Large-scale cytological profiling for functional analysis of bioactive compounds

Abstract

Cytological profiling (CP) is an unbiased image-based screening technique that uses automated microscopy and image analysis to profile compounds based on numerous quantifiable phenotypic features. We used CP to evaluate a library of nearly 500 compounds with documented mechanisms of action (MOAs) spanning a wide range of biological pathways. We developed informatics techniques for generating dosage-independent phenotypic "fingerprints" for each compound, and for quantifying the likelihood that a compound's CP fingerprint corresponds to its annotated MOA. We identified groups of features that distinguish classes with closely related phenotypes, such as microtubule poisons vs. HSP90 inhibitors, and DNA synthesis vs. proteasome inhibitors. We tested several cases in which cytological profiles indicated novel mechanisms, including a tyrphostin kinase inhibitor involved in mitochondrial uncoupling, novel microtubule poisons, and a nominal PPAR-gamma ligand that acts as a proteasome inhibitor, using independent biochemical assays to confirm the MOAs predicted by the CP signatures. We also applied maximal-information statistics to identify correlations between cytological features and kinase inhibitory activities by combining the CP fingerprints of 24 kinase inhibitors with published data on their specificities against a diverse panel of kinases. The resulting analysis suggests a strategy for probing the biological functions of specific kinases by compiling cytological data from inhibitors of varying specificities.

Integration of high-content screening and untargeted metabolomics for comprehensive functional annotation of natural product libraries

“…Cell culture, extraction, library preparation, and crude extract fractionation were performed as described by Schulze et al (15). Briefly, bacterial strains were isolated from marine sediment collected by hand using SCUBA from coastal areas of Panama at depths of 5-30 m. Bacterial isolates were grown under standard fermentation conditions (16), extracted with 1:1 methanol/ dichloromethane, and fractionated on a reverse-phase C 18 column with an eleutropic series of water and methanol [20,40,60,80, and 100% (vol/vol) methanol in water, and an ethyl acetate wash]. These extracts were concentrated to dryness in vacuo and resuspended in 1 mL of DMSO.…”

“…Methods for cell culture and staining were used as previously reported (15,18). HeLa cells were plated into two clear-bottom 384-well plates at a target density of 2,500 cells per well.…”

“…The plates were incubated for 24 h under 5% CO 2 at 37°C, 150 nL of extract was pinned into the culture plates, and the plates were incubated for 19 h under 5% CO 2 at 37°C. The plates were then fixed and stained with either cell cycle or cytoskeletal stain sets, which report on the number of cells in S-phase or mitosis and amount and distribution of tubulin and actin, respectively (15,18). Both stain sets contained a nuclear stain (Hoechst), which was used to count the number of cells and segment the image.…”

“…The cytological profiling platform optimized by Schulze and coworkers characterizes the biological activities of extracts using untargeted phenotypic profiling. These phenotypic profiles are compared with natural products extracts and a training set of compounds with known modes of action to characterize the bioactivity landscape of the screening library (17,18). This cytological profiling tool forms the basis of the biological characterization component of the Compound Activity Mapping platform, as described below.…”

The Natural Product N-Palmitoyl-l-leucine Selectively Inhibits Late Assembly of Human Spliceosomes

“…To investigate how N-palmitoyl-L-leucine and its analogs affect cells, we used a cytological profiling assay (11,14). For the assay, HeLa cells cultured in 384-well plates were treated with test compounds or with DMSO alone for 20 h, after which they were fixed and stained with fluorescent probes targeting actin (phalloidin), tubulin (␣-tub), total DNA (Hoechst), newly synthesized DNA (EdU), and phosphohistone H3 (␣-pHH3).…”