Target profiling of zerumbone using a novel cell-permeable clickable probe and quantitative chemical proteomics

Kalesh, Karunakaran A.; Clulow, James A.; Tate, Edward W.

doi:10.1039/c4cc09527h

Cited by 30 publications

(17 citation statements)

References 18 publications

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“…Optimising probe concentration and other labelling conditions can sometimes help, and quantifying enrichment in the presence and absence of a competitor (typically the parent NP) is one approach widely used to test whether a protein is a probe-specific hit. This approach was used to help identify the specific targets of the NPs zerumbone, 91 eupalmerin acetate, 89 hypothemicin 80 and fimbrolides, 85 for example. Multiple probes with modifications at different positions can be used to reduce the risk of probe-related off-targets and to ensure coverage of as many targets as possible (an approach used to identify possible targets of acivicin, for example).…”

Section: Resultsmentioning

confidence: 99%

“…Spike-in SILAC was used by the Tate group to identify the targets of the NP zerumbone, for quantifying concentration-dependent competition of alkyne probe with the parent NP. 91 The cyclic sesquiterpene zerumbone, which has diverse anti-inflammatory activities, possess two electrophilic Michael acceptor systems with potential protein reactivity and so a probe 36 was synthesised with an alkyne attached at a distal site ( Table 2 ). A large batch of SILAC heavy cells were labelled with the probe, and subsequent triplicate samples in standard cell culture medium were prepared with probe and increasing concentrations of parent NP.…”

Section: Fishing For Targets: Probe-centric Approachesmentioning

confidence: 99%

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Chemical proteomics approaches for identifying the cellular targets of natural products

2016

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Section: Resultsmentioning

confidence: 99%

Section: Fishing For Targets: Probe-centric Approachesmentioning

confidence: 99%

Chemical proteomics approaches for identifying the cellular targets of natural products

2016

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“…While reports have been made regarding potential targets of curcumin, the approaches have generally been restricted in scope or otherwise limited in terms of their applicability to living cells 5 . Recently, clickable small molecule probes have been used in identifying the targets of many bio-active natural products or drugs 6 7 8 9 10 11 12 13 . In this study, we comprehensively identified in situ the specific and direct protein binding targets of curcumin in a colon cancer cell line (HCT116), via the synthesis of a cell-permeable clickable curcumin probe ( Cur-P ) combined with quantitative chemical proteomics methods ( Fig.…”

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confidence: 99%

In situ Proteomic Profiling of Curcumin Targets in HCT116 Colon Cancer Cell Line

Wang

Zhang

et al. 2016

Sci Rep

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To date, the exact targets and mechanism of action of curcumin, a natural product with anti-inflammatory and anti-cancer properties, remain elusive. Here we synthesized a cell permeable curcumin probe (Cur-P) with an alkyne moiety, which can be tagged with biotin for affinity enrichment, or with a fluorescent dye for visualization of the direct-binding protein targets of curcumin in situ. iTRAQTM quantitative proteomics approach was applied to distinguish the specific binding targets from the non-specific ones. In total, 197 proteins were confidently identified as curcumin binding targets from HCT116 colon cancer cell line. Gene Ontology analysis showed that the targets are broadly distributed and enriched in the nucleus, mitochondria and plasma membrane, and they are involved in various biological functions including metabolic process, regulation, response to stimulus and cellular process. Ingenuity Pathway AnalysisTM (IPA) suggested that curcumin may exert its anticancer effects over multiple critical biological pathways including the EIF2, eIF4/p70S6K, mTOR signaling and mitochondrial dysfunction pathways. Functional validations confirmed that curcumin downregulates cellular protein synthesis, and induces autophagy, lysosomal activation and increased ROS production, thus leading to cell death.

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“…Kalesh et al. demonstrates this method in their target identification study of zerumbone, a terpenoid compound derived from the Zingiber zerumbet plant with numerous possible therapeutic properties . Using a synthesized clickable cell‐permeable probe designated Yn‐Zer, HeLa cells were treated under normal conditions with a fixed Yn‐Zer concentration with increasing zerumbone concentrations, while an additional “heavy” labeled HeLa sample was treated with the same fixed concentration of Yn‐Zer.…”

Section: Quantitative Chemical Proteomicsmentioning

confidence: 99%

Target identification with quantitative activity based protein profiling (ABPP)

et al. 2016

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As many small bioactive molecules fulfill their functions through interacting with protein targets, the identification of such targets is crucial in understanding their mechanisms of action (MOA) and side effects. With technological advancements in target identification, it has become possible to accurately and comprehensively study the MOA and side effects of small molecules. While small molecules with therapeutic potential were derived solely from nature in the past, the remodeling and synthesis of such molecules have now been made possible. Presently, while some small molecules have seen successful application as drugs, the majority remain undeveloped, requiring further understanding of their MOA and side effects to fully tap into their potential. Given the typical promiscuity of many small molecules and the complexity of the cellular proteome, a high-flux and high-accuracy method is necessary. While affinity chromatography approaches combined with MS have had successes in target identification, limitations associated with nonspecific results remain. To overcome these complications, quantitative chemical proteomics approaches have been developed including metabolic labeling, chemical labeling, and label-free methods. These new approaches are adopted in conjunction with activity-based protein profiling (ABPP), allowing for a rapid process and accurate results. This review will briefly introduce the principles involved in ABPP, then summarize current advances in quantitative chemical proteomics approaches as well as illustrate with examples how ABPP coupled with quantitative chemical proteomics has been used to detect the targets of drugs and other bioactive small molecules including natural products.

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Target profiling of zerumbone using a novel cell-permeable clickable probe and quantitative chemical proteomics

Abstract: The first target profile for zerumbone in live cancer cells determined through quantitative competitive chemical proteomics with a cell-permeable probe.

Cited by 30 publications

References 18 publications

Chemical proteomics approaches for identifying the cellular targets of natural products

Chemical proteomics approaches for identifying the cellular targets of natural products

In situ Proteomic Profiling of Curcumin Targets in HCT116 Colon Cancer Cell Line

Target identification with quantitative activity based protein profiling (ABPP)

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