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.

Just-in-Time Compound Pooling Increases Primary Screening Capacity without Compromising Screening Quality.

Compound pooling, or multiplexing more than one compound per well during primary high-throughput screening (HTS), is a controversial approach with a long history of limited success. Many issues with this approach likely arise from long-term storage of library plates containing complex mixtures of compounds at high concentrations. Due to the historical difficulties with using multiplexed library plates, primary HTS often uses a one-compound-one-well approach. However, as compound collections grow, innovative strategies are required to increase the capacity of primary screening campaigns. Toward this goal, we have developed a novel compound pooling method that increases screening capacity without compromising data quality. This method circumvents issues related to the long-term storage of complex compound mixtures by using acoustic dispensing to enable "just-in-time" compound pooling directly in the assay well immediately prior to assay. Using this method, we can pool two compounds per well, effectively doubling the capacity of a primary screen. Here, we present data from pilot studies using just-in-time pooling, as well as data from a large >2-million-compound screen using this approach. These data suggest that, for many targets, this method can be used to vastly increase screening capacity without significant reduction in the ability to detect screening hits.