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Synthesis of the Pitstop family of clathrin inhibitors

Nature Protocols volume 9, pages 1592–1606 (2014)Cite this article

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Abstract

This protocol describes the synthesis of two classes of clathrin inhibitors, Pitstop 1 and Pitstop 2, along with two inactive analogs that can be used as negative controls (Pitstop inactive controls, Pitnot-2 and Pitnot-2-100). Pitstop-induced inhibition of clathrin TD function acutely interferes with clathrin-mediated endocytosis (CME), synaptic vesicle recycling and cellular entry of HIV, whereas clathrin-independent internalization pathways and secretory traffic proceed unperturbed; these reagents can, therefore, be used to investigate clathrin function, and they have potential pharmacological applications. Pitstop 1 is synthesized in two steps: sulfonation of 1,8-naphthalic anhydride and subsequent reaction with 4-amino(methyl)aniline. Pitnot-1 results from the reaction of 4-amino(methyl)aniline with commercially available 4-sulfo-1,8-naphthalic anhydride potassium salt. Reaction of 1-naphthalene sulfonyl chloride with pseudothiohydantoin followed by condensation with 4-bromobenzaldehyde yields Pitstop 2. The synthesis of the inactive control commences with the condensation of 4-bromobenzaldehyde with the rhodanine core. Thioketone methylation and displacement with 1-napthylamine affords the target compound. Although Pitstop 1–series compounds are not cell permeable, they can be used in biochemical assays or be introduced into cells via microinjection. The Pitstop 2–series compounds are cell permeable. The synthesis of these compounds does not require specialist equipment and can be completed in 3–4 d. Microwave irradiation can be used to reduce the synthesis time. The synthesis of the Pitstop 2 family is easily adaptable to enable the synthesis of related compounds such as Pitstop 2-100 and Pitnot-2-100. The procedures are also simple, efficient and amenable to scale-up, enabling cost-effective in-house synthesis for users of these inhibitor classes.

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Figure 1: Synthesis of Pitstop 1.
Figure 2: Synthesis of Pitstop 1 inactive control (Pitnot-1).
Figure 3: Synthesis of Pitstop 2 and Pitstop 2-100.
Figure 4: Synthesis of the Pitstop 2 series of inactive analogs.

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Acknowledgements

This work was supported by grants from the National Health and Medical Research Council (Australia), The Australia Research Council, The Australian Cancer Research Foundation, The Ramaciotti Foundation, The Children's Medical Research Institute and Newcastle Innovation Ltd.

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  1. Mark J Robertson and Fiona M Deane: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, Centre for Chemical Biology, School of Environmental and Life Sciences, University of Newcastle, Callaghan, New South Wales, Australia

    Mark J Robertson, Fiona M Deane & Adam McCluskey

  2. Leibniz Institut für Molekulare Pharmakologie & Freie Universität Berlin, Berlin, Germany

    Wiebke Stahlschmidt, Lisa von Kleist & Volker Haucke

  3. Cell Signalling Unit, Children's Medical Research Institute, The University of Sydney, Sydney, New South Wales, Australia

    Phillip J Robinson

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Contributions

M.J.R. and F.M.D. contributed equally to the synthesis of all analogs described in this work. L.K. and V.W. are responsible for conducting biological assays to ensure compound activity. V.H., P.J.R. and A.M. are responsible for the concept, design and use of the clathrin inhibitors reported herein.

Corresponding authors

Correspondence to Volker Haucke, Phillip J Robinson or Adam McCluskey.

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Competing interests

Pitstop is a trademark of Children's Medical Research Institute and Newcastle Innovation, Ltd. We have a commercial agreement with Abcam Biochemicals (Bristol, UK) for the sale of our Pitstop compounds. This includes many of the compounds listed in this paper.

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Robertson, M., Deane, F., Stahlschmidt, W. et al. Synthesis of the Pitstop family of clathrin inhibitors. Nat Protoc 9, 1592–1606 (2014). https://doi.org/10.1038/nprot.2014.106

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