A comparative cell-based high throughput screening strategy for the discovery of selective tyrosine kinase inhibitors with anticancer activity.

Growth factor receptor tyrosine kinases (RTK) have been implicated in tumor growth, metastasis and angiogenesis, and are thus considered promising targets for therapeutic intervention in malignant diseases. We present a novel drug discovery strategy to find inhibitors of RTKs based on comparative screening of compound libraries employing functional cellular assays. Cell lines stably expressing HER2 and the receptors for hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), insulin-like growth factor-I (IGF-I) and epidermal growth factor (EGF) have been established. All cell lines are based on FDC-P1, a murine myeloid progenitor cell line which allows a direct comparison of results obtained in primary screens. In addition, the same cell lines are suitable for compound optimization and for animal studies. Using this strategy we report the identification of promising lead candidates for further drug development which are highly selective, non-cytotoxic and cell permeable with potencies in the low micromolar range.

Effective inhibitors of hemagglutination by influenza virus synthesized from polymers having active ester groups. Insight into mechanism of inhibition.

Highly effective sialic acid-containing inhibitors of influenza virus X-31 were synthesized using poly[N-(acryoyloxy)succinimide] (pNAS), a polymer preactivated by incorporation of active ester groups. Polymers containing two and three different components were prepared by sequential reaction of pNAS with two and three amines, respectively. This preparation of co- and terpolymers was synthetically more efficient than methods involving copolymerization of different monomers and gave polymers that were more easily compared than those generated by copolymerization. Polymers in this study (prepared from a single batch of pNAS) had a constant degree of polymerization (DP approximately 2000) and probably had a distribution of components that was more random than analogous polymers prepared by copolymerization. Use of C-glycosides of sialic acid made it possible to investigate inhibition by different polymers at temperatures ranging from 4 to 36 degrees C without artifacts due to the hydrolytic action of neuraminidase. The inhibitors were, in general, more effective at 36 degrees C than at 4 degrees C. The hemagglutination (HAI) assay was used to measure the value of the inhibition constant KHAIi each polymer. The value of KHAIi for the two-component polymer containing 20% sialic acid on a polyacrylamide backbone at 4 degrees C was 4 nM (in terms of the sialic acid moieties present in solution) and was approximately 50-fold more effective than the best inhibitors previously described and 25-fold more effective than the best naturally occurring inhibitor. The most effective inhibitor synthesized in this work contained 10% benzyl amine and 20% sialic acid on a polyacrylamide backbone, and its value of KHAIi was 600 pM at 36 degrees C. Approximately 100 polymers that differed in one or two components were assayed to distinguish between two limiting mechanisms for inhibition of the interaction between the surfaces of virus and erythrocytes: high-affinity binding through polyvalency, and steric stabilization. The results suggest that both mechanisms play an important role. The system comprising polyvalent inhibitors of agglutination of erythrocytes by influenza provides a system that may be useful as a model for inhibitors of other pathogen-host interactions, a large number of which are themselves polyvalent.

Influence of the phenylalanine dose on the aromatic acid excretion in phenylketonuric heterozygotes and controls after oral loading.

Heterozygotes for phenylketonuria and controls were given oral loads of 100 mg and 200 mg L-phenylalanine per kilogram body weight. The concentrations of urinary aromatic acids were determined by gas-chromatography after isolation by ion-exchange chromatography and ethylacetate extraction. On an intake of 100 mg L-phenylalanine per kilogram, controls and carriers of classical phenylketonuria excreted nearly the same amounts of aromatic acids (P greater than 0.05). However on an intake of 200 mg per kilogram L-phenylalanine they could be distinguished from one another (P less than 0.001).