Screening library evolution through automation of solution preparation.

The quality of the compound library is a critical success factor in every high-throughput screening campaign. Screening solutions have to be prepared with a high level of process control to ensure the correct identity and initial concentration of each compound. However, even under optimized storage conditions, a certain level of degradation in solution cannot be avoided. Therefore, regular quality control and eventual removal of solutions from the screening deck is necessary. Because solution preparation, especially the weighing of compounds, is a tedious and often manual task, a regular resolubilization of compounds is difficult to achieve. By complete automation of the solution preparation, the authors have laid the foundation for a life cycle management of screening solutions. They demonstrate how a combination of quality and process control leads to a continuous improvement of the screening library. In presenting an automation concept, they show how a series of innovative process optimizations led to a high-performance system that achieves full industrialization of solution preparation.

SpeedScreen: label-free liquid chromatography-mass spectrometry-based high-throughput screening for the discovery of orphan protein ligands.

A high-throughput screening methodology tailored to the discovery of ligands for known and orphan proteins is presented. With this method, labeling of neither target protein nor screened compounds is required, as the ligands are affinity selected by incubation of the protein with mixtures of compounds in aqueous binding buffer. Unbound small-molecular-weight compounds are removed from the target protein:ligand complex by rapid size-exclusion chromatography in the 96-well format. The protein fraction is analyzed subsequently by liquid chromatography-mass spectrometry for detection and identification of the bound ligand. This screening method was validated with known protein:ligand model systems and optimized for selection of high-affinity binders in an industrial screening environment. All sample handling steps and the analytics are rapid, robust, and largely automated, adopting this technology to the needs of present high-throughput screening processes. This affinity-selection technology, termed SpeedScreen, is currently an integral part of our lead discovery process.

Infection of leaves of Arabidopsis thaliana by Botrytis cinerea: changes in ascorbic acid, free radicals and lipid peroxidation products.

Infection of leaves of Arabidopsis thaliana with conidial suspensions of the necrotrophic pathogen Botrytis cinerea resulted in a large decrease in the level of ascorbic acid and increases in intensity of a single-peak free radical and Fe(III) (g=4.27) signals in electron paramagnetic resonance (EPR) spectra. These changes were not confined to the spreading lesions or associated areas of chlorosis, but extended to other apparently healthy tissues in the infected leaves. They are, therefore, consistent with the existence of high levels of oxidative stress being generated as a result of the infection process. The expected accompanying increases in levels of the aldehydic products of lipid peroxidation, malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE), were not observed, and in the case of MDA the levels in tissue from infected plants were appreciably lower than in the healthy controls. These last findings are surprising and demonstrate a difference in the response of A. thaliana to infection with B. cinerea compared with tissues from other plant families studied previously.

Botrytis cinerea induces the formation of free radicals in fruits of Capsicum annuum at positions remote from the site of infection.

Free radical adducts of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone have been observed by electron paramagnetic resonance spectroscopy in detached fruits of Capsicum annuum investigated 5 days after infection with Botrytis cinerea. The spectra of these adducts were at a maximum within the soft rot lesion, but they could also be detected at distances up to 50 mm from the edge of the lesion in samples following main vascular bundles. At distances greater than 40 mm, the spectrum of the ascorbate radical was also seen, and at greater distances from the lesion it was the only radical detected. With samples taken from parenchyma tissue adjacent to the vascular bundles there was little adduct formation and the ascorbate radical could be detected, albeit with reduced intensity compared to healthy tissue, at distances as small as 10 mm from the edge of the lesion. This observation of chemical changes at considerable distances from the infected tissue is in contrast to previous observations on the behaviour of other markers of oxidative stress (e.g., 4-hydroxynonenal, malondialdehyde, single-peak free radical, and Fe(III) (g = 4.27) electron paramagnetic resonance signals), where their levels decreased rapidly outside of the soft rot.

Botrydial is produced in plant tissues infected by Botrytis cinerea.

The fungal metabolite botrydial was detected for the first time in ripe fruits of sweet pepper (Capsicum annuum) wound-inoculated with conidial suspensions of Botrytis cinerea and also in leaves of Phaseolus vulgaris and Arabidopsis thaliana inoculated without wounding. This phytotoxin was produced in soft rot regions of the infection. In C. annuum, the most aggressive isolate produced the highest botrydial concentrations in planta. The levels of botrydial produced by this isolate did not correlate with the reported relative susceptibilities of four P. vulgaris genotypes. The results suggest that botrydial is a pathogenicity factor for this fungus, but not a primary determinant of pathogenicity.

Markers for oxidative stress associated with soft rots in French beans (Phaseolus vulgaris) infected by Botrytis cinerea.

The role of active oxygen species has been studied in spreading soft-rot lesions caused by the necrotrophic fungal pathogen Botrytis cinerea Pers.:Fr. in leaves of four genotypes of French bean (Phaseolus vulgaris L.). Large increases were observed for the aldehydic end-products of oxidative damage, malondialdehyde and 4-hydroxy-2-nonenal, as a result of infection in each of the genotypes studied. Similar increases were found in a stable free radical and g=4.27 Fe(III) signals, but not Mn(II) signals, in electron paramagnetic resonance spectra. These changes were accompanied by large decreases in ascorbic acid levels, with changes in the antioxidant glutathione being genotype dependent.

