective butyrylcholinesterase inhibitors

The spacer structure of homobivalent quinazolinimes acting as potent acetyl-(AChE)- and butyrylcholinesterase (BChE) inhibitors was chemically modified introducing tertiary amine and acyl-amide moieties, and the activities at both ChEs were evaluated. Molecular docking was applied to explain the data and probe the capacity of the mid-gorge site of both ChEs. The novel spacer structures considerably alter the biological profile of bivalent quinazolinimines with regard to both inhibitory activity and selectivity. Mutual interaction of binding to the various sites of the enzymes was further investigated by applying also different spacer lengths and ring sizes of the alicycle of the tricyclic quinazolinimines. In order to achieve selectivity toward BChE and to improve inhibitory activities, the spacer structure was optimized and identified a highly potent and selective BChE inhibitor. (C) 2010 Elsevier Ltd. All rights reserved.

Proteasome inhibitors in cancer therapy

The ubiquitin proteasome pathway plays a critical role in regulating many processes in the cell which are important for tumour cell growth and survival. Inhibition of proteasome function has emerged as a powerful strategy for anti-cancer therapy. Clinical validation of the proteasome as a therapeutic target was achieved with bortezomib and has prompted the development of a second generation of proteasome inhibitors with improved pharmacological properties. This review summarises the main mechanisms of action of proteasome inhibitors in cancer, the development of proteasome inhibitors as therapeutic agents and the properties and progress of next generation proteasome inhibitors in the clinic.

Novel Inhibitors of the Pseudomonas aeruginosa Virulence Factor LasB: a Potential Therapeutic Approach for the Attenuation of Virulence Mechanisms in Pseudomonal Infection

Pseudomonas elastase (LasB), a metalloprotease virulence factor, is known to play a pivotal role in pseudomonal infection. LasB is secreted at the site of infection, where it exerts a proteolytic action that spans from broad tissue destruction to subtle action on components of the host immune system. The former enhances invasiveness by liberating nutrients for continued growth, while the latter exerts an immunomodulatory effect, manipulating the normal immune response. In addition to the extracellular effects of secreted LasB, it also acts within the bacterial cell to trigger the intracellular pathway that initiates growth as a bacterial bio?lm. The key role of LasB in pseudomonal virulence makes it a potential target for the development of an inhibitor as an antimicrobial agent. The concept of inhibition of virulence is a recently established antimicrobial strategy, and such agents have been termed “second-generation” antibiotics. This approach holds promise in that it seeks to attenuate virulence processes without bactericidal action and, hence, without selection pressure for the emergence of resistant strains. A potent inhibitor of LasB,N-mercaptoacetyl-Phe-Tyr-amide (Ki 41 nM) has been developed, and its ability to block these virulence processes has been assessed. It has been demonstrated that thes compound can completely block the action of LasB on protein targets that are instrumental in bio?lm formation and immunomodulation. The novel LasB inhibitor has also been employed in bacterial-cell-based assays, to reduce the growth of pseudomonal bio?lms, and to eradicate bio?lm completely when used in combination with conventional antibiotics.

The Effect of Liposome Encapsulation on the Pharmacokinetics of Recombinant Secretory Leukocyte Protease Inhibitor (rSLPI) Therapy after Local Delivery to a Guinea Pig Asthma Model

Inhaled recombinant Secretory Leukocyte Protease Inhibitor (rSLPI) has shown potential for treatment of inflammatory lung conditions. Rapid inactivation of rSLPI by cathepsin L (Cat L) and rapid clearance from the lungs have limited clinical efficacy. Encapsulation of rSLPI within 1,2-Dioleoyl-sn-Glycero-3-[Phospho-L-Serine]:Cholesterol liposomes (DOPS-rSLPI) protects rSLPI against Cat L inactivation in vitro. We aimed to determine the effect of liposomes on rSLPI pharmacokinetics and activity in vitro and after local delivery to the airways in vivo.