A chemical proteomics based enrichment technique targeting the interactome of the PDE5 inhibitor PF-4540124.

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 inhibitorPF-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 PDE6δ. 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.