Pathway Reporter Assays Reveal Small Molecule Mechanisms of Action

Abstract

C ell-based, phenotypic screening of small molecules often identifies compounds with provocative biological properties. However, determining the cellular target(s) and/or mechanism of action (MoA) of lead compounds remains an extremely challenging and timeconsuming exercise. To provide insights into a compound's cellular action and greatly reduce the time required for MoA determination, we have developed a screening platform consisting of an extensive series of reporter gene assays (RGAs). A collection of O11,000 compounds of known MoA (e.g., World Drug Index entries) were screened against the entire panel. The output provided evidence that an RGA signature could be ascribed to numerous, biologically diverse MoAs. The reference database generated suggested novel biological activity for particular compounds. For example, the profiling data led to the prediction that the cellular target of the natural product terprenin was dihydroorotate dehydrogenase (DHODH), which was confirmed experimentally. The screening methodology developed for this endeavor renders it amenable to the future examination of compounds with unknown MoA, in an automated, inexpensive, and time-efficient manner. ( JALA 2009;14:374-82)

Identifying compound efficacy targets in phenotypic drug discovery

“…Higher throughput has been achieved by using a signature of $100 genes maximally responsive to compound treatment [47]. Promoter signature profiling (PSP) uses a collection of >40 high-throughput luciferase-based reporter gene assays [48]. Taken together, the resulting profile provides a snapshot of the cellular signaling response to compound treatment and has proven in our hands to be valuable for large-scale correlation to profiles of compounds with known MoA.…”

Evidence-Based and Quantitative Prioritization of Tool Compounds in Phenotypic Drug Discovery

“…Furthermore, targets affecting multiple downstream signaling pathways, or induction of apoptosis, are less likely to be specifically probed in a phenotypic assay setup, as shown in the example of vorinostat (Dokmanovic et al, 2007), and dinaciclib (Pei and Xiong, 2005) (Figure 3). Using better and more specific tool compounds would not have improved the fishing of these targets from our RGA experiments due to the design of the reporters and the corresponding pathways (King et al, 2009). Therefore, careful evaluation of historical experimental data and setting up ongoing phenotypic assays is as important as the tool compound itself for the success of target hypothesis validation.…”

“…Phenotypic screening, as an alternative to target-based approaches, has proved its value in the discovery of over half of small-molecule first-in-class new molecular entities (Moffat et al, 2014;Swinney, 2013). Phenotypic assays are widely used for target and lead discovery, as well as to profile for compound mechanism of action (MOA) (Feng et al, 2009;Hart, 2005;Hopkins, 2008;Jones and Diamond, 2007;King et al, 2009;Lee et al, 2012;Moffat et al, 2014;Schirle and Jenkins, 2015;Swinney, 2013). For example, reporter gene assays (RGAs) are a representative type of high-throughput, low-cost phenotypic screening technology whereby pathway response to compound treatment can be observed in reporter-transfected cells typically driving expression of a fluorescent protein (King et al, 2009).…”

The opportunities of mining historical and collective data in drug discovery

“…At the Genomic Institute of the Novartis Research Foundation (GNF), King et al established a reporter gene assay (RGA) panel using luciferase activity as the readout [12]. The 40 different RGAs in the panel were designed to cover a broad biology of the cell and capture diverse pathway activities.…”

“…The captured RGA signatures yielded an information-rich reference panel for comparison with compounds of unknown MoA. As a proof-of-principle, they validated dihydroorotate dehydrogenase (DHODH) as a target for the immunosuppressive agent terprenin in vitro, based on the observation that terprenin had shown a similar RGA signature to the known DHODH inhibitor brequinar [12].…”