Pertti Hurskainen

Homogeneous Time-Resolved Fluorescence Quenching Assay (LANCE) for Caspase-3

In addition to kinases and G protein—coupled receptors, proteases are one of the main targets in modern drug discovery. Caspases and viral proteases, for instance, are potential targets for new drugs. To satisfy the current need for fast and sensitive high-throughput screening for inhibitors, new homogeneous protease assays are needed. We used a caspase-3 assay as a model to develop a homogeneous time-resolved fluorescence quenching assay technology. The assay utilizes a peptide labeled with both a luminescent europium chelate and a quencher. Cleavage of the peptide by caspase-3 separates the quencher from the chelate and thus recovers europium fluorescence. The sensitivity of the assay was 1 pg/μl for active caspase-3 and 200 pM for the substrate. We evaluated the assay for high-throughput usage by screening 9600 small-molecule compounds. We also evaluated this format for absorption/distribution/metabolism/excretion assays with cell lysates. Additionally, the assay was compared to a commercial fluorescence caspase-3 assay.

Novel detection strategies for drug discovery.

The Human Genome Project is expected to increase the number of potential drug targets from the current figure of 500 to approximately 3,000-4,000. This increased number of targets, and increasing knowledge of signaling-pathway networks and their complexities, sets new demands for efficiency on HTS assay technologies. Assessment of the total efficacy of a given drug candidate requires not only the classical assays, but also a wide variety of assays related to signaling cascades and cellular functions. Discrete functional assays traditionally involved Ca(2+) flux, kinases and cAMP, but today extend to the whole signaling network, from ligand binding to expression. This review discusses emerging novel non-radioisotopic assays, such as ligand-stimulated GTP-binding, the inositol triphosphate assay, cellular receptor trafficking, and protein-protein interactions.

Nonradioactive GTP binding assay to monitor activation of g protein-coupled receptors.

GPCRs represent important targets for drug discovery because GPCRs participate in a wide range of cellular signaling pathways that play a role in a variety of pathological conditions. A large number of screening assays have been developed in HTS laboratories for the identification of hits or lead compounds acting on GPCRs. One type of assay that has found relatively widespread application, due to its at least in part generic nature, relies on the use of a radioactive GTP analogue, [(35)S]GTPgammaS. The G-protein alpha subunit is an essential part of the interaction between receptor and G proteins in transmembrane signaling, where the activated receptor catalyzes the release of GDP from Galpha, thereby enabling the subsequent binding of GTP or a GTP analogue. [(35)S]GTPgammaS allows the extent of this interaction to be followed quantitatively by determining the amount of radioactivity associated with cell membranes. However, with the increased desire to move assays to nonradioactive formats, there is a considerable need to develop a nonradioactive GTP binding assay to monitor ligand-induced changes in GPCR activity. The Eu-GTP binding assay described here is based on TRF that exploits the unique fluorescence properties of lanthanide chelates, and provides a powerful alternative to assays using radioisotopes. In this article, we have used the human alpha(2A)-AR as a model GPCR system to evaluate the usefulness of this Eu-GTP binding assay.

The use of europium (Eu3+) labelled primers in PCR amplification of specific target DNA

The polymerase chain reaction (PCR) has many potential applications in the field of DNA probe diagnostics. Here we describe a method that utilizes PCR and time-resolved fluorometry (TRF) for the detection of specific target DNA. First the DNA segment to be detected is amplified according to standard procedures. Then a pair of europium (Eu3+) and biotin-labelled primers nested within the amplified fragment is incorporated in a few additional PCR cycles. Thus amplified DNA fragments are generated that contain an affinity label (biotin) and a detectable label (europium). The doubly-labelled amplified DNA fragments are collected onto streptavidin coated microtitration strips and the bound Eu3+ is measured in a time-resolved fluorometer. We show here the application of this method to the detection of HIV-1 DNA. As few as five copies of HIV-1 DNA could readily be detected using this assay. The method described here is sensitive, rapid and easy to employ. In addition it lends itself to automation.

Derivatization of 11-α-Hydroxyprogesterone using Phosphoramidite Chemistry

The well-known phosphoramidite chemistry routinely used in solid-phase oligonucleotide synthesis is exploited here in the preparation of steroid conjugates in solution. The applicability of the method is tested by conjugating one nucleosidic and one non-nucleosidic phosphoramidite building block to 11-alpha-hydroxyprogesterone. The suitability of one of the conjugates synthesized (5) for a competitive binding assay is also demonstrated.