Treatment of inflammatory bowel disease by chemokine receptor-targeted leukapheresis.

Leukapheresis removes circulating leukocytes en route to the target organ. Hitherto unspecific matrixes have been used to remove leukocytes in inflammatory bowel disease (IBD). This report describes a novel selective leukapheresis column based on chemokine-chemokine receptor interaction. We found an increased expression of the gut homing chemokine receptor CCR9 on CD14(+) monocytes and on CD3(+) T lymphocytes from IBD patients. Biologically active CCL25 was coupled to a Sepharose matrix and demonstrated to selectively remove CCR9-expressing cells leaving other cell populations largely unaffected. A patient with active ulcerative colitis, was subjected to CCL25-column leukapheresis. Four days after treatment, he experienced clinical improvement and stable disease improvement ensued. The study illustrates that specific cells can be targeted using high affinity interactions, i.e., CCL25-CCR9 interactions to remove pathogenic gut-homing cells. Leukapheresis using the bCCL25 column should be investigated in a clinical phase I trial of patients with inflammatory bowel disease.

Mixed SAMs and MALDI-ToF MS: preparation of N-glycosylamine derivative and thioctic acid methyl ester bearing 1,2-dithiolane groups and detection of enzymatic reaction on Au.

Herein, we report an enzymatic galactosylation reaction of ß-glucopyranosylamide 4 and thioctic acid methyl ester 5 bearing 1,2-dithiolane groups to form a new system of mixed self-assembled monolayers (SAMs) on gold. Characterization of the enzymatic activity was conveniently achieved by mass spectrometry.

9-Aminoacridine peptide derivatives as versatile reporter systems for use in fluorescence lifetime assays.

A novel long lifetime fluorescence reporter based on 9-aminoacridine was designed, the lifetime of which can be modulated in a defined manner when in proximity to a tryptophan residue enabling fluorescence lifetime based biochemical assays to be configured.

A fluorescence lifetime-based assay for serine and threonine kinases that is suitable for high-throughput screening.

We describe the development of a novel method for the assay of serine/threonine protein kinases based on fluorescence lifetime. The assay consists of three generic peptides (which have been used by others in the assay of >140 protein kinases in various assay formats) labeled with a long lifetime fluorescent dye (14 or 17 ns) that act as substrates for protein kinases and an iron(III) chelate that modulates the fluorescence lifetime of the peptide only when it is phosphorylated. The decrease in average fluorescence lifetime as measured in a recently developed fluorescence lifetime plate reader (Edinburgh Instruments) is a measure of the degree of phosphorylation of the peptide. We present data showing that the assay performs as well as, and in some cases better than, the "gold standard" radiometric kinase assays with respect to Z' values, demonstrating its utility in high-throughput screening applications. We also show that the assay gives nearly identical results in trial screening to those obtained by radiometric assays and that it is less prone to interference than simple fluorescence intensity measurements.

Real-Time Imaging of Protease Action on Substrates Covalently Immobilised to Polymer Supports.

We report for the first time single bead spatially resolved activity measurements of solid-phase biocatalytic systems followed in real-time. Trypsin cleavage of Bz-Arg-OH and subtilisin cleavage of Z-Gly-Gly-Leu-OH each liberate a free amino group on aminocoumarin covalently immobilised to PEGA(1900) beads [a co-polymer of poly(ethylene glycol) with molecular mass of 1900 cross-linked with acrylamide]. This restores fluorescence which is imaged in optical sections by two-photon microscopy. For trypsin cleavage, fluorescence is restricted initially to surface regions, with more than 1 hour needed before reaction is fully underway in the bead centre, presumably reflecting slow enzyme diffusion. In contrast, for subtilisin cleavage fluorescence develops throughout the bead more quickly.

Controlling protein retention on enzyme-responsive surfaces.

The ability to change the properties of solid surfaces on demand is a key component of a multitude of established and emerging technologies. Stimuli that have previously been used to trigger changes in surface properties include changes in solvent, light, pH, ionic strength, temperature and magnetic or electric fields. We are interested in developing surfaces that can be triggered by the catalytic action of enzymes. We demonstrate the selective protease (alpha-chymotrypsin and thermolysin) catalysed peptide hydrolysis of surface-tethered fluorenylmethoxycarbonyl-dipeptides. We highlight some of the challenges evident from surface analysis in overcoming enzyme retention to the surface addressed by physical adsorption of soluble PEG(200) to the surface prior to enzyme exposure. Analysis by ToF-SIMS and XPS shows that alpha-chymotrypsin is deposited and retained on the surfaces and that thermolysin, a much more stable enzyme, selectively cleaves the tethered peptides as intended, and is removed from the surface by washing.

Enzyme-cleavable linkers for peptide and glycopeptide synthesis.

Hydroxymethylphenoxy linkers that are commonly used in solid phase peptide synthesis are surprisingly susceptible to efficient cleavage by the protease chymotrypsin with a broad range of amino acid residues being tolerated at the scissile bond; this enzyme-cleavable linker system has been applied to peptide and glycopeptide synthesis.

Optimized polymer-enzyme electrostatic interactions significantly improve penicillin G amidase efficiency in charged PEGA polymers.

Hydrolytic yields as high as 80% were obtained by using penicillin G amidase (PGA) on substrates anchored on optimized positively charged PEGA polymers. By increasing the amount of permanent charges inside the polymer, electrostatic interactions between the positively charged PEGA(+) and the negatively charged PGA (pI = 5.2-5.4) were strengthened, thus favouring the accessibility of the bulky enzyme (MW = 88 kDa) inside the pores. The effect of different amounts of charges on polymer swelling and protein retention inside the polymer was investigated and correlated to the enzyme efficiency demonstrating that electrostatic interactions predominate over swelling properties in determining enzyme accessibility.

Synthesis of N-linked glycopeptides on solid support and their evaluation as protease substrates.

A range of glycopeptides containing protease cleavage sites were synthesized on solid support using Fmoc-based solid phase glycopeptide synthesis. The immobilized peptides were studied as substrates for the proteases chymotrypsin and thermolysin. For chymotrypsin, N-glycosylation of an Asn residue at the P(2) site appears to reduce hydrolysis whereas glycosylation of the P(1) site does not appear to affect peptide hydrolysis by thermolysin.

Synthesis and modifications of carbohydrates, using biotransformations.

Enzymes continue to be used as important catalysts, for the generation of rare and 'unnatural' monosaccharides and for the selective formation of glycosidic linkages. Multi-enzyme systems have been employed in one-pot strategies for multistep reaction sequences and for co-factor regeneration. The efficiency of glycosidases for glycosylation reactions has been dramatically increased by active-site mutagenesis to generate glycosynthases. First reports have detailed the expansion and optimization of glycosynthase substrate specificity by directed evolution. Novel glycosyltransferases are being identified from genomic databases and have been shown to glycosylate complex metabolites, such as glycopeptide antibiotics, with exquisite selectivity and in good yields. An emerging field is the application of glycosynthases and glycosyltransferases to reactions on solid support, generating potential applications in microarrays.