Searching for the Protein Targets of Bioactive Molecules

Abstract

The identification of all protein targets of a given drug or bioactive molecule within the human body is a prerequisite for an understanding of its beneficial and deleterious activities. Current approaches to reveal protein targets often fail to reveal physiologically relevant interactions. Here we review a recently introduced yeast-based approach for the identification of the binding partners of small molecules. We discuss the advantages and limitations of the approach using the clinically approved drug sulfasalazine as an example.

Yeast “N”-hybrid systems for protein–protein and drug–protein interaction discovery

“…Testing of each identified target in parallel against the original small molecule derivative used in the screen and a control molecule ensures that only specific interactors of the small molecule of interest are retained and virtually all false positive interactions are removed from the screen. Moreover, Chidley et al take an interesting approach by ''pre-screening'' cDNA libraries in the absence of a small molecule to eliminate false positives from a library, prior to the actual yeast three-hybrid screen [28]. Nevertheless, we and others [28] have observed a certain percentage of drug-target interactions which appear to be reproducible in a yeast three-hybrid assay, yet fail in in vitro confirmation assays, such as pulldown or co-immunoprecipitation studies.…”

“…Moreover, Chidley et al take an interesting approach by ''pre-screening'' cDNA libraries in the absence of a small molecule to eliminate false positives from a library, prior to the actual yeast three-hybrid screen [28]. Nevertheless, we and others [28] have observed a certain percentage of drug-target interactions which appear to be reproducible in a yeast three-hybrid assay, yet fail in in vitro confirmation assays, such as pulldown or co-immunoprecipitation studies. At present, the reason for this phenomenon is unclear, but a possible explanation could be the sensitivity of the yeast three-hybrid system, which allows detection of relatively weak interactions [29].…”

Affinity purification in target identification: the specificity challenge

“…In principle, one may accomplish a target ID project with any of these methods, but in practice, people have realized an integrated approach, combining multiple strategies and methods are instrumental to success. Details of various target identification strategies and methods, their respective advantages and limitations, and representative examples have been extensively covered by numerous excellent reviews (Lindsay 2003;Burdine and Kodadek 2004;Parsons et al 2004;Hart 2005;Luesch et al 2005;Parsons et al 2006;Boshoff and Dowd 2007;Perlstein et al 2007;Terstappen et al 2007; Leslie and Hergenrother 2008;Sleno and Emili 2008;Bantscheff et al 2009;Rix and Superti-Furga 2009;Bottcher et al 2010;Chan et al 2010;Sato et al 2010;Chidley et al 2011a;Das et al 2011;Lomenick et al 2011b;Raida 2011;Wierzba et al 2011;Cho and Kwon 2012;Cong et al 2012;Sakamoto et al 2012;Futamura et al 2013;Lee and Bogyo 2013;Park et al 2013;Su et al 2013;Sumranjit and Chung 2013;Ziegler et al 2013;Blagg and Workman 2014;Kawatani and Osada 2014). Here we elect to only focus on the classic biochemical method: affinity purification.…”

Target Abundance-Based Fitness Screening (TAFiS) Facilitates Rapid Identification of Target-Specific and Physiologically Active Chemical Probes

“…Whole-cell-based screens identify compounds that induce or correct a disease-relevant phenotype (e.g., inhibition of microbial growth), but the molecular target and mechanism of action (MOA) of each hit are unknown. Identification of the molecular target then requires a substantial investment of time and resources, without which further development and optimization of promising lead compounds toward a viable therapeutic is severely restricted ( 4 , 5 ). Thus, with either strategy, the identification of pharmacologically active agents that act via a defined MOA is a multistep process.…”