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Available corrector drugs are unable to effectively rescue the folding defects of CFTR-ΔF508 (or CFTR-F508del ), the most common disease-causing mutation of the cystic fibrosis transmembrane conductance regulator, a plasma membrane (PM) anion channel, and thus to substantially ameliorate clinical phenotypes of cystic fibrosis (CF). To overcome the corrector efficacy ceiling, here we show that compounds targeting distinct structural defects of CFTR can synergistically rescue mutant expression and function at the PM. High-throughput cell-based screens and mechanistic analysis identified three small-molecule series that target defects at nucleotide-binding domain (NBD1), NBD2 and their membrane-spanning domain (MSD) interfaces. Although individually these compounds marginally improve ΔF508-CFTR folding efficiency, function and stability, their combinations lead to ~50–100% of wild-type-level correction in immortalized and primary human airway epithelia and in mouse nasal epithelia. Likewise, corrector combinations were effective against rare missense mutations in various CFTR domains, probably acting via structural allostery, suggesting a mechanistic framework for their broad application.

Ablation of the large tumour suppressor kinases 1 and 2 promotes a luminal breast cell phenotype through stabilization of oestrogen receptor-α, thereby changing human breast cell fate.

There has been increasing interest in the development of cellular behavior models that take advantage of three-dimensional (3D) cell culture. To enable assessment of differential perturbagen impacts on cell growth in 2D and 3D, we have miniaturized and adapted for high-throughput screening (HTS) the soft agar colony formation assay, employing a laser-scanning cytometer to image and quantify multiple cell types simultaneously. The assay is HTS compatible, providing high-quality, image-based, replicable data for multiple, co-cultured cell types. As proof of concept, we subjected colorectal carcinoma colonies in 3D soft agar to a mini screen of 1528 natural product compounds. Hit compounds from the primary screen were rescreened in an HTS 3D co-culture matrix containing colon stromal cells and cancer cells. By combining tumor cells and normal, nontransformed colon epithelial cells in one primary screening assay, we were able to obtain differential IC50data, thereby distinguishing tumor-specific compounds from general cytotoxic compounds. Moreover, we were able to identify compounds that antagonized tumor colony formation in 3D only, highlighting the importance of this assay in identifying agents that interfere with 3D tumor structural growth. This screening platform provides a fast, simple, and robust method for identification of tumor-specific agents in a biologically relevant microenvironment.

Reporter assays are commonly used for high-throughput cell-based screening of compounds, cDNAs, and siRNAs due to robust signal, ease of miniaturization, and simple detection and analysis. Among the most widely used reporter genes is the bioluminescent enzyme luciferase, which, when exposed to its substrate luciferin upon cell lysis, yields linear signal over a dynamic range of several orders of magnitude. Commercially available luciferase assay formulations have been developed permitting homogeneous, single-step cell lysis and reporter activity measurements. Assay conditions employed with these formulations are typically designed to minimize well-to-well luminescence variability due to variability in dispensing, evaporation, and incomplete sample mixing. The authors demonstrate that incorporating a microplate orbital mixing step into 96- and 384-well microplate cell-based luciferase reporter assays can greatly improve reporter readouts. They have found that orbital mixing using commercially available mixers facilitates maximal luciferase signal generation from high cell density–containing samples while minimizing variability due to partial cell lysis, thereby improving assay precision. The authors fully expect that widespread availability of mixers with sufficiently small orbits and higher speed settings will permit gains in signal and precision in the 1536-well format as well.

The cataloguing of the human genome has provided an unprecedented prospectus for target identification and drug discovery. A current analysis indicates that slightly more than 3000 unique protein encoding loci are potentially amenable to pharmacological intervention (the ‘druggable genome’, which can be queried at http://function.gnf.org/druggable). However, the assessment of genome sequence data has not resulted in the anticipated acceleration of novel therapeutic developments. The basis for this shortfall lies in the significant attrition rates endemic to preclinical/clinical development, as well as the often underestimated complexity of gene function in higher order biological systems. To address the latter issue, a number of strategies have emerged to facilitate genomics-driven target identification and validation, including cellular profiling of gene function, in silico modelling of gene networks, and systematic analyses of protein complexes. The expectation is that the integration of these and other systems-based technologies may enable the conversion of potential genomic targets into functionally validated molecules, and result in practicable gene-based drug discovery pipelines.