Doug Auld, Ph.D. Novartis Institutes for BioMedical Research
This study outlines an approach to improved use of reporter genes in high-throughput screening.
Rapid assignment of compound activity to either wanted biological pathway/target modulation activity or unwanted interference with the assay system is critical to efficient progression of chemical matter arising from screens in chemical biology or drug discovery efforts. The most powerful assay for confirming that a compound’s activity is due to relevant biological activity is an orthogonal assay. An orthogonal assay aims to replicate the exact same biology as the primary assay but differs in that a single component is switched, such as the method of detection, which shouldn’t affect the targeted biology. Therefore, a positive result in an orthogonal assay provides evidence for target activity (e.g., the activity does not depend on the detection technology and is likely related to a mechanism relevant to the biological system under study). On the other hand, a negative result is a strong indication that the primary screen activity was due to the method of detection.
An orthogonal assay is therefore a much more direct approach to addressing assay reporter-based artifacts than using counter-screens in which a negative result is the desired outcome because negative results can arise from a number of assay format differences (such as the level of the reporter expression, incubation times, and other modifications of assay components necessary to create the counter-screen). For reporter-gene assays (RGAs) that employ enzymes to generate the assay signal a large portion of compounds can interfere with the assay by acting as enzyme inhibitors, which can lead to many confounding effects that complicate the interpretation of the results. For orthogonal RGAs, the goal is to provide two reporters in which the enzymes differ in substrate requirements, enzymatic mechanisms, and inhibitory profiles. Ideally these reporters are produced in an isogenic background.
The most optimal and efficient method to produce two orthogonal RGAs is to express the reporters in the same cell line. However, methods to express multiple reporters (such as the use of an internal ribosomal entry site [IRES] elements, bidirectional promoters, and coinfection with multiple vectors) are often plagued by inefficient and unpredictable expression of both reporters. For example, incorporation of an IRES sequence often results in more efficient expression of the upstream open reading (ORF) frame compared to the downstream ORF. This article* provides a solution to efficient co-expression of multiple reporters (or any other set of proteins) by exploiting the mechanism by which polyproteins are cleaved to produce multiple proteins in Picornaviridae.
The virus uses an 18-residue peptide sequence (so-called P2A peptide) that trips up the ribosome, causing translation to skip over this sequence and express the next ORF, thereby producing multiple separate proteins at near equimolar amounts. The authors validated this system using a construct wherein firefly luciferase (FLuc) and Renilla luciferase (RLuc) were separated by the P2A peptide sequence under control of a CRE response element (Figure). Coincidence expression of both reporters using this construct in HEK293 showed near equal expression of FLuc and RLuc as separate proteins with little detectable fusion protein (Figure).
In the assay, activity from both reporters can be measured in the same assay well using standard detection reagents (e.g., DualGlo, Promega). A small library of known drugs was screened (the Library of Pharmacology Active Substance; LOPAC, Sigma) and it was demonstrated that compounds annotated as agonists of CRE-dependent signaling were identified as active with both FLuc and RLuc signals; however, compounds known to specifically inhibit either FLuc or RLuc were only found when measuring the activity of the reporter that was inhibited. Many reporter inhibitors actually lead to increased reporter signal in RGAs because reporter inhibitors can act in cells to stabilize the reporter enzyme, leading to increased enzyme levels that mimic gene activation. This counter-intuitive result can greatly complicate the interpretation of compound activity arising from RGAs, but the coincidence expression demonstrated here rapidly flagged these compounds, which were detected as active in only one of the reporter responses (see also www.reportergene.com).
This system should greatly facilitate the production of orthogonal RGAs as well as other assay systems in which production of multiple proteins is required.
*Summary from Nature Methods 2012, Vol. 9: 709–736
Originally developed as sentinels of transcriptional activity to map the regulatory function of genetic elements, reporter gene assays have been extensively used in high-throughput screening (HTS) to identify chemical modulators of cellular pathways (Michelini E et al., Anal Bioanal Chem 2010;398:227–238). However, HTS chemical libraries consist of structurally diverse small molecules that frequently interact directly with the reporter, thus skewing data interpretation and complicating candidate selection. To distinguish compounds that target a biological pathway from those that interfere with a reporter, we designed a coincidence biocircuit based on the principle that it is easier to tell signal from noise when the signal is reported by two or more detectors. We conclude that coincidence reporter strategies rapidly discriminate compounds of relevant biological activity from those interfering with reporter function and stability, using a single assay platform. This study outlines an approach to improved use of reporter genes in HTS with numerous coincidence combination types and stoichiometries possible.
Doug Auld, Ph.D., is affiliated with the Novartis Institutes for BioMedical Research.
ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a “Literature Search and Review” column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the “Literature Search and Review” column, a paper published in Nature Methods titled “A coincidence reporter-gene system for high-throughput screening.” Authors of the paper are Cheng KC and Inglese J.