Elsevier

Journal of Chromatography A

Volume 1372, 12 December 2014, Pages 34-41
Journal of Chromatography A

Preparation and characterization of vorinostat-coated beads for profiling of novel target proteins

https://doi.org/10.1016/j.chroma.2014.10.098Get rights and content

Highlights

  • A vorinostat-based small molecule probe was designed, synthesized and immobilized onto the surface of sepharose beads.

  • Vorinosta-coated beads based proteomics approach for profiling of novel target proteins of vorinostat.

  • ENO-1 was detected and confirmed as a novel, potential target of vorinostat.

Abstract

Inhibitors of histone deacetylases (HDACs) have been considered to be new anticancer agents. As a key inhibitor of HDAC, vorinostat can cause growth arrest and death of a broad of transformed cells and interact with a variety of substrates. A comprehensive analysis of proteins interacting with HDAC inhibitors is of great importance in understanding molecular mechanisms of the drugs. Here, we reported the preparation and characterization of vorinostat-coated beads for profiling of novel target proteins of vorinostat (a key HDAC inhibitor). The enriched proteins were further analyzed by HPLC–MS/MS. Besides the known substrates, there were also several novel enriched protein candidates, one of which was a metalloenzyme α-enolase (ENO-1). According to our best knowledge, it is the first time that ENO-1 has been detected as a potential target of vorinostat through chemoproteomics approach. Further competition analysis indicated that ENO-1 may be co-enriched as a substrate complex. Our results demonstrated that the chemical probe combined with proteomics approach may be developed as a potential tool to identify target proteins of drugs.

Introduction

Protein lysine acetylation, including histone acetylation and non-histone protein acetylation, plays a major role in regulating chromatin structure, transcriptional activity and metabolic pathways, thus contributing to diverse cellular process like transcription, cell cycle regulation, apoptosis and senescence [1], [2], [3], [4]. This dynamic and reversible post-translational modification (PTM) is regulated by histone deacetylases (HDACs) and histone acetyl transferases (HATs) [5], [6]. Three major classes of mammalian HDACs have been extensively described, of which classes I and II are zinc-dependent metallohydrolases and class III are NAD+-dependent deacetylases [4], [5]. Recent advances have shown that the development of cancer is intimately associated with HDAC expression [7], [8], [9]. Treatment of tumor cells with HDAC inhibitors (HDACIs) results in growth arrest, differentiation and/or apoptosis of many cancer cells [7], [10]. HDACIs have emerged as exciting anticancer agents and several classes of HDACIs have been found to have potent and specific anticancer activities [10], [11], [12].

To understand the anticancer activity mechanisms of HDACIs, it is necessary to elucidate the target proteins of the drugs [13]. Small molecular probe-based proteomics approach has become a potential tool for profiling targets of the drugs [14]. A series of novel target proteins of HDACI have been revealed by combining affinity capture and mass spectrometry analysis [14], [15]. These studies further indicate that the HDACIs mode of action could be considerably broader and more complicated than original understanding of altering epigenetic changes [16], [17].

Suberoylanilide hydroxamic acid (SAHA, also named Vorinostat or Zolinza), is a key second-generation hydroxamate HDACI of classes I and II for the treatment of refractory cutaneous T-cell lymphoma [13], [18]. Vorinostat can cause growth arrest and death of a broad of transformed cells and have little or no toxic effects on normal cell [13]. Recent evidence indicates that, vorinostat may interact with a variety of substrates including chromatin proteins, transcription factors, metabolic enzymes, and cell structure proteins [19].

In this study, we aimed to profile potential targets and substrate complexes of vorinostat in a whole-cell lysate combining vorinostat-coated beads and HPLC–MS/MS analysis. A vorinostat-based small molecule probe was designed and synthesized, then immobilized onto the surface of ECH sepharose beads. We investigated the effect of different conditions on proteomics profiling and further identified 58 protein candidates using the probe in the optimized condition. Bioinformatic analysis indicated that most identified proteins were involved in metabolic process, protein biosynthesis, cell cycle and differentiation. Besides known complexes and substrates, there were also several novel enriched proteins in this analysis, one of which was a metalloenzyme α-enolase (ENO-1). As a novel, potential target of vorinostat, ENO-1 was detected and confirmed in the study. Further western blot competition analysis indicated that ENO-1 may be co-enriched as a substrate complex rather than direct interaction with vorinostat. This study showed that the probe-based approach holds a great potential for identification of novel candidates interacting with HDACIs.

Section snippets

Reagents and materials

Suberic acid monomethyl ester was purchased from Suzhou BEC Biological Technology Co., Ltd (Suzhou, China). Hydroxybenzotriazole (HOBt), dicyclocarbodiimide (DCC) and p-phenylenediamine were obtained from Sinopharm Chemical Reagent Beijing Co., Ltd. (Beijing, China). ECH Sepharose 4B was purchased from GE Healthcare Life Sciences (Pittsburgh, Pennsylvania, USA). N-(3-Dimethylaminopropy)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were both purchased from J&K

Design, synthesis and characterization of SAHA-probe

To explore target proteins of vorinostat, we designed a SAHA-probe to capture these proteins. According to the early studies of structure-activity relationship, the vorinostat pharmacophore consists of (a) a functional group that chelates to the Zn2+ cation in HDAC active sites and blocks the enzymatic activity, (b) an aliphatic chain that mimics the substrate and occupies the enzymatic channel, and (c) a cup domain that contacts with the rim of the binding pocket [21], [22], [23]. Considering

Conclusions

In this study, we designed and developed vorinosta-coated beads, which successfully enriched the target proteins and complexes interacting with vorinostat. After optimizing the enrichment method, we identified 58 protein candidates by HPLC–MS/MS. Bioinformatic analysis showed that identified proteins were enriched in metabolic process, one of which, ENO-1 was detected as a novel target of vorinosta through chemoproteomic approach and confirmed by western blot analysis. Competition analysis

Acknowledgements

This work was supported by National Basic Research Program of China (grants 2012CB910601 and 2013CB910903) and National Natural Science Foundation of China with grants (21275077 and 90919008) and the Tianjin Municipal Science and Technology Commission (no. 14JCYBJC24000).

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