Group assay development

Many groups have developed immunoassays for sulfonamides. These compounds were a problem in the late 1980s when they were commonly found in the milk supplies in the USA. Subsequently, a great deal of effort has been required to develop rapid, reliable assays for the large number of chemical variations of sulfonamides. 

In most cases, in consultation with the HTS group, the research area laboratory will develop a benchtop assay that is at least compatible with the HTS format of choice for their target. This tends to facilitate project transitions and provides a tool that the research area laboratory will use later to follow up hits and develop SAR. In other cases, the HTS assay development group will assume all responsibility for assay development. The formality of the transfer of the project from the research area to the HTS group varies between organizations, but the outcomes are quite similar. All of the details of the prototype assay are reviewed by both teams, and, where applicable, reagents, protocols, and even plates or pipette tips are exchanged. 

Kinases are enzymes that place a phosphate group on a serine/threonine or a tyrosine residue of a protein or peptide. All kinase reactions use ATP as the phosphate source. Therefore there have been assays developed that monitor the loss or gain of the peptide/protein substrate (LANCE, ULight) [23], the loss of ATP (easylite luminescence kinaseGlo, Perkin Elmer) [20], or the gain of ADP (Tran-screener TR-FRET) [24]. Many of these formats are applicable to cell based assays. 

The active 2A and 3C proteases from different HRV serotypes have been overproduced in bacterial cells and purified by several groups [18,28-42], The availability of large quantities of the recombinant viral proteases has greatly facilitated their biochemical characterization, assay development, and efforts to identify specific inhibitors against these enzymes. We have worked on the purification and assay development for the two proteases from HRV serotype-14 (HRV 14) as part of our efforts in antiviral development. The experimental procedures described below may provide a general approach toward the purification and development of high-throughput assays for the 2A and 3C proteases of other HRV serotypes as well as other members of the picomaviral family. 

We thank our coworkers in Molecular Modeling/Cheminformatics at Roche, Basel, for scientifically stimulating discussions and valuable contributions. The fruitful collaboration with colleagues from the Lead Generation, Medicinal Chemistry, Roche Compound Depository, Assay Development HTS departments and the Molecular Properties Group is gratefully acknowledged. 

In the classic two-hybrid system, protein interaction is assayed in the nucleus, a suboptimal environment for the detection of many protein-protein interactions— particularly those involving membrane proteins. As an alternative to nuclear two-hybrid assays, several groups have developed membrane-associated... 

Herg blockade is mesmerising the pharmaceutical industry, not just due to the implications for drug discovery, but also because the difficulty is understanding the interplay between displacement of dofetilide, patch clamp assays, dog telemetry and the occurrence of toursade des pointes in humans. Several groups have developed... 

Ultimately it may be cost effective for a company to subcontract assay development to a clinical division in-house or to a third party. However, if this is done before the parent company has the in-house expertise to monitor assay development, this can be a very dangerous and expensive process. Often the expertise on the chemistry of the compound class is not transferred and inferior assays are developed at great expense. If it is necessary to develop early assays outside the company, it is important that the assay development is approached as a collaborative project with a group possessing an established record in the development of assays for environmental samples. Involvement of scientists from the clinical field can be very useful since they have decades of accumulated knowledge on assay formatting and development. However, the involvement of scientists with an appreciation of matrix effects, metabolism, and the regulatory questions posed is critical. 

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