Target identification of biologically active small molecules via in situ methods

Affinity purification in target identification: the specificity challenge

“…The authors have successfully accomplished the inter-subunit crosslinking of egg-white avidin tetramer with this probe (Hatanaka et al 1994). Over the next two decades, covalent affinity probes have been extensively employed not only in target identification (Adam et al 2002b;Takaoka et al 2006;MacKinnon et al 2007;Ban et al 2010;Koteva et al 2010;Wurdak et al 2010;Eirich et al 2011;Cisar and Cravatt 2012;Park et al 2012;Shi et al 2012;Park et al 2013;Su et al 2013), but also in affinity labeling of proteins (Dorman and Prestwich 2000;Hatanaka and Sadakane 2002;Chen et al 2003;Hosoya et al 2004;Lee et al 2005;Koshi et al 2008; Tanaka et al Tsukiji et al 2009a;Das 2011;Wacker et al 2011;Wang et al 2011;Hayashi and Hamachi 2012;Tamura et al 2012;Vinkenborg et al 2012;Willems et al 2012) and activity-based proteomic profiling (ABPP) (Kozarich 2003;Chan et al 2004;Ballell et al 2005;Evans and Cravatt 2006;Sadaghiani et al 2007;Salisbury and Cravatt 2007;Cravatt et al 2008;Salisbury and Cravatt 2008;Uttamchandani et al 2008;Nomura et al 2010;…”

“…These ''all-in-one'' probes have high potential to reduce the level of non-specific binding in affinity purification. In 2013, the Yao laboratory designed a type of ''minimalist probes'' containing a terminal alkyne, a diazirine crosslinker, and a functional group for conjugation with the small molecule (Li et al 2013c;Su et al 2013). Without the ligand, these probes are only seven atoms long so that they have minimal perturbation on the binding property of the ligand.…”

“…(Koteva et al 2010;Eirich et al 2011). We refer the reader to a number of excellent reviews for more specific examples (Burdine and Kodadek 2004;Leslie and Hergenrother 2008;Sato et al 2010;Das et al 2011;Lomenick et al 2011b;Futamura et al 2013;Schenone et al 2013;Su et al 2013;Ziegler et al 2013;Kawatani and Osada 2014).…”

“…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.…”

Small-molecule probes elucidate global enzyme activity in a proteomic context

“…While medicinal chemists have attempted to design drug molecules that interact with single target proteins, most drug molecules suffer from off-target effects, and their binding partners have not been identified. Consequently, the Yao group developed ABPs for known drug molecules to reveal the targets in situ (65,66). They synthesized ABPs with Dasatinib using alkyne and photoactivatable tags; this anti-cancer drug molecule targets protein kinases.…”