Lipid peroxidation and the oxidative burst associated with infection of capsicum annuum by botrytis cinerea

A combination of electron paramagnetic resonance (EPR) spectroscopy and analytical chemistry has been used to study the changes in free radical content, transition metal ion status and lipid peroxidation following inoculation of fruits of sweet pepper (Capsicum annuum) with Botrytis cinerea. EPR detected a high concentration of an unidentified free radical associated with the spreading lesion that extends into the surrounding, healthy tissues. In addition, the EPR-detectable iron(III) was highest at the centre of the lesion, again displaying a gradient out into the surrounding tissues. Analyses for aldehydic products of lipid peroxidation were performed to assess the accumulation and potential of these compounds to contribute to the cell death associated with necrotrophic pathogens. In contrast to the spectrum of aldehydes typically observed within peroxidized biological samples, no accumulation of malondialdehyde nor n-hexanal was observed. Instead, high levels of two hydroxyalkenals (4-hydroxy-2-hexenal and 4-hydroxy-2-nonenal) were detected at concentra- tions up to 4000 and 20 000 pmol g- 1, respectively, at the host-pathogen interface. These results are discussed in terms of the likely mechanisms of formation of these aldehydes.

Quantitation of hyaluronidases by the Morgan-Elson reaction: comparison of the enzyme activities in the plasma of tumor patients and healthy volunteers.

The Morgan-Elson reaction, a method for the determination of hyaluronidase activity, was optimized for the quantitation of the enzyme in biological material. Based on HPLC and spectrometric (UV-Vis, LC-MS) studies, the structure of the red-colored product (mesomeric forms of N3-protonated 3-acetylimino-2-(4-dimethylaminophenyl)methylidene-5-(1,2-++ +dihydroxyethyl)furane) formed by condensation of chromogen III with p-dimethylaminobenzaldehyde is proposed. Activities corresponding to > or = 0.1 IU of endogenous and therapeutically administered hyaluronidase can be detected in 50 microl samples. Application of the method for the determination of the enzyme in plasma of tumor patients revealed no difference in activity levels, interindividual variability and pH profile compared to healthy volunteers.

Pharmacokinetics and tissue distribution of bovine testicular hyaluronidase and vinblastine in mice: an attempt to optimize the mode of adjuvant hyaluronidase administration in cancer chemotherapy.

The influence of the route of administration (i.v., i.p. and s.c.) on pharmacokinetics and tissue distribution of bovine testicular hyaluronidase and vinblastine was studied in mice (plasma, skeletal muscle, liver, kidney and human melanoma). After i.v. injection, hyaluronidase was accumulated in liver and kidney, whereas i.p. and s.c. administration led to almost equal distribution in plasma, muscle, liver and kidney. In melanoma, the highest levels of hyaluronidase were found after s.c. injection of the enzyme close to the tumor. Hyaluronidase s.c. increased the intratumoral concentration of s.c. co-administered vinblastine most efficiently, making local simultaneous application as in interstitial chemotherapy most promising.

Hyaluronidase pretreatment produces selective melphalan enrichment in malignant melanoma implanted in nude mice.

Preclinical and clinical observations suggest that the administration of hyaluronidase (Hyase) shortly before that of chemotherapy increases the access and, thus, the effectiveness of anticancer drugs in tumors. To examine this hypotheses as well as the selectivity of such a therapeutic approach potentially beneficial in isolated limb perfusion, the Hyase-induced distribution of melphalan was measured in tumor-bearing nude mice with respect to the mode of drug administration using RP-18 ion-pair high-performance liquid chromatography (HPLC) with fluorimetric detection. Melphalan alone (50 micromol/kg) or a combination of melphalan (50 micro mol/kg) and Hyase (100,000 IU/kg) was injected either i.p. or s.c. in the vicinity of the tumors. The s.c. melphalan injection caused a 4-fold rise in melphalan concentration (59 microM) in the tumors as compared with i.p. application (15 microM). Only minor effects were observed with respect to the route of melphalan application on its distribution in other tissues (ca. 13 microM in plasma, 15 microM in muscle, 30 microM in the liver, 26 microM in the kidney, and 21 microM in the testicle). Irrespective of the route of Hyase coadministration, the enzyme increased the concentration of i.p. injected melphalan in all tissues to ca. 20 microM in the tumor, 15 microM in plasma, 27 microM in muscle, 40 microM in the liver, 29 microM in the kidney, and 28 microM in the testicle. In contrast, s.c. injected melphalan was selectively accumulated by the tumors after both s.c. and i.p. Hyase administration (462 and 388 microM, respectively). Melphalan enrichment in the tumors was higher (16- to 32-fold higher than in the other tissues) after i.p. administration of Hyase since, in contrast to s.c. injection of the enzyme, its i.p. administration caused a decrease in the concentration of the cytostatic in all other tissues as compared with the s.c. administration of melphalan alone.