Discovery of novel cathepsin S inhibitors by pharmacophore-based virtual high-throughput screening

The cysteine protease cathepsin S (CatS) is involved in the pathogenesis of autoimmune disorders, atherosclerosis, and obesity. Therefore, it represents a promising pharmacological target for drug development. We generated ligand-based and structure-based pharmacophore models for noncovalent and covalent CatS inhibitors to perform virtual high-throughput screening of chemical databases in order to discover novel scaffolds for CatS inhibitors. An in vitro evaluation of the resulting 15 structures revealed seven CatS inhibitors with kinetic constants in the low micromolar range. These compounds can be subjected to further chemical modifications to obtain drugs for the treatment of autoimmune disorders and atherosclerosis.

Experimental validation of the performance of a microreactor for the high-throughput screening of catalytic coatings

In this paper, the results of computational fluid dynamics simulations of flow, temperature, and concentration distributions used in the design of a microreactor for the high-throughput screening of catalytic coatings (Mies et al., Chem. Eng. J. 2004, 101, 225) are compared with experimental data, and good agreement is obtained in all cases. The experimental results on flow distribution were obtained from laser Doppler anemometry measurements in the range of Reynolds numbers from 6 to 113. The measured flow nonuniformity in the separate reactor compartments was below 2%. The temperature distribution was obtained from thermocouple measurements. The temperature nonuniformity between the reactor compartments was below 3 K at a maximum heat production rate of 1.3 W in ethylene oxidation at 425 degrees C over CuO/Al2O3/Al coatings. With respect to concentration gradients, a deviation from the average rate of reaction of only 2.3% was obtained at realistic process conditions in the ethylene ammoxidation process over identical Co-ZSM-5 coatings in all reactor compartments. The cross talking noise between separate compartments does not exceed 0.1% when the reactor parts have a smooth surface finish. This illustrates the importance of ultraprecision machining of surfaces in microtechnology, when interfaces cannot be avoided.

Synthesis and SAR of novel, 4-(phenylsulfamoyl)phenylacetamide mGlu(4) positive allosteric modulators (PAMs) identified by functional high-throughput screening (HTS)

Herein we disclose the synthesis and SAR of a series of 4-(phenylsulfamoyl)phenylacetamide compounds as mGlu(4) positive allosteric modulators (PAMs) that were identified via a functional HTS. An iterative parallel approach to these compounds culminated in the discovery of VU0364439 (11) which represents the most potent (19.8 nM) mGlu(4) PAM reported to date. (C) 2010 Elsevier Ltd. All rights reserved.

Molecular diagnosis for heterogeneous genetic diseases with high-throughput DNA sequencing applied to retinitis pigmentosa

BACKGROUND:
The genetic heterogeneity of many Mendelian disorders, such as retinitis pigmentosa which results from mutations in over 40 genes, is a major obstacle to obtaining a molecular diagnosis in clinical practice. Targeted high-throughput DNA sequencing offers a potential solution and was used to develop a molecular diagnostic screen for patients with retinitis pigmentosa.
METHODS:
A custom sequence capture array was designed to target the coding regions of all known retinitis pigmentosa genes and used to enrich these sequences from DNA samples of five patients. Enriched DNA was subjected to high-throughput sequencing singly or in pools, and sequence variants were identified by alignment of up to 10 million reads per sample to the normal reference sequence. Potential pathogenicity was assessed by functional predictions and frequency in controls.
RESULTS AND CONCLUSIONS:
Known homozygous PDE6B and compound heterozygous CRB1 mutations were detected in two patients. A novel homozygous missense mutation (c.2957A?T; p.N986I) in the cyclic nucleotide gated channel ß1 (CNGB1) gene predicted to have a deleterious effect and absent in 720 control chromosomes was detected in one case in which conventional genetic screening had failed to detect mutations. The detection of known and novel retinitis pigmentosa mutations in this study establishes high-throughput DNA sequencing with DNA pooling as an effective diagnostic tool for heterogeneous genetic diseases.

Development of a high-throughput enzyme-linked immunosorbent assay for the routine detection of the carcinogen acrylamide in food, via rapid derivatisation pre-analysis

The spontaneous formation of the neurotoxic carcinogen acrylamide in a wide range of cooked foods has recently been discovered. These foods include bread and other bakery products, crisps, chips, breakfast cereals, and coffee. To date, the diminutive size of acrylamide (71.08Da) has prevented the development of screening immunoassays for this chemical. In this study, a polyclonal antibody capable of binding the carcinogen was produced by the synthesis of an immunogen comprising acrylamide derivatised with 3-mercaptobenzoic acid (3-MBA), and its conjugation to the carrier protein bovine thyroglobulin. Antiserum from the immunised rabbit was harvested and fully characterised. it displayed no binding affinity for acrylamide or 3-MBA but had a high affinity for 3-MBA-derivitised acrylamide. The antisera produced was utilised in the development of an ELISA based detection system for acrylamide. Spiked water samples were assayed for acrylamide content using a previously published extraction method validated for coffee, crispbread, potato, milk chocolate and potato crisp matrices. Extracted acrylamide was then subjected to a rapid 1-h derivatisation with 3-MBA, pre-analysis. The ELISA was shown to have a high specificity for acrylamide, with a limit of detection in water samples of 65.7 mu g kg(-1), i.e. potentially suitable for acrylamide detection in a wide range of food commodities. Future development of this assay will increase sensitivity further. This is the first report of an immunoassay capable of detecting the carcinogen, as its small size has necessitated current analytical detection via expensive, slower, physico-chemical techniques such as Gas or Liquid Chromatography coupled to Mass Spectrometry. (c) 2007 Elsevier B.V. All rights reserved.