Discovery of a Highly Selective Tankyrase Inhibitor Displaying Growth Inhibition Effects against a Diverse Range of Tumor Derived Cell Lines.

The availability of high quality probes for specific protein targets is fundamental to the investigation of their function and their validation as therapeutic targets. We report the utilization of a dedicated chemoproteomic assay platform combining affinity enrichment technology with high-resolution protein mass spectrometry to the discovery of a novel nicotinamide isoster, the tetrazoloquinoxaline 41, a highly potent and selective tankyrase inhibitor. We also describe the use of 41 to investigate the biology of tankyrase, revealing the compound induced growth inhibition of a number of tumor derived cell lines, demonstrating the potential of tankyrase inhibitors in oncology.

Thermal profiling reveals phenylalanine hydroxylase as an off-target of panobinostat.

We describe a two-dimensional thermal proteome profiling strategy that can be combined with an orthogonal chemoproteomics approach to enable comprehensive target profiling of the marketed histone deacetylase inhibitor panobinostat. The N-hydroxycinnamide moiety is identified as critical for potent and tetrahydrobiopterin-competitive inhibition of phenylalanine hydroxylase leading to increases in phenylalanine and decreases in tyrosine levels. These findings provide a rationale for adverse clinical observations and suggest repurposing of the drug for treatment of tyrosinemia.

Screening Pools of Compounds against Multiple Endogenously Expressed Targets in a Chemoproteomics Binding Assay.

Chemoproteomics-based competition-binding assays allow the screening of compounds against endogenous proteins in cell or tissue extracts, but these assays are hampered by low throughput and high cost. Using compound pools rather than single compounds in a screening campaign holds the promise of increased efficiency and substantial cost reduction. Previous attempts to screen compounds in pools often fell short due to complex data tracking, deconvolution issues, compound interferences, and automation problems. The desire to screen compounds in a high-throughput chemoproteomics format sparked a reassessment of compound pooling. Through the integration of acoustic dispensing, we enabled a flexible pooling process, allowing mixture creation by combining randomized or specific samples to create defined pools. Automation enabled end-to-end tracking, using barcode scan check points and output files to track data and ensure integrity during the mixture creation process. The compound pooling approach proved to be highly compatible with the chemoproteomics assay technology. Pools of 10 compounds in a single well did not show compound interference effects or increased false-positive/negative rates. In the present study, four targets, TBK1, PI3Kδ, PI3Kγ, and mTOR, were screened using a chemoproteomics approach against pools of 10 compounds per well, resulting in robust hit identification.