A novel high-content phenotypic screen to identify inhibitors of mitochondrial DNA maintenance in trypanosomes

Kinetoplastid parasites cause diverse neglected diseases in humans and livestock, with an urgent need for new treatments. The survival of kinetoplastids depends on their uniquely structured mitochondrial genome (kDNA), the eponymous kinetoplast. Here, we report the development of a high-content screen for pharmacologically induced kDNA loss, based on specific staining of parasites and automated image analysis. As proof of concept, we screened a diverse set of ∼14,000 small molecules and exemplify a validated hit as a novel kDNA-targeting compound

Application of a high-content screening assay utilising primary human lung fibroblasts to identify antifibrotic drugs for rapid repurposing in COVID-19 patients

Lung imaging and autopsy reports among COVID-19 patients show elevated lung scarring (fibrosis). Early data from COVID-19 patients as well as previous studies from severe acute respiratory syndrome, Middle East respiratory syndrome, and other respiratory disorders show that the extent of lung fibrosis is associated with a higher mortality, prolonged ventilator dependence, and poorer long-term health prognosis. Current treatments to halt or reverse lung fibrosis are limited; thus, the rapid development of effective antifibrotic therapies is a major global medical need that will continue far beyond the current COVID-19 pandemic. Reproducible fibrosis screening assays with high signal-to-noise ratios and disease-relevant readouts such as extracellular matrix (ECM) deposition (the hallmark of fibrosis) are integral to any antifibrotic therapeutic development. Therefore, we have established an automated high-throughput and high-content primary screening assay measuring transforming growth factor-β (TGFβ)-induced ECM deposition from primary human lung fibroblasts in a 384-well format. This assay combines longitudinal live cell imaging with multiparametric high-content analysis of ECM deposition. Using this assay, we have screened a library of 2743 small molecules representing approved drugs and late-stage clinical candidates. Confirmed hits were subsequently profiled through a suite of secondary lung fibroblast phenotypic screening assays quantifying cell differentiation, proliferation, migration, and apoptosis. In silico target prediction and pathway network analysis were applied to the confirmed hits. We anticipate this suite of assays and data analysis tools will aid the identification of new treatments to mitigate against lung fibrosis associated with COVID-19 and other fibrotic diseases

A multicomponent reaction platform towards multimodal near-infrared BODIPY dyes for STED and fluorescence lifetime imaging

We report a platform combining multicomponent reaction synthesis and automated cell-based screening to develop biocompatible NIR-BODIPY fluorophores. From a library of over 60 fluorophores, we optimised compound NIRBD-62c as a multimodal probe with suitable properties for STED super-resolution and fluorescence lifetime imaging. Furthermore, we employed NIRBD-62c for imaging trafficking inside cells and to examine how pharmacological inhibitors can alter the vesicular traffic between intracellular compartments and the plasma membrane

A synergistic anti-cancer FAK and HDAC inhibitor combination discovered by a novel chemical-genetic high-content phenotypic screen

We mutated the focal adhesion kinase (FAK) catalytic domain to inhibit binding of the chaperone Cdc37 and ATP, mimicking the actions of a FAK kinase inhibitor. We re-expressed mutant and wild-type FAK in squamous cell carcinoma (SCC) cells from which endogenous FAK had been deleted, genetically fixing one axis of a FAK inhibitor combination high-content phenotypic screen to discover drugs that may synergize with FAK inhibitors. Histone deacetylase (HDAC) inhibitors represented the major class of compounds that potently induced multiparametric phenotypic changes when FAK was rendered kinase-defective or inhibited pharmacologically in SCC cells. Combined FAK and HDAC inhibitors arrest proliferation and induce apoptosis in a sub-set of cancer cell lines in vitro and efficiently inhibit their growth as tumors in vivo. Mechanistically, HDAC inhibitors potentiate inhibitor-induced FAK inactivation and impair FAK-associated nuclear YAP in sensitive cancer cell lines. Here we report the discovery of a new, clinically actionable, synergistic combination between FAK and HDAC inhibitors

Naturally Inspired Peptide Leads: Alanine Scanning Reveals an Actin-Targeting Thiazole Analogue of Bisebromoamide

Systematic alanine-scanning of the linear peptide bisebromoamide (BBA), isolated from a marine cyanobacterium, is enabled by targeting the solid phase peptide synthesis of thiazole analogues. The synthetic Tz-BBA analogues have comparable cytotoxicity (nM) to bisebromoamide and cellular morphology assays indicate that they target the actin cytoskeleton. Pathway inhibition in human colon tumour (HCT-116) cells has been explored using reverse phase protein array (RPPA) analysis, which shows a dose-dependent response of IRS-1 expression. Alanine-scanning reveals a structural dependence to the cytotoxicity, actin-targeting and pathway inhibition, and allows a new readily-synthesised lead to be proposed.Johnston, Heather; Boys, Sarah; Makda, Ashraff; Carragher, Neil; Hulme, Alison. (2016). Naturally Inspired Peptide Leads: Alanine Scanning Reveals an Actin-Targeting Thiazole Analogue of Bisebromoamide, [dataset]. University of Edinburgh. School of Chemistry.. http://dx.doi.org/10.7488/ds/1417