Preparation and characterization of vorinostat-coated beads for profiling of novel target proteins
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).
References (31)
- et al.
Acetylation and deacetylation of non-histone proteins
Gene
(2005) - et al.
HDAC inhibitor-based therapies: can we interpret the code?
Mol. Oncol.
(2012) - et al.
Ferritin H induction by histone deacetylase inhibitors
Biochem. Pharmacol.
(2010) - et al.
Omega-alkoxy analogues of SAHA (vorinostat) as inhibitors of HDAC: a study of chain-length and stereochemical dependence
Bioorg. Med. Chem. Lett.
(2007) - et al.
The large subunit of replication factor C interacts with the histone deacetylase, HDAC1
J. Biol. Chem.
(2002) - et al.
Protein lysine acetylation analysis: current MS-based proteomic technologies
Analyst
(2013) - et al.
Lysine acetylation targets protein complexes and co-regulates major cellular functions
Science
(2009) - et al.
Regulation of cellular metabolism by protein lysine acetylation
Science
(2010) - et al.
HATs and HDACs: from structure, function and regulation to novel strategies for therapy and prevention
Oncogene
(2007) - et al.
Mutations in SIRT2 deacetylase which regulate enzymatic activity but not its interaction with HDAC6 and tubulin
Mol. Cell. Biochem.
(2007)
Targeting histone deacetylase in cancer therapy
Med. Res. Rev.
HDACs and their inhibitors in immunology teaching anticancer drugs new tricks
Immunol. Cell Biol.
Histone deacetylases and cancer: causes and therapies
Nat. Rev. Cancer
Effects of treatment with histone deacetylase inhibitors in solid tumors: a review based on 30 clinical trials
Future Oncol.
HDAC inhibitors in cancer biology: emerging mechanisms and clinical applications
Immunol. Cell. Biol.
Cited by (7)
Mechanism of action for HDAC inhibitors—Insights from omics approaches
2019, International Journal of Molecular SciencesUnderstanding epigenetic alterations in Alzheimer’s and Parkinson’s disease: Towards targeted biomarkers and therapies
2017, Current Pharmaceutical DesignStudying epigenetic complexes and their inhibitors with the proteomics toolbox
2016, Clinical Epigenetics