Identification of HSP60 as a Primary Target of o-Carboranylphenoxyacetanilide, an HIF-1α Inhibitor

Abstract

We succeeded in the design and synthesis of multifunctional chemical probes of the HIF-1alpha inhibitor carboranylphenoxyacetanilide (1) that combine photoaffinity labeling and click reaction to identify the target protein. HSP60 was identified as a primary target protein of 1 using the chemical probes 2 and 3. Furthermore, HSP60 inhibitor 4 suppressed hypoxia-induced HIF activation, indicating that HSP60 affects HIF-1alpha accumulation directly or indirectly.

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;…”

Chaperonotherapy for Alzheimer’s Disease: Focusing on HSP60

“…Interestingly, a hypoxic-inducible factor 1 alpha inhibitor (13) containing a pharmacophorically unusual carboranyl moiety was found to primarily target HSP60, although the actual binding site is still undefined [138]. Recently, one of carboranylphenoxyacetanilide derivatives (13) (Fig.…”

Hunting Molecular Targets for Anticancer Reagents by Chemical Proteomics

“…Over the past several years, these potent tools have brought revolutionary changes to many fields. 57-61 Nacamura et al 62,63 developed two multifunctional photoaffinity probes to profile the target proteins of the HIF-1a inhibitor o-carboranylphenoxyacetanilide. Hypoxia response element (HRE) reporter gene assay with two such probes, followed by immunoblot analysis, confirmed that the modification to an efficient functional group (i.e., boronic acid) of o-carboranylphenoxyacetanilide in the probe synthesis correlated with negative reductions on HIF-1a activation to some extent.…”