Kutchukian, P. S. et al. (2017) Iterative focused screening with biological fingerprints identifies selective Asc-1 inhibitors distinct from traditional high throughput screening. ACS Chemical Biology, 12(2), pp. 519-527. (doi:10.1021/acschembio.6b00913) This is the author's final accepted version.There may be differences between this version and the published version. You are advised to consult the publisher's version if you wish to cite from it.http://eprints.gla.ac.uk/146530/

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The primary goal of high-throughput screening (HTS) is to rapidly survey a broad collection of compounds, numbering from tens of thousands to millions of members, and identify those that modulate the activity of a therapeutic target of interest. For nearly two decades, mass spectrometry has been used as a label-free, direct-detection method for HTS and is widely acknowledged as being less susceptible to interferences than traditional optical techniques. Despite these advantages, the throughput of conventional MS-based platforms like RapidFire or parallel LC-MS, which typically acquire data at speeds of 6–30 s/sample, can still be limiting for large HTS campaigns. To overcome this bottleneck, the field has recently turned to chromatography-free approaches including MALDI-TOF-MS and acoustic droplet ejection-MS, both of which are capable of throughputs of 1 sample/second or faster. In keeping with these advances, we report here on our own characterization of an acoustic droplet ejection, open port interface (ADE-OPI)-MS system as a platform for HTS using the membrane-associated, lipid metabolizing enzyme diacylglycerol acyltransferase 2 (DGAT2) as a model system. We demonstrate for the first time that the platform is capable of ejecting droplets from phase-separated samples, allowing direct coupling of liquid–liquid extraction with OPI-MS analysis. By applying the platform to screen a 6400-member library, we further demonstrate that the ADE-OPI-MS assay is suitable for HTS and also performs comparably to LC-MS, but with an efficiency gain of >20-fold.

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The estrogen-related receptor a (ERRa) was one of the first orphan receptors identified through a search for genes encoding proteins related to the steroid nuclear receptor, Estrogen Receptor a (ERa). The physiological role of ERRa has not yet been established nor has a natural ligand been elucidated. Importantly, research indicates that ERRa may be a novel drug target to treat breast cancer and/ or metabolic disorders. A homogeneous time-resolved fluorescence (HTRF) assay has been developed to screen for ERRa-specific antagonists. This assay uses the ERR ligand binding domain and the coactivator interaction domain of Proliferator-activated Receptor g Coactivator-1a (PGC-1a) to examine the ability of compounds to antagonize the constitutive interaction between ERRa and the coactivator. A dissociationenhanced lanthanide fluorescence immunoassay (DELFIA) was also created to counter screen compounds identified in the HTRF screen. Here we report the discovery of high-affinity ERRa subtype selective antagonists. Additionally, a homology model of ERRa in an antagonist conformation has been developed and after subsequent docking studies, we offer a model showing the molecular determinants that suggest why our novel tri-cyclic antagonist, N-[(2Z)-3-(4,5-dihydro-1,3-thiazol-2-yl)-1,3-thiazolidin-2-yl idene]-5 H dibenzo [a,d][7]annulen-5-amine, binds to ERRa with high affinity but does not bind to either ERRb or ERRg. Drug Dev Res 69:203-218, 2008.