Publications by authors named "David G Tew"

3 Publications

  • Page 1 of 1

Development of an insect-cell-based assay for detection of kinase inhibition using NF-kappaB-inducing kinase as a paradigm.

Biochem J 2009 Apr;419(1):65-73

Biological Reagents & Assay Development, GlaxoSmithKline R&D, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW, UK.

Identification of small-molecule inhibitors by high-throughput screening necessitates the development of robust, reproducible and cost-effective assays. The assay approach adopted may utilize isolated proteins or whole cells containing the target of interest. To enable protein-based assays, the baculovirus expression system is commonly used for generation and isolation of recombinant proteins. We have applied the baculovirus system into a cell-based assay format using NIK [NF-kappaB (nuclear factor kappaB)-inducing kinase] as a paradigm. We illustrate the use of the insect-cell-based assay in monitoring the activity of NIK against its physiological downstream substrate IkappaB (inhibitor of NF-kappaB) kinase-1. The assay was robust, yielding a signal/background ratio of 2:1 and an average Z' value of >0.65 when used to screen a focused compound set. Using secondary assays to validate a selection of the hits, we identified a compound that (i) was non-cytotoxic, (ii) interacted directly with NIK, and (iii) inhibited lymphotoxin-induced NF-kappaB p52 translocation to the nucleus. The insect cell assay represents a novel approach to monitoring kinase inhibition, with major advantages over other cell-based systems including ease of use, amenability to scale-up, protein expression levels and the flexibility to express a number of proteins by infecting with numerous baculoviruses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BJ20081646DOI Listing
April 2009

Highly potent inhibitors of methionine aminopeptidase-2 based on a 1,2,4-triazole pharmacophore.

J Med Chem 2007 Aug 18;50(16):3777-85. Epub 2007 Jul 18.

Department of Medicinal Chemistry, Enzymology, Oncology, and Structural Biology, GlaxoSmithkline, King of Prussia, PA 19406, USA.

High-throughput screening for inhibitors of the human metalloprotease, methionine aminopeptidase-2 (MetAP2), identified a potent class of 3-anilino-5-benzylthio-1,2,4-triazole compounds. Efficient array and interative synthesis of triazoles led to rapid SAR development around the aniline, benzylthio, and triazole moeities. Evaluation of these analogs in a human MetAP2 enzyme assay led to the identification of several inhibitors with potencies in the 50-100 picomolar range. The deleterious effects on inhibitor potency by methylation of the anilino-triazole nitrogens, as well as the X-ray crystal structure of triazole 102 bound in the active site of MetAP2, confirm the key interactions between the triazole nitrogens, the active site cobalt atoms, and the His-231 side-chain. The structure has also provided a rationale for interpreting SAR within the triazole series. Key aniline (2-isopropylphenyl) and sulfur substituents (furanylmethyl) identified in the SAR studies led to the identification of potent inhibitors (103 and 104) of endothelial cell proliferation. Triazoles 103 and 104 also exhibited dose-dependent activity in an aortic ring tissue model of angiogenesis highlighting the potential utility of MetAP2 inhibitors as anticancer agents.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jm061182wDOI Listing
August 2007

Determination of phosphate in nanomolar range by an enzyme-coupling fluorescent method.

Anal Biochem 2003 Sep;320(2):292-8

GlaxoSmithKline Pharmaceuticals, Centro de Investigación Básica, Santiago Grisolía 4, Tres Cantos 28760, Madrid, Spain.

We have successfully configured a new ultrasensitive fluorescent phosphate assay that detects free phosphate in solution through the formation of the fluorescent product resorufin. The phosphate assay relies on coupling phosphate generation to purine nucleoside phosphorylase, xanthine oxidase, and horseradish peroxidase. The response is excellent in the nanomolar range, being linear between 50 nM and 5 microM phosphate. This method is more sensitive (more than 10-fold) than other reported methods and is amenable to miniaturization. In particular, we have demonstrated the utility of this new method in a format suitable for ultra-high-throughput screening.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/s0003-2697(03)00400-7DOI Listing
September 2003