Cell-Based Assay Design for High-Content Screening of Drug Candidates

Abstract

To reduce attrition in drug development, it is crucial to consider the development and implementation of translational phenotypic assays as well as decipher diverse molecular mechanisms of action for new molecular entities. High-throughput fluorescence and confocal microscopes with advanced analysis software have simplified the simultaneous identification and quantification of various cellular processes through what is now referred to as highcontent screening (HCS). HCS permits automated identification of modifiers of accessible and biologically relevant targets and can thus be used to detect gene interactions or identify toxic pathways of drug candidates to improve drug discovery and development processes. In this review, we summarize several HCS-compatible, biochemical, and molecular biology-driven assays, including immunohistochemistry, RNAi, reporter gene assay, CRISPR-Cas9 system, and protein-protein interactions to assess a variety of cellular processes, including proliferation, morphological changes, protein expression, localization, post-translational modifications, and protein-protein interactions. These cell-based assay methods can be applied to not only 2D cell culture but also 3D cell culture systems in a high-throughput manner.

Keywords: 3D cell culture system; Cell-based Assay; Drug discovery; High content screening.

Fig. 1

Schematics of (A) introduction of GFP into a specific target locus (Exon 2) and (B) the construction of GFP reporter cell lines using BAC technology for chemical safety assessment (ToxTracker Assay). PGK, phosphoglycerate kinase promoter; IRES, internal ribosomal entry site; NEO, neomycin resistance gene.

Fig. 2. One-step incorporation of fluorescent protein (FP) into the specific locus of an endogenous target gene (e.g., Nanog)

The CRISPR-Cas9 genome editing system requires the co-expression of a Cas9 protein with a guide RNA vector expressed from the human U6 polymerase III promoter. LHA, left homologous arm; RHA, right homologous arm; CMV, constitutive cytomegalovirus promoter; gRNA; guide RNA.

Fig. 3. Schematics of cell-based assay for identifying proteinprotein interactions

(A) Mammalian two-hybrid system. Interaction between the two test proteins (Bait and Prey), expressed as DNA binding domain (BD-Bait) and RNA polymerase activation domain (PolAD-Prey) fusion constructs, results in an increase in the expression of reporter genes over the negative controls. (B) Protein-fragment complementation assays (PCAs). Interacting proteins are fused to either of the two complementary fragments of a reporter protein (blue and red). Interaction of the two proteins brings the unfolded reporter-protein fragments into proximity, allowing them to fold into their active conformation. (C) Fluorescent indicators for protein phosphorylation in living cells. Upon phosphorylation of the substrate domain (SubD) within the fusion protein by the protein kinase, the adjacent phosphorylation recognition domain (PhosRD) binds with the phosphorylated substrate domain, which changes the efficiency of FRET between the GFP mutants. CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; P in an open circle, the phosphorylated residue.

Fig. 4. HCS strategies in 3D cell culture system

(A) In-cell immunofluorescence assay of biomarkers (target proteins) in 3D cell culture on the microarray chip [34, 64]. (B) Micropillar/microwell chip platform for RNAi delivery into 3D cell culture [64]. The micropillar chip can be transferred to a microwell chip containing a target compound after incubation with lentiviruses carrying the RNAi library.