A broad activity screen in support of a chemogenomic map for kinase signalling research and drug discovery.

Despite the development of a number of efficacious kinase inhibitors, the strategies for rational design of these compounds have been limited by target promiscuity. In an effort to better understand the nature of kinase inhibition across the kinome, especially as it relates to off-target effects, we screened a well-defined collection of kinase inhibitors using biochemical assays for inhibitory activity against 234 active human kinases and kinase complexes, representing all branches of the kinome tree. For our study we employed 158 small molecules initially identified in the literature as potent and specific inhibitors of kinases important as therapeutic targets and/or signal transduction regulators. Hierarchical clustering of these benchmark kinase inhibitors on the basis of their kinome activity profiles illustrates how they relate to chemical structure similarities and provides new insights into inhibitor specificity and potential applications for probing new targets. Using this broad dataset, we provide a framework for assessing polypharmacology. We not only discover likely off-target inhibitor activities and recommend specific inhibitors for existing targets, but also identify potential new uses for known small molecules.

In vitro high-throughput screening assay for modulators of transcription.

We developed a 96-well microtiter-plate high-throughput screening (HTS) assay for the detection of modulators of transcription. This HTS assay consists of three steps: (1) the in vitro transcription reaction; (2) modification and hybridization of RNA products; and (3) washing and quantification. During the first step, a DNA template containing the promoter of interest upstream of a cassette lacking guanosine residues in one of its strands (G-less cassette) is incubated with nuclear extract and the necessary cofactors/activators and substrates. During the second step, the in vitro synthesized transcripts are digested with RNase T1 and hybridized to two DNA oligonucleotides. One oligonucleotide is biotinylated for trapping of the RNA products to a streptavidin-coated plate, and the other is europium-labeled for detection by time-resolved fluorescence. We show that this assay is highly reproducible and robust, yielding results comparable to those obtained by standard methodologies employing radioactive nucleotide incorporation and gel electrophoresis while offering a very significant advantage in terms of throughput (>2000 assay points per operator per day). We demonstrate the usefulness of the assay for the discovery of small molecule inhibitors of transcription, and applications of this approach for the high-throughput discovery of transcriptional modulators are discussed.

Human papillomavirus type 6: classification of clinical isolates and functional analysis of E2 proteins.

Human papillomaviruses (HPVs) cause a variety of clinical manifestations, including the most prevalent viral sexually transmitted disease, genital warts. HPV-6 is found in a greater number of genital warts than any other HPV. To increase our understanding of the structural and functional relationships between HPV-6 isolates and to provide information for epidemiological studies, the sequences of the E2, E6 and E7 coding regions of HPV-6 genomes in clinical samples were determined. This sequence analysis was performed on isolates originally designated HPV-6a on the basis of analysis of patterns generated by restriction enzyme digestion. It was found that the designation of subtype on the basis of restriction enzyme digestion correlated poorly with the designation of subtype on the basis of sequence comparison; in fact, the clinical isolates were clearly categorized into HPV-6a and HPV-6b groups, with the previously described HPV-6vc being a member of the HPV-6a group. It was also found that the HPV-6a E2 protein is a much less potent activator of transcription than the HPV-16 E2 protein, generalizing our previous results with the HPV-6b E2 protein to this second HPV-6 E2 protein. These studies indicate that the amino acid differences observed between these natural variants of the HPV-6 E2 protein do not affect its function.