title: Use of a Machine Learning-Based High Content Analysis Approach to Identify Photoreceptor Neurite Promoting Molecules.
abstract: High content analysis (HCA) has become a leading methodology in phenotypic drug discovery efforts. Typical HCA workflows include imaging cells using an automated microscope and analyzing the data using algorithms designed to quantify one or more specific phenotypes of interest. Due to the richness of high content data, unappreciated phenotypic changes may be discovered in existing image sets using interactive machine-learning based software systems. Primary postnatal day four retinal cells from the photoreceptor (PR) labeled QRX-EGFP reporter mice were isolated, seeded, treated with a set of 234 profiled kinase inhibitors and then cultured for 1 week. The cells were imaged with an Acumen plate-based laser cytometer to determine the number and intensity of GFP-expressing, i.e. PR, cells. Wells displaying intensities and counts above threshold values of interest were re-imaged at a higher resolution with an INCell2000 automated microscope. The images were analyzed with an open source HCA analysis tool, PhenoRipper (Rajaram et al., Nat Methods 9:635-637, 2012), to identify the high GFP-inducing treatments that additionally resulted in diverse phenotypes compared to the vehicle control samples. The pyrimidinopyrimidone kinase inhibitor CHEMBL-1766490, a pan kinase inhibitor whose major known targets are p38α and the Src family member lck, was identified as an inducer of photoreceptor neuritogenesis by using the open-source HCA program PhenoRipper. This finding was corroborated using a cell-based method of image analysis that measures quantitative differences in the mean neurite length in GFP expressing cells. Interacting with data using machine learning algorithms may complement traditional HCA approaches by leading to the discovery of small molecule-induced cellular phenotypes in addition to those upon which the investigator is initially focusing.
authors.affiliation: Ophthalmology, Molecular Biology & Genetics, Neuroscience, and Institute of Genetic Medicine, Wilmer Eye Institute, Johns Hopkins University School of Medicine, 400 N Broadway, Smith 3001, 21231, Baltimore, MD, USA. jfulle19@jhmi.edu.
authors.forename: John A
authors.initials: JA
authors.lastname: Fuller
authors.affiliation: Ophthalmology, Molecular Biology & Genetics, Neuroscience, and Institute of Genetic Medicine, Wilmer Eye Institute, Johns Hopkins University School of Medicine, 400 N Broadway, Smith 3001, 21231, Baltimore, MD, USA. cdoughe1@jhmi.edu.
authors.forename: Cynthia A
authors.initials: CA
authors.lastname: Berlinicke
authors.affiliation: NCATS, NHGRI, National Institutes of Health, 9800 Medical Center Drive, MSC 3370, 20850, Rockville, MD, USA. jinglese@mail.nih.gov.
authors.forename: James
authors.initials: J
authors.lastname: Inglese
authors.affiliation: Ophthalmology, Molecular Biology & Genetics, Neuroscience, and Institute of Genetic Medicine, Wilmer Eye Institute, Johns Hopkins University School of Medicine, 400 N Broadway, Smith 3001, 21231, Baltimore, MD, USA. donzack@gmail.com.
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Zack
mesh_terms: D000465:Algorithms; D000818:Animals; D058948:Cell Tracking / Q000379:methods*; D002478:Cells, Cultured; D019295:Computational Biology / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D000069550:Machine Learning; D008822:Mice, Transgenic; D016501:Neurites / Q000187:drug effects / Q000378:metabolism*; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020419:Photoreceptor Cells, Vertebrate / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D061251:Primary Cell Culture; D015203:Reproducibility of Results; D054852:Small Molecule Libraries / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D054852:Small Molecule Libraries; D049452:Green Fluorescent Proteins; D010770:Phosphotransferases
keywords: High content analysis; Imaging; Inhibitor; Machine learning; Neuritogenesis; Phenotypic screening; Photoreceptor; Protein kinase; qHTS