Optimising Phenotypic Screening
Optimising Phenotypic Screening - Single-cell analysis versus 3D multicell analysis

Optimising Phenotypic Screening - Single-cell analysis versus 3D multicell analysis

By Dr Richard M. Eglen

In light of the high failure rate of compounds when they are subjected to clinical testing, we are seeing a renaissance in phenotypic screening in drug discovery. However, most phenotypic screening is based on the use of cellular assays and here we debate the advantages and disadvantages of single-cell versus 3D multi-cell analyses.

The phenotypic screening of novel drug candidates determines whether a small molecule (or biologic) exerts the desired pharmacology, either in vitro (isolated cells, organoids, tissues) or in vivo. This functional approach is ‘unbiased’ given that the molecular target, and therefore molecular mechanism of action (MMOA), is only determined following lead identification and preclinical optimisation. By contrast to phenotypic screening, target-based screening commences with a defined MMOA implicated in a specific disease pathology and utilises discrete compound libraries designed against this presumed molecular target. In this respect, target-based screening is a ‘biased’ approach. In general, phenotypic screening can identify ‘first-in-class’ compounds against novel targets, while target-based screening is optimal to identify ‘best-in-class’ compounds.

Despite the historical success of phenotypic screening techniques, target-based screening (often directly measuring the biochemical affinity between chemical compound and the biological target) has predominated compound screening campaigns. This is due to the efficiency of highly-automated and ultra-high throughput biochemical assay systems. By contrast, phenotypic approaches are comparatively inefficient given the high costs per sample and the low throughput assays frequently employed. Consequently, the approach is usually incompatible with the screening of large compound libraries. This is perhaps ironic, given that unbiased screening (eg phenotypic screening) should arguably be conducted against the broadest compound chemical library possible, with the goal of gaining the broadest range of lead chemical structures.

A common feature of both target-based and phenotypic-based screening assays is the extensive use of human primary cells, often derived from the patient with the disease under investigation. Such cells, which can provide robust understanding of disease pathologies, are optimal for drug discovery, as they express the targets, disease-causing mutations and signal pathways involved in the disease pathology. The widespread availability of such cells has contributed to a renaissance in phenotypic screening, facilitated by the rapid development of robust, high throughput, high content cellular imaging systems. This is particularly relevant in assays directed at difficult drug targets as well as in diseases for which the molecular mechanisms are unknown. Furthermore, rapid technological advances in signal detection and microfluidic handling systems now enable phenotypic assays to be conducted using human cells in two mutually exclusive formats. The first is single-cell analysis, in which several analytical studies can be directed at a single-cell (genomic, proteomic, etc). The second is in multicellular populations, where aggregate responses are measured. In this format, the cells are cultured in three-dimensional assemblies (spheroid, organoids, tissues, etc) in a manner that more accurately resembles, or translates to, human tissues in vivo (1).

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