Design of Phenotypic Screens for Bioactive Chemicals and Identification of their Targets by Genetic and Proteomic Approaches

Abstract

Cell-based screening using phenotypic assays is a useful means of identifying bioactive chemicals for use as tools to elucidate complex cellular processes. However, the chemicals must display sufficient selectivity and their targets have to be identified. We describe how cell-based screening assays can be designed to maximize the likelihood of discovering selective compounds through the choice of positive readouts, low chemical concentrations and long incubation periods. Examining the potency, efficacy and activity range of chemicals can further help set apart those likely to act more specifically. Identifying the cellular targets of active chemicals can be especially demanding. Secondary screens and the cautious use of the candidate approach can help narrow down their mechanisms of action, but biased approaches may lead to the identification of secondary or even irrelevant targets. We discuss strategies for unbiased target identification by sampling potential targets at the genome-wide and proteome-wide levels.

A High-Throughput Assay for DNA Replication Inhibitors Based upon Multivariate Analysis of Yeast Growth Kinetics

“…In principle, such limitations can be addressed using a cell-based HTS approach. 11 One particularly fruitful method is based upon synthetic growth inhibition. 12 As defined genetically in yeast and bacteria, synthetic growth inhibition occurs when two nonallelic mutations, which individually cause little or no growth defect relative to the corresponding wild-type alleles, synergistically cause lethality or sickness when combined in a double mutant strain.…”

Effects of chemical manipulation of mitotic arrest and slippage on cancer cell survival and proliferation

“…The inclusion of a positive readout in cell-based screening is an effective way to filter out compounds acting non-specifically. [25][26][27] In independent work, DeMoe et al, Salmela et al and Stolz et al recently reported similar screens for chemical inducers of mitotic slippage using different cell lines and chemical collections. 28,29,47 The screen for inhibitors of mitotic slippage identified several microtubule-targeting agents, which was not surprising given the observation that increasing the concentration of paclitaxel strengthened mitotic arrest.…”

“…A positive readout (increased mitotic arrest) was selected over a negative readout (decreased slippage) because positive readout assays are less prone to artefactual results from toxic chemicals. [25][26][27] The paclitaxel concentration was selected to provide ~10% mitotic arrest (20-50 nM), compared to 1-2% for untreated controls (Fig. 4A), to provide a large window to identify chemicals that increase mitotic arrest.…”

HTS and hit finding in academia – from chemical genomics to drug discovery

“…Hit discovery, therefore, is ripe for innovation to produce the screening platforms that can address the large repertoire of undrugged targets. Cell-based screening has emerged as a more physiological alternative to isolated target-based approaches, with the advantage of not presuming which target on a particular pathway will be best to target [40], yet its full adoption has been hindered by robustness issues, difficulties in target deconvolution [41] and the undercurrent of reluctance among medicinal chemists to deal with the potential for complex SARs in the emergent chemical matter. Hit discovery in this sector should have the confidence and freedom to tackle pathway-based assays that provide a repertoire of potential targets and intervention points for chemical matter.…”