Exemplification of the challenges associated with utilising fluorescence intensity based assays in discovery

Abstract

Importance of the field: Despite the advances in the understanding of biological processes, significant challenges still face those engaged in small molecule drug discovery. To complicate matters further, researchers are often overwhelmed with a range of off-the-shelf as well as bespoke assay formats to choose from when initiating a drug discovery programme. Although fluorescence intensity based assays have traditionally been adopted in drug discovery programmes for a wide range of target classes, it is essential to fully validate the chosen readouts to confirm that they accurately reflect the underlying biological mechanism under investigation.

Areas covered in this review: This review exemplifies the challenges that are often encountered with fluorescence intensity based assays and particular attention is paid to compound interference, the protease, deacetylating enzyme and kinase enzyme target classes.

What the reader will gain: Designing a critical path in early stage drug discovery, which combines several diverse and minimally overlapping readout modes, will maximise the chance that compound activities will translate between the primary assay (utilised in the initial screening campaign) and secondary assay (utilised to evaluate the confirmed hits identified in the primary assay, usually a cell based assay) formats in a meaningful way. However, this is not always the case as is amply demonstrated across both academia and the pharmaceutical industry. Paying insufficient attention to these points can lead to the early termination of drug discovery programmes, not for want of resources or confidence in the rationale underlying the target, but instead because decision making has been driven by assay data originating from a different biological mechanism than the one under investigation.

Take home message: Although fluorescence intensity based assays are likely to remain popular for many target classes in drug discovery, in particular in small molecule screening campaigns, it is essential that at the outset they are sufficiently well validated so that compounds are likely to exhibit profiles that are confirmed in subsequent assays.

Keywords::

• compound interference
• deacetylating enzymes
• drug discovery
• fluorescence intensity
• furin
• high-throughput screening
• kinase
• PCSK9
• protease

Notes

This box summarises key points contained in the article.