Chemical proteomics approaches for identifying the cellular targets of natural products.

M H Wright,S A Sieber

doi:10.1039/c6np00001k

M H Wright, S A Sieber

Open Access

https://doi.org/10.1039/c6np00001k

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Abstract

Covering: 2010 up to 2016Deconvoluting the mode of action of natural products and drugs remains one of the biggest challenges in chemistry and biology today. Chemical proteomics is a growing area of chemical biology that seeks to design small molecule probes to understand protein function. In the context of natural products, chemical proteomics can be used to identify the protein binding partners or targets of small molecules in live cells. Here, we highlight recent examples of chemical probes based on natural products and their application for target identification. The review focuses on probes that can be covalently linked to their target proteins (either via intrinsic chemical reactivity or via the introduction of photocrosslinkers), and can be applied "in situ" - in living systems rather than cell lysates. We also focus here on strategies that employ a click reaction, the copper-catalysed azide-alkyne cycloaddition reaction (CuAAC), to allow minimal functionalisation of natural product scaffolds with an alkyne or azide tag. We also discuss 'competitive mode' approaches that screen for natural products that compete with a well-characterised chemical probe for binding to a particular set of protein targets. Fuelled by advances in mass spectrometry instrumentation and bioinformatics, many modern strategies are now embracing quantitative proteomics to help define the true interacting partners of probes, and we highlight the opportunities this rapidly evolving technology provides in chemical proteomics. Finally, some of the limitations and challenges of chemical proteomics approaches are discussed.

Highlights

We focus here on strategies that employ a click reaction, the copper-catalysed azide–alkyne cycloaddition reaction (CuAAC), to allow minimal functionalisation of natural product scaffolds with an alkyne or azide tag
Two-step activity and affinity-based protein pro ling Chemical proteomics and bioorthogonal ligation chemistry Using intrinsic chemical reactivity Photoaffinity labelling Design and validation of probes Probe design Intrinsic reactivity of alkynes Probe validation Chemical proteomic work ows Proteomic identi cation of hits Quantitative chemoproteomics Cleavable linkers in chemical proteomics Alternative enrichment/detection strategies Target validation and mode of action Fishing for targets: probe-centric approaches Early in situ approaches to identify the targets of electrophilic natural products Antibacterials
We aim to survey and analyse studies from the last ve years on chemical proteomics technologies for mode of action, focusing on the following aspects: natural products, ‘discovery’ chemical proteomics, in situ approaches using covalent or photoprobes, two-step labelling protocols that exploit bioorthogonal ligation chemistry, and nally the advantage that can be taken of recent advances in quantitative mass spectrometry-based proteomics methods

Summary

Introduction

Two-step activity and affinity-based protein pro ling Chemical proteomics and bioorthogonal ligation chemistry Using intrinsic chemical reactivity Photoaffinity labelling Design and validation of probes Probe design Intrinsic reactivity of alkynes Probe validation Chemical proteomic work ows Proteomic identi cation of hits Quantitative chemoproteomics Cleavable linkers in chemical proteomics Alternative enrichment/detection strategies Target validation and mode of action Fishing for targets: probe-centric approaches Early in situ approaches to identify the targets of electrophilic natural products Antibacterials. Cytotoxic and anti-cancer NPs Going gel-free and quantitative Studies using label-free methods Label-based approaches: quanti cation at the peptide level Label-based approaches: SILAC Alternative quanti cation approaches Photoaffinity labelling Competitive activity-based protein screening Screening platforms Competitive mode for natural product target elucidation Conclusions Limitations, pitfalls and potential solutions Current and future challenges Acknowledgements Notes and references. Drug discovery typically takes one of two Review approaches: a target-based approach, where a compound is developed to target a particular enzyme, and phenotypic screening, where libraries of small molecules are screened against live cells with a phenotypic read-out. An added complexity is that compounds may have different modes of action in different organisms or contexts, or may have pleiotropic effects

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