In-cell click labelling of small molecules to determine subcellular localisation
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In-cell click labelling of small molecules to determine subcellular localisation Lyn H. Jones & David Beal & Matthew D. Selby & Owen Everson & George M. Burslem & Peter Dodd & Jared Millbank & Thien-Duc Tran & Florian Wakenhut & Emily J. S. Graham & Paul Targett-Adams
Received: 22 September 2010 / Accepted: 1 November 2010 / Published online: 18 November 2010 # Springer-Verlag 2010
Abstract Small molecule fluorometric boron dipyrromethene probes were developed to bind hepatitis C virusencoded NS5A protein and aid subcellular distribution studies. These molecules did not co-locate with NS5A, therefore alternative ‘silent’ azide reporters were used to obtain a more relevant picture of their distribution. Following pre-incubation with replicon cells, click chemistry was used to append a fluorophore to the azide that confirmed the co-localisation of the small molecule with the NS5A protein, thus providing greater insight into the antiviral mode of action of this chemotype. Keywords Hepatitis C virus . Click chemistry . Labelling . Small molecules
Introduction Hepatitis C virus (HCV) infection is a global public health problem and it has been estimated that 3% of the world’s population are infected with HCV (http://www.who.int/csr/ Electronic supplementary material The online version of this article (doi:10.1007/s12154-010-0047-1) contains supplementary material, which is available to authorized users. L. H. Jones (*) : D. Beal : M. D. Selby : O. Everson : G. M. Burslem : P. Dodd Chemical Biology Group, Sandwich Chemistry, Pfizer, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK e-mail: [email protected] J. Millbank : T.-D. Tran : F. Wakenhut : E. J. S. Graham : P. Targett-Adams New Opportunities Unit, Pfizer, Ramsgate Road, Sandwich, Kent CT13 9NJ, UK
disease/hepatitis/whocdscsrlyo2003/en/index1.html). Gao et al. [1] have previously described a chemical proteomics strategy to identify NS5A, an HCV-encoded zinc-binding phosphoprotein, as the target of the potent inhibitor BMS790052, a clinical candidate for the treatment of HCV. In this work, the structurally-related biotinylated stilbene derivative 1 was incubated with genotype 1b replicon cells and NS5A was affinity-isolated using streptavidin– agarose beads following cell lysis, to aid identification of NS5A as the relevant biological target of the inhibitor (Fig. 1) [1]. In tissue culture cells replicating HCV RNA, NS5A is sequestered in replication complexes, which are essentially factories for HCV genome synthesis [2, 3]. Replication complexes are thought to form from invaginations in the membrane of the endoplasmic reticulum (ER) to create vesicles that house HCV-encoded proteins, specific host factors required by the virus for RNA synthesis, and replicating HCV RNA [2–4]. Consequently, NS5A is localised to the ER in cells replicating HCV RNA [2]. We decided to conjugate the boron dipyrromethene (BODIPY®) fluorophore to the stilbene template in 1 to assess the subcellular distribution of this chemotype in the 1b replicon cells and to confirm the co-localisation wi
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