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

Abstract

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.

Theory and applications of differential scanning fluorimetry in early-stage drug discovery

“…R universal gas constant DSF has a direct application in fragment-based ligand design (FBLD) due to the ease of use in high-throughput screening. In this approach, small molecule building blocks (100-150 Da) are potentially pooled (3-5 molecules per pool) and screened (Elkin et al 2015;Valenti et al 2019). Although these small molecular mass compounds are unlikely to possess high affinity by themselves, this pooled approach allows for a significant reduction in the number of experiments that need to be performed to screen a large library.…”

Creating and screening natural product libraries

“…A nal iteration on multiplexing HTS is library pooling. 176,177 In this strategy, the screening library itself is pooled and screened as normal and then any potential leads are then rescreened as individual components during secondary follow-up. This approach is most amenable to compound screening with assays that have very low hit rates and thus attempts to address the "dark-matter" problem (the majority of screened wells are inactive) encountered in large screening campaigns.…”

Cell-Based Screening to Identify Cytoprotective Compounds

“…Drug combinations may provide a further option to reduce the number of assays to be run [51,52]. If you expect that the number of active compounds is low, you may consider pooling multiple compounds and testing them in combination.…”