Prospective Assessment of Virtual Screening Heuristics Derived Using a Novel Fusion Score.

High-throughput screening (HTS) is a widespread method in early drug discovery for identifying promising chemical matter that modulates a target or phenotype of interest. Because HTS campaigns involve screening millions of compounds, it is often desirable to initiate screening with a subset of the full collection. Subsequently, virtual screening methods prioritize likely active compounds in the remaining collection in an iterative process. With this approach, orthogonal virtual screening methods are often applied, necessitating the prioritization of hits from different approaches. Here, we introduce a novel method of fusing these prioritizations and benchmark it prospectively on 17 screening campaigns using virtual screening methods in three descriptor spaces. We found that the fusion approach retrieves 15% to 65% more active chemical series than any single machine-learning method and that appropriately weighting contributions of similarity and machine-learning scoring techniques can increase enrichment by 1% to 19%. We also use fusion scoring to evaluate the tradeoff between screening more chemical matter initially in lieu of replicate samples to prevent false-positives and find that the former option leads to the retrieval of more active chemical series. These results represent guidelines that can increase the rate of identification of promising active compounds in future iterative screens.

Parsimonious discovery of synergistic drug combinations.

Combination therapies that enhance efficacy or permit reduced dosages to be administered have seen great success in a variety of therapeutic applications. More fundamentally, the discovery of epistatic pathway interactions not only informs pharmacologic intervention but can be used to better understand the underlying biological system. There is, however, no systematic and efficient method to identify interacting activities as candidates for combination therapy and, in particular, to identify those with synergistic activities. We devised a pooled, self-deconvoluting screening paradigm for the efficient comprehensive interrogation of all pairs of compounds in 1000-compound libraries. We demonstrate the power of the method to recover established synergistic interactions between compounds. We then applied this approach to a cell-based screen for anti-inflammatory activities using an assay for lipopolysaccharide/interferon-induced acute phase response of a monocytic cell line. The described method, which is >20 times as efficient as a naïve approach, was used to test all pairs of 1027 bioactive compounds for interleukin-6 suppression, yielding 11 pairs of compounds that show synergy. These 11 pairs all represent the same two activities: ß-adrenergic receptor agonists and phosphodiesterase-4 inhibitors. These activities both act through cyclic AMP elevation and are known to be anti-inflammatory alone and to synergize in combination. Thus we show proof of concept for a robust, efficient technique for the identification of synergistic combinations. Such a tool can enable qualitatively new scales of pharmacological research and chemical genetics.

A 1,536-well ultra-high-throughput siRNA screen to identify regulators of the Wnt/beta-catenin pathway.

High-throughput siRNA screens are now widely used for identifying novel drug targets and mapping disease pathways. Despite their popularity, there remain challenges related to data variability, primarily due to measurement errors, biological variance, uneven transfection efficiency, the efficacy of siRNA sequences, or off-target effects, and consequent high false discovery rates. Data variability can be reduced if siRNA screens are performed in replicate. Running a large-scale siRNA screen in replicate is difficult, however, because of the technical challenges related to automating complicated steps of siRNA transfection, often with multiplexed assay readouts, and controlling environmental humidity during long incubation periods. Small-molecule screens have greatly benefited in the past decade from assay miniaturization to high-density plates such that 1,536-well nanoplate screenings are now a routine process, allowing fast, efficient, and affordable operations without compromising underlying biology or important assay characteristics. Here, we describe the development of a 1,536-well nanoplate siRNA transfection protocol that utilizes the instruments commonly found in small-molecule high throughput screening laboratories. This protocol was then successfully demonstrated in a triplicate large-scale siRNA screen for the identification of regulators of the Wnt/beta-catenin pathway.