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Phosphatidylethanolamine-Binding Proteins, Including RKIP, Exhibit Affinity for Phosphodiesterase-5 Inhibitors

Authors

  • Poupak Dadvar ,

    1. Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht (NL), Fax: (+31) 30-253-6919
    2. Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht (NL)
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  • Duangnapa Kovanich ,

    1. Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht (NL), Fax: (+31) 30-253-6919
    2. Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht (NL)
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  • Gert E. Folkers Dr.,

    1. Department of NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht (NL)
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  • Klaus Rumpel Dr.,

    1. Pfizer Global Research and Development, Sandwich (UK)
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  • Reinout Raijmakers  Dr.,

    1. Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht (NL), Fax: (+31) 30-253-6919
    2. Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht (NL)
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  • Albert J. R. Heck Prof. Dr.

    1. Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht (NL), Fax: (+31) 30-253-6919
    2. Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht (NL)
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Abstract

Identifying protein “interactors” of drugs is of great importance to understand their mode of action and possible cross-reactivity to off-target protein binders. In this study, we profile proteins that bind to PF-3717842, a high-affinity phosphodiesterase-5 (PDE5) inhibitor, by using a refined affinity pulldown approach with PF-3717842 immobilized beads. By performing these pulldowns in rat testis tissue lysate, we strongly and specifically enriched for PDE5 and a few other PDEs. In addition to these expected affinity-enriched proteins we also detect rodent-specific phosphatidylethanolamine-binding protein 2 (PEBP2), as a putative binder to the PDE5 inhibitor. By using recombinant forms of the related murine mPEBP2, mPEBP1 and human hPEBP1 (also known as Raf kinase inhibitor protein or RKIP) we confirm that they all can bind strongly to immobilized as well as soluble PF-3717842. As the phosphatidylethanolamine-binding proteins are involved in various important signal transduction pathways, the synthetic PDE5 inhibitor used here might form a platform to synthesize enhanced binders/inhibitors of the family of PEBP proteins. Our approach shows how chemical proteomics might be used to profile the biochemical space (interactome) of small molecule inhibitors.

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