Screening


by economics and technical shortcomings in assay throughput and sensitivity. This is principally due to the high equipment costs and throughput required to characterise large cell numbers in very small volumes. Nonetheless, recent advances in automated liquid handling, coupled with precision microfluidics, are beginning to mitigate these shortcomings, as they provide increased sensitivi- ty, economy of scale and precise automation. Indeed, microfluidic engineering is now founda- tional in manipulating single-cells for assays and analysis5. Part of the reason for this is that the handling of extremely small volumes and low con- centrations of target molecules that is required when working with individual cells necessitates precise manipulation, high precision of assay con- ditions and highly-sensitive signal detection. Specifically, microfluidic chips are being combined with several analytical techniques to assess cell morphology and a range of cell behaviours, including growth dynamics, migration, prolifera- tion, differentiation and apoptosis6.


Another answer: multicell analysis While single-cell analysis can provide an unprece- dented view of pharmacology and physiology from a single rare cell – unmasked by heterogeneous pop- ulation responses – each cell in the assay must be isolated from the microenvironment. A significant disadvantage of this approach, therefore, is that sin- gle-cell pharmacology in vitro may differ from that present in a larger population, particularly in an architecture that is not three-dimensional (3D), where cell:cell contact is critical. In contrast, 3D cell assembles, including spheroids and organoids, pro- vide a much more physiologically-relevant context for compound screening. Indeed, 3D cell culture has been rapidly adopted in compound identifica- tion and optimisation, as they better model in vivo human physiology and pathophysiology. Many techniques are now in routine use and recent reviews emphasise the use of 3D cultures as preclin- ical models as a common practice in both drug dis- covery and fundamental disease research7. To take a recent example, ultra-high throughput


screening (uHTS) using 1536-well microtiter plate formats was used to study cellular spheroids against compounds targeting a critical KRAS kinase muta- tion involved in several cancers8. Previously, direct- ly targeting oncogenic group RAS mutations had been challenging due to the enzyme biology and the complexity pathways involved, including down- stream effectors and upstream regulatory networks. As importantly, confounding factors associated with uHTS in 2D adherent monolayer cell cultures


40


resulted in false assay negatives, with many active compounds being undetected. In contrast, uHTS using 3D spheroid culture allowed the identifica- tion of Proscillaridin A as a selective inhibitor of cells harbouring the oncogenic KRasG12V allele. Importantly, Proscillaridin A identified by the 3D screening platform was not identified by standard 2D culturing methods. Multicellular organoids are self-organised,


three-dimensional tissue cultures often derived from adult stem cells or induced pluripotent stem cells (iPSCs). They can replicate a degree of organ complexity to a greater degree than seen with sin- gle-cells, 2D culture and simple 3D cultures such as spheroids. Furthermore, as organoids are cultured using cells from a specific individual, they can be used to predict individual patient responsivity to a specific drug or a specific dose. Related to this is the advantage that cancers and many other dis- eases can be modelled in vivo using biopsy samples and be subsequently used for compound screening (Table 1). As an example, a recent study presents data for more than 80 tested compounds, regis- tered or in clinical evaluation, that are screened against a colon cancer organoid ‘biobank’ and demonstrate the utility of a high throughput approach9.


Conclusion The high failure rate of preclinical compounds in clinical study remains a key problem in modern drug discovery. Innovative technologies will only be adopted if they can improve efficiency and, ulti- mately, the probability of clinical translation. In drug discovery, the renaissance in phenotypic screening, particularly against the non-classical, previously ‘undruggable’ drug targets and/or sig- nalling pathways, provides novel opportunities for a new generation of medicines. However, most phenotypic-based screening,


and, to a large extent, target-based screening, is based on the use of cellular assays, increasingly with primary stem cells from the individual patient. Should the cell-based assay, therefore, be predicated on single-cell techniques, or multicellu- lar techniques? Single-cell analysis is critical to understanding cell:cell variability and the impact of measuring responses based on population-aver- age measurements. Interrogating individual cells, as opposed to measuring the average population response, enables meaningful understanding of the interactions and contributions from low numbers of cell subpopulations in the organoid or tissue. By contrast, the growing adoption of 3D cultures is also increasing clinical predictability of compound


Drug Discovery World Fall 2019


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