The starting point for the discovery and development of new drugs is the design of molecules that bind to their target proteins with high specificity. Here we describe a systematic chemical proteomics based approach, whereby we use a novel PDE5 inhibitor as bait in mice lung tissue. The compound N-(6-aminohexyl)-3-(1-ethyl-3-methyl-7-oxo-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-4-propoxybenzenesulfonamide (or PF-4540124), which binds to phosphodiesterase-5 (PDE5) with high affinity, was therefore immobilized on an affinity support. Initial affinity enrichment revealed the binding of hundreds of proteins to this immobilized PDE5 inhibitor. Therefore, selective pre-clearing and elution protocols were designed and used in combination with differential stable-isotope labeling to discriminate between the specific binding of low abundant proteins and less specific binding of high abundant proteins. The optimized method allowed us to selectively analyze the "interactome" of the PDE5 inhibitor PF-4540124 and enabled us to identify different isoforms of PDE5 present in mouse lung. Additionally, we enriched for the prenyl binding protein PrBP, which is also known as PDE6delta. Further analysis, applying in vitro binding assays allowed us to verify PrBP as a novel interactor of PF-4540124. The presented method provides a generic highly-specific chemical proteomics based enrichment technique for analyzing drug-protein interactions in mammalian tissue lysates.

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.

Inhibitors of phosphodiesterase 5 (PDE5) are widely used for the treatment of erectile dysfunction and pulmonary hypertension. The commercially available inhibitors are effective, well-tolerated drugs, but differ in their phosphodiesterase specificity. To explore and manipulate the specificity of PDE5 inhibitors, a small library of four inhibitors was synthesized using the structure of known PDE5 inhibitors as a scaffold. Their inhibitory potency towards PDE5 and related family members was evaluated. Next, they were immobilized on a matrix to perform affinity pull-down assays in rat testis tissue, followed by mass spectrometric (MS) analysis. By using unique peptide spectral counts of identified proteins in the MS analysis, we were able to assess the relative binding of these inhibitors to a large set of proteins, allowing the determination of their selectivity profiles in vitro. For selected proteins of interest, the results were verified using quantitative isotopic dimethyl labeling and immunoblotting, and isothermal titration calorimetry (ITC). For the PDE5 inhibitors, our data reveal that even slight chemical modifications can bias their selectivity significantly towards other interacting proteins, opening up the potential of these compounds to be used as scaffolds for the development of inhibitors for new protein targets. In a broad sense, we demonstrate that the combination of chemical proteomics and unique peptide spectral counting allows for the confident and facile analysis of the differential interactome of bioactive small molecules.

The cover picture shows the structure of an immobilized, clinically used phosphodiesterase-5 (PDE5) inhibitor. On p. 2654 ff., A. J. R. Heck et al. discuss how they used beads functionalized with this inhibitor to affinity enrich and identify interacting proteins directly in mammalain tissue (i.e., rat testis, shown in the background). Mass spectrometric analysis showed that not only the known target, PDE5, but also several members of the PEBP family of proteins, including the Raf kinase inhibitory protein (RKIP), bound to the drug.